Lithuanian University of Health Sciences Veterinary Academy

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Vytautė Šakienė

BIOTECHNOLOGICAL SOLUTIONS FOR PREPARATION OF SUSTAINABLE AND SAFE PLANT PROTEINS THROUGH THE USE OF LOW-WASTE AND/OR NON-WASTE TECHNOLOGIES

Doctoral Dissertation Agricultural Sciences, Zootechnics (03A)

Kaunas, 2018

Dissertation has been prepared at the Department of Food Safety and Quality of Veterinary Academy of Lithuanian University of Health Sciences during the period of 2014–2018.

Scientific Supervisor Prof. Dr. Elena Bartkienė (Lithuanian University of Health Sciences, Veterinary Academy, Agricultural Sciences, Zootechnics – 03A).

Dissertation is defended at the Zootechnics Research Council of the Veterinary Academy of Lithuanian University of Health Sciences:

Chairperson Assoc. Prof. Dr. Agila Daukšienė (Lithuanian University of Health Sciences, Agricultural Sciences, Zootechnics – 03A).

Members: Prof. Dr. Asta Racevičiūtė-Stupelienė (Lithuanian University of Health Sciences, Agricultural Sciences, Zootechnics – 03A); Dr. Violeta Razmaitė (Lithuanian University of Health Sciences, Agri- cultural Sciences, Zootechnics – 03A); Assoc. Prof. Dr. Vilma Kaškonienė (Vytautas Magnus University, Physical Sciences, Chemistry – 03P); Dr. Damian Escribano Tortosa (Autonomy University of Barcelona, Agricultural Sciences, Veterinary – 02A).

Dissertation will be defended at the open session of the Zootechnics Research Council of Lithuanian University of Health Sciences on the 7th of September, at 12:00 a.m. in Dr. S. Jankauskas Auditorium of the Veterinary Academy. Address: Tilžės 18, LT-47181 Kaunas, Lithuania. 2

LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS VETERINARIJOS AKADEMIJA

Vytautė Šakienė

BIOTECHNOLOGINIAI SPRENDIMAI TVARIŲ IR SAUGIŲ AUGALINIŲ BALTYMŲ IŠGAVIMUI TAIKANT BEATLIEKINES IR MAŽAATLIEKINES GAMYBOS TECHNOLOGIJAS

Daktaro disertacija Žemės ūkio mokslai, Zootechnika (03A)

Kaunas, 2018

Disertacija rengta 2014–2018 metais Lietuvos sveikatos mokslų universitete Veterinarijos akademijos Maisto saugos ir kokybės katedroje.

Mokslinė vadovė Prof. Dr. Elena Bartkienė (Lietuvos sveikatos mokslų universitetas, Vete- rinarijos akademija, Žemės ūkio mokslai, Zootechnika – 03A).

Disertacija ginama Lietuvos sveikatos mokslų universiteto Veterina- rijos akademijos Zootechnikos mokslo krypties taryboje:

Pirmininkė Doc. Dr. Agila Daukšienė (Lietuvos sveikatos mokslų universitetas, Žemės ūkio mokslai, Zootechnika – 03A).

Nariai: Prof. Dr. Asta Racevičiūtė-Stupelienė (Lietuvos sveikatos mokslų universitetas, Žemės ūkio mokslai, Zootechnika – 03A); Dr. Violeta Razmaitė (Lietuvos sveikatos mokslų universitetas, Žemės ūkio mokslai, Zootechnika – 03A); Doc. Dr. Vilma Kaškonienė (Vytauto Didžiojo universitetas, Fiziniai mokslai, Chemija – 03P); Dr. Damián Escribano Tortosa (Barselonos autonominis universitetas, Žemės ūkio mokslai, Veterinarija – 02A).

Disertacija ginama viešame Zootechnikos mokslo krypties tarybos posė- dyje 2018 m. rugsėjo 7 d. 12 val. Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos Dr. S. Jankausko auditorijoje. Adresas: Tilžės g. 18, LT-47181 Kaunas, Lietuva.

CONTENTS

ABBREVIATIONS ...... 8 INTRODUCTION ...... 9 1. LITERATURE REVIEW ...... 13 1.1. The Perspectives of the Lupine Seeds in Europe ...... 13 1.2. The Chemical Composition of Lupine Seeds ...... 13 1.3. The Processes of Increasing the Nutritional Value of Lupine Seeds...... 17 1.4. Lactic Acid Fermentation in Feed Industry – Formation of Desirable and Undesirable Compounds ...... 18 1.5. Technologies for Preparing Protein Isolates/Concentrates of Lupine Seeds ...... 22 1.6. Lupine Seeds as the Material for Animal Feed ...... 25 2. MATERIALS AND METHODS ...... 30 2.1. Investigation Venue ...... 30 2.2. Materials ...... 30 2.2.1. Plant Material...... 30 2.2.2. Microorganisms Used for Experiments ...... 30 2.3. The Lupine Wholemeal Biotreatment and Protein Isolation ...... 30 2.3.1. The Lupine Wholemeal Fermentation ...... 30 2.3.2. The Lupine Protein Isolation ...... 31 2.4. The Methods Used for Analysis of Lupine Seeds and Their Bioproducts .. 31 2.4.1. The Evaluation of Lupine Seeds Proximate Composition ...... 31 2.4.2. The Analysis of Fatty Acids Composition in Lupine Seeds ...... 32 2.4.3. The Analysis of Macro- and Microelements in Lupine Seeds ...... 32 2.4.4. The Analysis of Alkaloids Content in Lupine Seeds ...... 33 2.4.5. The Microbiological Analysis of Fermented Lupine Wholemeal .... 33 2.4.6. The Determination of Acidity Parameters of Fermented Lupine Wholemeal ...... 33 2.4.7. The Analysis of Amino Acid Profile in Lupine Wholemeal and Protein Isolates/Concentrates ...... 34 2.4.8. The Analysis of Biogenic Amines Content in Treated and Untreated Lupine Seeds ...... 35 2.4.9. The Determination of Protein and Protein Isolates/Concentrates of Lupine Seeds Wholemeal Digestibility in Vitro ...... 36 2.4.10. The Determination of the Total Phenolic Compounds Content and Antioxidants Properties of Lupine Products ...... 36 2.4.11. The Evaluation of the Lupine Protein Solubility and Content in Isolates/Concentrates ...... 36 2.4.12. The Determination of Molecular Weight of Lupine Protein Isolates/Concentrates by Applying Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) ...... 37 2.4.13. The Analysis of Isoflavones Content in Lupine Bioproducts ...... 37 2.5. The Statistical Analysis ...... 38

3. RESULTS AND DISCUSSION ...... 39 3.1. The Proximate Composition of Lupine Seeds ...... 39 3.2. The Effectiveness of Lupine Seeds Fermentation – Acidity Parameters and Lactic Acid Bacteria Count ...... 45 3.2.1. The Main Parameters of Fermentation Process ...... 54 3.3. The Lupine Protein Solubility at Different pH and Their Yield in Isolates/Concentrates ...... 56 3.4. The Influence of Fermentation on Molecular Weights of Lupine Protein Fractions ...... 66 3.4.1. The Main Lupine Protein Fractions ...... 77 3.5. The Modulation of Amino Acids Profile in the Lupine Seeds Wholemeal and Protein Isolates/Concentrates ...... 78 3.5.1. The Total Amino Acids Content in Lupine Wholemeal Samples .... 78 3.5.2. The Free Amino Acids Content in Lupine Wholemeal Samples ...... 94 3.5.3. The Amino Acids Profile in Untreated and Biotreated Lupine Protein Isolates/Concentrates ...... 111 3.5.4. The Essential and Nonessential Amino Acids – Possibility to Modulate Their Profile in Lupine Seeds wholemeal and Their Isolates/Concentrates ...... 129 3.6. The Influence of Technological Factors on Formation of Biogenic Amines in Lupine Products ...... 132 3.6.1. The Biogenic Amines Content in Untreated and Biotreated Lupine Wholemeal ...... 132 3.6.2. The Influence of Fermentation on the Biogenic Amines Content in Lupine Protein Isolates/Concentrates ...... 143 3.6.3. The Formation of Biogenic Amines in Plant Based Substrates and Factors Influencing Their Formation ...... 152 3.7.The Total Phenolic Compounds Content in Lupine Wholemeal and Protein Isolates/Concentrates and Their Antioxidant Properties ...... 155 3.7.1. The Formation of Antioxidant Properties Showing Compounds and Their Modulation by Using Fermentation ...... 160 3.8. Influence of Technological Factors on Isoflavones Content in Lupine Bioproducts ...... 162 3.8.1. Isoflavones in Lupine Seeds and Their Possible Changes During Technological Processes ...... 164 3.9. The Trypsin Inhibitors Activity in Lupine Products ...... 166 3.9.1. The Perspectives of Reduction of Trypsin Inhibitors in Plant Based Material ...... 168 3.10. The Digestibility In Vitro of Lupine Wholemeal Protein and Protein Isolates/Concentrates ...... 168 3.10.1. The Digestibility of Lupine Seeds Protein and Technologies to Improve It ...... 171 CONCLUSIONS ...... 173 PRACTICAL RECOMMENDATIONS ...... 176

REFERENCES ...... 177 PUBLICATIONS ...... 203 SUMMARY ...... 301 ANNEX ...... 331 CURRICULUM VITAE ...... 333 ACKNOWLEDGEMENTS ...... 334

ABBREVIATIONS

AA – Amino Acids ANFs – Antinutritional Factors BAs – Biogenic Amines BIOR – Institute of Food Safety, Animal Health and Environment B.w. – Body Weight CAD – Cadaverine CFU – Colony-Forming Units DM – Dry Matter DW – Dry Water GC/MS – Gas Chromatography – Mass Spectrometry HPLC-MS/MS – High-Performance Liquid Chromatography – Mass Spectrometry HIS – Histamine GC-FID – Gas Chromatography-Flame Ionization Detector TRP – Tryptamine PEA – Phenylethylamine VAL – Valine ALA – Alanine GLY – Glycine ILE – Isoleucine LEU – Leucine THR – Threonine HIS – Histamine TYM – Tyramine KTU – Kaunas University of Technology LAB – Lactic Acid Bacteria Ls – Lactobacillus sakei LUHS VA – Lithuanian University of Health Sciences Veterinary Academy MRS – de Man Rogosa Sharpe Pa – Pediococcus acidilactici PHE – Phenylethylamine Pp – Pediococcus pentosaceus PUT – Putrescine SMF – Submerged Fermentation SPRM – Spermine SPRMD – Spermidine SSF – Solid State Fermentation NF – Nonfermented samples TIA – Trypsin inhibitors activity TTA – Total Titratable Acidity SER – Serine PRO – Proline ASP – Asparagine TYR – Tyramine MET – Methionine LYS – Lysine 8

INTRODUCTION

Global climate change will present ever increasing challenges in marginal, already-stressed agricultural ecosystems; also, the climate change is another factor affecting the goal of feeding the world [1–4]. Globally, 76% of the population derives most of their daily protein obtained from plants [4]. Nevertheless, many people are suffering from a lack or protein malnutrition, especially children [5]. With projected population growth to 9.5 billion by 2050 [6], alongside dietary and demographic changes, future nutritional demands may overwhelm global production of crop [7]. Animal proteins have a competitive advantage over plant-based proteins in terms of their nutritional and functional properties; protein ingredient market is intensively seeking for alternative, underutilized sources of concentrated plant proteins in order to satisfy the demands of consumers with different ethnic, religious, dietary and moral preferences associated with consumption of animal-based products. The demand of the ever-growing world population for protein foods is no longer sustainable through animal products alone. To compensate this deficiency, soya bean has become the prevalent source of plant proteins used for food/feed. Plant proteins are generally of a lower nutritional quality compared to animal proteins due to limited essential amino acids (AA) (lysine in cereals, methionine in legumes) and poor digestibility [8, 9], while animal proteins such as eggs and meat are highly digestible [9]. In recent years, the interest in plant protein sources that could replace soybean meal used for animal nutrition has increased. Soybean meal is the most common plant protein source in animal nutrition. It can be used as the only protein source in diets due to the low level of antinutritional substances and high level of crude protein [10], as well as good amino acid profile, however, soybean is genetically modified [11]. Lupine seeds is a significant alternative to soybean in animal nutrition [12], and the growth of plant protein utilisation has led to the development of new technologies, created to increase bio-availability of plant based protein. There are numerous reasons for increasing global demand of novel, sustainable sources of proteins. Moreover, a high consumption of animal proteins increases gas emission and thus represents an ecological issue [13]. To compare plant protein and animal protein production , the latter is more expensive due to water and energy resources; therefore it is important to find a balance between animal and plant protein in sustainable food/feed systems [14]. The growing demand of plant protein isolates/ concentrates is based on their good functional and technological properties, such as solubility, foaming properties, emulsion stability and viscosity. The

9 functional properties of protein can be affected by different protein extraction techniques, protein composition etc. Accordingly, Europe has become heavily dependant on soya bean imports, entailing trade agreements and quality standards that do not fully satisfy the European citizen’s expectations, hence lupine could be established as fundamental crop in various agro-climatic zones and marginal lands of Europe, and their yields and adaptation could be genetically improved to ensure the continuous supply of high-quality grain [15]. Lupine, an autochthonous European legume crop, represents a significant alternative to soya bean due to the following characterisics: it has elevated high-quality protein content (up to 44%); it is well accepted by European consumers, partially as it is not genetically modified; it has high potential in health benefits; and it is well suited for sustainable cultivation. Lupine is a successful protein crop in Australia, where an important industry related to lupine protein has spawned. However, its cultivation in Baltic countries remains insufficient to guarantee a steady supply to the food/feed industry, which in turn must be developed and innovated to produce attractive lupine-based protein-rich food/feed. Yellow lupines were bred in Lithuania until 1995. The narrow- leaved lupine breeding program took off in 1995. The breeding was done in three directions: first, low-alkaloid narrow-leaved lupine varieties were bred for food industry, second – low-alkaloid narrow-leaved lupines were bred for animal feeding, and third – narrow lupines were bred for green manure. The use of low alkaloid narrow-leaved lupine varieties for production of higher value food would be very important for all Baltic region countries. Lupine is a source of seeds that are rich in protein and could be used for animal feeding. However, the high content of alkaloids (from 5 g/kg to 40 g/kg) has been one of the factors limiting the use of white lupine (L. albus) in the past. Alkaloids are compounds that have negative effects on feed intake and nutrient utilisation. Over recent decades, plant breeders have succeeded in developing lupine cultivars with alkaloid content which is close to zero (0.08 g/kg to 0.12 g/kg) [16]. Also, lupine can be considered as an alternative protein-rich crop, capable of promoting socio-economic growth and environmental benefits in Europe [15]. Lupine seeds contain large amount of soluble and insoluble non-starch polysaccharides, oligosaccharides, phytates and tannins that have anti-nutritional effects including reduced digestion and absorption of amino acids [17, 18]. The antinutritional factors (ANFs), such as trypsin inhibitors, phytic acid, saponins, heamagglutinins and tannins are undesirable components in legumes that could hinder utilization of important minerals, particularly calcium, magnesium, iron and zinc. It interferes with their absorption and utilization and thereby contributes to mineral deficiency [19]. Processing of legumes can 10 reduce ANFs. Milling can reduce the levels of iron, zinc and phytate, however, the remaining iron and zinc is more bioavailable; fermentation can degrade phytate and enchance iron and zinc absorption because during the fermentation process low molecular weight organic acids are produced [20– 22]. The isolation and/or concentration of plant proteins reduce ANFs, also the extraction of proteins can improve digestibility, which is similar to animal-based protein [9].

The Aim of the Study

The aim of this work was to evaluate detailed chemical composition of the lupine seed varieties bred newly and locally; and to develop high-value, sustainable and safe plant protein stock/products by applying biotechnological treatment for whole seed (non-waste technology) and protein isolates/concentrates (low-waste technology).

Objectives of the Study

1. To select samples from the lupine seed varieties that are newly bred in Lithuania and contain the lowest concentration of alkaloids, and to evaluate their detailed chemical composition. 2. To select the most effective conditions of treatment to increase protein digestibility and reduce activity of trypsin inhibitors, by applying submerged and solid state fermentation with bacteriocins that produce lactic acid bacteria strains. 3. To evaluate the influence of technological factors on the changes of total phenolic compounds and isoflavones contents, as well as on antioxidant properties of the treated substrate. 4. To perform protein isolation/concentration by analysing their solubility at different pH conditions to obtain the highest protein yield. 5. To evaluate the influence of microbial hydrolysis on the amino acids profile and molecular weight of protein in lupine seeds wholemeal and protein isolates/concentrates. 6. To indicate the influence of technological factors on biogenic amines and formation of D(–) lactic acid isomers in lupine bioproducts.

The Scientific Novelty and Practical Usefulness

The combination of innovative and sustainable processing proposed in this disertation work will allow the researcher to select newly bred lupine varieties and microorganisms used to produce highly proteinaceous products 11 with improved properties, as well as potentially novel products with high biofunctionality. Sustainable agriculture that could satisfy the growing European and Global demand for food/feed is becoming a manifest neces- sity in the light of the concerns of growing population, land-use and food/ feed safety. The results of this work will reformulate the prototypes of food/feed stock with high biological value. The newly developed stock with high biological value and their preparation technologies are expected to rapidly reach the populations of defined niche, those who are rejecting the consumption of genetically modified plants and those who prefer convenient and natural food. The main consideration of this scientific novelty and practical benefits of this work are the following: the food/feed stock of higher value can be produced in an environmentally and socio-economically sustainable and beneficial manner, ensuring its safety at global level and its acceptance by the consumer, resulting in impact on the overall sustainability of our society.

1. LITERATURE REVIEW

1.1. The Perspectives of the Lupine Seeds in Europe

The most important lupine varieties for the food and feed industries are L. angustifolius, L. albus, L. luteus and L. mutabilis [23]. Unlike soybean, lupine seeds can be grown in moderate European climate [15]. The technological properties of lupine seeds are suitable for industrial processing [24]. It is reported in various publications that lupine is high in protein (20– 48%), high in fibre,low in fat and has negligible amount of starch; also, lupine seeds are good sources of vitamins, minerals and bioactive compounds [25–31]. Röder et al. [32] reported that protein content in blue lupine seed is 22–29.8%, 30.1–31.2% in white lupine and 35.2–38.1% in yellow lupine. The high protein content is mainly composed of seed storage proteins, particularly globulins and represent approximately 87% of the total protein content [33]. Seed storage proteins (α-conglutin, β-conglutin, γ-conglutin, and δ-conglutin) account for the main protein fractions in legumes [27, 33, 34]. The globulins are further subdivided into α-conglutin (3–4%), β-conglutin (43–45%), γ-conglutin (5–6%), and δ-conglutin (3– 13%) [27, 33, 34]. However, lupine can cause severe allergic reactions [35– 37], and Jimenez-Lopez et al. [38] reported that β-conglutin has been identified as a major allergen in L. angustifolius seeds. Lupine, an autochthonous European legume crop, represents a significant alternative to the soya bean for the following benefits: it is rich in high- quality protein content; it is well accepted by European consumers as it is not genetically modified; it has a good potential in health benefits; and it is highly suitable for sustainable cultivation. Lupine is a successful protein crop in Australia, where it has spawned an important industry, related to lupine protein. However, its cultivation in European countries remains insufficient to ensure a steady supply to the food/feed industry, which in turn must be developed and innovated in order to produce attractive lupine- based protein-rich food/feed.

1.2. The Chemical Composition of Lupine Seeds

The main nutrients of lupine seeds. Bähr et al. [39] has reported that lupine seeds can be an alternative to soya, as they contain comparable amounts of protein of a similar AAs profile, but higher fiber content, compared to beans, which is favorable from the dietary point of view for humans, but not for animals. The lupine seeds typically involve 33–47% of

13 proteins and 6–13% of oil [40, 16], while the content in dietary fiber ranges from 34–39% [41]. In comparison with other bioactive compounds present in lupine seeds, phenolic compounds are primarily responsible for their antioxidant capacity [42]. The most abundant phenolic compounds detected in lupine seeds belong to the subclasses of phenolic acids, flavones and isoflavones [43]. Lupine is rich in phytochemicals (polyphenols, phytosterols, squalene) compared to other legumes [44]. Fiber and protein in lupine seeds are associated with prevention of chronic diseases and improvement of animal health [42]. The digestible carbohydrate content in lupine is lower than in most legumes and comprises oligosaccharides mostly, whereas starch is absent or scarce. The fat content is variable, falling in the interval of 8–12% depending on species, with a high presence of α-linolenic acid (about 8–10% of the oil) [45, 46]. The polysaccharide content in lupine cotyledons mainly consists of galactan and the hull consists of cellulose and/or hemicellulose mostly. The lupine hull comprises 25% of the whole seed and is low in lignin [48]. Lupine kernel is also an excellent source of fibre, containing up to 39% of fibre, composed of 75–80% of soluble fibre, 18–25% of insoluble fibre, and 5–9% of hemicellulose [39]. Apart from its useful nutritional features, it is claimed that lupine is beneficial to management of hyperglycemia [47], prevention of hyperten- sion [49] and cholesterol lowering [50]; what is more, protein seems to be relevant to these beneficial effects [25]. Antinutritional compounds and/or factors (ANF) in lupine seeds. The bioactive phytochemicals in legumes include: enzyme inhibitors, phytoestrogens, oligosaccharides, phytosterols, saponins, phytates, phenolic acids and flavanoids. The proteinaceous antinutritonal factors (ANF) include lectins, protease (trypsin, chymotrypsin) and amylase inhibitors and lipoxy- genase. Non-proteinaceous compounds include phytic acid, α-galactosides, phenolics, tannins, saponins, cyanogens and toxic AAs [4]. The amount of ANFs, such as alkaloids, saponins, tannins, phytate, trypsin inhibitors (TIs) and lectins is lower in lupine than in other legumes [51]. The polysaccharide content in lupine cotyledons mainly consists of galactan and the hull mainly consists of cellulose or hemicelluloses [52]. Flavonoids and phenolic acids are rich in antioxidant and good physiological and biological properties [53], however, phytic acid is one of the most ANFs in plants. The antinutritional activity of phytic acid can be eliminated by adding phytase. Phytic acid or phytate in legume seeds is bound with phosphorus, calcium and magnesium, trace elements, such as iron and zinc, protein compounds and AAs [54]. Saponins are amphiphilic compounds composed of saccharide chains (hexoses and pentoses) soluble in polar water with a non-polar (fat soluble) 14 aglycones attached to them [55]. Its glycosylated compounds or glycosides are divided into three main groups, according to the carbon skeleton, of non- polar aglycone region: triterpenoidal glycosides, steroidal glycosides and steroid-alkaloid glycosides [56]. Lupine, beans and peas are the main sources of dietary saponins [57]. Trypsin inhibitors (TIs) are one of the most relevant ANF, because they reduce digestion and absorption of dietary proteins [58]. TIs strongly inhibit the activity of key pancreatic enzymes, particularly trypsin and chymotrypsin, thereby reducing digestion and absorption of dietary proteins by the formation of complexes that are indigestible even in the presence of high amounts of digestiveenzymes [59]. Other ANFs in lupine seeds are alkaloids. The varieties of L. albus with favorable agronomic characteristics contain toxic quinolizidine alkaloids (1.9–2.7%), which are not suitable for animal consumption [60]. The ANF presence in seeds limits levels of inclusion of legume seeds in diets, especially for young, growing animals [61, 62]. Total alkaloid content in sweet white lupine cultivars has been significantly reduced during the process of domestication and breeding and currently does not exceed 0.02% [63]. Analysis of different qualitative composition of major alkaloids in the seeds of narrow-leafed lupine and was performed, with then following results (% of total alkaloids): lupanine – 46.4%, hydroxylupanine – 35.6%, angustifoline – 15.5%, and α-isolupanine – 2.5% [64]. Under the environmental condition of high temperature, the accumulation of alkaloids may vary and the alkaloid quantity in sweet lupine seeds may exceed the alkaloid limit for using lupine seeds as a feedstuff; however, pigs are particularly sensitive to alkaloids [65]. Apart from possible reduction of alkaloids during fermentation process, further benefits, such as physiological effects of lactic acid silages of moist lupine grains are the following: the positive effects of lactic acid in terms of physiology of nutrition, for example, the acidification of certain parts of the digestive tract to prevent the proliferation of clostridia and other pathogenic microorganisms; the improvement of the feed quality due to the reduction of other ANF such as oligosaccharides [66, 67]; the elevated contents of essential AA following proteolysis (only when desmolysis can be prevented) [68]; the improvement of feed intake and digestion [69]; the improvement of digestibility of AA (demonstrated for LYS and MET [69]; the possibility to upgrade the quantity of lupine grains in daily ration (broiler, 10%; weaned piglets, 12%) resulting in lower consumption of soy products [70]. Lupine (L. angustifolius) has been identified as a potential source of protein of locally produced plants that could be fed to animals at a positive economic profit and could replace soybean oilcake meals as a raw material 15 in ostrich diets. Currently, the price of L. angustifolius is estimated to be 56% of the value of soybean oilcake meal, making it a worthwhile economic alternative. If nutrition expenses could be reduced, this would have a major impact on the profits of the commercial ostrich production enterprise. Concerns have been raised due to the high presence of alkaloids in lupine seeds. Alkaloids are compounds that have a bitter taste which reduces the palatability of the feed. However, there are sweet (low in alkaloids, <0.1%) and bitter (alkaloid-rich, 0.1–4%) varieties within the species [71]. Nonethe- less, lupine can still be included only up to certain levels to be utilized efficiently and to prevent undesirable effects [72]. Lupanine, 13α-hydroxylupanine and angustifoline are the main alkaloids of L. angustifolius seeds [73]. Other group of specific compounds in lupine seeds are phytoestrogens. Phytoestrogens are non-steroidal plant compounds and have structure similar to mammalian estrogen (17-β estradiol). Phytoestrogens and their mammalian metabolites can bind to estrogen receptors found in animal and human cells, and cause a weak estrogenic or anti-estrogenic effect. Intake of phytoestrogens may impair sheep fertility, whereas effects in cattle reproduction are not consistent. Isoflavones and lignans are metabolised to equol and enterolactone in rumen [74]. Isoflavones exist in plant tissues as the variety of O- or C-glycosylated derivatives, often acylated with malonyl group on sugar moieties. Free aglycones are released in the cells that are under abiotic or biotic stress [75]. Genistein belongs to isoflavones, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants [76]. The Chemical structure of main isoflavones present in lupine seeds is given in figure 1.2.1. Genistein is a phyto- estrogen, a family of plant-derived compounds that exhibit effects similar to, albeit weaker than, those of mammalian estrogens [77]. Schreihofer et al. [78] has reported that genistein protects neurons from cerebral ischemic injury in rat hippocampus, thus exerting neuroprotective effects in stroke- like injury in vitro. In the brain, genistein can improve spine thickness, as well as their cognitive function, synapse development and regulate the transcription factor of neurotrophic genes in the hippocampal region of adult animals [79]. Also, isoflavones exhibit antifungal activities [80].

Biochanin-A Daidzein

Genistein Formononetin

Fig. 1.2.1. The chemical structure of main isoflavones present in lupine seeds

1.3. The Processes of Increasing the Nutritional Value of Lupine Seeds

The nutritional value of legumes depends upon the processing methods, presence or absence of anti-nutritional and/or toxic factors and possible interaction of nutrients with other food/feed components [81]. Roasting improves colour, extends life of shelf, enhances flavor, reduces the ANFs and denatures proteins thereby improving their digestibility [82]. Neverthe- less, treatments such as heat, germination, soaking and fermentation have been reported to reduce the ANFs [83]. It could lead to conclusion that the methods of mechanical, physical and biological processing can help to improve the nutritional value of lupine seeds used in pig nutrition [84, 85], or even affect the nutritional value of complex diets. Other studies have shown that the reduction of the particle size [86], germination [87], hydro- thermal treatment [88] or dehulling [89] can improve nutrient digestibility of lupine seeds in the ileal and total tract in pigs. Despite the above- mentioned information regarding the nutrient digestibility of differentially processed blue lupine meals, there is a lack of information regarding the breakdown of NSP structures and the resulting proximate nutrient, AA and NSP digestibility in complex diets along with gastrointestinal tract of the pig. As indicated by Zijlstra et al. [90], feed processing and enzyme technologies can be valuable tools to enhance digestive utilization of nutrients in NSP-rich feedstuffs. Rutkowski et al. [91] has published that the use of extrusion of yellow lupine seeds had led to relatively minimal changes in nutrient composition, including AAs and ANFs.

1.4. Lactic Acid Fermentation in Feed Industry – Formation of Desirable and Undesirable Compounds

Lactic acid bacteria (LAB) is a wide variety of bacteria that can produce bacteriocins, and it is mainly Gram-positive [92]. LAB are classified as either homofermentative or heterofermentative based on the metabolism of glucose. Although the terms are typically applied to LAB, many other organisms that generate lactic acid share the features of the same pathways and can be considered to be homofermentative or heterofermentative. As the name implies, a homofermentative lactic acid process can potentially generate lactate [92]. Fermentation is an easy and economic method to improve nutritional value of feed [93]. Lactobacilli is the most important bacteria for industrial applications related to feed and animal health [94], as well as, fermentation is a major process used in the production of foods from soybeans [95]. Fermentation leads to changes of the physicochemical and sensory properties (colour, flavour) and bioactive compounds of soybean [96]. Fermentation reduces undesirable compounds in legumes and other feed ingredients [97] and lowers the content of flatulence-causing factors in legumes; what is more, it increases protein concentration and improves protein digestibility through the hydrolysis of high-molecular- weight proteins into peptides and AA [98]. According to Amadou et al. [100], fermentation increases trypsin digestibility in vitro and nitrogen solubility under alkaline conditions. A wide variety of microorganisms, mainly yeasts and fungi, are used in the fermentation process. LAB, including Lactobacilllus, Lactococcus, Streptococcus, Leuconostoc and Pediococcus, are also applied due to their unique characteristic to formate precursors of aromatic compounds and texture forming compounds [99, 100]. During the fermentation of plant based substrates, the growth of LAB enhances conversion of phenolic compounds such as flavonoids to biologically active metabolites via expression of glycosyl hydrolase, esterase, decarboxylase, and phenolic acid reductase [101]. The subsequent reaction of these metabolites with anthocyanidins results in formation of pyrano- anthocyanidins or 3-desoxypyranoanthocyanidins [102]. Some of these alkyl catechols potently activate Nrf2 (NFE2L2), the main regulator of response to oxidative stress in mammalian cells and thereby induce the expression of antioxidant and detoxifying enzymes protecting against oxidative and chemical damage. Additionally, fermentation can result in the removal of toxic or undesirable constituents such as phytic acid. This plant- associated, anti-nutritional compound chelates divalent metal ions. Fermentation reduces the pH of fermentable substrate, which optimizes the activity of endogenous phytase thus removing the most phytic acid [103, 18

104]. AA and its derivatives together with neurotransmitter (e.g. g-ami- nobutyric acid) and immunomodulatory functions are also synthesized during fermentation [105]. Fermentation process can also result in the new compounds with potential health-modulating effects. In addition, fermentation can increase the total amount of acids, free AA, antioxidants and other biologically active substances [106]. Fermentation results in the degradation of plant cell walls, which can release antioxidants [107, 108]. Modifi- cation of proteins also can be performed by using fermentation processes. For this reason, fermentation is a useful technology for increasing the amount of natural bioactive compounds [110]. The main microbial agents in food and feed technology are LAB, which play an important role in the modification of isoflavone conjugates. It is indicated in the literature sources that fermentation increases the bioavailability of isoflavones [111]. However, some technological processes can also result in significant losses of isoflavones [112]. Lactic acid is the main metabolite in LAB fermentations that is synthesized in amounts often exceeding 1%. Lactic acid was recently evi- denced to reduce pro-inflammatory cytokine secretion of toll-like receptor- activated, bone-marrow-derived macrophages and dendritic cells in the dose-dependent manner [113, 114]. Lactic acid also alters redox status by reducing the burden of reactive oxygen species in intestinal enterocytes [114]. Through LAB metabolic activities (e.g., lipolysis and proteolysis), they also produce important aroma and ﬂavor compounds, while contribut- ing to the texture formation (e.g., by the production of exopolysaccharides) [115, 116]. Lactic acid imparts a sour taste which is an important sensory attribute of LAB fermentation [117]. Supplementation of LAB in neonatal piglets can regulate the formation of the piglet gut microflora, thus benefiting the health of piglets [118]. LAB can inhibit pathogenic bacteria by competing for nutrients in the gut or for binding sites on the intestinal epithelium [119]. However, the metabolic activity of the LAB may also rise the formation of biogenic amines (BAs), and the concentration of AA has an effect on the overall formation of BAs [120]. From this point of view, the fermentation process should be controlled to minimize potential enhan- cement effects on BAs formation; in this case, safe microbial starters should be used [121]. The main metabolite of LAB is the lactic acid, which can be obtained as two optical isomers, particularly L(+) and D(-) and/or their mixture. Increased levels of D(-) isomer in plasma and urine have been demonstrated in cases of intestinal ischaemia, short bowel and appendicitis, and are considered as the indicator of dysbiosis and/or increased intestinal permeability [122]. Therefore, the desirable lactic acid isomer in fermentation of food 19 and feed is L(+) [123]. LAB also produce organic acids with several compounds, hydrogen peroxide, diacetyl, acetaldehyde, D(-) isomer of AA, reuterin and bacteriocins [124]. D(-) isomer can be produced during fermentation by using Lactobacillus spp., hence glucose and glycerol are the main sources of carbon used for fermentation of D(-) isomer [125]. The silage used for ruminant nutrition may contain considerable amounts of D(-) isomer [2]. However, the main fraction is metabolised or converted to the L(+) in rumen which seldom leads to D(-) acidosis and neurotoxity. D(-) isomers in the mammalian body are mainly of microbiological origin and they are often located in the digestive tract. Consequently, the control of D(- ) in feed is very important. Several analytical procedures for D(-) isomer have been introduced, but it is absolutely mandatory to distinguish this metabolite from the much more abundant and naturally occurring L(+) stereoisomer. When enzymatic analytical methods are used, it is consequently essential to eliminate the response from L(+) and the dehydrogenase of ubiquitous enzyme L(+), which will interfere with the D(–) isomer determination heavily [3]. The fermentation is very important in the process of ensiling because it affects the nutritional quality of the silage and the animal performance. If the fermentation is not effective, it will result in a completely spoiled feed that has potential risk for animal health. Well- fermented silage can be used in diets for ruminant animals without any risk for their health and without compromising the productive performance [126]. The silage metabolites, such as organic acids (lactic acid, acetic acid etc.) formation is important for the fermentation quality of ensiled forage [127]. The main genera of LAB commonly associated with silage fermentation are Lactobacillus, Pediococcus, Leuconostoc and Lactococcus [126, 128]. The lactic acid production and the rate of pH decrease are responsible for the disappearance of enterobacterial and clostridial secondary fermentations. Others studies have shown that spontaneous fermentation results in higher concentrations of both acetic acid and BAs which adversely affect the palatability of fermented feed [129, 130]. However, it is not necessary to use spontaneous fermentation, as the quality of spontaneously fermented feed can be improved by adding copper to the fermentation medium which speeds up the production of lactic acid [131]. BA are non- volatile low-molecular-weight nitrogenous organic bases, derived through decarboxylation of corresponding AAs. They can be both formed and degraded as a result of normal metabolic activities in animals, plants and microorganisms. Putrescine and cadaverine are known to potentiate these effects. Moreover, these amines are thermo-stable [132]. BAs can produce a wide range of toxicological effects [133], histamine and tyramine being the main BA, regarding their toxic effect. BAs in the gastrointestinal tract are 20 important metabolites of dietary protein and AAs due to their support to digestive enzymes and microbes in gut, which play a crucial role in the regulation of intestinal functions, including digestion, absorption and local immunity. However, high concentrations of BAs can induce adverse reactions and are harmful to animal health [134]. Özyurt et al. [135] reported that due to the activity of various endogenous and bacterial decarboxylase enzymes, fish silage contains considerable levels of FAA that constitute the precursors for BAs. In fish silages, the concentration of spermidine, tryptamine, phenylethylamine and spermine was lower than the concentration of putrescine, cadaverine, histamine, tyramine and agmatine. Formation of BAs can be reduced by restricting fermentation in the silage or by achieving rapid acidification during the first phase of ensiling [136]. Bacterial proteolysis, the presence of BAs is associated with a decrease in the protein content and nutritional value of the silage. Negative effects of BAs on animal health have been reported; the BAs have been implicated to be causative factors in ketonemia, and absorption of ruminal histamine should be considered a major cause of systemic histaminosis in acidotic ruminants [138, 139]. Although hyperketonemia may result from consumption of silage with high content of butyric acid, high concentrations of putrescine or other amines in the feed may contribute to the development of ketonemia both in early and further period of lactation. BAs notably decrease palatability of silage and reduce dry matter intake and cattle performance [140]. However, Van Os et al. [137] and Mao et al. [141] have shown that in sheep adapted to silage with high levels of BAs, amine accumulation in the rumen is prevented by the increase in the amine degrading capacity of rumen microbes [142]. Formation of BAs can be affected by several factors such as temperature, rapidity of pH decrease during the initial stage of fermentation, and oxygen availability. Effects of BAs on cow health also differ depending on the composition of the mixture; TRP, PUT and CAD form a group of highly intercorrelated substances with similar effects [143]. Exogenous dietary putrescine can increase the growth rates of neonatal animals under nutritional stress. Dietary putrescine prevents damage to the intestinal mucosa and has beneficial effects on the height and width of villus in tissues [144]. Addition of putrescine could stimulate the intestinal epithelial DNA, RNA, and protein synthesis [145] and accelerate the development of small intestinal of ducks during the posthatch period [146]. Spermine modulates depression, shortens the immobility time of animals in a dosage-dependent manner, presumably by activating N-methyl D-aspartate receptors [147]. Supplementing newly hatched chicks with putrescine has shown no positive or even adverse effects on development of small intestine [148]. The proper concentration of 21 dietary protein and AA is very important to ensure optimal nutrition of livestock [134].

1.5. Technologies for Preparing Protein Isolates/Concentrates of Lupine Seeds

More than 90% of protein is considered to be isolate and lower than 90% of protein is considered to be concentrate [149], however, other opinion exists stating that lupine protein isolates contain >85% of protein and concentrates contain >60% of protein content. The main protein extraction processes can be classified into dry and wet methods. In general, wet extraction methods can be applied for preparing both: protein concentrates and isolates at levels of 70% and 90% protein and higher, respectively. However, it should be noted that currently there is no universal classification scheme which separates concentrate from an isolate for all the legumes. The various wet extraction processes include acid/alkaline extraction – isoelectric precipitation, ultrafiltration and salt extraction. Legume flour dispersed in aqueous solutions typically shows high solubility when subjected to alkaline or acidic extraction conditions at pH 8-10 and below pH 4, respectively [150, 151]. Important conditions for the development of plant-based alternatives are the latter: the availability of highly functional plant protein concentrates and isolates produced in a sustainable manner. Current protein extraction processes are inefficient due to the use of organic solvents, acids, bases and large amounts of water, resulting in little environmental gain which is even lower than theoretically possible [149]. Conven- tional plant protein production methods involve the use of solvents, concentrated acids and alkali that can result in denaturation of protein and loss of solubility, thereby reducing the quality and functionality of protein ingredients [152, 153]. There are two techniques usually applied in isolation of protein: the alkaline extraction with subsequent isoelectric precipitation and the salt-induced extraction followed by dilutive precipitation. The alkaline extraction and the salt-induced extraction techniques provide protein isolates with different properties [154]. The consistency of lupine protein precipitate obtained by applying isoelectric technique is rough, compact and curdy and its microstructure is unfolded and has large protein agglomerates, while protein precipitate obtained by applying dilutive precipitation is smooth, pasty, mellifluent at room temperature and features a fat-like texture [154, 155]. Muranyi et al. [156] compares alkaline extraction and isoelectric precipitation with salt-induced extraction followed by dilutive precipitation. It was shown that alkaline extraction has had a higher protein denaturation, and summarized that depending on the desired 22 properties of the products,it is an appropriate isolation technique. Muranyi et al. [156] has reported that lupine protein isolates extracted in alkaline solution have resulted in higher protein content (26.4–31.7%), compared to salt-induced extraction (19.8–30%) and combined alkaline salt-induced extraction (23.3-25.6%). Other Can Karaca’s et al. studies [157] have proven that isolates prepared from faba bean, chickpea, lentil, pea and soybean by applying an alkaline extraction using isoelectric technique have had higher overall protein content (85.6%), compared to those prepared applying a salt extraction method (78.4%). Moreover, it was reported that both legume source and protein extraction method along with their interaction have had significant effects on protein levels of the isolates, and also on physicochemical and emulsifying properties. The overall surface charge, solubility, hydrophobicity and creaming stability of isolates produced via isoelectric technique was higher, compared to isolates produced applying salt extraction. Acid extraction involves the preliminary extraction of proteins under acidic conditions. This technique could result in high solubilization of proteins prior to protein recovery (isoelectric technique, ultrafiltration), as proteins tend to be more soluble under acidic conditions below pH 4.0 [82]. Sussmann et al. [158] reported that protein isolation method based on salt-induced extraction followed by a dilutive precipitation has shown relevant processing parameters; different raw materials have been investigated systematically to obtain high yields of protein preparations with characteristic of fatlike textural properties. In the case of full-fat lupine ﬂakes, a protein yield of 38% was achieved. The sensory proﬁle of the lupine protein isolate revealed unique creamy, smooth and fatlike characteristics due to the formation of micellar aggregate [154, 159]. The membrane separation methods have shown protein isolates with higher functionality and were effective in reducing levels of anti-nutritional compounds, which include protease and amylase inhibitors, lectins and polyphenols [160, 161]. Ultrafiltration and microfiltration are membrane- based methods of fractionation using pressure as the driving force for separation [162]. For preparation of protein by using ultrafiltration, alkaline or acidic extraction is followed by the processing of supernatant using either ultrafiltration or diafiltration together to isolate the protein material, also ultrafiltration is often combined with diafiltration to improve protein recovery [163]. To increase the life of shelf of the protein isolate, ultrafiltration may be followed by spray drying. If desired, the oil, which ends up for 0.5–0.6 g in the fibre-rich fraction, can be recovered by an additional oil extraction step. Overall, it seems that scope exists to lower the environmental impact on the extraction of water- and dilute salt-soluble proteins from legume materials. This is required to facilitate the transition 23 from animal-based protein towards plant-based protein in a sustainable manner [164]. The aqueous fractionation without appliance of an organic solvent but with defatting using and organic solvent can be used for isolation of lupine protein; what is more, the aqueous fractionation is a sustainable alternative, because the oil extraction step is omitted and thereby the organic solvent is not used [165] for defatting using an organic solvents, such as hexane. The defatted flour is then solubilized in water or a buffer at alkaline pH; later on, insoluble parts are separated from the protein-rich supernatant. However, since oil is not removed prior to purification of protein, the oil might become oxidized during processing and have a negative effect on the quality of the obtained protein isolate. The protein is separated from other soluble solids, such as sugars, by isoelectric precipitation of the protein. Wet fractionation of lupine seeds without oil extraction results in lupine protein isolates containing a few percents of oil together with functional properties similar to those of wet fractionated lupine protein isolates, which generally do not contain oil [166]. The lupine protein isolates obtained by applying wet fractionation techniques have a low capacity of gelling and high heat stability [167]. The fractionation is followed by drying process to stabilise the protein isolate. This might not be necessary if the final application contains or requires water. Therefore, it was explored whether the process of drying could be omitted [166]. Dry and wet fractionation processes differ in their separation principle, the use of resources, the unit operations needed, and the yield and composition of obtained fractions. The efficiency of these fractionation processes is evaluated through calculating and visualizing mass, energy and exergy flows. Dry fractionation by fine milling is based on the physical disen- tanglement of protein bodies from fibres and other cellular components, which allows their subsequent separation by air classification [82, 168]. Wet fractionation is based on the differences in solubility of different components in organic solvents, water and saline solutions. Dry fractionation of lupine seeds leads to protein-enriched flours (>50 g protein/100 g). Wet fractionation of lupine seeds can yield protein concentrates (>70 g protein/100 g) and further fractionation leads to protein isolates (>90 g protein/100 g). Papalamprou et al. [169] have reported that milder processing techniques, rather than the composition of the protein isolate, improved the functional properties of chickpea protein isolates in terms of increased protein solubility, reduced minimum protein concentration needed for gel formation, and improved gel elasticity. The effect of the drying method on protein functionality depends on the drying method and on the type of protein. Freeze-drying influences the morphology and size of the protein particles and the surface hydrophobicity of proteins by partial denaturation, 24 due to such stresses as low temperature, freezing stress (e.g. phase separation, pH change and formation of ice crystals) and drying stress [170, 171]. Spray drying has reduced the solubility of a lentil protein isolate less than vacuum drying, however, it has thermally damaged lupine protein isolates [172]. Since freeze-drying is generally perceived as the mildest form of drying, this drying method was chosen for comparison with ultrafiltration [167, 173].

1.6. Lupine Seeds as the Material of Animal Feed

Legumes belong to the family of Leguminosae or Fabacae and are considered the second most important crop worldwide, cereals being the first. They are grown on about 180 million hectares, equivalent to 12-15% of Earth’s arable land [174]. In contrast to many crops, the lupine is reasonably yielding even on sandy soils with a low pH value, also it improves the soil structure by mobilizing soil-bound phosphorus and fixing atmospheric nitrogen, thus providing nutrients to the succeeding crop. Despite its value in crop rotations, lupine so far is an underutilized crop due to its low grain yield stability. To improve yield stability and lupine yield potential, intense breeding efforts are required [175]. Global legume production is currently growing due to the increasing nutritional and economic significance of legume seeds [176]. In animal nutrition, especially in intensive livestock systems, soya bean is the most utilized protein source, mainly administered as extract of meal solvent, a by-product of the oil industry, where soya bean seeds are treated with high temperature and organic solvents. However, recently some obstacles have limited the use of soya bean: the ban in organic livestock [177] due to the chemical treatment, its cost and availability strongly related to the price developments of agricultural commodities on the world market. Ensiling lupine seeds may be an economical and ecologically advantageous alternative to produce a high- protein feed of local origin that can be used in both conventional and organic farming [178]. Also, lupine seeds are important for both animal feed and human foodstuff due to the production of lupine flour and isolate proteins, whereas yellow lupine is mostly used in the livestock chain [179, 180]. Nevertheless, they are still considered to be rich in nutrients and phytochemicals, which makes them an important, inexpensive seed in many developing countries not just a for feed uses, but also for supplementing human diets based on legumes, cereals and roots; consequently, it is suggested to be one of the best solutions to malnutrition in these countries [181, 182].

Lupine Seeds for Poultry Feeding. The growing interest in cultivation and the introduction of legume seeds into poultry diets have been observed in recent years. It was discovered that biologically active legume proteins, rich in lysine and owing antioxidant potential can be a good source for production of animal feed [183–189]. Legume seeds have been used for broiler chickens [190, 191], turkeys [192, 193] and laying hens [194, 195] feeding. The inclusion of lupine seed meal in feed mixtures for broilers did not have a negative effect on the chemical composition of breast and femoral musculature. Unlike the control group, the experimental group of chickens taking lupine in the proportion of n-3 and n-6 fatty acids, had the outcome of breast and femoral musculature narrowing for both pullets and cockerels, which is a proof of an increased dietetic value of musculature. On the other hand, replacement of soybean meal with yellow lupine seeds meal in turkey nutrition (0%, 8%, 16%, and 24%) did not have a positive effect on feed intake and body weight gain. Increase in the concentration of polyunsaturated fatty acids (PUFA) was noted in turkey meat of those fed with lupine-based feed; this did not change the n-6/n-3 PUFA ratio, but improved the value of the atherogenic and thrombogenic indices [196]. In the previous studies during which yellow lupine seeds were used (raw and extruded) in broiler diets in a share of 5– 30%, it was found that the extrusion improved digestibility of fats from seeds as well as nitrogen retention in chickens, and enhanced the apparent metabolizable energy correcting it to zero value of N balance in seeds [197]. However, the inclusion of 25% or 30% of either raw or extruded yellow lupine seeds into diets significantly decreased the performance index of broiler chickens. Using 10% or 20% of extruded seeds positively affected the ratio of growth and feed conversion of birds in comparison to those fed with raw seeds [197]. The lack of information on the inclusion of lupine in ostrich feeding and the ways in which dietary inclusion might affect the production and quality of the meat, leather and feathers of slaughter birds. However, the use of locally produced feed sources would benefit the local legume industry and the ostrich industry. In an attempt to decrease the feeding costs of ostriches, animal nutritionists apply low cost diet formulations and the use of lupine as a raw material could contribute to these formulations [198]. Zdunczyk et al. [199] have analysed performance of growing, gastrointestinal function and meat quality in growing-finishing turkeys that were on diets with different levels (6%, 12% and 18%) of L. luteus seed meal. During the first phase of feeding, yellow lupine meal has decreased feed intake and body weight gain linearly due to significant deterioration in the feed conversion ratio. An opposite trend was noted in the second phase of feeding. Body weight gain 26 and feed conversion ratio improved significantly. The effects of dietary replacement of soybean meal with blue lupine meal on the gastrointestinal tract function, growth performance and meat quality in growing-finishing turkeys were studied by Mikulski et al. [200]. Lupine Seeds for Pig Feeding. The use of lupine in weaner diets has been limited from 50 to 100 g/kg on the basis of idea that pigs would have limited ability to deal with the high fibre content in lupine wholemeal. Other studies have shown that yellow lupine seeds could be included at up to 150 g/kg in weaner diets without compromising performance of pigs [201]. In this regard, it is possible that a similar or greater amount of lupine seeds could be used in a weaner diet. Moreover, there is a general perception that removal of the hull, which is indigestible, from lupines may offer the opportunity for even higher levels of inclusion (i.e. >150 g/kg) as increased amounts of insoluble fibre may physically limit the quantity of lupine stock that can be incorporated in a diet for weaner pigs. However, despite a relatively low concentration of anti-nutritional factors such as alkaloids in lupine varieties, the use of blue lupine meal over soybean meal in swine diets may have some limitations due to the profile of imbalanced AAs, which leads to lower protein digestibility, lower proximate nutrient and putative negative interaction between the relatively high concentrations of non starch polisaccharides (NSP) and the digestion process of other nutrients [202]. Use of sweet lupine wholemeal up to 240 g/kg in diets for weaner pigs did not affect performance and indices of intestinal health most likely because of the insoluble and non-soluble polysacharides in the hull, that in turn could have possibly altered physico-chemical properties of the digesta. However, feeding animals with a diet containing greater than 180 g/kg of dehulled lupine seeds significantly compromised feed intake and hence growth of the pigs, and stimulated the proliferation of β-haemolytic strains of E. coli in the gastrointestinal tract [203]. However, the data about lupine feed stock influence on the efficiency of production of pigs are not homogenous. Reduced feed intake and growth of pigs fed a diet containing 150–430 g/kg of L. albus seeds have been reported by Zettl et al. [204]. Applying the inclusion of 30% of white lupine in the feed mixture, they have noticed a reduced feed intake, lower conversion of nutrients and growth of depression; however, they have not observed a positive effect of dehulling and supplementation with AAs. Conversely, in pigs fed a diet with L. angustifolius, compared to a barley- and soy-based diet, Gdala et al. [205] have not observed growth of depression. Positive results of feeding with yellow lupine seeds of Juno variety were achieved by Flis et al. [206]. The nutritional value of diets with various contents of cultural lupines (L. angustifolius and L. albus) in fattened pigs have been studied by Zralý et al. 27

[207], who proved that the animal protein or soybean could be completely replaced by lupine in the diet for fattened pigs. Also, it was reported that limitation of AA was balanced and the nutritional value was increased by supplementation of fat and lupine seed dehulling. No antinutritional effect was observed in the applied representation of lupine seeds in experimental diets. Effects of feeding finisher pigs with chicory or lupine seeds for one or two weeks before slaughter with respect to levels of Bifidobacteria and Campylobacter have been studied by Jensen et al. [208]. This study has shown that even a short-term strategy of alternative feeding with probiotics added in the diet of pre-slaughter pigs elicited changes in the composition of the intestinal microbiota, where lupine has increased the level of bifidobacteria in the caecum and reduced the level of Campylobacter spp. excretion after one week. Lupine Seeds for Sheep and Lamb Feeding. Yilkal et al. [209] have reported positive responses of washera sheep on a hay based supplementation of different forms of processed lupine seeds. However, Lestingi‘s et al. [210] experiment has shown that the lupine protein is highly degradable in the rumen and this may partly explain the poor performance of lambs observed. It is possible that combined use of the lupine seeds and faba beans in lamb feeding could achieve a better balance and thus improve the animal performances. The use of lupine and faba bean seeds as the main protein sources provides lamb growth performance and slaughtering data comparable to those obtained when faba beans are used alone. However, when faba seeds were used as the sole protein source in the diet, the half-carcasses presented a higher percentage of loin than the half-carcasses of lambs fed with the two protein supplements combined. The use of lupine and faba bean protein sources together improved lamb growth performances and decreased the percentage of leg bone compared to lupine used alone. Blood parameters were little affected by dietary treatments. Both lupine and faba protein sources in lamb feed have a positive effect on lamb growth and slaughtering data [211]. Lupine Seeds for Ruminants Feeding. The lupine could be an excellent protein and energy source for ruminants, and it can be fed as wholemeal, ground seeds, whole plant – silage. However, its bitter taste due to a high alkaloid content remains to be a big challenge [212]. Homolka et al. [213] have reported that lupine can be used in the feed rations of milk cows and in fattening of bullocks. Inclusion of lupine seed meal in the feed rations for high-utility milk cows requires crushing and flaking of seeds and their inclusion in the feed mixture for bullocks up to 30% (0.5 kg/100 kg of live weight), for milk cows up to 20% (0.4 kg/100 kg live weight). Their advantage is that unlike soybeans, they do not need heat treating. Depending on 28 the lupine variety, the degradability of lupine seed protein in rumen ranges from 71 to 79%. Cattle can even use whole fodder plants as a fresh fodder or ensilage. In sheep, Somchit-Assavacheep et al. [214] have monitored the effect of short-term nutritional supplementation with lupine seeds (L. luteus) on folliculogenesis, concentrations of hormones and glucose in plasma and follicular fluid. The numbers of follicles have increased in the group fed by lupine, glucose and insulin levels were also higher. Lupine Seeds for Fish Feeding. Ranjan and Bavitha (2015) have reported that lupine kernels is a suitable meal for fish. The lupine kernels is a cheap source of protein, which shows high protein digestibility, high phosphorus retention, does not cause enteritis is salmon unlike soya, and protein in it is not damaged by heating processes [215]. By replacing 60% of fish meal with Lactobacillus spp. fermented lupine wholemeal, the performance of barramundi was improved [216]. Phytic acid is ANFs in plant based feedstuffs. Most of the fish do not have endogenous enzymes to break down the phytate and release nutrients, and the feedstuff is not completely digestable. However, the advantages of phytase are the following: phytase reduces the release of nutrients into the environment by making more bound phosphorus available to fish for growing; phytase added to diets improves protein and amino acid digestibility in fishes; it can improve the metabolic energy of feeds by breaking down the phytate-lipid complex; plant protein sources can be substituted with animal protein sources (e.g., fishmeal), reducing feed cost [217]. Hoerterer et al. [217] have reported that lupine wholemeal and lupine kernel meal have great potential as a sustainable, locally produced replacement for fishmeal in diets for the carnivorous European sea bass with no negative effects on their growth.

2. MATERIALS AND METHODS

2.1. Investigation Venue

The experiments were conducted between 2014 and 2018 at the Lithuanian University of Health Sciences Veterinary Academy (LSMU VA) Department of Food Safety and Quality (Kaunas, Lithuania); Institute of Food Safety, Animal Health and Environment – “BIOR“ (Ryga, Latvia); University of Leipzig Institute of Food Hygiene (LU IFH) (Leipzig, Germany).

2.2. Materials

2.2.1. Plant Material. The seeds of narrow-leafed lupine varieties Vilciai and Vilniai, as well as hybrid lines No.1700, No.1701, No.1702, No.1072, No.1734 and No.1800 (Lupinus angustifolius L.) seeds were obtained from the Lithuanian Institute of Agriculture (Voke, Lithuania) in 2014. 2.2.2. Microorganisms Used for Experiments. Lactobacillus sakei KTU05-6, Pediococcus acidilactici KTU05-7, Pediococcus pentosaceus KTU05-8, KTU05-9 and KTU05-10 were selected due to their good technological, functional and antimicrobial properties, above mentioned microorganisms were obtained from the Kaunas University of Technology, Department of Food Science and Technology, Cereal and Cereal Products research group collection [218].

2.3. The Lupine Wholemeal Biotreatment and Protein Isolation

2.3.1. The Lupine Wholemeal Fermentation

Lupine seeds were crushed (particle size 3 mm) using a household mill (Braun, Germany). The water content was calculated with reference to the moisture content of raw materials and the required 45% moisture content of the solid state fermentation (SSF) end product and 65% moisture content of the submerged fermentation (SMF) end product. Fermentation was carried out for 48 h at the optimal temperature for the cultivation of Lactobacillus sakei (30 °C), Pediococcus acidilactici (30 °C), Pediococcus pentosaceus strains KTU05-8, KTU05-9, and KTU05-10 (35 °C). Ten different samples from each seeds variety were prepared by using different LAB strains and different fermentation technologies (SSF or SMF). Nonfermented lupine seeds were used as a control.

2.3.2. The Lupine Protein Isolation

Lupine protein was isolated according to procedure described by Muranyi et al. [156]. For preparation of isoelectric lupine protein isolates full-fat lupine seeds where crushed using a Grindomix GM 200 (Retsch GmbH, Haan, Germany) and suspended in DI water at a ration 1:8 (w/v). For alkaline protein extraction the pH value was adjusted to pH 10 with 0.5 mol/L NaOH and this suspension stirred for 1 h at room temperature to maximise the extend of solubilized proteins. After separation through a sieve (mesh size: 1.5 mm) removing the fibre fraction, the suspension was centrifuged at 3300 g for 10 min., and the supernatant was removed and acidfied to pH 4.5 with 0.5 mol/L HCl. For an exhaustive protein precipitation, the crude protein suspension was left for 18 hours at 1 °C in refrigerator. The precipitate was recovered by centrifugation and the supernatant was discarded. The protein precipitate was washed 3 times with DI water (ration 1:10 (w/v)) to eliminate effectively surplus salts. After a last centrifugation step was the precipitated concentrate/isolate stored at - 20 °C. For futher analysis the protein isolate was directly frozen at -80 °C and lyophilized in a freeze-drier (Beta 18, Christ GmbH, Osterode, Germany). Protein isolates/concentrates were prepared from nonfermented and fermented seeds.

2.4. The Methods used for Analysis of Lupine Seeds and Their Bioproducts

2.4.1. The Evaluation of Lupine Seeds Proximate Composition

Chemical composition of lupine seeds was investigated according to the ICC standard methods. Moisture content was determined by drying the samples at 105 ± 2°C to constant weight (ICC 109/01:1976. Determination of the moisture content of cereals and cereal products). Ash content was determined by calcinations at 900°C (ICC 104/1:1990. Determination of ash in cereals and cereal products). Nitrogen content was determined using Kjeldahl method with a factor of 5.7 to determine protein content (ICC 105/2:2001. Determination of crude protein in cereals and cereal products for food and for feed). The total lipid content was determined by extraction in the Soxhlet apparatus (“Boeco”, Germany) with hexane technical grade (Fisher Scientific, USA) (ICC 136:1984. Cereals and cereal products – Determination of total fat content). Carbohydrates content in lupine seeds was calculated by the following formula: 100 − (weight in grams [protein + fat + water + ash] in 100 g of seeds). Energy value was calculated by 31 multiplying the content of protein, fat and carbohydrates by the appropriate factor – 4, 4 and 9 for protein, carbohydrates and fat, respectively.

2.4.2. The Analysis of Fatty Acids Composition in Lupine Seeds

Fatty acid (FA) composition of lupine seed oil was determined using a gas chromatography-flame ionization detector (GC-FID), gas chromatograph Agilent 6890N (Agilent Technologies, USA). Methyl esters of FAs were dissolved in anhydrous 99.5% (Sigma-Aldrich, Germany) cyclohexane (100 mg in 4 mL) and were prepared by transmethylation using 8 mL 1.5% sulphuric acid (≥95%, Sigma-Aldrich, Germany) in the pure (99.9%) methanol (Sigma-Aldrich, Germany), and kept at 60°C for 12 h in the dark. Samples were cooled, shaken for 30 s and centrifuged for 10 min, at 3000 relative centrifugal force at 17°C and injected (100 µL of the upper part of supernatant, diluted before in cyclohexane 1:9, respectively) into a capillary BPX90 column (60 m × 0.32 mm, ID × 0.25 µm film thickness) (SGE, USA). The following conditions were used: flame ionization detector – 280°C, H2 flow – 40 ml/min, air flow – 450 ml/min, helium (carrier gas) flow – 1 ml/min , injector – 250°C (split 1:10), oven temperature – 50°C (2 min), 4°C ml/min to 245°C and 245°C for 15 min. The identification of FA was carried out by retention times and expressed as percentage of the total peak area of all the fatty acids in the oil sample.

2.4.3. The Analysis of Macro- and Microelements in Lupine Seeds

Determination was carried out using inductively coupled plasma mass spectrometry (ICP-MS). The seeds were milled and homogenised (final particle size ≤150 µm). For the analysis the following chemicals were used: nitric acid (concentration ≥69.0%), for-trace element analysis (Sigma- Aldrich, France), hydrogen peroxide, 30% w/w (weight/weight), extra pure (Scharlau, Spain), multielement standard solution V for ICP-MS calibration (Sigma-Aldrich, France). Agilent 7700x ICP-MS (Agilent Technologies, Japan), software Mass Hunter Work Station for ICP-MS, version B.01.01 (Agilent Technologies, Japan) were used for analysis. For sample preparation for ICP-MS analysis, 0.3 g of milled lupine seeds was accurately weighed in a microwave vessel. 2 mL of de-ionized water, 8 mL of concentrated nitric acid and 2 mL of concentrated hydrogen peroxide were added and waited for 2–8 h for reaction stabilization until the formation of bubbles had finished. The vessel was sealed and heated in the microwave system. The following thermal conditions were applied: 150 °C temperature was reached in approx. 20 min and remained such for 30 min, and then 32

200°C was reached in approx. 20 min and remained such for 30 min for the completion of specific reactions. After cooling (approx. 40 min) the prepared solution was filtered through the filter with a pore size of 8–10 μm. The solution was transferred to a 50 mL volumetric flask and filled with water to 50 mL volume. The following operating conditions of Agilent 7700x ICP-MS were used for the analysis of the samples: plasma mode – normal, robust; RF forward power 1300 W; sampling depth 8.00 mm; carrier gas flow 0.6 L/min; dilution gas flow 0.4 L/min; spray chamber temperature 2°C; extraction lens 1 V; kinetic energy discrimination 3V.

2.4.4. The Analysis of Alkaloids Content in Lupine Seeds

Extraction of lupine seeds was carried out according to the modified method of Harris and Wilson (1988) [219]. Finely ground and dried lupine seeds (0.5 g) were extracted with 5 mL of 5% trichloroacetic acid (TCA) in ultrasonic bath three times for 20 min. The crude TCA extract mixture was centrifuged at 3500 × g for 20 min. The supernatant was pipetted into a separatory funnel and made basic with 1 mL of 10 M NaOH. The aqueous basic layer was extracted three times with 15 mL of methylene chloride (DCM). Combined DCM extracts were dried (anhydrous Na2SO4) and filtered. The solvent was removed under vacuum. The residue was dissolved in 1 mL of methanol. 1 μL of this mixture was injected into capillary gas chromatograph. The alkaloids were analyzed using a GC-MS instrument (Agilent; 6890N) equipped with an auto-sampler (Agilent; 7973 Series). The extracts (2 μL) were injected at 290°C into a Zebron column (ZB-AAA, 30 m, 0.25 µm in diameter) programmed from 180–300°C at 32°C/min.

2.4.5. The Microbiological Analysis of Fermented Lupine Wholemeal

For the microbiological analysis, 10 g of a sample were homogenized with 90 mL of saline (0.9%). The suspension was diluted, the 10-4–10-8 solutions were inoculated in the MRS agar CM0361 (Oxoid Ltd, Basing- stoke, United Kingdom) and incubated under anaerobic conditions at 30°C for 72 h (for the LAB count evaluation). The final number of bacteria was calculated and expressed as a log10 of CFU/ml of the sample.

2.4.6. The Determination of Acidity Parameters of Fermented Lupine Wholemeal

The pH values were measured and recorded with a pH electrode (PP–15, Sartorius, Goettingen, Germany). The total titratable acidity (TTA) was determined on 10 g of a sample homogenized with 90 mL of distilled water and expressed as the volume (mL) of 0.1M NaOH to get a pH of 8.2 (TTA 33 assessed in the Neiman degrees, °N). The concentration of L(+)- and D(-)- lactic acid in fermented plant products was determined using a rapid Megazyme test kit (Megazyme International Ireland, Wicklow, Ireland).

2.4.7. The Analysis of Amino Acid Profile in Lupine Wholemeal and Protein Isolates/Concentrates

The Analysis of free amino acids. Free amino acids (FAA) were extracted using 0.1 M HCl. The extracts were analyzed by gas chromatography with flame ionization detection after the ion-exchange solid phase extraction and chloroformate derivatization using EZ-Faast technology (Phenomenex). Standard solutions of the amino acids alanine (ALA), glycine (GLY), valine (VAL), leucine (LEU), isoleucine (ILE), threonine (THR), serine (SER), proline (PRO), asparagine (ASP), methionine (MET), glutamine (GLU), phenylalanine (PHE), lisine (LYS), histidine (HIS), and tyrosine (TYR), in addition of the internal standard (NVAL). Eluting and derivatization agents were all provided in an inclusive kit (EZ-Faast amino acid analysis kit for protein hydrolysates by GC-FID or GC-NPD). Hydrochloric acid (25%) and thioglycolic acid used were purchased from Sigma-Aldrich (Cat. No: T3758). Samples were milled using “Cross Beater Mill Pulverisette 16“ (Idar-Oberstein, Germany), and weighed (1.00 g) in 15 mL polypropylene test tubes with screw caps and mixed with 7.5 mL of 0.1 M HCl, and subjected to ultrasonification in a water bath (t = 40°C) for 15 minutes. The mixture was shaked and then centrifuged (3000 rpm, 15 min). And 2.5 mL aliquot of the mixture was transferred into another 15 mL polypropylene screw cap test tube and 7.5 mL of deionized water was added to 10 mL volume. Samples were then stored at -80°C until analysis. The derivatized amino acids were analysed using a GC-FID instrument (Agilent, 6890N) equipped with an auto-sampler (Agilent; 7683 Series). Aliquots of the derivatized amino acids (2 μL) were injected at 1:15 split ratio at 250°C into a Zebron column (ZB-AAA, 10 m, 0,25 mm in diameter) programmed from 110–320°C at 32°C/min. Helium was used as a carrier gas at 1.5 mL/min in constant flow mode and nitrogen was used as a make- up gas. The detector temperature was 320°C. Five different standard solutions with different concentrations (from 50 to 200 nmol/μL) of amino acid standards were used for the calibration of gas chromatograph. The Analysis of total amino acids. Amino acids (AA) were analyzed by gas chromatography with flame ionization detection after the ion-exchange solid phase extraction and chloroformate derivatization using EZ-Fast technology (Phenomenex). Standard solutions of the amino acids alanine (ALA), glycine (GLY), valine (VAL), leucine (LEU), isoleucine (ILE), 34 threonine (THR), serine (SER), proline (PRO), asparagine (ASP), methionine (MET), glutamine (GLU), phenylalanine (PHE), lysine (LYS), histidine (HIS), and tyrosine (TYR) were analysed, in addition to the internal standard (Nval). Samples (1.00 g) were weighed in 15 mL polypropylene test tubes with screw caps and mixed with 7.5 mL of 0.1 M HCl, and subjected to ultrasonification in a water bath (t = 40°C) for 15 minutes. The mixture was shaked and then centrifuged (3000 rpm, 15 min). An 2.5 mL aliquot of the mixture was transferred into another 15 mL polypropylene screw cap test tube and 7.5 mL of deionized water was added to 10 mL volume. Samples were then stored at -80°C until analysis. The derivatized amino acids were analyzed using a GC-FID instrument (Agilent; 6890N) equipped with an auto-sampler (Agilent; 7683 Series). Aliquots of the derivatized amino acids (2 μL) were injected at 1:15 split ratio at 250°C into a Zebron column (ZB-AAA, 10 m, 0.25 mm in diameter) programmed from 110-320°C at 32°C/min. Helium was used as a carrier gas at 1.5 mL/min in constant flow mode and nitrogen was used as a make-up gas. The detector temperature was 320 °C. Five different standard solutions with different concentrations (from 50 to 200 nmol/μL) of amino acid standards were used for the calibration of gas chromatograph.

2.4.8. The Analysis of Biogenic Amines Content in Treated and Untreated Lupine Seeds

Extraction and determination of BAs were carried out according to the procedures developed by Ben-Gigirey et al. [220]. Perchloric acid (0.4 mol/L, 10 mL) containing a known amount of 1.7-diaminoheptane used as an internal standard was added to a 3 g sample, and the mixture was homogenized with Ultra-Turrax (IKA Labortechnik, Staufen, Germany) and centrifuged at 3000 × g for 10 min. The residue was extracted again with an equal volume of 0.4 mol/L of perchloric acid. Both supernatants were combined, and the final volume was adjusted to 30 mL with 0.4 mol/L of perchloric acid. The extract was filtered through Whatman No. 1 paper. One milliliter of the extract or standard solution was mixed with 200 mL of 2 mol/L sodium hydroxide and 300 mL saturated sodium bicarbonate. 5-(dimethylamine) naphthalene-1-sulfonyl chloride (dansyl chloride reagent) (10 mg/mL, 2 mL) prepared in acetone was added to the mixture and incubated at 40°C for 45 min. The residual dansyl chloride was removed by adding 100 mL of 25 mg/L ammonium hydroxide. After incu- bation at room temperature for 30 min, the mixture was adjusted to 5 mL with acetonitrile. Finally, the mixture was centrifuged at 3000×g for 5 min; the supernatant was filtered through 0.2 μm filters (Millipore Co., Bedford, 35

USA) and stored at 225 °C until analysis by high-performance liquid chromatography (HPLC). An Agilent 1200 HPLC instrument (Carlsbad, USA), equipped with a diode-array detector and Chemstation LC software was employed. A Chromolith C18 HPLC column (100 mm × 4.6 mm × 4 mm, Merck KGaA/EMD Chemicals, Darmstadt, Germany) was used. Ammonium acetate (0.1 mol/L) and acetonitrile were used as the mobile phases, with a flow rate of 0.45 mL/min. The sample volume injected was 10 mL, and amines were monitored at nm. The detection limit for standard amine solutions was approximately 0.1 mg/kg.

2.4.9. The Determination of Protein and Protein Isolates/Concentrates of Lupine Seeds Wholemeal Digestibility In Vitro

Determination of in vitro protein digestibility was carried out according to Lqari et al. [221]. Samples containing 62.5 mg of protein were suspended in 10 mL of water, and the pH was adjusted to 8 with 0.1 mol/L of NaOH. An enzymatic solution containing 1.6 mg of trypsin (18 AU/mg), 3.1mg of -chymotrypsin (40 AU/mg) and 1.3 mg of protease (15 AU/mg) mL was added to the protein suspension in a 1:10 (v/v) ratio. The pH of the mixture was훼 measured exactly after 10 min and the in vitro digestibility calculated as a percentage of digestible protein (DP) using the equation DP = 210.464−18.103 × pH.

2.4.10. The Determination of the Total Phenolic Compounds Content and Antioxidants Properties of Lupine Products

The total content of phenolic compounds (TPC) in fermented lupine samples was determined by spectrophotometric method, as reported Vaher et al. [222]. The absorbance of samples was measured at 765 nm using spectrophotometer “J.P. SELECTA S.A. V-1100D“ (Barcelona, Spain). Antioxidant activity of lupine samples was evaluated according to the method reported by Zhu et. al. [223].

2.4.11. The Evaluation of the Lupine Protein Solubility and Protein Content in Isolates/Concentrates

Protein solubility was evaluated according to method described by King et al. [224]. Estimation of the soluble protein was done by the Bradford (1976) method [225]. The total protein content of lupine protein isolates/ concentrates based on nitrogen content was determined by the Kjeldahl method according to AACC [226] using the Nitrogen Analyzer (Vapodest, 36

Gerhardt GmbH Co KG, Königswinter, Germany). To determine the total protein content 5.7 factor was used (ICC 105/2) [227].

2.4.12. The Determination of Molecular Weight of Lupine Protein Isolates/Concentrates by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The molecular weights of the lupine protein fractions were investigated according to method described by Muranyi et al. [154]. The SDS-PAGE was carried out using SDS-polyacrylamide commercial vertical gel (Roti- PAGE Precast Gels, Roth GmbH, Germany). The resolving gradient gel ranged from 4% to 20%. The freeze-dried protein samples were prepared according to the method described by Magni et al. [228]. In order to determine the molecular weights of the protein fractions, a molecular weight standard from 10 kDa to 220 kDa (Bench Mark Unstained Protein Ladder, Thermo Fisher Scientific) was used and added at the first line on the gel. Gels were stained using Coomassie Blue (CBBG-250, Roth GmbH, Germany) and scanned with Multimage Light Cabinet (Vilber Lourmant, France). The molecular weight of each band was analyzed by software Gel Analyzer (2010).

2.4.13. The Analysis of Isoflavones Content in Lupine Bioproducts

Since in this study, the influence of microbial hydrolysis on the isoflavones content was evaluated, sample preparation procedure omits the need to hydrolyze the compound glycosides, the procedure was simplified to two-phase system liquid-solid extraction and reconstitution in an aqeous- organic solvent mixture. For the sample preparation 40 mg of sample was weighed and transfered to the 15 mL PP tube. For QCs procedure the standard was added, then sample was letted to dry in room temperature for 15–30 min and 500 uL of Milli-Q water was added and vortex for 15 s, and ultrasonicated for 5 minutes. Then, 3000 uL of diethyl ether was added and 10 sec. vortex. Sample was transfered 2000 uL aliqoute of diethylether. Dry residue were reconsituted in the 1000 uL of 0.1% formic acid in 20:80 water/acetonitrile mixture by vortexing – 10 s, ultrasonification – 3 min and repeated vortexing for 5 s. Than samples were centrifugated and 50 uL of final extract was transfered to to autosampler vials to perform the chromatographic analysis. For the chromatographic analysis Kinetex PFP 100 x 3.0 mm, 1.7 u column was used. Column oven temperature – 40°C, sampler temperature – 10°C, injection volume - 2 uL, sample loop solvent – 37

20:80 ACN/H2O, sample injection solvent – 0.1% FA in 20:80 ACN/H2O. Mobile phase composition and gradient were A – 0.1% FA in H2O, B – 0.1% FA in ACN, flow – constant 400 uL/min. Chromatograpic runs were performed whilst operating the Orbitrap-MS in low resolution setting for faster scan speeds and polarity switching was enabled to evaluate the signal strength in each polarity mode for all 4 of the analytes.

2.5. The Statistical Analysis

Physical chemical analysis were repeated three times by analysing two parallel samples, the microbiological analysis were repeated five times, by analysing two parallel samples. The data was subjected to analysis of variance (ANOVA) and the Tukey HDS test as a post-hoc test (statistical program R 3.2.1, Core Team 2015) by using statistical package SPSS for Windows (Ver.15.0, SPSS, Chicago, Illinois, USA), with the significance of results at p≤0.05. In order to evaluate the influence of various factors (fermentation method, the type of LAB strain applied for the fermentation, lupine variety and protein isolation process) on the analysed lupine product parameters, a univariate analysis of variance was performed.

3. RESULTS AND DISCUSSION

3.1. The Proximate Composition of Lupine Seeds

The proximate composition of wholemeal seeds of lupine varieties Vilciai, Vilniai, hybrid lines No.1072, No.1734, No.1701, No.1700, No.1800 and No.1702 are presented in Table 3.1.1. The highest protein content was found in Vilciai lupine seeds (40.8 ± 0.10%), while in samples of hybrids No.1700 and No.1800 protein content has ranged from 20.10 to 25.70%, respectively, lower. The content of carbohydrates in lupine seeds varied from 41.60 ± 0.10% to 50.80 ± 0.30% (in Vilciai and hybrid No.1800, respectively). The highest content of minerals was found in Vilciai seeds (4.30 ± 0.20%), whereas the lowest content was observed in hybrid No.1800 (2.93%). The lowest fat content was found in variety Vilciai seeds (4.40 ± 0.10%), while the highest content was in hybrid No.1702 (7.00 ± 0.10%). The alkaloids content in seeds of hybrids was observed to be lower, in comparison with Vilniai variety seeds, and varied from 0.0107 to 0.0121%, whereas in Vilniai and Vilciai seeds the content of alkaloids was 0.0300% and 0.0210%, respectively. Results of the ANOVA test have indicated that the proximate composition of lupine seeds was significantly affected by the selection of lupine variety (on protein content (F(483.139)= 244.136, p=0.0001), on fat (F(138.794)=14.816, p≤0.0001), on carbohydrates (F(686.995)=174.986, p≤0.0001), on minerals (F(23.574)=3.956, p≤0.0001) and on alkaloids (F(148.527)=0.001, p≤0.0001)). According to literature, there is considerable variation of protein content among lupine species (from 24% to 61%), for example yellow lupine seeds contain 39– 47% and blue lupine seeds contain 31–38% of proteins. Moreover, there are variations in the protein content between species and cultivars, growing conditions and soil types [229]. According to Hudson [237], L. mutabilis sweet seeds are one of the richest in fats (13-23%), whereas the content of fat in other species such as L. albus (5-14%), L. luteus (5–7%) and L. angus- tofilius (4-8.5%) was found to be lower. In general, the content of fat in lupine is relatively high and only a few pulses, such as soybean exceeds lupine in this respect. According to Uzun et al. [230], the content of fat in lupine is ranked to be the third among the legumes, following ground nuts (Arachis hypogeae L.) and soybean (Glycine max L. Merril). High fat content confers a high energy value on lupine meal as food/feed. As dietary oil, lupine compares favourably with soybean and rape seed oils.

Table 3.1.1. Proximate composition (%) of lupine varieties Vilciai, Vilniai, hybrid lines No.1072, No.1734, No.1701, No.1700, No.1800 and No.1702 seeds wholemeal Lupine samples Proteins Fat Carbohydrates Moisture Ash Alkaloids Vilciai 40.80±0.10c 4.40±0.10a 41.60±0.10a 9.00±0.10a 4.30±0.20c 0.021±0.005c Vilniai 32.20±0.10b 5.80±0.10c 49.30±0.10b 9.70±0.10b 3.00±0.20a 0.030±0.003d No.1701 31.00±0.10a 5.20±0.10b 50.20±0.20bc 10.40±0.10bc 3.20±0.20b 0.012±0.002b No.1072 31.90±0.70ab 5.80±0.10c 47.40±0.20b 11.80±0.10e 3.20± 0.10ab 0.012±0.002b No.1700 30.30±0.10a 6.40±0.10d 49.20±0.10b 11.10±0.20d 3.00± 0.20a 0.011±0.002a No.1702 32.00±0.10ab 7.00±0.10de 46.90±0.20b 11.10±0.20d 2.90±0.20a 0.012±0.002b No.1734 32.60±0.10b 6.70±0.10d 47.70±0.20b 9.90±0.20b 3.10±0.10ab 0.011±0.002a No.1800 30.30±0.10a 5.60±0.20c 50.80±0.30bc 10.40±0.10bc 2.90± 0.10a 0.012±0.002b Data expressed as a mean values (n = 3) ± SD; SD – standard deviation. a-e The mean values within a column with different letters are significantly different (p≤0.05).

Fatty acid (FA) composition in wholemeal seeds of lupine varieties Vilciai, Vilniai, hybrid lines No.1072, No.1734, No.1701, No.1700, No.1800 and No.1702 is presented in Table 3.1.2. The dominant FA in seeds of lupine hybrids are unsaturated fatty acids such as oleic acid (C18:1) and linoleic acid (C18:2), an average content of 33.20 ± 3.90 and 38.40 ± 4.50% respectively, and less content of saturated FA was found. Palmitic acid (C16:0) content in lupine varieties was on average 10.80 ± 2.70% of total FA content, whereas the content of other saturated FA was found to be significantly lower. Monounsaturated FA – paullinic acid (C20:1) and 10- pentadecenoic acid methyl ester (C20:1) were found only in variety of Vilciai seeds (1.30 ± 0.09%). The results of the ANOVA test have indicated that the FA composition of lupine seeds was significantly affected by the selection of lupine variety (on palmitic acid (C16:0) (F(854.266)=148.201, p=0.0001), on stearic acid (C18:0) (F(260.178)=89.696, p≤0.0001), on oleic acid (C18:1) (F(1450.286)=310.905, p≤0.0001), on linoleic acid (C18:2) (F(1064.961)=432.840, p≤0.0001), on arachidic acid (C20:0) (F(157.827)= 9.225, p≤0.0001), on α-linolenic acid (C18:3) (F(128.186)=19.785, p≤0.0001), on behenic acid (C22:0) (F(203.897)=61.605, p≤0.0001), and on lignoceric acid + eicosapentaenoic acid (C24:0+C20:5) (F(17.512)=0.285, p≤0.0001)); however, the selection of lupine variety had no significant influence on ginkgolic acid (C15:1) and eicosapentaenic acid (C20:1). Similar results were reported by Uzun et al. [230], as they found that among the unsaturated FA from L. albus seed, oleic and linolenic FA were dominant. High content of unsaturated FA indicates that lupine can be a potential source of considerable amount of useful fats. Moreover, the high content of linoleic and oleic FA make lupine seeds a good source of essential FA. The content of macroelements (mg/g d.m.) in the seeds of lupine varieties Vilciai, Vilniai, hybrid lines No.1072, No.1734, No.1701, No.1700, No.1800 and No.1702 are presented in Table 3.1.3. The highest content of Na was observed in hybrids No.1700, No.1701, No.1702, No.1072, No.1734 and No.1800 (ranging from 1.07 ± 0.04 mg/g to 1.19 ± 0.04 mg/g). The highest content of Mg was observed in variety Vilciai, ranging 3.44 ± 0.09 mg/g. The content of Mg has ranged from 2.00 ± 0.11 mg/g (in Vilniai seeds) to 3.44 ± 0.09 mg/g (in Vilciai seeds). The lowest content of K and Ca was found in variety Vilniai – 12.60 ± 0.09 mg/g and 1.46 ± 0.14 mg/kg, respectively. The results of the ANOVA test have indicated that the macroelements content in lupine seeds was significantly affected by the selection of lupine variety (on Na (F(6.798)=0.178, p≤0.0001), on Mg (F(22.331)= 3.959, p≤0.0001), K (F(20.526)=4.436, p≤0.0001) and on Ca (F(17.913)= 2.776, p≤0.0001)).

Table 3.1.2. Fatty acid composition (%) of total fatty acids in lupine varieties Vilciai, Vilniai, hybrid lines No.1072, No.1734, No.1701, No.1700, No.1800 and No.1702 seeds wholemeal. Lupine Fatty acid composition (%) of total fatty acids samples C16:0 C18:0 C18:1 C18:2 C20:0 C18:3 C22:0 C24:0+C20:5 C15:1 C20:1 Vilciai 4.20±0.09a 2.40±0.28a 25.20±0.24a 48.00±0.31g 2.90±0.22c 7.90±0.18d 6.90±0.31d 0.80±0.06b 0.40±0.05b 1.30±0.09b Vilniai 11.40±0.07 6.50±0.14b 30.80±0.18b 41.70±0.27f 0.90±0.07a 6.10±0.13c 2.10±0.16b 0.50±0.08a - - No.1701 11.60±0.10c 8.00±0.23c 32.60±0.11 37.50±0.24c 1.20±0.08b 6.40±0.17c 2.30±0.11b 0.40±0.04a - - No.1072 12.00±0.11d 8.20±0.16c 33.40±0.23c 37.30±0.22c 1.00±0.06a 5.50±0.22a 2.10±0.10b 0.50±0.02a - - No.1700 11.70±0.15c 9.10±0.21d 36.00±0.09d 34.40±0.29a 1.10±0.03b 5.10±0.09a 2.10±0.13b 0.50±0.04a - - No.1702 12.00±0.21d 7.50±0.17b 34.00±0.13c 38.30±0.17e 1.00±0.04a 4.90±0.10a 1.80±0.21a 0.50±0.05a - - No.1734 11.50±0.16c 8.20±0.22c 36.20±0.17d 35.20±0.18b 1.10±0.03b 5.30±0.14a 2.00±0.27a 0.50±0.04a - - No.1800 11.60±0.31c 7.40±0.31b 37.20±0.19e 34.80±0.21a 1.00±0.01a 5.40±0.11a 2.10±0.26b 0.50±0.03a - - Average 10.80±2.70b 7.20±2.10b 33.20±3.90c 38.40±4.50c 1.30±0.70b 5.80±1.00b 2.70±1.70c 0.50±0.10a 0.050±0.10a 0.20±0.10a C16:0 – palmitic acid; C18:0 – stearic acid; C18:1 – oleic acid; C18:2 – linoleic acid; C20:0 – arachadic acid; C18:3 – α-linoleic acid; C22:0 – behenic acid; C24:0+C20:5 – lignoseric+eicosapentaenoic acid; C15:1 – ginkgolic acid; C20:1 - eicosapentaenoic acid; a-g The mean values within a column with different letters are significantly different (p≤0.05).

Macroelements such as Na, Mg, K and Ca are essential for a wide variety of metabolic and physiological processes in the human body; therefore should be included in daily diets to prevent chronic diseases [231, 232]. Na is an essential nutrient, dietary intakes in Europe today far exceed nutritional requirements. Daily Na intakes of populations in Europe range from around 3-5 g and are well in excess of dietary needs (around 1.5 g per day for adults, according to Dietary reference intakes [233]. The content of Na in lupine seeds did not exceed the recommended level; therefore the tested lupine seeds are not harmful for human health. Porres et al. [234] have reported similar Mg and K values in L. angustifolius seeds. Dietary reference intake [233] recommendations for adults (31-50 years old) are 420, 4700 and 100 mg of Mg, K and Ca per day respectively. The lowest coefficient of variation in lupine seeds was found for K (3.61%).

Table 3.1.3. The content of macroelements (mg/g d.m.) in lupine varieties Vilciai, Vilniai, hybrid lines No.1072, No.1734, No.1701, No.1700, No.1800 and No.1702 seeds wholemeal. Minerals Lupine Na Mg K Ca samples g/kg Vilciai 0.93±0.06a 3.44±0.09a 13.9±0.12c 1.93±0.25b Vilniai 1.01±0.09b 2.00±0.11a 12.6±0.09b 1.46±0.14a No.1701 1.07±0.04b 2.45±0.14c 12.7±0.21b 2.23±0.09c No.1072 1.11±0.07b 2.33±0.15b 12.9±0.15b 2.53±0.15d No.1700 1.12±0.04c 2.48±0.12c 13.6±0.17b 1.92±0.12b No.1702 1.18±0.02c 2.14±0.21a 13.4±0.23a 1.65±0.20b No.1734 1.18±0.09c 2.29±0.23b 13.5±0.20a 2.41±0.08d No.1800 1.19±0.04c 2.44±0.17c 13.3±0.19a 2.04±0.06c Data values are expressed as means with the standard deviations (n = 3). Na – natrium; Mg – magnessium; K – potassium; Ca – calcium; a-d The mean values within a column with different letters are significantly different (p≤0.05).

The microelements (µg/g d.m.) in the seeds of lupine varieties Vilciai, Vilniai, hybrid lines No.1072, No.1734, No.1701, No.1700, No.1800 and No.1702 are presented in Table 3.1.4. The highest concentration of Cr was observed in hybrid No.1702 seeds (1.81 ± 0.03 µg/g), whereas the lowest concentration was in variety Vilciai seeds (0.73 ± 0.01 µg/g). The highest iron and Zn concentration 59.84 ± 3.22 µg/g was found in variety Vilciai. The highest Fe concentrations (73.52 ± 4.77 µg/g) were found in variety Vilciai. The concentration of Ni in lupine seeds varied from 1.25 ± 0.04 µg/g (hybrid no. 1701) to 2.31 ± 0.02 µg/g (hybrid No.1702). Concentration of Sr varied from 9.09 ± 0.09 µg/g (hybrid No.1701 seeds) to 28.49 ± 0.21 µg/g (hybrid No.1072). The concentration of Ag was discovered to be below 43 the detection limit of the apparatus in all lupine samples. Concentration of Co in lupine seeds varied from 0.06 ± 0.02 µg/g (hybrid No.1800) to 0.18 ± 0.03 µg/g (hybrid No.1702). The highest content of Se was found in hybrid No.1702 and Vilciai seeds (0.13 ± 0.01 µg/g). Concentration of Cd varied from 0.026 ± 0.01 µg/g (hybrid No.1701) to 0.049 ± 0.02 µg/g (hybrid No.1734). Concentration of Pb varied from 0.06 ± 0.02 µg/g (variety Vilniai) to 0.11 ± 0.03 mg/kg (hybrid No.1072) in lupine seeds. The concentration of As varied from 0.015 ± 0.01 µg/g (hybrid No.1701) to 0.033 ± 0.01 µg/g (variety Vilciai). The results of the ANOVA test have indicated that the microelement content in lupine seeds was significantly affected by the selection of lupine variety on Al (F(363.070)=93.559, p=0.0001), on Cr (F(22.357)=2.422, p≤0.0001), on Mn (F(90216.207)= 34772.709, p≤0.0001), on Fe (F(4508.221)=1164.079, p=0.0001), on Co (F(10.218)=0.046, p=0.0001), on Ni (F(26.274)=2.506, p=0.0001), on Cu (F(118.689)=15.931, p=0.0001), on Zn (F(7794.620)=1553.663, p=0.001), on Se (F(10.759)=0.014, p=0.001) and on Sr (F(6341.740)=1044.511, p≤0.0001). However, the selection of lupine variety had no significant influence on As, Cd and Pb content. In humans and animals, Cr is an essential nutrient that plays a significant role in the metabolism of glucose, fat and protein through potentiation of insulin action. There is limited data on which to tolerable daily intakes can be based. Shah et al. [235] has found that deficiency of Ni can lead to anaemia as Ni is considered to be synergistic to Fe by promoting its intestinal absorption. The highest concentration of Ni was found in the two lines, particularly Nos. 1703 and 1734. Deficiencies of only four elements, particularly cobalt as vitamin B12, I, Fe and Zn, occur with known sufficient frequency in humans, which is a concern to health professionals [236]. Se is an essential trace element and integral part of many antioxidant enzymes, such as glutathioneperoxidase and selenoprotein P in humans and animals, therefore the deficiency of Se leads to various clinical consequences including cancer, cardiovascular diseases, type 2 diabetes and lung disorders [238]. Cd is toxic to a wide range of organs and tissues, and a variety of toxicological endpoints (repro- ductive toxicity, neurotoxicity, carcinogenicity) have been observed in experimental animals and subsequently investigated in human populations [239]. However, during the meeting in June 2010, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) has withdrawn the provisional tolerable weekly intake and established a provisional tolerable monthly intake of 25 µg/kg b.w. [240]. A monthly intake was established taking into the consideration the long half-life of Cd, and consequently small to negligible influence of daily ingestion on overall exposure.

Table 3.1.4. The microelements (µg/g d.m.) in lupine varieties Vilciai, Vilniai, hybrid lines No.1072, No.1734, No.1701, No.1700, No.1800 and No.1702 seeds wholemeal. Lupine Vilciai Vilniai No.1701 No.1072 No.1700 No.1702 No.1734 No.1800 samples µg/kg 1.19 1.73 0.94 6.00 4.00 2.99 6.27 1.79 Al ±0.02b ±0.03c ±0.03b ±0.07da ±0.05c ±0.05c ±0.08c ±0.08b 0.73 1.19 1.38 1.40 1.32 1.81 1.51 0.91 Cr ±0.01b ±0.02c ±0.04c ±0.02b ±0.03b ±0.03b ±0.03c ±0.02b 147.65 88.16 28.13 122.82 90.19 123.23 118.84 48.68 Mn ±8.95d ±5.77f ±0.25e ±7.88f ±5.91e ±6.14f ±5.13f ±1.29d 73.52 53.06 52.54 63.97 64.27 55.62 63.59 54.05 Fe ±4.77 ±3.41e ±3.22f ±4.01d ±3.62d ±3.44e ±2.77e ±1.47e 0.14 0.12 0.03 0.09 0.10 0.18 0.12 0.06 Co ±0.01a ±0.02a ±0.01a ±0.02a ±0.02a ±0.03a ±0.02b ±0.02a 1.80 1.61 1.25 1.51 1.44 2.31 1.95 1.40 Ni ±0.03c ±0.04b ±0.04b ±0.05c ±0.04b ±0.02b ±0.09c ±0.06b 8.07 5.53 5.10 6.15 6.46 6.44 6.40 6.03 Cu ±0.08d ±0.08c ±0.07d ±0.07c ±0.08e ±0.09c ±0.05b ±0.09b 59.84 36.90 35.15 53.66 40.92 39.79 45.17 39.95 Zn ±3.22d ±0.44f ±2.79f ±3.77f ±3.22f ±1.14e ±1.33d ±1.02e 0.03 0.03 0.02 0.02 0.02 0.02 0.03 0.02 As ±0.01a ±0.01a ±0.01a ±0.01a ±0.01a ±0.01a ±0.01a ±0.01a 0.13 0.09 0.10 0.10 0.09 0.16 0.09 0.09 Se ±0.01a ±0.02b ±0.02a ±0.03a ±0.02b ±0.02b ±0.02b ±0.02b 16.60 11.26 9.09 26.10 14.21 20.89 28.49 12.95 Sr ±0.12d ±0.12f ±0.09c ±0.22d ±0.35c ±0.21e ±0.29e ±0.15 Ag <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 <0.005 0.04 0.04 0.03 0.03 0.04 0.04 0.05 0.04 Cd ±0.02b ±0.02b ±0.01a ±0.01a ±0.02a ±0.01a ±0.02a ±0.01a 0.07 0.06 0.06 0.11 0.07 0.08 0.08 0.08 Pb ±0.02b ±0.02b ±0.01b ±0.03b ±0.02b ±0.02a ±0.03b ±0.02b Data values are expressed as means with the standard deviations (n = 3). Al – aluminum; Cr – chromium; Mn – manganese; Fe – iron; Co – cobalt; Ni – nickel; Cu – copper; Zn – zinc; As – arsenic; Se – selenium; Sr – strontium; Ag – silver; Cd – cadmium; Pb – lead. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

3.2. The Effectiveness of Lupine Seeds Fermentation – The Acidity Parameters and Lactic Acid Bacteria Count

The acidity parameters and LAB counts in lupine seeds Vilciai, Vilniai, hybrid lines No.1072 and No.1734 wholemeal treated by SMF and SSF with L. sakei and P. acidilactici strains are presented in Table 3.2.1. The pH values of SMF lupine samples have ranged from 3.60 ± 0.03 to 3.90 ± 0.03 (in lupine seeds No.1734 fermented with P. acidilactici and in lupine seeds variety Vilniai fermented with L. sakei, respectively); and SSF lupine samples ranging from 4.00 ± 0.02 to 4.63 ± 0.03 (in seeds Vilniai and seeds

No.1734 fermented with P. acidilactici, respectively). The results of the ANOVA test have indicated that the pH values of fermented lupine seeds were significantly affected by the selection of lupine variety (F(0.110)= 177.608, p≤0.0001) and fermentation method (F(2.220)=3600.162, p≤0.0001), and the interaction of these factors was significant (interaction lupine variety × type of LAB × fermentation method F(0.027)=44.554, p≤0.0001). The TTA of SMF samples was found to be on average 8.60 ºN, and SSF samples to be on average 10.79 ºN. The TTA values of fermented lupine seeds were significantly affected by the selection of lupine variety (F(3.289)=1118.157, p≤0.0001), fermentation method (F(68.046)= 23137.029, p≤p<0.0001), the types of LAB applied for the fermentation (F(1.320)=448.895, p<0.0001), and the interaction of these factors was significant (interaction lupine variety × type of LAB × fermentation method F(0.253)=86.182, p≤0.0001). In all the cases higher content of L(+) lactic acid in lupine samples was found, while D(-), and L(+) lactic acid content has ranged from 6.02 ± 0.02 mg/kg to 8.69 ± 0.16 mg/kg, in SMF with L. sakei lupine seeds variety Vilciai and in SSF with P. acidilactici variety Vilniai, respectively. The L(+) lactic acid content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(3.585)= 145.486, p≤0.0001), fermentation method (F(4.796)=194.634, p≤0.0001), the types of LAB applied for the fermentation (F(0.922)=37.400, p<0.0001), and the interaction of these factors was significant (F(0.348)=14.130, p≤0.0001). D(-) lactic acid content in fermented lupine samples has ranged from 1.10 ± 0.20 mg/kg to 3.12 ± 0.10 mg/kg, in SSF with P. acidilactici variety Vilciai and in SMF with L. sakei hybrid line No.1072 seeds, respectively. The D(-) lactic acid content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(0.274)=11.606, p≤0.0001) and fermentation method (F(0.416)=17.597, p≤0.0001), and the interaction of these factors was significant (F(5.032)=212.843, p≤0.0001). On average, LAB count in SMF samples was 9.29 log10 CFU/g, and in SSF samples on average 9.32 log10 CFU/g, and the highest LAB count was established to be in SMF with P. acidilactici hybrid line No.1072 seeds wholemeal (9.76 ± 0.04 log10 CFU/g). The LAB count in fermented lupine seeds was significantly affected by the selection of lupine variety (F(0.432)=488.575, p≤0.0001), the type of LAB applied for fermentation (F(0.051)=57.311, p≤0.0001), and the interaction of these factors was significant (F(0.095)=108.085, p≤0.0001). A weak correlation between the pH and LAB count values in fermended lupine samples (r = 0.2142; P = 0.0361) was found, while no correlation between TTA and LAB count was observed.

Table 3.2.1. The acidity parameters and LAB counts in lupine seeds Vilciai, Vilniai, hybrid lines No.1072 and No.1734 wholemeal treated by SMF and SSF with L. sakei and P. acidilactici strains (pH, TTA (°N), and L(+) and D(-)-lactic acid (mg/kg)). Lupine Vilciai Vilniai No.1072 No.1734 samples pH after 48 hours of fermentation Ls SMF 3.69±0.02a 3.90±0.03b 3.67±0.04a 3.64±0.01a Pa SMF 3.65±0.03a 3.83±0.01a 3.64±0.02a 3.60±0.03a Ls SSF 4.55±0.02c 4.02±0.03c 4.02±0.02b 4.40±0.03b Pa SSF 4.46±0.01b 4.00±0.02c 4.31±0.03c 4.63±0.03a TTA (ºN) after 48 hours of fermentation Ls SMF 7.88±0.03a 8.75±0.03a 8.89±0.04a 8.14±0.03a Pa SMF 8.22±0.04b 9.01±0.04b 8.77±0.04a 9.16±0.04b Ls SSF 11.89±0.10c 12.34±0.05c 11.18±0.05c 9.97±0.04c Pa SSF 11.01±0.09c 12.14±0.05c 9.47±0.05b 8.29±0.03a L(+)-lactic acid (mg/kg) Ls SMF 6.02±0.02a 7.11±0.10a 6.87±0.27a 7.04±0.22a Pa SMF 6.42±0.11a 7.38±0.09a 6.99±0.06a 7.13±0.14a Ls SSF 6.89±0.08b 7.52±0.14b 7.61±0.15c 7.88±0.12b Pa SSF 7.02±0.11c 8.69±0.16c 7.44±0.32b 8.02±0.14c D(-)-lactic acid (mg/kg) Ls SMF 1.46±0.19a 1.14±0.09a 3.12±0.10c 1.32±0.19a Pa SMF 2.89±0.10c 1.24±0.19a 2.01±0.14b 2.74±0.22b Ls SSF 2.67±0.14b 2.87±0.15c 1.41±0.18a 1.23±0.18a Pa SSF 1.10±0.20a 2.62±0.18b 2.48±0.13b 3.02±0.28c

LAB count (log10 CFU/g) after 48 hours of fermentation Ls SMF 9.45±0.04c 9.12±0.04b 9.21±0.03a 9.38±0.04b Pa SMF 9.14±0.03a 9.15±0.02d 9.76±0.04c 9.12±0.02a Ls SSF 9.16±0.02a 9.32±0.03c 9.42±0.02a 9.43±0.04b Pa SSF 9.21±0.03b 9.10±0.01a 9.63±0.03b 9.21±0.03a Data values are expressed as means with the standard deviations (n = 3). SMF – submerged fermentation; SSF – solid state fermentation; Ls – Lactobacillus sakei; Pa – Pediococcus pentosaceus. a-dThe mean values within a column with different letters are significantly different (p≤0.05).

The acidity parameters and LAB counts in lupine seeds of hybrid lines No.1700, No.1701, No.1800 and No.1702 treated by SMF and SSF with L. sakei and P. acidilactici strains are presented in Table 3.2.2. The pH values of SMF lupine samples have ranged from 3.60 ± 0.03 to 3.76 ± 0.02 (in lupine seeds No.1701 fermented with P. acidilactici and in hybrid line No. 1702 seeds L. sakei, respectively). The pH values of SSF lupine samples 47 have ranged from 3.99 ± 0.02 to 4.84 ± 0.03 (in hybrid line No.1702 and No.1700 seeds fermented with L. sakei, respectively). The results of the ANOVA test have indicated that the pH values of fermented lupine seeds were significantly affected by the selection of lupine variety (F(0.166)= 232.658, p≤0.0001) and fermentation method (F(9.639)=13528.658, p≤0.0001), and the interaction of these factors was significant (F(0.042)= 59.535, p≤0.0001). On average, TTA of SMF samples was found to be 10.09 ºN, and of SSF on average 10.85 ºN. The TTA of fermented lupine seeds was significantly affected by the selection of lupine variety (F(10.347)=2565.262, p≤0.0001), fermentation method (F(3.255)=807.076, p≤0.0001), the type of LAB applied for fermentation (F(2.576)=638.711, p≤0.0001), and the interaction of these factors was significant (F(1.864)=462.260, p≤0.0001). In all the cases higher content of L(+) lactic acid in lupine samples was found, compared to D(-), and L(+) lactic acid content, ranging from 6.20 ± 0.20 mg/kg to 9.01 ± 0.14 mg/kg, in SMF with L. sakei in hybrid line No.1701 seeds and in SSF with L. sakei in hybrid line No.1700 seeds, respectively. The L(+) lactic acid content in fermented lupine seeds was affected by the selection of lupine variety (F(9.089)= 325.994, p≤0.0001), fermentation method (F(4.896)=325.994, p≤0.0001), the types of LAB applied for fermentation (F(1.566)=104.271, p≤0.0001), and the interaction of these factors was significant (F(0.024)=1.569, p≤0.0001). D(-) lactic acid content in fermented lupine wholemeal has ranged from 1.03 ± 0.22 mg/kg to 2.97 ± 0.26 mg/kg, in SSF with P. Acidi- lactici in hybrid line No.1800 seeds and in SSF with L. sakei in hybrid line No.1701 seeds, respectively. The D(-) lactic acid content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(0.966)=22.546, p≤0.0001) and fermentation method (F(6.156)=143.647, p≤0.0001), and the interaction of these factors was significant (F(0.880)= 20.537, p≤0.0001). The highest content of LAB in SSF with P. acidilactici was found to be in hybrid line No.1800 lupine seeds (10.04 ± 0.02 log10 CFU/g). The LAB count in fermented lupine seeds was significantly affected by the selection of lupine variety (F(0.223)=260.051, p≤0.0001), the type of LAB applied for the fermentation (F(0.117)=136.664, p≤0.0001), and the interaction of these factors was significant (F(0.100)=117.277, p≤0.0001).

Table 3.2.2. The acidity parameters and LAB counts in lupine seeds hybrid lines No.1700, No.1701, No.1800 and No.1702 wholemeal treated by SMF and SSF with L. sakei and P. acidilactici strains (pH, TTA (°N), and L(+) and D(-)-lactic acid (mg/kg)). Lupine No.1700 No.1701 No.1800 No.1702 samples pH after 48 hours of fermentation Ls SMF 3.70±0.02a 3.72±0.02a 3.67±0.01a 3.76±0.02a Pa SMF 3.64±0.02a 3.60±0.03a 3.64±0.01a 3.65±0.02a Ls SSF 4.84±0.03b 4.20±0.02b 4.37±0.03b 3.99±0.02b Pa SSF 4.75±0.04b 4.30±0.03b 4.89±0.04c 4.21±0.03c TTA (ºN) after 48 hours of fermentation Ls SMF 9.82±0.05a 11.04±0.08b 11.04±0.08c 9.52±0.07a Pa SMF 9.70±0.04a 10.18±0.07a 10.18±0.07a 9.20±0.06a Ls SSF 10.80±0.08c 11.20±0.08c 11.20±0.08c 11.13±0.08c Pa SSF 10.00±0.07b 10.99±0.07b 10.99±0.07b 10.49±0.08b L(+)-lactic acid (mg/kg) Ls SMF 8.01±0.15a 6.20±0.20a 7.87±0.06c 6.88±0.22a Pa SMF 8.12±0.07a 6.71±0.09b 8.44±0.13a 7.48±0.09b Ls SSF 9.01±0.14c 6.41±0.07a 8.21±0.05a 7.67±0.26c Pa SSF 8.99±0.05b 7.22±0.08c 8.89±0.08b 7.89±0.32c D(-)-lactic acid (mg/kg) Ls SMF 2.44±0.14b 2.01±0.22a 1.21±0.18a 2.29±0.12b Pa SMF 1.17±0.17a 1.88±0.26a 1.03±0.22a 2.08±0.17a Ls SSF 1.09±0.15a 2.97±0.26b 2.89±0.11b 2.88±0.09c Pa SSF 2.25±0.18b 2.87±0.24b 3.21±0.23c 2.12±0.13a

LAB count (log10 CFU/g) after 48 hours of fermentation Ls SMF 9.78±0.02c 9.43±0.03a 9.00±0.03a 9.90±0.02b Pa SMF 9.49±0.03b 9.12±0.02a 9.26±0.02a 9.64±0.02a Ls SSF 9.15±0.03a 10.02±0.04c 10.03±0.02b 10.05±0.02c Pa SSF 9.48±0.02b 9.70±0.04b 10.04±0.02b 9.82±0.03b Data values are expressed as means with the standard deviations (n = 3). SMF – submerged fermentation; SSF – solid state fermentation; Ls – Lactobacillus sakei; Pa – Pediococcus pentosaceus. TTA – total titratable acidity; CFU – colony forming units. a-d The mean values within a column with different letters are significantly different (p≤0.05).

The acidity parameters and LAB counts in lupine seeds Vilciai, Vilniai, hybrid lines No.1072 and No.1734 treated by SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains are presented in Table 3.2.3. The lowest pH of SSF with P. pentosaceus was found to be in No.10 Vilniai variety seeds (3.60 ± 0.01), the highest pH of hybrid line No.1734 SSF with P. pentosaceus was observed to be in No.8 (5.00 ± 0.03). The results of the 49

ANOVA test have indicated that the pH values of fermented lupine seeds were significantly affected by the selection of lupine variety (F(0.353)=1049.658, p≤0.0001) and fermentation method (F(1.284)= 388.745, p≤0.0001), and the interaction of these factors was significant (F(0.083)=247.252, p≤0.0001). The TTA of fermented lupine samples has ranged from 8.27 ± 0.04 ºN to 13.25 ± 0.02 ºN (SMF with P. pentosaceus in No.9 Vilciai seeds and SMF with P. pentosaceus in No.10 variety Vilniai samples, respectively). The TTA values of fermented lupine seeds were significantly affected by the selection of lupine variety (F(4.966)=1776.461, p≤0.0001), fermentation method (F(32.555)=11645.726, p≤0.0001), the type of LAB applied for fermentation (F(2.350)=840.786, p≤0.0001), and the interaction of these factors was significant (F(4.092)=1463.841, p≤0.0001). In all the cases higher content of L(+) lactic acid was found to be in SSF lupine samples, while D(-), and L(+) lactic acid concentration has ranged from 6.64 ± 0.13 mg/kg to 8.94 ± 0.19 mg/kg, in SMF with P. pentosaceus in No.10 hybrid line No.1072 and in SSF with P. pentosaceus in No.9 variety Vilciai seeds. The L(+) lactic acid content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(1.378)= 49.120, p≤0.0001), fermentation method (F(11.747)=418.771, p≤0.0001), the type of LAB applied for fermentation (F(0.517)=18.436, p≤0.0001). The D(-) lactic acid concentration in fermented lupine samples has ranged from 1.23 ± 0.02 mg/kg to 2.89 ± 0.08 mg/kg, in SMF with P. pentosaceus in No.8 variety Vilciai lupine seeds and in SSF with P. pentosaceus in No.10 hybrid line No.1734 lupine seeds. The D(-) lactic acid content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(0.162)=28.221, p≤0.0001) and fermentation method (F(8.646)=1506.111, p≤0.0001), the type of LAB applied for fermentation (F(0.529)=92.205, p≤0.0001), and the interaction of these factors was significant (F(0.125)=21.726, p≤0.0001). In all the cases, LAB count in fermented lupine samples was found to be higher than 10 log10 CFU/g, and the highest LAB count was obtained in SMF with P. pentosaceus No.9 in hybrid line No.1734 samples (11.40 log10 CFU/g). The LAB count in fermented lupine seeds was significantly affected by the selection of lupine variety (F(0.637)=2061.098, p≤0.0001), fermentation method (F(1.477)=4777.126, p≤0.0001), the type of LAB applied for fermentation (F(1.466)=4743.696, p≤0.0001), and the interaction of these factors was significant (F(0.350)=1133.647, p≤0.0001). Moderate negative correlations were discovered between LAB count and acidity parameters (between LAB count and pH r=−0.37935, between LAB count and TTA r=−0.21672).

Table 3.2.3. The acidity parameters and LAB counts in lupine seeds Vilciai, Vilniai, hybrid lines No.1072 and No.1734 treated by SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains (pH, TTA (°N), and L(+) and D(-)-lactic acid (mg kg)). Lupine Vilciai Vilniai No.1072 No.1734 samples pH after 48 hours of fermentation Pp8 SMF 4.14±0.01b 3.69±0.02a 3.72±0.02a 4.18±0.01b Pp9 SMF 3.62±0.01a 3.75±0.02b 3.72±0.03a 3.70±0.01a Pp10 SMF 3.67±0.01a 3.72±0.02b 3.82±0.01a 3.82±0.01a Pp8 SSF 4.34±0.02d 3.99±0.02c 4.00±0.02b 5.00±0.03d Pp9 SSF 4.05±0.01b 3.78±0.01b 4.01±0.02b 4.22±0.01b Pp10 SSF 4.39±0.01c 3.60±0.01a 4.14±0.02c 4.72±0.01c TTA (ºN) after 48 hours of fermentation Pp8 SMF 9.13±0.07b 9.23±0.05a 9.61±0.04a 10.34±0.02a Pp9 SMF 8.27±0.04a 10.04±0.07b 10.23±0.02b 10.22±0.05a Pp10 SMF 9.08±0.03b 13.25±0.02d 9.41±0.04a 10.15±0.03a Pp8 SSF 10.76±0.04c 11.52±0.04c 10.87±0.07b 11.72±0.03b Pp9 SSF 11.15±0.03d 11.53±0.07c 11.41±0.03c 12.14±0.03c Pp10 SSF 12.15±0.05d 11.21±0.05c 11.36±0.04c 11.43±0.05b L(+)-lactic acid (mg kg) Pp8 SMF 7.74±0.15a 6.92±0.13a 7.25±0.14b 7.44±0.15b Pp9 SMF 7.82±0.16b 7.21±0.15b 7.32±0.15b 7.32±0.14a Pp10 SMF 7.69±0.14a 7.11±0.14b 6.64±0.13a 6.98±0.12a Pp8 SSF 8.19±0.17b 7.99±0.16c 8.14±0.16c 8.46±0.16c Pp9 SSF 8.94±0.19d 8.12±0.17d 8.72±0.17d 8.52±0.17c Pp10 SSF 8.62±0.18c 8.41±0.18d 8.64±0.16d 8.71±0.19d D(-)-lactic acid (mg kg) Pp8 SMF 1.23±0.02a 1.61±0.03b 1.30±0.02a 1.52±0.05a Pp9 SMF 1.72±0.04b 1.35±0.02a 1.25±0.02a 1.48±0.04a Pp10 SMF 1.39±0.03a 1.44±0.04a 1.47±0.03b 1.69±0.06b Pp8 SSF 2.03±0.05c 2.12±0.05c 2.51±0.04c 2.19±0.07c Pp9 SSF 2.26±0.06c 2.57±0.06c 2.67±0.05d 2.68±0.07d Pp10 SSF 2.33±0.07d 2.83±0.07d 2.77±0.06d 2.89±0.08d

LAB count (log10 CFU/g) after 48 hours of fermentation Pp8 SMF 10.41±0.01a 11.31±0.02b 10.72±0.01a 11.21±0.02ab Pp9 SMF 10.40±0.01a 11.11±0.02b 11.01±0.03b 11.40±0.01b Pp10 SMF 10.92±0.02b 11.22±0.02b 10.92±0.02ab 11.32±0.01b Pp8 SSF 10.21±0.02a 10.41±0.01b 11.23±0.01b 10.53±0.01a Pp9 SSF 10.63±0.01a 10.43±0.01a 10.31±0.01a 10.81±0.02a Pp10 SSF 10.12±0.02a 10.32±0.01a 10.32±0.01a 10.52±0.01a Data values are expressed as means with the standard deviations (n = 3). SMF – submerged fermentation; SSF – solid state fermentation. Pp8 – Pediococcus pentosaceus No. 8; Pp9 – Pediococcus pentosaceus No. 9; Pp10 – Pediococcus pentosaceus No. 10. TTA – total titratable acidity; CFU – colony forming units. a-d The mean values within a column with different letters are significantly different (p≤0.05).

The acidity parameters and LAB counts in lupine hybrid lines No.1700, No.1701, No.1800 and No.1702 seeds treated by SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains are presented in Table 3.2.4. The pH in lupine samples was ranging from 3.69 ± 0.06 to 4.86 ± 0.10, in SMF with P. pentosaceus No.9 in hybrid line No.1700 seeds and in SSF with P. pentosaceus No.8 in hybrid line No.1701 wholemeal. Results of the ANOVA test have indicated that the pH of fermented lupine seeds was significantly affected by the fermentation method (F(4.500)=85.967, p=0.0001), the type of pediococci applied for the fermentation (F(0.255)=4.865, p=0.012), and the interaction of these factors was significant (F(0.522)=9.973, p=0.0001). Selection of the hybrid line was not significant on the pH of lupine samples. The TTA of lupine samples has ranged from 8.99 ± 0.18 °N to 12.21 ± 0.25 °N, of SMF with P. pentosaceus No.8 in hybrid line No.1700 lupine seeds and of SSF with P. pentosaceus No.10 in hybrid line No.1702 lupine seeds, respectively. The TTA was significantly affected by the fermentation conditions (F(83.722)=239.134, p≤0.0001), the type of pediococci applied for the fermentation (F(2.132)=6.089, p=0.004), lupine hybrid line (F(1.831)=5.231, p=0.003), and the interaction was significant (F(1.052)= 3.006, p=0.039). From all the analyzed factors, only fermentation conditions have a significant influence on LAB count in fermented lupine wholemeal (F(1.653)=6.599, p=0.013), and moderate positive correlations between the LAB count and acidity parameters (pH and TTA) were established (r=0.6519 and r=0.5827, respectively). In all the cases, higher content of L(+) lactic acid, compare to D(-),was observed in fermented lupine wholemeal, and L(+) lactic acid concentration has ranged from 6.79 ± 0.13 mg/kg to 8.92 ± 0.17 mg/kg, in SMF with P. pentosaceus No.8 in hybrid line No.1700 lupine seeds, and in SSF with P. pentosaceus No.8 in hybrid line No.1700, respectively. D(-) lactic acid concentration in fermented lupine samples has ranged from 1.22 ± 0.02 mg/kg to 2.90 ± 0.07 mg/kg, in SMF with P. pentosaceus No.8 in hybrid line No.1700 lupine seeds, and in SSF with P. pentosaceus No.10 in hybrid line No.1702 seeds. The LAB count in lupine samples was ranging from 10.04 ± 0.10 log10 CFU/g to 10.68 ± 0.14 log10 CFU/g, in SMF with P. pentosaceus No.8 in hybrid line No.1700 seeds, and in SSF with P. pentosaceus No.10 in hybrid line No.1800 seeds. To compare LAB count in samples treated with SMF and SSF conditions, in all the cases higher LAB count was found in SSF samples.

Table 3.2.4. The acidity parameters and LAB counts in lupine hybrid lines No.1700, No.1701, No.1800 and No.1702 seeds treated by SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains (pH, TTA (°N), and L(+) and D(-)-lactic acid (mg/kg)). Lupine samples No.1700 No.1701 No.1800 No.1702 pH after 48 hours of fermentation Pp8 SMF 3.78±0.07a 3.89±0.08a 3.74±0.07a 3.76±0.07a Pp9 SMF 3.69±0.06a 3.78±0.07a 3.75±0.08a 3.77±0.08a Pp10 SMF 3.70±0.06a 4.10±0.08b 3.80±0.09a 3.84±0.09b Pp8 SSF 4.41±0.09c 4.86±0.10d 4.30±0.11c 4.35±0.11c Pp9 SSF 4.37±0.08c 4.25±0.09c 4.18±0.10b 4.80±0.12d Pp10 SSF 4.14±0.07b 4.00±0.08b 4.05±0.09b 3.87±0.10b TTA (ºN) after 48 hours of fermentation Pp8 SMF 8.99±0.18a 9.25±0.20a 9.40±0.22b 9.11±0.19a Pp9 SMF 9.06±0.20a 9.66±0.21b 9.02±0.20a 9.38±0.20b Pp10 SMF 9.31±0.21b 9.31±0.23a 9.67±0.21b 9.15±0.18a Pp8 SSF 9.87±0.23b 11.18±0.25c 11.29±0.22c 11.49±0.22c Pp9 SSF 10.21±0.25c 11.24±0.26c 12.14±0.24d 11.88±0.23c Pp10 SSF 11.85±0.28d 12.01±0.29d 11.82±0.23d 12.21±0.25d L(+)-lactic acid (mg kg) Pp8 SMF 6.79±0.13a 7.22±0.15b 6.90±0.13a 7.07±0.14a Pp9 SMF 7.28±0.15b 7.09±0.14a 7.26±0.15b 7.46±0.16b Pp10 SMF 7.41±0.16b 6.93±0.13a 7.29±0.16b 7.19±0.15a Pp8 SSF 8.92±0.17d 8.65±0.16c 7.99±0.17c 7.69±0.16b Pp9 SSF 8.51±0.15c 8.42±0.15c 8.05±0.18c 7.89±0.17c Pp10 SSF 8.64±0.16c 8.77±0.16d 8.27±0.17d 8.06±0.18d D(-)-lactic acid (mg kg) Pp8 SMF 1.22±0.02a 1.28±0.03a 1.35±0.03a 1.41±0.04a Pp9 SMF 1.55±0.03b 1.34±0.04b 1.43±0.04a 1.39±0.03a Pp10 SMF 1.67±0.04b 1.29±0.03a 1.52±0.05b 1.57±0.04b Pp8 SSF 2.01±0.05c 2.24±0.05c 2.14±0.06c 2.62±0.05c Pp9 SSF 2.18±0.07c 2.36±0.06c 2.19±0.06c 2.78±0.06c Pp10 SSF 2.34±0.07d 2.48±0.07d 2.26±0.07d 2.90±0.07d

LAB count (log10 CFU/g) after 48 hours of fermentation Pp8 SMF 10.04±0.10a 10.22±0.12b 10.08±0.10a 10.16±0.14b Pp9 SMF 10.19±0.21a 10.18±0.11a 10.15±0.20a 10.10±0.12a Pp10 SMF 10.13±0.20a 10.16±0.13a 10.05±0.14a 10.03±0.09a Pp8 SSF 10.61±0.13c 10.31±0.20b 10.41±0.14b 10.40±0.14c Pp9 SSF 10.56±0.15b 10.27±0.18a 10.38±0.13b 10.63±0.17c Pp10 SSF 10.25±0.16b 10.54±0.12c 10.68±0.14b 10.29±0.15b Data values are expressed as means with the standard deviations (n = 3). SMF – submerged fermentation; SSF – solid state fermentation. Pp8 – Pediococcus pentosaceus No. 8; Pp9 – Pediococcus pentosaceus No. 9; Pp10 – Pediococcus pentosaceus No. 10. TTA – total titratable acidity; CFU – colony forming units. a-d The mean values within a column with different letters are significantly different (p≤0.05).

3.2.1. The Main Parameters of Fermentation Process

LAB is one of the most important groups of bacteria/probiotics with health benefits that are thought to partially result from their production of lactic acid, their major fermentation product [219, 240]. The LAB count of lupine wholemeal was significantly affected by the lupine variety (F(53.650)=0.813, p≤0.0001), the type of microorganism applied for the fermentation (F(1073.521)=16.258, p≤0.0001) and the interaction of analysed factors was significant (F(14.018)=0.212, p≤0.0001). The decrease of pH occurred due to hydrolysis of carbohydrates during the fermentation which was followed by the production of organic acids [242]. The pH of lupine wholemeal was significantly affected by the lupine variety (F(146.151)=0.403, p≤0.0001), the fermentation method (F(6463.010)= 17.833, p≤0.0001), the type of microorganism applied for the fermentation (F(94.132)=0.260, p≤0.0001) and the interaction of analysed factors was significant (F(35.572)=0.098, p≤0.0001). The most important characteristic of fermented products include not only pH, but TTA as well [243]. Usually, the growth of LAB in fermentable media is limited by pH, but not by TTA, and substrates with buffer capacity enable the preparation of products with high values of TTA [244]. The TTA of lupine wholemeal was significantly affected by the lupine variety (F(114.392)=3.600, p≤0.0001), the fermentation method (F(5469.105)=172.098, p≤0.0001), the type of microorganism applied for the fermentation (F(163.958)=5.159, p≤0.0001) and the interaction of analysed factors was significant (F(71.304)=2.244, p≤0.0001). Lactobacilli are ubiquitous in nature and are usually found in carbohydrate rich environments. They also are a part of the normal flora in the intestinal tract of many animals [241]. The main metabolite of LAB lactate has two optical isomers, which are L-lactate and D-lactate. The L-lactate is produced from pyruvate applying the dehydrogenase of enzyme lactate during normal anaerobic metabolism, whereas the D-lactate is produced by many commensal bacteria in the colon. Increased levels of D-lactate in plasma and urine have been demonstrated in intestinal ischaemia; short bowel and appendicitis and are considered as a marker of dysbiosis and/or increased intestinal permeability [166]. Therefore, desirable lactic acid isomer in food and feed fermentation is the L-lactate. The L(+) lactic acid and D(-) lactic acid concentration in lupine wholemeal was significantly affected by the lupine variety (on L(+) lactic acid F(73.380)=1.986, p≤0.0001; on D(-) lactic acid F(10.880)=0.454, p≤0.0001), the fermentation method (on L(+) lactic acid F(1920.232)=51.973, p≤0.0001; on D(-) lactic acid F(970.818)= 40.486, p≤0.0001), the type of microorganism applied for the fermentation (on L(+) lactic acid F(84.787)=2.295, p≤0.0001; on D(-) lactic acid 54

F(23.126)=0.964, p≤0.0001) and the interaction of analysed factors was significant (on L(+) lactic acid F(6.734)=0.182, p≤0.0001; on D(-) lactic acid F(21.911)=0.914, p≤0.0001). The lupine would be a good alternative source of protein, allowing the nutritional enrichment of foods and making them economically viable for underserved populations. Thus, it creates great potential for its use in the food industry [245]. Strains intended for the use should be systematically monitored during the process of their manufacture for BA and D(-) lactate production capacity to avoid the accumulation of these toxins and thus produce safer products. The most important parameters, which have significant effect on LAB viability, are medium pH and acidity [246]. Particularly pH but not the organic acid concentration limits the growth of LAB in substrate [247]. Recently, the development of new starter cultures of LAB with an industrially important functionality has been started. The latter can contribute to the microbial safety or offer one or several organoleptic, technological, nutritional, or health advantages. Rele- vant important factors that affect the growth of LAB include composition of substrate, method of processing, substrate fermentation, growth capability of the organisms, rate of productivity of starter culture, strain stability during storage, sensory properties of the product and nutritional value of the product [167]. Lactate exists a form of two isomers, in particular D(-)- and L(+)-lactate [248]. D(-)-lactate is an isomer of lactate that is not produced by the human body, but released by intestinal bacteria [249]. The well- known L(+)-lactate is produced by anaerobic glycolysis, and rarely known D-lactate is only produced by intestinal bacteria [250]. The release of organic acids by LAB during fermentation results in a lower pH, which is associated with enhanced activity of LAB proteases, further leading to protein hydrolysis [251]. Similar characteristics of fermented lupine were reported by Fritsch et al. [252]. As indicated in their publication, after 48 h of fermentation with P. pentosaceus, the pH value and LAB count in sweet lupine flour were 4.2 and 5.8–8.2 log10 CFU/mL respectively; while the pH value and LAB count in bitter lupine flour were 4.3 and 6.2–8.4 log10 CFU/mL respectively; and the results of this analysis in lupine protein isolate were 4.6 and 5.7–8.8 log10 CFU/mL respectively. LAB are commonly used microorganisms for the fermentation of food and feed, because their metabolic activity is generally recognized to be safe, on top of that, they rapidly produce organic acids. P. pentosaceus, Lactobacillus plantarum, and Lactococcus lactis are the most popular strains for the fermentation of lupine flour and lupine protein isolates [238].

3.3. The Lupine Protein Solubility at Different pH and Their Yield in Isolates/Concentrates

The protein solubility (%) at different pH values of nonfermented, SMF and SSF with L. sakei, P. acidilactici, P. pentosaceus No.8, No.9 and No.10 strains in the seed protein isolates/concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072, and No.1734 is presented in Table 3.3.1. The highest solubility of nonfermented lupine protein was established to be at pH 10.0, and it was ranging from 80.43 ± 1.42% to 87.97 ± 1.53% (of nonfermented hybrid No.1072 and hybrid No.1734 protein respectively). The highest solubility of SMF and SSF isolates/concentrates was found at pH 10.0, and it has ranged from 70.82 ± 0.82% to 92.75 ± 1.52% (SMF with P. pentosaceus No.10 Vilciai and with L. sakei hybrid No.1072 protein, respectively), and from 72.90 ± 1.09% to 91.12 ± 1.37% (SSF with L. sakei Vilciai and with P. pentosaceus No.9 hybrid No.1072 protein, respectively), respectively. In all samples of SMF and SSF, the solubility of lupine protein isolates/concentrates at pH 4.0 and pH 6.0 was found to be on average 52.32% and 42.90% lower , in compare with their solubility at pH 10.0. The results of the ANOVA test have indicated that the solubility at different pH values was significantly influenced by the analysed factors and their interaction (analysed factors were lupine variety, type of microorganism, fermentation method). The protein solubility at pH 2.0 was significantly affected by the fermentation method (F(1299.719)=21.600, p≤0.0001) and the type of microorganism applied for the fermentation (F(472.902)=7.859, p≤0.0001) and the interaction of analysed factors was significant (F(272.220)=4.524, p≤0.0001); however, the selection of lupine variety had no significant influence on protein solubility at pH 2.0. The protein solubility at pH 4.0 was significantly affected by the selection of lupine variety (F(241.494)=5319.060, p≤0.0001), the fermentation method (F(360.193)=53460.898, p≤0.0001) and the interaction of analysed factors was significant (F(33.826)=4.013, p≤0.0001); nevertheless, the type of LAB applied for the fermentation had no significant influence on protein solubility at pH 4.0. The protein solubility at pH 10.0 was significantly affected by the fermentation method (F(150.088)=28.291, p≤0.0001), the type of LAB applied for the fermentation (F(129.591)=21.600, p≤0.0001) and the interaction of these factors (F(80.499)=15.174, p≤0.0001); nonethe- less, the selection of lupine variety had no significant influence on protein solubility at pH 10.0. The protein solubility at pH 6.0 and pH 8.0 was not influenced by analysed factors.

Table 3.3.1. The protein solubility (%) at different pH values of nonfermented, SMF and SSF with L. sakei, P. acidilactici, P. pentosaceus No.8, No.9 and No.10 strains in the seed protein isolates/concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072, and No.1734. Lupine samples pH 2.0 pH 4.0 pH 6.0 pH 8.0 pH 10.0 Nonfermented samples Vilciai 71.24±1.32b 32.84±0.57c 34.06±0.59b 77.56±1.44b 82.89±1.49b Vilniai 66.68±1.21c 31.66±0.54b 32.06±0.57c 77.07±1.51b 82.87±1.54b No.1072 69.12±1.24a 29.24±0.43a 34.13±0.54b 74.10±1.47a 80.43±1.42a No.1734 65.25±1.18a 23.44±0.24a 22.21±0.44a 79.03±1.55c 87.97±1.53c Submerged fermented samples Vilciai Ls 63.26±1.10b 23.47±0.28b 34.64±0.41e 64.40±1.14d 86.60±1.48c Vilciai Pa 70.52±1.15d 19.24±0.23a 26.76±0.32d 66.73±1.15c 83.11±1.44b Vilciai Pp8 45.16±0.42b 16.87±0.12a 22.73±0.27b 61.79±0.88b 80.55±1.24c Vilciai Pp9 54.15±0.55b 22.49±0.19c 17.37±0.18a 58.01±0.71a 75.52±0.84a Vilciai Pp10 59.60±0.62c 23.45±0.22b 37.52±0.44c 59.96±0.63a 70.82±0.82a Vilniai Ls 76.98±1.18b 19.93±0.22b 40.21±0.43d 62.45±1.12c 84.24±1.42b Vilniai Pa 69.29±1.04b 19.91±0.25a 34.89±0.31b 63.17±1.07b 87.57±1.37c Vilniai Pp8 55.49±0.57a 18.06±0.17a 37.05±0.42b 66.35±0.97b 84.62±0.93b Vilniai Pp9 55.23±0.59d 10.97±0.09a 32.63±0.38a 65.02±0.68c 80.81±0.92a Vilniai Pp10 59.24±0.63b 10.14±0.08a 30.65±0.27b 61.80±0.62c 82.19±0.87b No.1072 Ls 63.26±1.09a 22.32±0.26 32.79±0.37c 74.61±1.15d 92.75±1.52d No.1072 Pa 62.87±1.02b 19.75±0.21a 29.19±0.25a 63.61±1.09b 84.26±1.42c No.1072 Pp8 61.53±0.71b 22.41±0.23c 33.04±0.32b 62.84±0.73b 81.76±0.98b No.1072 Pp9 61.01±0.68a 22.50±0.25 21.00±0.24a 61.46±0.60c 83.92±1.02c No.1072 Pp10 61.38±0.56b 18.78±0.16a 22.44±0.29b 61.76±0.67c 89.54±0.85d No.1734 Ls 58.32±1.05b 19.32±0.23b 23.33±0.24c 61.31±1.07d 84.35±1.44d No.1734 Pa 68.89±1.08c 20.70±0.29a 23.13±0.28b 64.02±1.17c 77.26±1.31b No.1734 Pp8 77.27±0.69d 21.46±0.24a 16.50±0.17a 62.34±0.81b 80.85±0.74c No.1734 Pp9 54.44±0.52b 23.82±0.29d 22.86±0.25b 59.31±0.58a 86.94±0.83d No.1734 Pp10 54.67±0.54a 24.34±0.25d 21.22±0.21a 62.69±0.66b 78.64±0.68c Solid state fermented samples Vilciai Ls 82.39±1.48b 18.62±0.21a 38.90±0.28c 49.12±0.39d 84.13±1.32c Vilciai Pa 82.21±1.35b 21.82±0.25b 35.01±0.39d 39.45±0.32c 79.35±1.28b Vilciai Pp8 51.06±0.69a 10.66±0.08a 31.86±0.36c 50.53±0.47e 78.80±0.87b Vilciai Pp9 60.41±0.82a 15.27±0.17a 33.99±0.42c 45.33±0.42c 82.34±1.05c Vilciai Pp10 59.58±0.75a 23.01±0.26b 35.96±0.48c 50.81±0.56d 82.13±1.09d Vilniai Ls 47.44±0.57a 20.05±0.24b 20.37±0.18b 26.05±0.25a 72.90±1.09b Vilniai Pa 57.07±0.62b 25.77±0.29c 35.92±0.35d 60.67±0.72d 76.18±1.28b Vilniai Pp8 46.53±0.42a 17.89±0.18a 28.32±0.29c 57.91±0.64c 76.45±0.95c Vilniai Pp9 74.50±0.88c 15.60±0.12a 25.03±0.24c 64.73±0.79d 90.95±1.25d Vilniai Pp10 65.99±0.75d 21.86±0.26b 32.35±0.43d 70.80±0.88d 81.73±1.01d No.1072 Ls 35.54±0.23b 15.90±0.17a 25.84±0.24b 79.92±1.27d 70.45±1.02c No.1072 Pa 49.86±0.47c 19.65±0.21a 26.92±0.21c 72.16±0.98c 83.68±1.41c No.1072 Pp8 48.01±0.39c 22.85±0.29b 22.71±0.22a 74.06±0.85c 78.95±0.85b No.1072 Pp9 47.49±0.35d 23.72±0.25c 33.92±0.41c 71.76±0.82c 91.12±1.37d No.1072 Pp10 59.50±0.69b 19.61±0.19b 30.22±0.34b 69.06±0.77b 75.46±0.82c No.1734 Ls 60.15±0.98b 19.72±0.19b 31.45±0.31b 66.31±1.03b 76.33±1.16c No.1734 Pa 56.54±0.61a 14.55±0.14a 18.57±0.16a 76.30±1.31d 78.05±1.22d No.1734 Pp8 44.89±0.48a 22.51±0.22c 24.26±0.23a 70.24±0.83d 78.37±0.93d No.1734 Pp9 28.30±0.25a 21.16±0.20c 23.54±0.25a 64.66±0.68c 79.58±0.96d No.1734 Pp10 41.40±0.44b 17.50±0.18a 28.17±0.29c 63.32±0.65c 79.26±0.81c Data expressed as means (n = 5) ± SD; SD – standard deviation; p significant, when p ≤ 0.05; a-e The mean values within a column with different letters are significantly different (p≤0.05).

Table 3.3.3. The protein solubility (%) at different pH values of nonfermented, SMF and SSF with L. sakei, P. acidilactici, P. pentosaceus No.8, No.9 and No.10 strains in the seed protein isolates/concentrates of lupine hybrid lines No.1700, No.1701, No.1800, and No.1702. Lupine samples pH 2.0 pH 4.0 pH 6.0 pH 8.0 pH 10.0 Nonfermented samples No.1700 60.56±1.09b 21.07±0.22b 27.60±0.38a 72.16±1.41b 77.87±1.41a No.1701 74.07±1.47b 19.71±0.19a 32.57±0.43b 74.79±1.42b 78.78±1.42a No.1800 58.16±1.04a 21.51±0.21b 28.83±0.38a 74.39±1.43b 82.27±1.52c No.1702 65.64±1.09b 22.28±0.23b 31.26±0.41b 70.85±1.38a 80.73±1.45b Submerged fermented samples No.1700 Ls 65.69±0.89c 24.49±0.22c 19.43±0.25b 54.83±0.63a 83.58±0.98c No.1700 Pa 70.49±0.67d 18.06±0.18a 21.04±0.26c 60.78±0.75c 72.30±0.85a No.1700 Pp8 66.03±0.72b 16.69±0.16a 22.28±0.23c 63.37±0.72d 77.26±0.84b No.1700 Pp9 71.95±0.89d 26.50±0.25d 19.22±0.18b 67.67±0.82d 92.24±0.98d No.1700 Pp10 65.74±0.68c 24.82±0.23d 15.29±0.14a 50.44±0.51b 84.07±0.92c No.1701 Ls 55.62±0.52b 17.54±0.12a 19.64±0.22b 77.56±0.89d 81.36±0.95c No.1701 Pa 54.77±0.58b 17.80±0.15a 26.98±0.29d 69.25±0.78d 75.28±0.78b No.1701 Pp8 61.89±0.62c 25.48±0.29c 13.51±0.12a 62.64±0.63c 75.89±0.89b No.1701 Pp9 59.36±0.55c 27.02±0.32d 12.84±0.11a 60.70±0.61b 87.85±0.95d No.1701 Pp10 59.27±0.48c 26.82±0.28d 23.00±0.24c 62.67±0.58b 78.48±0.78c No.1800 Ls 66.99±0.77d 20.75±0.23b 15.73±0.16a 64.95±0.67c 88.36±1.08d No.1800 Pa 63.54±0.72d 18.10±0.19a 12.82±0.11a 61.43±0.64b 89.78±1.11d No.1800 Pp8 56.10±0.49c 16.94±0.18a 24.05±0.27c 68.48±0.73d 72.36±0.82c No.1800 Pp9 55.17±0.55c 15.40±0.12a 32.33±0.36d 54.25±0.55a 69.36±0.76a No.1800 Pp10 68.43±0.77d 20.87±0.22b 20.32±0.21c 55.38±0.59b 88.05±0.97d No.1702 Ls 61.80±0.69d 16.14±0.17a 17.45±0.15b 65.76±0.73c 84.43±0.99d No.1702 Pa 71.53±0.85d 19.09±0.20b 15.67±0.13a 58.39±0.58c 83.30±0.86c No.1702 Pp8 62.07±0.71c 21.17±0.21c 22.55±0.23b 65.19±0.69c 74.02±0.83b No.1702 Pp9 63.70±0.69c 18.18±0.19b 14.35±0.15a 74.63±0.88d 83.02±0.88d No.1702 Pp10 64.99±0.73c 18.55±0.20b 13.26±0.13a 59.51±0.52b 78.60±0.77c Solid state fermented samples No.1700 Ls 49.71±0.44a 15.32±0.15a 24.69±0.32b 63.81±0.74a 80.77±0.89c No.1700 Pa 47.66±0.38a 21.81±0.22b 19.06±0.22a 65.76±0.68b 76.55±0.82b No.1700 Pp8 55.05±0.58b 19.05±0.19a 23.56±0.23b 64.34±0.71c 81.87±0.92c No.1700 Pp9 63.86±0.77b 20.97±0.22b 31.02±0.36c 65.80±0.75c 83.80±0.97c No.1700 Pp10 56.34±0.59c 18.44±0.18a 31.11±0.32c 71.47±0.82d 75.64±0.85b No.1701 Ls 46.96±0.32b 19.67±0.19b 18.97±0.18a 65.82±0.61c 71.33±0.63a No.1701 Pa 47.43±0.41c 19.09±0.18a 19.86±0.21a 68.29±0.79d 80.97±0.91b No.1701 Pp8 48.66±0.48c 24.09±0.28d 33.38±0.37d 69.95±0.68d 84.08±0.98b No.1701 Pp9 36.38±0.32a 22.96±0.23c 22.76±0.23b 64.86±0.62c 78.97±0.82b No.1701 Pp10 52.43±0.62b 20.12±0.21a 19.98±0.21a 66.98±0.70d 91.17±0.99d No.1800 Ls 42.57±0.42b 19.00±0.16b 20.45±0.23b 67.38±0.76d 81.46±0.83c No.1800 Pa 72.12±0.68d 20.93±0.23c 22.43±0.25b 62.32±0.81c 77.32±0.72b No.1800 Pp8 78.68±0.88d 10.68±0.11a 32.06±0.35c 61.51±0.63b 68.15±0.69a No.1800 Pp9 59.07±0.67c 10.96±0.13a 42.11±0.54d 64.29±0.60c 78.73±0.87b No.1800 Pp10 69.54±0.73c 16.55±0.16c 36.04±0.39d 70.13±0.77d 88.77±0.92d No.1702 Ls 73.89±0.76d 14.56±0.13b 19.86±0.17a 58.62±0.62b 82.98±0.83a No.1702 Pa 53.47±0.52b 9.96±0.08a 21.22±0.23b 58.16±0.57b 78.21±0.77b No.1702 Pp8 53.03±0.52b 9.11±0.09a 21.21±0.22b 63.19±0.67c 82.14±0.90c No.1702 Pp9 73.60±0.82c 21.93±0.24b 22.75±0.25c 73.60±0.89d 78.97±0.83b No.1702 Pp10 58.78±0.58b 20.96±0.22b 21.93±0.23b 72.31±0.85d 89.22±0.94c Data expressed as means (n = 5) ± SD; SD – standard deviation; p significant, when p ≤ 0.05; a-d The mean values within a column with different letters are significantly different (p≤0.05).

The protein solubility at pH 2.0 was significantly affected by the selection of lupine variety (F(255.525)=9.185, p≤0.0001) and the interaction of analysed factors was significant (F(243.074)=8.737, p≤0.0001), however, the selection of lupine variety, the fermentation method and the type of LAB applied for the fermentation had no significant influence on protein solubility at pH 2.0. The protein solubility at pH 4.0 was significantly affected by the selection of lupine variety (F(21.884)=11.592, p≤0.0001), the fermentation method (F(295.115)=156.321, p≤0.0001), the type of LAB applied for the fermentation (F(62.954)=33.346, p≤0.0001) and the interaction of analysed factors was significant (F(53.843)=28.520, p≤0.0001). The protein solubility at pH 6.0 was significantly affected by the selection of lupine variety (F(37.516)=8.146, p≤0.0001), the fermentation method (F(1045.781)=227.059, p≤0.0001), the type of LAB applied for the fermentation (F(93.972)=20.403, p≤0.0001) and the interaction of analysed factors was significant (F(82.642)=17.943, p≤0.0001). The protein solubility at pH 8.0 was significantly affected by the fermentation method (F(1045.782)=227.620, p≤0.0001) and the interaction of analysed factors was significant (F(81.979)=10.122, p≤0.0001). The protein solubility at pH 10.0 was significantly affected by the fermentation method (F(150.088)= 28.291, p=0.0001), the type of LAB applied for the fermentation (F(175.597)=14.725, p≤0.0001) and the interaction of analysed factors was significant (F(108.833)=9.12, p≤0.0001), however, the selection of lupine variety had no significant influence on protein solubility at pH 10.0. The most important functional properties of protein include its solubility, water- and fat-binding capacities, gel forming and rheological behaviours, as well as emulsifying capabilities, foaming and whipping abilities. These properties relate to the way in which proteins interact with large (carbohydrates, lipids and proteins) and small (gases, salts, volatiles and water) molecules, as well as the molecular size, the structure (primary amino acid sequences, secondary and tertiary conformations), and the charge distribution in the protein molecules [253]. Protein solubility in aqueous solutions is often a prerequisite for its other functional properties such as emulsiﬁcation and foaming. Factors that affect protein solubility are the following: pH, ionic strength, type of solvent and temperature. Proteins are the least soluble at their isoelectric point. A common method used to isolate most soluble plant proteins (largely, albumins and/or globulins) is based on this isoelectric point principle, i.e. proteins are solubilised using acid, alkali or solvent (with or with salt) away from their isoelectric point and then precipitated out by adjusting the pH of the protein extract to the targeted isoelectric point. The isolated proteins (e.g. protein isolates prepared from soy, pea, lupine and canola) have good protein solubility at neutral pH. However, most plant 60 proteins, particularly the cereal proteins that contain high levels of prolamins and glutelins, have extremely low solubility at neutral pH due to the low content of charged AA residues [253]. Protein solubility can be strongly affected by pH due to a large decrease in droplet size as a result of high pressure application in an alkaline pH [254, 255]. Solubility directly affects other techno-functional properties of incorporated substrates, such as emulsifying, foaming, and gelling properties, as well as viscosity; therefore, solubility is a key functional property for developing new formulations of functional additives [256]. The most widely applied procedure in preparing protein isolates is isoelectric precipitation. After alkaline solubilization of the proteins, ranging from pH 8-10 and removal of the insoluble material by centrifuging, proteins are precipitated by adding acid with pH 4-6 in order to reach the isoelectric point [257]. Polyphenol and protein interaction reduces the solubility of protein profile. Polyphenols induce crosslinking between proteins, which changes the net charge on the surface of protein molecule and hence affects the solubility of proteins [258]. The protein content (%) of nonfermented, SMF and SSF with L. sakei, P. acidilactici, P. pentosaceus No.8, No.9 and No.10 strains in seed protein isolates/concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072, and No.1734is presented in Table 3.3.4. In nonfermented lupine protein isolates/concentrates, protein content was ranging from 82.39 ± 0.79% to 88.24 ± 0.97% (in nonfermented hybrid No.1734 and nonfermented Vilciai concentrates, respectively). In SMF samples protein content was ranging from 80.43 ± 0.92% to 89.72 ± 0.93% (in SMF with P. pentosaceus No.9 Vilniai and in SMF with P. acidilactici hybrid No.1734 concentrates, respectively), and in SSF samples from 82.19 ± 0.69% to 92.27 ± 0.89% (in SSF with P. pentosaceus No.9 hybrid No.1734 concentrate and in SSF with L. sakei hybrid No.1734 isolates, respectively). To compare SMF and SSF samples, higher content of protein was found in all SSF samples. The results of the ANOVA test have indicated that the protein content in lupine isolates/concentrates was significantly affected by the selection of lupine variety (F(16.138)=21.319, p≤0.0001), the fermentation method (F(28.548)=37.298, p≤0.0001), the type of LAB applied for the fermentation (F(69.282)=90.516, p≤0.0001) and the interaction of these factors (F(24.821)=32.428, p≤0.0001).

Table 3.3.4. The protein content (%) of nonfermented, SMF and SSF with L. sakei, P. acidilactici, P. pentosaceus No.8, No.9 and No.10 strains in seed protein isolates/concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072, and No.1734. Lupine Protein content (% d.m.) samples Vilciai Vilniai No.1072 No.1734 NF 88.24±0.97c 86.98±0.94b 88.18±0.90c 82.39±0.79a SMF Ls 85.69±0.89b 89.68±0.99d 85.20±0.82b 89.33±0.91d SMF Pa 84.51±0.92a 88.00±0.95c 88.44±0.84c 89.72±0.93d SMF Pp8 87.92±1.01d 88.90±0.97c 86.98±1.02c 88.64±0.93d SMF Pp9 88.24±0.99d 80.43±0.92a 88.91±1.01d 82.87±0.88b SMF Pp10 86.98±0.98c 86.94±0.99c 86.94±0.96c 86.92±0.79c SSF Ls 89.42±0.95b 90.11±0.92c 89.12±0.82b 92.27±0.89d SSF Pa 91.88±0.99d 90.02±0.85c 87.91±0.77a 87.26±0.71a SSF Pp8 82.58±0.94a 86.98±0.78b 86.98±0.79b 89.98±0.71b SSF Pp9 82.49±0.85a 89.08±0.69c 86.94±0.67b 82.19±0.69a SSF Pp10 86.98±0.69b 86.06±0.74b 82.39±0.62a 88.91±0.83c Data expressed as means (n = 3) ± SD; SD: standard deviation; p significant, when p ≤ 0.05. NF – nonfermented samples; SMF – submerged fermented; SSF – solid state fermented. BI – before protein isolation; AI – after protein isolation; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici; Pp8 – Pediococcus pentosaceus; Pp9 – Pediococcus pentosaceus; Pp10 – Pediococcus pentosaceus. a-d The mean values within a column with different letters are significantly different (p≤0.05).

The protein content (%) in nonfermented, SMF and SSF with L. sakei, P. acidilactici, P. pentosaceus No.8, No.9 and No.10 strains in protein isolates/concentrates of lupine hybrid lines No.1700, No.1701, No.1800, and No.1702 is presented in Table 3.3.5. In nonfermented lupine protein isolates/concentrates, protein content was ranging from 87.85 ± 0.82% to 89.71 ± 0.97% (in nonfermented hybrid No.1702 and nonfermented hybrid No.1800 concentrates respectively). In SMF samples protein content was ranging from 84.68 ± 0.69% to 90.43 ± 1.11% (in SMF with P. pentosaceus No.10 hybrid No.1701 concentrates and in SMF with P. pentosaceus No.8 hybrid No.1701 isolates, respectively). In SSF samples protein content has ranged from 86.95 ± 0.89% to 92.97 ± 1.09% (in SSF with L. sakei hybrid No.1700 concentrates and in SSF with P. pentosaceus No.9 hybrid No.1701 isolates, respectively). In SSF samples, protein content was on average higher by 1.86%, compared to SMF samples. The protein content in lupine isolates/concentrates was significantly affected by the fermentation method (F(87.826)=104.190, p≤0.0001), the type of LAB applied for the fermentation (F(18.568)=22.027, p≤0.0001) and the interaction of analysed

62 factors (F(5.740)=6.809, p≤0.0001), however, the selection of lupine variety had no significant influence on protein content.

Table 3.3.5. The protein content (%) in nonfermented, SMF and SSF with L. sakei, P. acidilactici, P. pentosaceus No.8, No.9 and No.10 strains in protein isolates/concentrates of lupine hybrid lines No.1700, No.1701, No.1800, and No.1702. Protein content (% d.m.) Samples No.1700 No.1701 No.1800 No.1702 NF 88.87±0.84b 89.11±0.92c 89.71±0.97c 87.85±0.82a SMF Ls 88.31±0.79c 86.55±0.88a 89.99±0.94c 88.66±0.92d SMF Pa 87.90±0.69b 87.78±0.91b 90.03±0.97d 89.60±0.89cd SMF Pp8 87.80±0.76b 90.43±1.11d 87.72±0.79b 88.67±0.86b SMF Pp9 88.12±0.87c 88.81±0.85b 89.63±0.89c 90.11±0.97d SMF Pp10 88.07±0.81c 84.68±0.69a 90.35±1.07d 89.14±0.91c SSF Ls 86.95±0.89a 91.45±0.98c 91.17±0.97d 92.44±1.07d SSF Pa 87.18±0.91b 89.01±0.95b 90.50±1.02c 92.14±1.01d SSF Pp8 89.26±0.87b 91.68±1.02c 87.02±0.82a 87.60±0.83a SSF Pp9 91.71±0.97d 92.97±1.09d 91.68±0.86d 91.88±1.05c SSF Pp10 90.54±0.91c 91.05±0.95d 90.08±0.92b 90.27±0.98b Data expressed as means (n = 3) ± SD; SD: standard deviation; p significant, when p ≤ 0.05. NF – nonfermented samples; SMF – submerged fermented; SSF – solid state fermented. BI – before protein isolation; AI – after protein isolation; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici; Pp8 – Pediococcus pentosaceus; Pp9 – Pediococcus pentosaceus; Pp10 – Pediococcus pentosaceus. a-dThe mean values within a column with different letters are significantly different (p≤0.05).

Proteins are the main constituents of agricultural raw materials with two main (complementary) functions: bio- and techno-function. Biofunctionality of proteins is related to their nutritional and physiological properties, while techno-functionality is related to their physico-chemical properties affecting appearance, texture and stability of food products (e.g. solubility, viscosity, foaming, emulsifying and gelling ability, fat absorption capacity) [259– 261]. Prior defatting leads to protein isolates with improved purity as it minimizes the risk of co-extraction of lipids, saturated and unsaturated aldehydes and carboxylic acids which might contaminate the final protein and impair protein extraction [262]. Though aqueous extraction was performed to omit the use of organic solvent during defatting, co-extraction of oil components and long chain carbohydrates was observed [208], impairing protein purification. Therefore, aqueous fractionation without prior defatting is not suitable when the aim is to maximise protein purity. Moreover, the

63 removal of lipid fraction improves the oxidative stability of the flour and decreases the risk of off-flavour generation through oxidative rancidity. Hydrating the intact lupine kernels in excess water, with separation of the soak water prior to wet milling of the kernels has been reported [263]. This pre-treatment was performed to reduce the levels of low molecular weight compounds, including raffinose family oligosaccharides (RFO) which can later contaminate the protein fraction. However, during the hydration process some proteins were also lost so no increase in protein extraction yield was observed [263]. The utilization of novel technologies for protein extraction can improve protein extraction yield, their techno-functional and nutritional properties. Moreover, they are rated as affordable, safe, effective, and ecologically-friendly alternatives enabling clean label status [264]. In spite of numerous advantages, the application of novel technologies at industrial level is still limited [265]. The selection of the type of the process for protein extraction is dependent on the intended purpose of the resulting product, available resources and technical capabilities. For example, proteins obtained by alkali and enzymatic-extraction are applicable in the formulation of emulsion-based foodstuffs [266]. The resulting protein products are categorized on the basis of their protein content as protein flours (containing up to 65% of proteins), protein concentrates (containing up to 65–90% of proteins) and isolates (containing more than 90% of proteins) [267]. Despite the vast literature data about the production of different types of protein concentrates and isolates, only those originating from soybean [210] and rapeseed/canola are commercialized, unlike the different types of protein flours which are widely marketed. Protein extraction techniques can be divided into dry and wet techniques. Novel dry protein extraction techniques Sieving and/or air classification techniques for the fractionation of protein-rich and fiber-rich fractions are widely covered in the literature [268–273]. Although they are able to yield fractions with retained protein functionality and proved to be more energy efficient in comparison to wet fractionation processes, the main disadvantage of these processes is impurity of resulting fractions and agglomeration of particles, especially when it is subjected to oilseed meals due to the residual lipid content. To overcome the existing drawbacks, novel dry fractionation process has been proposed: electrostatic separation. It is performed in two steps involving charging the particles followed by the separation of charged particles in an electric field [274]. Wang et al. [275] and Tabtabaei et al. [276] utilized a tribo-electrostatic separation method for the fractionation of legume flour in order to obtain protein- and carbohydrate-enriched fractions, where it was found that protein enrichment by electrostatic separation is up to 15% higher than that obtained by air classification. Protein extraction 64 usually starts with solubilizing the protein rich source in a medium with the pH far from the isoelectric point, followed by their precipitation in medium with the pH close to the isoelectric point of the solubilized proteins. The other approach is to achieve protein solubilization using saline solutions followed by protein precipitation caused by salt removal by ultrafiltration and diafiltration. The protein produced in this way has a micellar structure before being dried, with preserved native state [277]. The variety of acid and alkaline protein extraction protocols have been reported so far depending on the type of plant protein source [278]. In general, acid aided extraction appears less promising because of inefficient cell wall degradation by acid which prevents protein diffusion to the medium. Moreover, the applied acid pH is closer to the protein isoelectric point than that of the alkaline experiments; therefore, the protein has less net charge providing lower protein solubility [279]. Generally, alkaline extraction shows better results. Currently, only soybean protein has been commercially extracted under alkaline conditions (pH 8–9) because the process provides high protein yields with a low price [280]. For the other potential sources of proteins, alkaline extraction doesn't provide such good yields and for that reason and due to extreme pH conditions which may cause protein denaturation as well as high consumption of acids, alkalis and water, alternative extraction methods have emerged. The combination of water and enzymes, water at subcritical conditions and extraction of proteins by reverse micelles is attracting more and more attention. The effect of the drying method on protein functionality depends on the drying method and on the type of protein. Waterbased plant protein dispersions are typically dried using freezedrying, spray-drying or vacuum-drying [281–283]. Freeze-drying is an expensive drying process, which is typically used for speciality ingredients and is perceived to be a relative mild drying process [284]. Spray drying is often applied in industry because of its scalability, continuous operation and standardized quality specifications[285]. Vacuum drying is a low cost process that is performed at low temperature, but requires long residence times [282]. Freeze-drying influences the morphology and size of the protein and the surface hydrophobicity of proteins by partial denaturation, due to stresses such as low temperatures, freezing stresses (e.g. phase separation, pH change and ice crystal formation) and drying stresses [215, 281]. Spray drying reduced the solubility of a lentil protein isolate less than vacuum drying [282] and can lead to thermal damage in case of lupin protein isolates [216]. Since freezedrying is generally perceived as the mildest form of drying, this drying method was chosen for comparison with ultrafiltration.

3.4. The Influence of Fermentation on Molecular Weights of Lupine Protein Fractions

The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 strains variety Vilciai protein isolates/concentrates are showed in Table 3.4.1. In compare fermented and nonfermented variety Vilciai seeds protein isolates/concentrates fractions, it was established that in SMF and SSF protein isolates/concentrates units of a lower molecular weight protein were not found (from 10 kDa to 20 kDa), except in SSF with P. acidilactici samples in which protein units at 13 kDa were visible, compare to control lane. In SMF with L. sakei and P. acidilactici samples was visible the higher molecular weight protein between 88 kDa to 102 kDa, in compare to control lane. The lower molecular weight protein units in all profiles of SSF with L. sakei, P. acidilactici and P. pentosaceus were not visble. A few units between 107 kDa to 122 kDa of SMF and SSF with P. pentosaceus No.9 protein isolates were disappeared. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 variety Vil- niai protein isolates/concentrates are showed in Table 3.4.2. In compare fermented and nonfermented protein isolates/concentrates fractions, it was established, that a lower molecular weight units in protein isolates/concentrates (from 10 kDa to 19 kDa) in SMF and SSF with P. acidilactici and P. pentosaceus strains samples, in compare to control lane, can be obtained. A few units from 122 kDa to 140 kDa in SMF with L. sakei lupine samples were disappeared. The molecular weight from 19 kDa to 31 kDa proteins in SSF with L. sakei lupine samples were not seen. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 hybrid line No.1072 protein isolates/concentrates are showed in Table 3.4.3. In compare nonfermented and fermented hybrid No.1072 protein isolates fractions, it was established that in SMF and SSF protein isolates a lower molecular weight protein units (between 10 kDa to 20 kDa) were not found. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 in protein isolates/concentrates of hybrid line No.1734 are shown in Table 3.4.4. In SMF with L. sakei samples visible protein units of the lower molecular weight (between 10 kDa and 20 kDa) were seen, in compare to control lane.

Table 3.4.1. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 strains variety Vilciai protein isolates/concentrates. Vilciai MW, kDa Marker NF SMF Ls SMF Pa SMF Pp9 220 160 120 100 90 80 70 60 50 40 30 20

220 160 120 100 90 80 70 60 50 40 30 20 10 MW – molecular weight; NF – nonfermented samples; SMF – submerged fermentation; SSF – solid state fermentation; Ls – Lactobacillus sakei; Pa – Pedioccoccus acidilactici; Pp9 – Pediococcus pentosaceus No.9.

Table 3.4.2. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 in protein isolates/concentrates of variety Vilniai. Vilniai MW, kDa Marker NF SMF Ls SMF Pa SMF Pp9 220 160 120 100 90 80 70 60 50 40 30 20

220 160 120 100 90 80 70 60 50 40 30 20

10 MW – molecular weight; Control NF – nonfermented samples; SMF – submerged fermentation; SSF – solid state fermentation; Ls – Lactobacillus sakei; Pa – Pedioccoccus acidilactici; Pp9 – Pediococcus pentosaceus No.9.

Table 3.4.3. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 in protein isolates/concentrates of hybrid line No.1072. No.1072 MW, kDa Marker NF SMF Ls SMF Pa SMF Pp9 220 160 120 100 90 80 70 60 50 40 30 20 10

Table 3.4.4. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 in protein isolates/concentrates of hybrid line No.1734. No. 1734 MW, kDa Marker NF SMF Ls SMF Pa SMF Pp9 220 160 120 100 90 80 70 60 50 40 30 20 10

220 160 120 100 90 80 70 60 50 40 30 20 10

In SMF with L. sakei lupine samples the protein units were heavily concentrated between 60 kDa and 90 kDa molecular weight, in compare to control lane. However, a few protein units between 85 kDa and 98 kDa in SMF with P. acidilactici and P. pentosaceus No.9 lupine samples were not found, in compare to control lane. An opposite tendencies in SSF with P. acidilactici were found, as higher intensity of protein fractions between 60 kDa and 95 kDa was visible, and the highest intensity of protein fraction between 60 kDa and 76 kDa in SSF with L. sakei samples was obtained. A few protein units in SSF with P. pentosaceus No.9 samples from 110 kDa to 220 kDa have disappeared. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 in protein isolates/concentrates of hybrid line No.1700 are shown in Table 3.4.5. After comparing fermented and nonfermented hybrid No.1700 lupine sample protein fractions, it was established that in SMF with L. sakei, P. acidilactici and P. pentosaceus No.9 samples the units of lower molecular weight proteins (between 10 kDa and 20 kDa) were not found. In SSF with P. pentosaceus No.9 hybrid No.1700 lupine protein samples the units of lower molecular weight (visible units from 15 kDa) were found, compared to control lane. Consequently, the low molecular weight proteins in profiles of SMF and SSF (except in SSF with P. pentosaceus No.9) lupine sample protein isolates/concentrates of hybrid No.1700 were not seen. However, SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 have shown that the protein units intensity from 55 kDa to 62 kDa, compared to control lane. A few units have disappeared from 88 kDa to 94 kDa in SMF with P. acidilactici lupine protein isolates/concentrates of hybrid No.1700. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 in protein isolates/concentrates of hybrid line No.1701 are shown in Table 3.4.6. In SMF with P. pentosaceus No.9 hybrid No.1702 lupine protein samples the units of lower molecular weight proteins (between 18 kDa and 24 kDa) were visble; however, units from 94 kDa to 220 kDa were not obtained, compared to control lane. In SMF with P. acidilactici lupine No.1701 isolates the lower intensity of protein units between 102 kDa and 109 kDa was found. In SSF with P. acidilactici lupine samples lane the lower intensity of protein units between 80 kDa and 88 kDa was visible, compared to control lane. The higher intensity of protein units in SSF with P. pentosaceus No.9 samples between 78 kDa and 101 kDa were found. In SSF with L. sakei, P. acidilactici and P. pentosaceus protein units between 102 kDa to 220 kDa of hybrid No.1701 have disappeared.

Table 3.4.5. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 in isolates/concentrates of hybrid line No.1700 protein. No. 1700 MW, kDa Marker NF SMF Ls SMF Pa SMF Pp9 220 160 120 100 90 80 70 60 50 40 30 20

Table 3.4.6. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 in protein isolates/concentrates of hybrid line No.1701. No. 1701 MW, kDa Marker NF SMF Ls SMF Pa SMF Pp9 220 160 120 100 90 80 70 60 50 40 30 20 10

The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 in lupine protein isolates/concentrates of hybrid line No.1800 lupine are shown in Table 3.4.7. In comparison of nonfermented and fermented protein isolates/ concentrates fractions of hybrid No.1800, it was established that in SMF and SSF protein isolates the lower molecular weight protein (from 10 kDa to 20 kDa) was not found. In all profiles of SMF hybrid No.1800 lupine samples the lower intensity of protein units from 102 kDa to 109 kDa were found, and in SSF with P. acidilactici and P. pentosaceus No.9 lupine isolates/ concentrates lane the lower intensity units from 80 kDa to 88 kDa were found, compared to control lane. In compare to control lane, in all profiles of SMF and SSF the protein units were heavily concentrated from 88 kDa to 92 kDa, except in lupine samples during SSF with P. acidilactici No.9, in which lower intensity was established. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 in protein isolates/concentrates of hybrid line No.1702 are shown in Table 3.4.8. After comparing nonfermented and fermented protein isolate fractions of hybrid No.1702, it was established that in SMF and SSF protein isolates the lower molecular weight proteins (between 10 kDa and 20 kDa) were not found. In SMF with P. acidilactici hybrid No.1734 lupine samples the protein units between 80 kDa and 89 kDa were heavily concentrated, in compare to control lane. In SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 protein units between 104 kDa and 220 kDa have dissapeared and were not visible, in compare to control lane.

Table 3.4.7. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 in protein isolates/concentrates of lupine hybrid No.1800. No. 1800 MW, kDa Marker NF SMF Ls SMF Pa SMF Pp9 220 160 120 100 90 80 70 60 50 40 30 20 10

Table 3.4.8. The protein fractions and molecular weights of the nonfermented, SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.9 in protein isolates/concentrates of hybrid line No.1702. No. 1702 MW, kDa Marker NF SMF Ls SMF Pa SMF Pp9 220 160 120 100 90 80 70 60 50 40 30 20

220 160 120 100 90 80 70 60 50 40 30 20

10 MW – molecular weight; NF – nonfermented samples; SMF – submerged fermentation; SSF – solid state fermentation; Ls – Lactobacillus sakei; Pa – Pedioccoccus acidilactici; Pp9 – Pediococcus pentosaceus No.9.

3.4.1. The Main Lupine Protein Fractions

The application of fermentation is mainly possible due to the degradation of the cell wall structure by microbial enzymes into unique metabolites through different pathways [286]. The monomeric molecular weight is around 47–48 kDa consisting of two polypeptide chains of 27–30 kDa and 16–18 kDa respectively [287], of which the larger chain (27–30 kDa) is glycosylated [288]. γ-Conglutin has higher levels of the essential amino acids, particularly methionine, cysteine and lysine than α-conglutin and β-conglutin indicating a higher nutritional value. The most peculiar characteristic of γ-conglutin is its isoelectric point (pI) in the basic range unlike other conglutins, which have an acidic pI. The most abundant lupine seed protein is β-conglutin (about 44% w/w of total conglutins) [289–291]. Its native molecular weight varies in the range of 143–260 kDa, thus suggesting a trimeric quaternary structure, as most vicilin-like globulins [292] and consists of precursor and proteolytically cleaved polypeptides of molecular weights of around 15–72 kDa. The second most abundant globulin, which is 33% w/w of total conglutins, is α-conglutin, a legumin-like globulin [291] which is very similar in terms of its solubility, amino acid composition, sedimentation∼ behaviour and charge in all lupine species [290]. It is a hexamer with the native molecular weight of 300–430kDa, made up of subunits ranging from 53 kDa to 74 kDa and consisting of acidic and basic chains which are bound together by disulphide linkage [293]. The third conglutin is δ-conglutin ( 12% w/w of total conglutins). In contradiction, it has been described variously as a globulin and as a composite albumin-globulin, but mostly as an albumin∼ [290]. The main lupine proteins that are able to bind IgE antibodies of allergic individuals are characterised by molecular weights in the range of 43–45 kDa [294– 296]. However, similar effects may also be caused by other lupine proteins characterised by different molecular weight, i.e., 13 kDa [294], 29 kDa [298], 34 kDa [299], 38 kDa [300], and 66 kDa [301]. The minor seed globulin is γ-conglutin which accounts for 4–5% w/w of total globulins [291]. γ-Conglutin has unique physicochemical properties, which makes it different amongst other lupin proteins [302].∼ The water soluble sulfur-rich albumins (conglutin δ) account for 2.6% of the total protein content of the lupin seed [303]. The molecular weight of conglutin δ varies depending on solvent composition and protein concentration. Conglutin δ (14 kDa) is a monomer composed of a heavy (9.5 kDa) and a light (4.5 kDa) polypeptide chains. At neutral pH, it forms a dimer (conglutin d1, 28 kDa) that associates via disulfide bonds to oligomeric aggregates of up to 56 kDa

[304]. α-Conglutin is also known for its characteristic glycosylation [305]. The legumin-like conglutin α and the vicilin-like conglutin β of lupin seeds present the main globulins [305] and account for 76% and 16.4%, respectively, of total lupin seed protein content [306–308]. Transition from a trimer to a hexamer occurs under limited proteolytic activity, as a result of shifts in the environment, such as a decrease in pH [306]. The subunits are further divided into alkaline polypeptides of 21–24 kDa and acidic polypeptides of 42–62 kDa linked together via intermolecular disulfide bonds. Lqari et al. (2004) have found trimeric protein structures of around 216 kDa [307]. Conglutin a is composed of three subunits connected non- covalently, with each exhibiting the sizes of 64 kDa, 72 kDa and 85 kDa [309]. Conglutin β is the only lupin protein free of disulfide bonds, which leads to identical band profiles in SDS-PAGE under both reducing and non- reducing conditions. The prevalent form of the 150–170 kDa oligomer is trimeric but conglutin b also forms tetramers. The sizes of the subunits range from 20 kDa to 70 kDa each of which is composed of 10 to 12 distinct and mostly glycosylated polypeptides [310, 311].

3.5. The Modulation of Amino Acids Profile in the Lupine Seeds Wholemeal and Protein Isolates/Concentrates

3.5.1. The Total Amino Acids Content in the Lupine Wholemeal Samples

The total essential amino acids (TEAA) content (%) from total protein hydrolysed in lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734 nonfermented wholemeal, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.1.1. In all the cases, in SMF with L. sakei and P. acidilactici wholemeal samples the contents of VAL (except for SMF with P. acidilactici hybrid No.1734 samples), THR and MET were increased, in compare with nonfermented wholemeal. In SSF lupine seeds wholemeal contents of LEU, THR (except in SSF with P. acidilactici variety Vilciai samples and in SSF with L. sakei and P. acidilactici hybrid line No.1734 samples) and MET have increased, in compare with nonfermented lupine wholemeal. However, content of MET in SSF wholemeal was on average lower by 62.40%, compared to SMF samples. The contents of LYS (except in SSF with P. acidilactici variety Vilciai wholemeal) and HIS (except in SSF with L. sakei and P. acidilactici variety Vilciai and in SSF with P. acidilactici hybrid line No.1734 wholemeal) in SSF samples have decreased, compared to the nonfermented samples. The results of the ANOVA test indicated that the LYS content in 78 fermented lupine wholemeal was significantly affected by the selection of lupine variety (F(6.029)=6.638, p=0.0001). However, the selection of lupine variety had no significant influence on VAL, ILE, LEU, THR, MET, PHE and HIS contents. The VAL, LEU, MET, PHE, LYS and HIS contents were significantly affected by applying the fermentation method (F(10.491)= 152.698, p≤0.0001; F(6.156)=20.771, p≤0.0001; F(2.637)=313.235, p≤0.0001; F(3.045)=15.725, p≤0.0001; F(10.859)=11.956, p=0.0001; F(7.922)=23.440, p≤0.0001, respectively). The type of microorganism applied for the fermentation had no signifacant influence on EAA profile mentioned above. The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine varieties Vilciai, Vilniai and hybrid lines No.1072, and No.1734 , SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.1.2. In all the cases, in SMF wholemeal contents of ALA, GLY, SER, PRO and GLU (except in SMF with P. acidilactici variety Vilciai wholemeal) were increased, in compare to nonfermented samples. Differet tendencies of GLU content in SMF wholemeal were established. The content of TYR in SMF wholemeal was decreased, compared to nonfermented samples. In SSF wholemeal higher content of PRO and ASP (except in SSF with P. acidilactici variety Vilciai samples) was established. Also, in SSF lupine wholemeal samples contents of ALA (except in SSF with L. sakei and P. acidilactici hybrid No.1734 samples), GLY (except in SSF with P. acidilacti variety Vilciai and hybrid No.1734 samples), SER (except in SSF with L. sakei and P. acidilactici hybrid No.1734 samples) and GLU (except in SSF with L. sakei and P. acidilactici variety Vilniai and hybrid No.1072 samples) were increased, in compare to nonfermented samples. However, in SSF wholemeal content of HIS was decreased, in compare to nonfermented one. The contents of ALA, GLY, SER and TYR in lupine wholemeal samples were significantly affected by the selection of lupine variety (F(0.943)=27.593, p≤0.0001; F(0.896)=6.100, p≤0.0001; F(1.547)=8.818, p≤0.0001; F(13.932)=22.508, p≤0.0001, respectively); however, the selection of lupine variety had no significant influence on PRO, ASP and GLU contents. The type of microorganism applied for the fermentation had significant influence on ALA content in lupine wholemeal (F(0.321)=9.397, p≤0.0001). The fermentation method was significantly affected GLY, PRO, ASP and TYR contents in lupine wholemeal (F(2.104)=14.327, p≤0.0001; F(3.746)=19.510, p≤0.0001; F(6.395)=15.217, p≤0.0001; F(36.575)=59.091, p≤0.0001, respectively), however, the fermentation method had no significant influence on ALA, SER and GLU contents. 79

Table 3.5.1.1. The total essential amino acids (TEAA) content (%) from total protein hydrolysed in lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734 nonfermented wholemeal, SMF and SSF with L. sakei and P. acidilactici strains. TEAA content (%) from total protein hydrolysed Lupine VAL ILE LEU THR MET PHE LYS HIS samples Nonfermented samples 4.35 4.99 8.34 3.37 0.16 5.52 6.42 5.17 Vilciai ±0.06a ±0.03b ±0.05e ±0.02a ±0.01b ±0.05d ±0.08c ±0.06e 4.32 4.83 7.05 3.45 0.15 4.75 7.16 4.34 Vilniai ±0.05a ±0.04a ±0.04a ±0.04b ±0.01a ±0.03a ±0.09f ±0.05a 4.56 5.10 7.39 3.75 0.15 5.10 7.66 4.40 No.1072 ±0.05b ±0.05d ±0.04c ±0.03c ±0.01a ±0.05c ±0.09f ±0.05b 4.69 5.12 8.43 4.26 0.17 5.58 5.83 4.30 No.1734 ±0.06c ±0.03d ±0.06e ±0.04e ±0.02c ±0.05e ±0.06b ±0.04a Submerged fermented samples 5.69 4.97 7.90 3.82 1.57 4.94 5.78 3.36 Vilciai Ls ±0.07a ±0.02c ±0.04c ±0.03a ±0.02c ±0.04c ±0.06e ±0.04b 4.75 5.77 9.47 3.58 0.89 6.20 7.19 4.58 Vilciai Pa ±0.05c ±0.08c ±0.11c ±0.02b ±0.01b ±0.06d ±0.05d ±0.04e 6.50 4.39 8.11 6.33 2.08 4.62 7.95 4.18 Vilniai Ls ±0.04b ±0.04a ±0.05d ±0.02e ±0.02f ±0.05b ±0.08f ±0.04d 4.58 5.23 9.45 4.53 0.73 6.03 4.20 5.57 Vilniai Pa ±0.03c ±0.05d ±0.12b ±0.04c ±0.01d ±0.06c ±0.03c ±0.05d No.1072 6.48 4.53 7.50 6.14 1.58 4.77 6.39 3.97 Ls ±0.04b ±0.04b ±0.05b ±0.02d ±0.03c ±0.04b ±0.07f ±0.03c No.1072 4.58 5.00 8.11 4.54 0.67 5.41 7.16 4.64 Pa ±0.02a ±0.04c ±0.08a ±0.04c ±0.01e ±0.04e ±0.06c ±0.04b No.1734 6.75 4.64 7.52 6.39 1.76 4.53 5.24 3.69 Ls ±0.05c ±0.04b ±0.05b ±0.04f ±0.03e ±0.04a ±0.05d ±0.03c No.1734 4.52 4.74 7.93 4.43 0.72 5.01 4.84 4.66 Pa ±0.02a ±0.05b ±0.06a ±0.04d ±0.01c ±0.05f ±0.04e ±0.05b Solid state fermented samples 4.22 4.82 9.44 3.88 0.72 5.03 5.31 3.86 Vilciai Ls ±0.04a ±0.07a ±0.08d ±0.04a ±0.04b ±0.06a ±0.05e ±0.03c 4.21 5.08 9.45 3.20 0.78 5.72 7.61 4.69 Vilciai Pa ±0.03b ±0.08b ±0.09c ±0.03b ±0.01a ±0.06b ±0.08c ±0.05d 5.32 4.65 9.42 6.54 0.81 5.92 6.10 5.75 Vilniai Ls ±0.03e ±0.04a ±0.08c ±0.04ef ±0.05b ±0.06c ±0.04f ±0.07e 4.79 5.28 9.33 4.42 0.83 6.10 4.59 5.49 Vilniai Pa ±0.04d ±0.06c ±0.07d ±0.06c ±0.02c ±0.05d ±0.05d ±0.05c No.1072 4.83 5.39 9.32 5.83 0.90 6.55 4.29 6.36 Ls ±0.05c ±0.05b ±0.08c ±0.05e ±0.04d ±0.07e ±0.04b ±0.07f No.1072 4.25 5.20 8.89 3.98 0.80 6.08 3.79 5.73 Pa ±0.03c ±0.04c ±0.08b ±0.04d ±0.03b ±0.06c ±0.03c ±0.06b No.1734 4.79 5.28 8.30 4.23 0.84 5.31 3.99 4.90 Ls ±0.05b ±0.05b ±0.07a ±0.04b ±0.04c ±0.05b ±0.03a ±0.04d No.1734 3.92 4.83 8.57 3.65 0.57 4.83 5.46 4.37 Pa ±0.04a ±0.04d ±0.06c ±0.03a ±0.02d ±0.04b ±0.05d ±0.05b TEAA – total essential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; VAL – valine; ILE – isoleucine; LEU – leucine; THR – threonine; MET – methionine; PHE – phenylethylamine; LYS – lysine; HIS – histamine. Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-f The mean values within a column with different letters are significantly different (p≤0.05). 80

Table 3.5.1.2. The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine varieties Vilciai, Vilniai and hybrid lines No.1072, and No.1734, SMF and SSF with L. sakei and P. acidilactici strains. TNAA content (%) from total protein hydrolysed Lupine ALA GLY SER PRO ASP GLU TYR samples Nonfermented samples 2.93 3.79 5.11 4.56 10.64 25.88 8.78 Vilciai ±0.03a ±0.03a ±0.05a ±0.05b ±0.11b ±0.14b ±0.05e 3.14 3.83 5.02 4.35 10.43 26.58 10.61 Vilniai ±0.04a ±0.04a ±0.05a ±0.04a ±0.10a ±0.16c ±0.10f 3.37 4.06 5.53 4.67 11.34 27.34 5.57 No.1072 ±0.03b ±0.04b ±0.06b ±0.05b ±0.12d ±0.15d ±0.04b 3.99 4.59 6.11 4.70 10.39 26.20 5.64 No.1734 ±0.04d ±0.05d ±0.07e ±0.06c ±0.11a ±0.17b ±0.06b Submerged fermented samples 3.70 4.33 6.09 4.87 11.12 26.45 7.08 Vilciai Ls ±0.02b ±0.05b ±0.06b ±0.05c ±0.11ab ±0.15c ±0.04f 3.38 4.31 5.73 5.32 10.21 23.19 5.43 Vilciai Pa ±0.01c ±0.05e ±0.06c ±0.06d ±0.12c ±0.14b ±0.05c 3.58 3.98 7.29 4.56 11.37 30.43 2.32 Vilniai Ls ±0.03b ±0.03a ±0.07e ±0.04a ±0.12b ±0.18e ±0.02a 3.55 5.08 7.50 6.05 13.72 21.07 2.74 Vilniai Pa ±0.02b ±0.06d ±0.09d ±0.07bc ±0.13a ±0.13c ±0.03d 3.39 4.08 5.57 4.72 11.48 27.99 5.55 No.1072 Ls ±0.03a ±0.04a ±0.05a ±0.05b ±0.10b ±0.16d ±0.05e 3.84 4.88 7.74 5.47 12.34 23.22 2.43 No.1072 Pa ±0.04d ±0.05d ±0.10c ±0.06c ±0.12a ±0.15e ±0.02e 4.00 4.62 6.17 4.78 10.88 26.96 5.60 No.1734 Ls ±0.04c ±0.05b ±0.06b ±0.04b ±0.11a ±0.15d ±0.05e 4.01 4.96 7.37 5.24 11.94 27.29 2.33 No. 1734 Pa ±0.05a ±0.06c ±0.09d ±0.05b ±0.12b ±0.16d ±0.02d Solid state fermented samples 3.51 4.46 7.04 5.03 12.86 27.04 2.77 Vilciai Ls ±0.02a ±0.03a ±0.04c ±0.04a ±0.12b ±0.16f ±0.02c 3.15 3.72 5.92 4.61 10.06 27.56 4.24 Vilciai Pa ±0.03b ±0.02ab ±0.03b ±0.03c ±0.10c ±0.17b ±0.03b 3.91 5.39 7.23 5.85 12.12 23.38 1.97 Vilniai Ls ±0.02d ±0.05e ±0.06d ±0.05b ±0.11a ±0.14d ±0.01a 4.15 5.67 7.36 6.40 13.19 20.57 1.85 Vilniai Pa ±0.05c ±0.06c ±0.09c ±0.05d ±0.14bc ±0.16b ±0.01b 3.88 5.75 7.12 6.76 13.33 18.57 2.37 No.1072 Ls ±0.03c ±0.06f ±0.05c ±0.06e ±0.13c ±0.17a ±0.02b 3.59 5.10 7.04 6.04 12.23 23.77 2.32 No.1072 Pa ±0.04d ±0.04c ±0.08d ±0.05c ±0.12c ±0.18c ±0.02c 3.82 5.09 7.67 5.78 13.04 25.43 2.11 No.1734 Ls ±0.04c ±0.05c ±0.07e ±0.05b ±0.12d ±0.18e ±0.01b 3.56 4.36 7.05 4.88 11.44 31.56 1.94 No. 1734 Pa ±0.03d ±0.03d ±0.06b ±0.04b ±0.11b ±0.21c ±0.01d TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; ALA – alanine; GLY – glycine; SER – serine; PRO – proline; ASP – asparagine; GLU – glutamine; TYR – tyramine. Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-f The mean values within a column with different letters are significantly different (p≤0.05). 81

The total essential amino acids (TEAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine varieties Vilciai, Vilniai and hybrid lines No.1072 and No.1734, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains is presented in Table 3.5.1.3. In all the cases, higher contents of LEU (except in SMF with P. pentosaceus No.9 and No.10 variety Vilciai wholemeal), THR (except in SMF with P. pentosaceus No.8 and No.10 hybrid line No.1734 wholemeal) and MET in SMF wholemeal, compared to nonfermented, were found. In all the tested SSF wholemeal samples the contents of ILE, LEU, THR (except in SSF with P. pentosaceus No.8 hybrid No.1734 samples), MET, PHE (except in SSF with P. pentosaceus No.8 and No.9 variety Vilciai samples and in SSF with P. pentosaceus No.8 hybrid No.1734 samples) were increased, compared to nonfermented samples. Also, in SSF samples contents of LYS (except in SSF with P. pentosaceus No.8 and No.10 variety Vilciai samples) and HIS (except in SSF with P. pentosaceus No.8, No.9 and No.10 strains variety Vilciai samples) were increased, in compare to nonfermented samples.

Table 3.5.1.3. The total essential amino acids (TEAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine varieties Vilciai, Vilniai and hybrid lines No.1072 and No.1734, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains. TEAA content (%) from total protein hydrolysed Lupine VAL ILE LEU THR MET PHE LYS HIS samples 1 2 3 4 5 6 7 8 9 Nonfermented samples 4.35 4.99 8.34 3.37 0.16 5.52 6.42 5.17 Vilciai ±0.06a ±0.03b ±0.05e ±0.02a ±0.01b ±0.05d ±0.08c ±0.06e 4.32 4.83 7.05 3.45 0.15 4.75 7.16 4.34 Vilniai ±0.05a ±0.04a ±0.04a ±0.04b ±0.01a ±0.03a ±0.09f ±0.05a 4.56 5.10 7.39 3.75 0.15 5.10 7.66 4.40 No.1072 ±0.05b ±0.05d ±0.04c ±0.03c ±0.01a ±0.05c ±0.09f ±0.05b 4.69 5.12 8.43 4.26 0.17 5.58 5.83 4.30 No.1734 ±0.06c ±0.03d ±0.06e ±0.04e ±0.02c ±0.05e ±0.06b ±0.04a Submerged fermented samples Vilciai 4.65 5.34 8.43 3.78 0.84 5.51 5.94 3.59 Pp8 ±0.07d ±0.09e ±0.12e ±0.06a ±0.03a ±0.07c ±0.08c ±0.05b Vilciai 4.45 4.99 7.63 3.82 0.77 4.82 6.26 2.99 Pp9 ±0.05c ±0.07c ±0.09d ±0.07a ±0.02b ±0.05a ±0.06e ±0.04a Vilciai 4.74 5.51 8.31 4.04 0.78 5.22 4.87 3.58 Pp10 ±0.06d ±0.11d ±0.11de ±0.06c ±0.02b ±0.06b ±0.05b ±0.05b Vilniai 3.82 4.43 8.12 3.69 0.62 4.78 7.29 4.12 Pp8 ±0.05b ±0.06b ±0.07d ±0.07ab ±0.03 ±0.08c ±0.09f ±0.06c Vilniai 4.34 4.93 9.05 4.26 0.77 5.43 5.44 4.60 Pp9 ±0.07c ±0.08fb ±0.09c ±0.08c ±0.04c ±0.09a ±0.06c ±0.08c Vilniai 4.51 5.29 9.96 4.29 0.75 6.40 3.81 5.93 Pp10 ±0.08d ±0.08d ±0.12b ±0.07c ±0.03c ±0.11b ±0.04a ±0.09d 82

Table 3.5.1.3 continued 1 2 3 4 5 6 7 8 9 No.1072 4.18 4.70 7.52 4.15 0.58 5.02 6.30 3.94 Pp8 ±0.06b ±0.06d ±0.07c ±0.06bc ±0.02a ±0.06d ±0.08d ±0.05b No.1072 4.32 4.72 7.58 4.23 0.65 4.91 5.91 3.77 Pp9 ±0.05c ±0.07de ±0.05c ±0.05d ±0.02ab ±0.05d ±0.07c ±0.04b No.1072 4.21 4.57 7.41 3.91 0.61 4.77 6.28 3.96 Pp10 ±0.06a ±0.06c ±0.06bc ±0.04b ±0.01a ±0.04c ±0.06d ±0.06c No.1734 4.47 4.92 7.95 4.12 0.69 5.10 5.91 4.20 Pp8 ±0.04bc ±0.08b ±0.09e ±0.07c ±0.02a ±0.08cd ±0.09c ±0.07d No.1734 4.92 5.15 8.60 4.76 0.79 5.72 4.65 5.26 Pp9 ±0.08b ±0.10d ±0.13e ±0.08e ±0.03c ±0.09d ±0.05b ±0.09e No.1734 4.33 4.68 7.58 4.22 0.63 4.66 6.79 4.29 Pp10 ±0.06a ±0.07b ±0.10d ±0.05c ±0.02b ±0.06c ±0.09de ±0.06c Solid state fermented samples Vilciai 4.20 5.15 8.99 3.67 0.80 5.31 7.13 3.87 Pp8 ±0.06a ±0.07b ±0.12e ±0.04a ±0.03d ±0.06a ±0.09d ±0.05a Vilciai 4.81 5.53 8.39 4.23 0.78 5.43 6.25 3.94 Pp9 ±0.08de ±0.09c ±0.08d ±0.06b ±0.02c ±0.08b ±0.06c ±0.06a Vilciai 4.52 5.09 9.10 3.91 0.85 5.64 7.52 4.39 Pp10 ±0.06c ±0.04c ±0.11c ±0.05b ±0.03d ±0.06b ±0.08d ±0.07b Vilniai 4.74 5.35 9.12 4.59 0.79 6.44 3.70 6.44 Pp8 ±0.07d ±0.06cd ±0.13c ±0.07c ±0.01c ±0.09d ±0.04b ±0.09d Vilniai 4.38 5.18 8.03 4.08 0.66 5.04 5.40 4.89 Pp9 ±0.05c ±0.08d ±0.09b ±0.06b ±0.01b ±0.07c ±0.07d ±0.06c Vilniai 4.17 4.99 10.40 3.80 0.81 7.36 2.87 7.13 Pp10 ±0.04a ±0.05a ±0.11e ±0.05a ±0.02c ±0.10de ±0.03a ±0.08d No.1072 4.63 5.11 8.63 4.53 0.82 6.08 3.79 5.73 Pp8 ±0.08d ±0.09c ±0.08d ±0.08d ±0.02c ±0.07c ±0.04c ±0.05c No.1072 4.47 5.15 9.11 4.34 0.72 6.47 3.85 6.04 Pp9 ±0.06c ±0.09bc ±0.10c ±0.07cd ±0.03c ±0.09c ±0.06c ±0.08b No.1072 4.53 6.34 9.48 4.31 0.75 6.70 2.92 6.30 Pp10 ±0.07b ±0.06e ±0.09d ±0.09e ±0.04c ±0.11d ±0.02a ±0.11b No.1734 4.18 5.20 8.50 4.22 0.68 5.36 4.47 4.90 Pp8 ±0.05b ±0.05a ±0.07b ±0.06e ±0.02b ±0.08a ±0.06cd ±0.05a No.1734 4.40 6.15 9.08 4.29 0.62 6.46 2.97 5.94 Pp9 ±0.06c ±0.08d ±0.09d ±0.07d ±0.01a ±0.09c ±0.03a ±0.09d No.1734 4.85 5.17 8.57 4.74 0.64 5.89 5.14 5.27 Pp10 ±0.08d ±0.07c ±0.08c ±0.10f ±0.03a ±0.08b ±0.07d ±0.07c TEAA – total essential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation. VAL – valine; ILE – isoleucine; LEU – leucine; THR – threonine; MET – methionine; PHE – phenylethylamine; LYS – lysine; HIS – histamine; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-f The mean values within a column with different letters are significantly different (p≤0.05).

Content of HIS in SSF was higher (on average by 22.59%), in compare with SMF wholemeal samples. The VAL, ILE, LEU, THR, MET, PHE, LYS and HIS contents in lupine seeds wholemeal were significantly influenced by the selection of lupine variety (F(0.139)=35.997, p≤0.0001; F(0.342)= 67.653, p≤0.0001; F(0.210)=25.436, p≤0.0001; F(0.810)= 204.403, p≤0.0001; F(0.010)=18.372, p≤0.0001; F(0.021)=3.781, p≤0.0001 and F(0.271)=60.099, p≤0.0001, F(0.060)=13.481, p≤0.0001, respectively), the fermentation method (F(0.110)=28.556, p≤0.0001; F(2.030)=401.718, p≤0.0001; F(10.329)=1250.858, p≤0.0001; F(0.259)=65.443, p≤0.0001;

F(0.024)=43.716, p≤0.0001; F(12.103)=2194.881, p≤0.0001, F(22.579)= 5013.621, p≤0.0001, F(26.682)=5986.237, p≤0.0001, respectively), the type of microorganism applied for the fermentation (F(0.158)=40.743, p≤0.0001; F(0.718)=142.031, p≤0.0001; F(1.560)=188.947, p≤0.0001; F(0.152)= 38.386, p≤0.0001; F(0.955)=173.114, p≤0.0001; F(2.092)=464.490, p≤0.0001; F(1.787)=400.896, p≤0.0001, respectively, except MET content) and their interaction (F(0.338)=87.468, p≤0.0001; F(1.093)=216.237, p≤0.0001; F(1.131)=136.973, p≤0.0001; F(0.260)=65.623, p≤0.0001; F(0.022)=39.712, p≤0.0001; F(0.816)=148.058, p≤0.0001; F(7.059)= 1567.451, p≤0.0001, F(1.163)=260.836, p≤0.0001, respectively). Negative strong correlations between the LAB count and VAL and between the LAB count and MET were established (r=-0.6231 and r=-0.7729, respectively). Positive moderate correlations between the LAB count and LEU and between the LAB count and PHE were found (r=0.4603 and r=-0.4949, respectively). The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine varieties Vilciai, Vilniai and hybrid lines No.1072, and No.1734, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains is presented in Table 3.5.1.4. In all the cases, in SMF and SSF samples SER and ASP contents were higher, compared to nonfermented samples. Also, in most of the SMF samples contents of ALA (except in SMF with P. pentosaceus No.8 and No.10 strains hybrid No.1734 samples) and PRO (except in SMF with P. pentosaceus No.9 variety Vilciai samples) were higher, compared to nonfermented wholemeal. However, in SMF samples content of TYR (except in SMF with P. pentosaceus No.8 variety Vilciai wholemeal, in which opposite tendencies of TYR content were found it was increased) was decreased. Similar tendencies of TYR in SSF wholemeal samples, compared to SMF, were found. In compare with nonfermented wholemeal, in all SSF samples higher contents of GLY and PRO were established. Also, in SSF lupine samples higher content of ALA (except in SSF with P. pentosaceus No.8, No.9 and No.10 strains hybrid No.1734 samples) was found, compared to nonfermented samples. The SER content was significantly influenced by the selection of lupine variety (F(0.318)=6.804, p≤0.0001), however, the selection of lupine variety had no significant influence on ALA, GLY, PRO, ASP, GLU and TYR contents. The PRO content was significantly affected by the fermentation method (F(2.079)=17.532, p≤0.0001), however, the fermentation method had no significant influence on ALA, GLY, SER, ASP, GLU and TYR contents. The ALA, GLY, SER, PRO, ASP, GLU and TYR contents were significantly influenced by the type of microorganism applied for the fermentation (F(4.594)=87.378, p≤0.0001; F(0.843)=7.982, 84 p≤0.0001; F(1.795)=38.439, p≤0.0001; F(9.940)=83.814, p≤0.0001; F(419.725)=2579.772, p≤0.0001; F(615.063)=40.355, p≤0.0001; F(6.032)= 23.695, p≤0.0001, respectively).

Table 3.5.1.4. The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine varieties Vilciai, Vilniai and hybrid lines No.1072, and No.1734, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains. TNAA content (%) from total protein hydrolysed Lupine seeds ALA GLY SER PRO ASP GLU TYR 1 2 3 4 5 6 7 8 Nonfermented samples 2.93 3.79 5.11 4.56 10.64 25.88 8.78 Vilciai ±0.03a ±0.03a ±0.05a ±0.05b ±0.11b ±0.14b ±0.05e 3.14 3.83 5.02 4.35 10.43 26.58 10.61 Vilniai ±0.04a ±0.04a ±0.04a ±0.04a ±0.10a ±0.16c ±0.10f 3.37 4.06 5.53 4.67 11.34 27.34 5.57 No.1072 ±0.03b ±0.04b ±0.06b ±0.05b ±0.12d ±0.15d ±0.04b 3.99 4.59 6.11 4.70 10.39 26.20 5.64 No.1734 ±0.04d ±0.05d ±0.07e ±0.06c ±0.11a ±0.17b ±0.06b Submerged fermented samples 3.40 4.24 6.04 4.93 11.23 23.11 8.98 Vilciai Pp8 ±0.05a ±0.06e ±0.07b ±0.04b ±0.10a ±0.12b ±0.08e 3.49 4.03 6.29 4.51 11.20 27.07 7.76 Vilciai Pp9 ±0.04b ±0.07b ±0.09a ±0.05c ±0.09b ±0.14f ±0.07c 3.67 4.34 6.58 5.11 12.46 27.52 3.26 Vilciai Pp10 ±0.05c ±0.04e ±0.08b ±0.06b ±0.11c ±0.14a ±0.03b 3.33 3.90 6.99 4.45 11.19 30.99 2.28 Vilniai Pp8 ±0.04b ±0.05a ±0.07c ±0.05d ±0.12d ±0.16f ±0.04b 3.68 4.62 7.52 5.37 13.20 24.44 2.36 Vilniai Pp9 ±0.05d ±0.06d ±0.08b ±0.06d ±0.09e ±0.11c ±0.03f 3.63 5.28 6.93 6.29 12.94 21.60 2.38 Vilniai Pp10 ±0.04c ±0.05e ±0.07d ±0.05c ±0.10f ±0.12b ±0.04a 3.58 4.30 7.27 4.83 12.02 29.13 2.47 No.1072 Pp8 ±0.05b ±0.04d ±0.08c ±0.04d ±0.09b ±0.11d ±0.03d 3.67 4.21 7.69 4.78 11.71 29.52 2.32 No.1072 Pp9 ±0.04b ±0.03c ±0.06b ±0.05e ±0.11c ±0.13e ±0.04e 3.70 4.24 7.46 4.80 11.44 30.30 2.39 No.1072 Pp10 ±0.03e ±0.04c ±0.05d ±0.06d ±0.10a ±0.16d ±0.05b 3.79 4.53 7.24 5.07 12.09 27.15 2.76 No.1734 Pp8 ±0.05c ±0.04e ±0.07e ±0.06e ±0.12b ±0.13d ±0.04d 4.10 5.45 7.57 6.07 13.37 21.03 2.56 No.1734 Pp9 ±0.06e ±0.05c ±0.08d ±0.07b ±0.15e ±0.14e ±0.04a 3.83 4.44 7.66 4.94 12.14 27.49 2.34 No.1734 Pp10 ±0.04c ±0.03b ±0.09f ±0.05e ±0.12d ±0.15e ±0.03c Solid state fermented samples 3.31 4.11 6.52 4.76 10.89 28.05 3.68 Vilciai Pp8 ±0.03b ±0.05a ±0.07c ±0.04b ±0.10b ±0.14f ±0.05d 3.48 4.55 6.84 5.33 12.31 24.69 6.34 Vilciai Pp9 ±0.04c ±0.06b ±0.08d ±0.06c ±0.12c ±0.16d ±0.05e 3.43 4.32 7.15 5.15 11.56 24.03 3.34 Vilciai Pp10 ±0.04b ±0.04b ±0.06a ±0.05a ±0.09d ±0.15c ±0.04d

Table 3.5.1.4 continued 1 2 3 4 5 6 7 8 3.66 5.76 7.12 6.68 12.64 20.85 2.12 Vilniai Pp8 ±0.02c ±0.05a ±0.05c ±0.06b ±0.11c ±0.13a ±0.02b 3.58 4.72 7.66 5.54 12.59 26.41 1.83 Vilniai Pp9 ±0.03a ±0.03c ±0.07a ±0.04d ±0.13e ±0.17f ±0.03a 3.44 5.26 6.01 6.30 10.92 24.24 2.48 Vilniai Pp10 ±0.04c ±0.05f ±0.05b ±0.06a ±0.11d ±0.15d ±0.04d 3.80 5.55 7.46 6.56 13.57 21.63 2.12 No.1072 Pp8 ±0.06e ±0.06d ±0.06d ±0.05b ±0.14c ±0.12b ±0.03b 3.78 5.69 7.17 6.70 13.28 21.05 2.22 No.1072 Pp9 ±0.05c ±0.05f ±0.08d ±0.05e ±0.12b ±0.10b ±0.02c 3.72 5.76 7.07 6.83 12.51 20.80 2.00 No.1072 Pp10 ±0.07b ±0.06e ±0.06b ±0.08b ±0.09c ±0.11a ±0.03b 3.73 4.99 7.38 5.54 12.83 26.53 2.26 No.1734 Pp8 ±0.05f ±0.05d ±0.07a ±0.06b ±0.12d ±0.14e ±0.04c 3.79 5.91 7.67 6.94 13.11 20.58 2.09 No.1734 Pp9 ±0.06c ±0.04d ±0.08b ±0.07c ±0.09e ±0.11a ±0.03b 3.97 5.33 7.12 6.10 13.99 23.06 1.10 No.1734 Pp10 ±0.04e ±0.06e ±0.07c ±0.06d ±0.13e ±0.15c ±0.04a TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; ALA – alanine; GLY – glycine; SER – serine; PRO – proline; ASP – asparagine; GLU – glutamine; TYR – tyramine; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10; a-f The mean values within a column with different letters are significantly different (p≤0.05).

The total essential amino acids (TEAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.1.5. In all the cases, in SMF samples content of ILE (except in SMF with P. acidilacti hybrids No.1700 and No.1702 wholemeal) was increased, in compare with nonfermented samples. Also, in SMF samples higher contents of LEU, THR and MET were established (except in SMF with L. sakei and P. acidilactici hybrid No.1700 wholemeal samples), in compare with nonfermented samples. Content of THR in SSF samples was found to be higher on average by 21.59% , in compare to SMF samples. In SSF wholemeal higher contents of THR, MET (except in SSF with P. acidilactici hybrid No.1700 samples) and PHE (except in SSF with L. sakei and P. acidilactici hybrid No.1700 samples) were established, in compare to nonfermented wholemeal. The MET, LYS and HIS contents in lupine wholemeal were significantly affected by the selection of lupine variety (F(0.880)=21.871, p≤0.0001; F(3.995)=24.898, p≤0.0001; F(13.273)=25.382, p≤0.0001, respectively), however, the selection of lupine variety had no significant influence on VAL, ILE, LEU, THR and PHE contents. The fermentation method significantly affected VAL (F(8.645)=18.504, p≤0.0001), THR F(15.527)= 66.640, p≤0.0001) and LYS (F(2.350)=14.643, p≤0.0001) contents,

Table 3.5.1.5. The total essential amino acids (TEAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains. TEAA content (%) from total protein hydrolysed Lupine LEU VAL ILE THR MET PHE LYS HIS samples Nonfermented samples 4.74 5.05 7.36 3.87 0.15 5.06 6.40 4.47 No.1701 ±0.04d ±0.05c ±0.04b ±0.03d ±0.01a ±0.05c ±0.06c ±0.05c 6.07 6.04 9.34 4.36 1.38 6.41 3.63 9.27 No.1700 ±0.08e ±0.07e ±0.07f ±0.05d ±0.02d ±0.06f ±0.04a ±0.05f 4.50 4.89 7.32 3.76 0.17 4.85 6.61 4.37 No.1800 ±0.04b ±0.04a ±0.05b ±0.03c ±0.01c ±0.05b ±0.07e ±0.04b 4.54 5.09 7.47 3.69 0.15 5.17 6.47 4.78 No.1702 ±0.04b ±0.05c ±0.05d ±0.03c ±0.01a ±0.06d ±0.07d ±0.05d Submerged fermented samples No.1701 8.76 5.69 8.64 4.15 1.43 5.63 4.90 4.54 Ls ±0.06f ±0.06d ±0.06e ±0.02b ±0.02b ±0.06e ±0.05c ±0.04e No.1701 4.89 5.68 9.15 4.54 0.81 5.92 4.88 4.62 Pa ±0.05b ±0.05c ±0.05f ±0.05d ±0.03b ±0.04c ±0.04b ±0.05c No.1700 5.22 9.34 6.04 4.32 0.94 6.34 3.58 4.84 Ls ±0.05a ±0.08f ±0.06a ±0.04c ±0.01a ±0.06f ±0.03a ±0.04e No.1700 5.02 5.98 9.12 4.34 0.85 6.23 4.44 4.54 Pa ±0.04b ±0.06c ±0.07c ±0.03c ±0.02c ±0.05c ±0.05d ±0.06d No.1800 7.68 6.43 9.67 3.77 1.97 6.59 5.73 5.73 Ls ±0.05d ±0.07e ±0.08f ±0.04a ±0.02e ±0.07f ±0.06e ±0.06f No.1800 4.28 5.01 8.03 3.81 0.60 5.05 5.71 2.98 Pa ±0.03f ±0.05f ±0.06d ±0.05c ±0.03b ±0.06e ±0.05c ±0.03c No.1702 8.42 5.33 8.79 4.30 1.62 5.32 4.20 2.87 Ls ±0.05e ±0.06d ±0.06e ±0.04c ±0.04d ±0.05d ±0.04b ±0.02a No.1702 4.58 4.90 7.99 3.88 0.73 5.08 4.53 2.83 Pa ±0.04c ±0.04f ±0.08f ±0.05d ±0.02c ±0.05c ±0.05c ±0.03c Solid state fermented samples No.1701 5.08 6.15 15.41 3.98 0.88 6.92 4.31 4.75 Ls ±0.05d ±0.07f ±0.06f ±0.03b ±0.03c ±0.07f ±0.04c ±0.04d No.1701 5.38 5.95 9.30 4.39 1.08 6.97 3.59 5.13 Pa ±0.06c ±0.05d ±0.07c ±0.05d ±0.04e ±0.08e ±0.03d ±0.06c No.1700 6.83 5.74 11.78 5.50 1.54 5.81 5.31 3.36 Ls ±0.07f ±0.06c ±0.08e ±0.04c ±0.04f ±0.06b ±0.04c ±0.02b No.1700 5.11 5.49 4.40 5.56 0.88 4.46 4.92 4.99 Pa ±0.05e ±0.04b ±0.04b ±0.04b ±0.02d ±0.05c ±0.05c ±0.05d No.1800 5.01 5.66 8.92 5.51 0.67 6.07 4.30 3.44 Ls ±0.05d ±0.06d ±0.07b ±0.04c ±0.04a ±0.06d ±0.03b ±0.03a No.1800 4.96 5.75 3.57 6.42 0.71 5.42 4.08 3.19 Pa ±0.03d ±0.07c ±0.05d ±0.06b ±0.03d ±0.05e ±0.03c ±0.04b No.1702 5.39 5.03 8.19 5.28 1.07 5.83 4.76 3.34 Ls ±0.05e ±0.04b ±0.06a ±0.05d ±0.05e ±0.06c ±0.04d ±0.03a No.1702 4.30 5.01 4.02 5.61 0.65 6.29 3.23 4.25 Pa ±0.04c ±0.06c ±0.04c ±0.07c ±0.02b ±0.07d ±0.03e ±0.05c TEAA – total essential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; VAL – valine; ILE – isoleucine; LEU – leucine; THR – threonine; MET – methionine; PHE – phenylethylamine; LYS – lysine; HIS – histamine. Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-fThe mean values within a column with different letters are significantly different (p≤0.05). 87 however, the fermentation method had no significant influence on ILE, LEU, MET, PHE and HIS contents. The type of microorganism applied for the fermentation had no significant influence on VAL, ILE, THR, MET, PHE, LYS and HIS contents. The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.1.6. In compare with nonfermented samples, in SMF and SSF lupine seeds wholemeal higher contents of ALA, SER and PRO were established. Similar tendencies of GLY and ASP contents in most of the SMF and SSF samples were established, and in most of the fermented samples higher contents of GLY (except in SMF with P. acidilactici hybrid No.1800 samples) and ASP (except in SSF with P. acidilactici hybrid No.1800 samples) were established, in compare with nonfermented wholemeal. The content of TYR in SMF lupine wholemeal was decreased, only in SMF with P. acidilactici hybrid No.1700 samples and in 3 of the 12 analysed SSF samples (in SSF with L. sakei and P. acidilactici hybrid No.1700 wholemeal and in SSF with L. sakei hybrid No.1702 wholemeal) TYR content was found to be higher, in compare with nonfermented samples. The ASP and TYR contents were significantly influenced by the selection of lupine variety (F(14.573)=87.797, p≤0.0001; (F(38.539)=150.695, p≤0.0001, respectively), but the selection of lupine variety had no significant influence on ALA, GLY, SER, PRO and GLU contents. The SER, PRO, ASP and TYR contents were significantly affected by the type of microorganism applied for the fermentation (F(0.394)=8.412, p≤0.0001; F(0.666)=5.604, p≤0.0001; F(6.121)=38.877, p≤0.0001; F(21.034)=82.245, p≤0.0001, respectively); however, the above mentioned factor had no significant influence on ALA, GLY and GLU contents. The fermentation method had no significant influence on ALA, GLY, SER, PRO, ASP, GLU and TYR contents. The total essential amino acids (TEAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains is presented in Table 3.5.1.7.

Table 3.5.1.6. The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains. TNAA content (%) from total protein hydrolysed Lupine ALA GLY SER PRO ASP GLU TYR samples Nonfermented samples 3.26 4.19 5.72 4.72 11.77 24.86 8.40 No.1701 ±0.03b ±0.04c ±0.05d ±0.04c ±0.12d ±0.16a ±0.07 3.64 4.38 5.90 5.08 12.16 26.25 3.58 No.1700 ±0.02c ±0.05c ±0.06d ±0.05d ±0.13e ±0.18b ±0.02a 3.27 3.99 5.63 4.53 11.31 27.80 7.00 No.1800 ±0.03b ±0.04b ±0.06c ±0.05b ±0.11c ±0.17e ±0.06d 3.22 4.05 5.38 4.69 11.04 27.54 6.74 No.1702 ±0.04b ±0.05b ±0.04b ±0.04b ±0.12c ±0.19d ±0.05c Submerged fermented samples 4.79 5.36 6.96 5.56 12.61 21.40 3.97 No.1701 Ls ±0.05d ±0.06d ±0.07d ±0.06c ±0.13c ±0.13b ±0.03b 4.05 5.10 7.21 6.01 13.93 19.85 2.97 No. 1701 Pa ±0.04c ±0.05b ±0.07b ±0.06b ±0.14b ±0.14c ±0.03d 4.18 5.27 6.65 6.43 12.95 19.85 3.88 No.1700 Ls ±0.04b ±0.05d ±0.06c ±0.07f ±0.14e ±0.17a ±0.04b 4.01 5.04 6.49 6.14 12.90 17.48 7.44 No. 1700 Pa ±0.03d ±0.04c ±0.05c ±0.06c ±0.12e ±0.12b ±0.05c 3.80 4.63 6.32 5.95 11.45 19.36 4.71 No.1800 Ls ±0.04b ±0.05b ±0.06b ±0.07e ±0.11c ±0.16a ±0.04c 3.43 4.01 6.40 4.97 12.66 30.04 3.02 No.1800 Pa ±0.03b ±0.04e ±0.06d ±0.05d ±0.13e ±0.22d ±0.04b 4.63 4.87 6.55 5.37 12.86 23.76 5.23 No.1702 Ls ±0.05c ±0.04c ±0.06c ±0.06d ±0.12d ±0.18c ±0.05d 3.51 4.18 6.37 5.16 12.63 28.49 5.14 No.1702 Pa ±0.04b ±0.03b ±0.05b ±0.05f ±0.13b ±0.23e ±0.04e Solid state fermented samples 3.40 5.07 6.18 6.67 12.89 20.53 3.87 No.1701 Ls ±0.02a ±0.04c ±0.06a ±0.07e ±0.11b ±0.19c ±0.03d 4.03 5.73 6.51 6.79 12.04 19.29 3.86 No.1701 Pa ±0.03f ±0.05d ±0.08b ±0.07a ±0.12a ±0.15a ±0.04b 4.25 4.84 7.27 5.78 13.60 19.28 6.50 No.1700 Ls ±0.04e ±0.03b ±0.07d ±0.05b ±0.13c ±0.17b ±0.06f 3.74 4.93 6.77 5.87 12.23 5.49 4.99 No.1700 Pa ±0.02d ±0.03c ±0.09a ±0.06d ±0.12b ±0.05a ±0.05b 3.79 4.91 6.40 6.15 13.53 21.91 4.93 No.1800 Ls ±0.03b ±0.04b ±0.05b ±0.05c ±0.13c ±0.15c ±0.04e 3.59 4.51 5.78 5.74 11.19 25.35 3.19 No.1800 Pa ±0.04e ±0.04e ±0.06c ±0.05c ±0.11d ±0.20d ±0.03c 4.27 5.16 7.68 6.24 12.43 19.23 7.50 No.1702 Ls ±0.05f ±0.05d ±0.06e ±0.06d ±0.11a ±0.14b ±0.05f 3.68 4.52 6.78 5.68 13.22 24.63 4.25 No.1702 Pa ±0.04d ±0.05c ±0.05f ±0.06a ±0.13b ±0.21b ±0.04a TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; ALA – alanine; GLY – glycine; SER – serine; PRO – proline; ASP – asparagine; GLU – glutamine; TYR – tyramine. Ls - Lactobaccilus sakei; Pa – Pediococcus acidilactici. a-f The mean values within a column with different letters are significantly different (p≤0.05). 89

Table 3.5.1.7. The total essential amino acids (TEAA) content (%) from total protein hydrolysed innonfermented seeds wholemeal of lupine hybrid lines No.1701, No.1700, No. 1800 and No.1702, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains. TEAA content (%) from total protein hydrolysed Lupine VAL ILE LEU THR MET PHE LYS HIS samples 1 2 3 4 5 6 7 8 9 Nonfermented samples 4.74 5.05 7.36 3.87 0.15 5.06 6.40 4.47 No.1701 ±0.04d ±0.05c ±0.04b ±0.03d ±0.01a ±0.05c ±0.06c ±0.05c 6.07 6.04 9.34 4.36 1.38 6.41 3.63 9.27 No.1700 ±0.08e ±0.07e ±0.07f ±0.05d ±0.02d ±0.06f ±0.04a ±0.05f 4.50 4.89 7.32 3.76 0.17 4.85 6.61 4.37 No.1800 ±0.04b ±0.04a ±0.05b ±0.03c ±0.01c ±0.05b ±0.07e ±0.04b 4.54 5.09 7.47 3.69 0.15 5.17 6.47 4.78 No.1702 ±0.04b ±0.05c ±0.05d ±0.03c ±0.01a ±0.06d ±0.07d ±0.05d Submerged fermented samples No.1701 5.27 2.80 4.31 6.99 0.83 3.07 5.49 3.20 Pp8 ±0.07c ±0.05bc ±0.06d ±0.09e ±0.03a ±0.06d ±0.06c ±0.05b No.1701 5.18 2.22 3.29 6.34 1.02 1.95 4.98 3.53 Pp9 ±0.05b ±0.03b ±0.04c ±0.07d ±0.04b ±0.03c ±0.06d ±0.06c No.1701 6.17 3.26 4.95 7.22 1.13 1.50 4.90 2.78 Pp10 ±0.06c ±0.06b ±0.07d ±0.06cd ±0.05ab ±0.04bc ±0.05de ±0.04c No.1700 4.77 2.83 4.55 6.66 0.62 2.92 5.48 4.65 Pp8 ±0.07bc ±0.05c ±0.06d ±0.07d ±0.02b ±0.05d ±0.07bc ±0.05c No.1700 4.46 2.20 3.55 5.80 0.56 1.83 5.31 4.32 Pp9 ±0.05b ±0.04b ±0.05b ±0.05b ±0.03c ±0.03b ±0.05b ±0.04c No.1700 5.57 3.66 6.93 6.20 0.95 1.45 2.50 3.79 Pp10 ±0.05d ±0.06d ±0.09e ±0.06c ±0.04b ±0.02b ±0.03a ±0.03b No.1800 4.42 1.85 1.78 5.72 0.74 2.64 6.64 3.43 Pp8 ±0.04b ±0.03c ±0.04a ±0.05c ±0.03b ±0.05c ±0.09c ±0.05a No.1800 5.62 1.74 2.73 6.70 0.94 2.40 6.13 4.61 Pp9 ±0.06c ±0.03b ±0.06b ±0.09d ±0.02c ±0.04bc ±0.06c ±0.08b No.1800 7.27 4.05 3.20 6.91 1.62 3.04 5.45 2.56 Pp10 ±0.08d ±0.06c ±0.04b ±0.11de ±0.04bc ±0.06d ±0.07d ±0.05b No.1702 3.96 2.09 3.41 5.24 1.04 1.90 4.53 2.72 Pp8 ±0.05b ±0.04b ±0.05bc ±0.06c ±0.03c ±0.04c ±0.05c ±0.06b No.1702 3.25 1.79 3.24 4.94 0.63 1.05 4.05 2.64 Pp9 ±0.04a ±0.03a ±0.03c ±0.04b ±0.02b ±0.03a ±0.04d ±0.04c No.1702 5.83 3.32 5.75 6.75 1.12 1.31 4.75 2.78 Pp10 ±0.08c ±0.05b ±0.08d ±0.08c ±0.04c ±0.02a ±0.06b ±0.05c Solid state fermented samples No.1701 5.08 5.90 6.05 4.59 0.98 6.60 5.65 4.98 Pp8 ±0.08c ±0.09d ±0.07c ±0.05b ±0.03b ±0.09d ±0.07d ±0.08c No.1701 4.81 5.72 6.51 4.19 0.68 6.61 5.85 4.91 Pp9 ±0.06b ±0.07c ±0.05cd ±0.04a ±0.02a ±0.09d ±0.09e ±0.07d No.1701 4.09 6.00 6.97 4.18 0.76 6.63 4.28 4.96 Pp10 ±0.05a ±0.05e ±0.11e ±0.03a ±0.04a ±0.04c ±0.04c ±0.09c No.1700 5.12 6.02 6.78 4.45 0.88 3.73 5.01 5.36 Pp8 ±0.06d ±0.06e ±0.09e ±0.07b ±0.04c ±0.04a ±0.08d ±0.09d No.1700 5.26 5.99 6.65 4.73 0.87 5.74 4.50 4.72 Pp9 ±0.07c ±0.04c ±0.07c ±0.09c ±0.05b ±0.06bc ±0.04c ±0.05b

Table 3.5.1.7 continued 1 2 3 4 5 6 7 8 9 No.1700 5.23 6.05 6.36 4.52 0.78 4.38 4.07 3.31 Pp10 ±0.05bc ±0.07d ±0.06b ±0.04b ±0.04b ±0.05b ±0.03b ±0.03a No.1800 4.90 5.42 5.61 4.26 0.79 6.08 3.40 3.81 Pp8 ±0.04b ±0.08c ±0.05a ±0.03b ±0.04b ±0.09b ±0.04a ±0.04a No.1800 5.52 5.99 6.53 3.66 0.99 5.90 4.31 5.55 Pp9 ±0.06d ±0.05cd ±0.08b ±0.05a ±0.05c ±0.07c ±0.05b ±0.06c No.1800 5.13 6.04 6.47 4.33 0.88 3.85 4.81 7.35 Pp10 ±0.07c ±0.08d ±0.09b ±0.04b ±0.03b ±0.04a ±0.07c ±0.09d No.1702 4.21 4.72 5.39 4.05 0.59 4.96 3.06 4.55 Pp8 ±0.04ab ±0.06a ±0.07b ±0.06b ±0.02a ±0.06b ±0.05a ±0.05b No.1702 4.08 4.27 5.09 3.96 0.57 3.58 2.65 5.98 Pp9 ±0.07b ±0.07a ±0.05a ±0.05a ±0.02a ±0.04a ±0.04a ±0.07c No.1702 5.74 5.03 5.89 5.07 1.13 7.09 3.96 8.52 Pp10 ±0.08d ±0.09c ±0.08c ±0.07c ±0.04d ±0.11c ±0.06b ±0.12d TEAA – total essential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; VAL – valine; ILE – isoleucine; LEU – leucine; THR – threonine; MET – methionine; PHE – phenylethylamine; LYS – lysine; HIS – histamine. Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-f The mean values within a column with different letters are significantly different (p≤0.05).

In all the cases, the higher contents of THR and MET in SMF wholemeal, in compare with nonfermented one, were found. However, in all SMF with P. pentosaceus No.8, No.9 and No.10 strains wholemeal samples contents of ILE, LEU and PHE were decreased, in compare to nonfermented samples. Similar tendencies in SMF lupine wholemeal samples were established, and, in compare to nonfermented samples, lower contents of LYS (in 9 samples out of 16 analysed) and HIS (in 11 samples out of 16 analysed) were found. In most of the SSF samples, in compare to nonfermented samples, the higher content of THR (in 11 samples out of 16 analysed) was found. In SSF samples contents of ILE, LEU and PHE, in compare to SMF samples, were established to be on average 47.41%, 64.13% and 38.38% higher, respectively. The VAL, ILE, LEU, THR, MET, PHE, LYS and HIS contents in lupine wholemeal were significantly affected by the selection of lupine variety (F(2.532)=703.979, p≤0.0001; F(1.324)=393.848, p≤0.0001; F(3.696)=867.448, p≤0.0001; F(0.677)= 190.590, p≤0.0001; F(0.424)= 390.158, p≤0.0001; F(0.614)=190.868, p≤0.0001; F(8.252)=2369.762, p≤0.0001 and F(5.580)=1461.497, p≤0.0001, respectively), the fermentation method (F(0.781)=217.229, p≤0.0001; F(156.468)=46557.974, p≤0.0001; F(88.512)=20773.867, p≤0.0001; F(68.640)=19328.769, p≤0.0001; F(0.211)= 194.572, p≤0.0001; F(200.901)=62433.167, p≤0.0001, F(9.374)= 2692.150, p≤0.0001, F(66.068)=17304.868, p≤0.0001, respectively), the type of microorganism applied for the fermentation (F(6.100)=1696.214, p≤0.0001; F(5.844)= 1738.793, p≤0.0001; F(9.655)=2266.031, p≤0.0001; F(2.283)=642.997,

91 p≤0.0001; F(0.507)=466.640, p≤0.0001; F(0.945)=293.755, p≤0.0001; F(2.010)=577.331, p≤0.0001, F(1.406)=391.912, p≤0.0001, respectively) and their interaction (F(1.088)=302.577, p≤0.0001; F(0.655)=194.776, p≤0.0001; F(2.849)=668.686, p≤0.0001; F(1.146)=322.634, p≤0.0001; F(0.137)=126.180, p≤0.0001; F(5.262)=1635.098, p≤0.0001; F(1.904)= 546.785, p≤0.0001, F(6.402)=1676.790, p≤0.0001, respectively). The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains is presented in Table 3.5.1.8. In all SMF and SSF wholemeal samples contents of ALA, GLY and PRO were increased, in compare with nonfermented samples. Different tendencies of SER content in SMF and SSF wholemeal samples were found (higher content of SER was found in 11 samples out of 16 SMF samples analysed, and in 7 samples out of 16 SSF samples analysed,), in compare with nonfermented. In SMF with P. pentosaceus No.8, No.9 and No.10 strains hybrid No.1700 and No.1702 wholemeal TYR content was decreased, in compare with nonfermented wholemeal samples. The content of GLU in SMF and SSF samples was found to be lower (except in SMF with P. pentosaceus No.9 hybrid No.1702 samples), in compare to nonfermented samples. To compare SMF and SSF wholemeal samples, in SSF samples TYR content was on average 67.91% higher. The ALA, GLY, SER, PRO, ASP, GLU and TYR contents in lupine wholemeal were significantly affected by the selection of lupine variety (F(0.583)=216.267, p≤0.0001; F(0.698)=180.963, p≤0.0001; F(0.481)=103.100, p≤0.0001; F(1.184)= 264.439, p≤0.0001; F(14.456)= 1126.519, p≤0.0001; F(37.940)=998.710, p≤0.0001 and F(35.898)= 7983.564, p≤0.0001, respectively), the fermentation method (F(0.035)=13.022, p≤0.0001; F(0.336)=87.163, p≤0.0001; F(0.151)=32.427, p≤0.0001; F(0.324)=72.347, p≤0.0001; F(2.904)=226.311, p≤0.0001; F(458.742)=12075.564, p≤0.0001, F(95.704)=21284.362, p≤0.0001, respectively), the type of microorganism applied for the fermentation (F(0.271)=100.554, p≤0.0001; F(0.025)=6.537, p≤0.0001; F(0.179)=38.280, p≤0.0001; F(0.208)=46.480, p≤0.0001; F(5.065)=394.723, p≤0.0001; F(23.295)=613.186, p≤0.0001; F(1.536)= 341.579, p≤0.0001, respectively) and their interaction (F(0.057)=21.246, p≤0.0001; F(0.357)=92.567, p≤0.0001; F(0.204)=43.776, p≤0.0001; F(0.246)=54.827, p≤0.0001; F(4.623)=360.265, p≤0.0001; F(34.092)= 897.418, p≤0.0001; F(13.757)= 3059.583, p≤0.0001, respectively).

Table 3.5.1.8. The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains. TNAA content (% ) from total protein hydrolysed Lupine samples ALA GLY SER PRO ASP GLU TYR 1 2 3 4 5 6 7 8 Nonfermented samples 3.26 4.19 5.72 4.72 11.77 24.86 8.40 No.1701 ±0.03b ±0.04c ±0.05d ±0.04c ±0.12d ±0.16a ±0.07 3.64 4.38 5.90 5.08 12.16 26.25 3.58 No.1700 ±0.02c ±0.05c ±0.06d ±0.05d ±0.13e ±0.18b ±0.02a 3.27 3.99 5.63 4.53 11.31 27.80 7.00 No.1800 ±0.03b ±0.04b ±0.06c ±0.05b ±0.11c ±0.17e ±0.06d 3.22 4.05 5.38 4.69 11.04 27.54 6.74 No.1702 ±0.04b ±0.05b ±0.04b ±0.04b ±0.12c ±0.19d ±0.05c Submerged fermented samples 3.82 5.43 6.34 6.64 13.72 17.68 3.46 No.1701 Pp8 ±0.04a ±0.06c ±0.08c ±0.07b ±0.19c ±0.22b ±0.05d 4.60 5.01 6.59 6.20 12.24 20.36 3.54 No.1701 Pp9 ±0.06c ±0.04b ±0.09d ±0.09d ±0.13b ±0.25e ±0.04c 3.89 5.30 6.33 5.68 12.75 20.29 3.38 No.1701 Pp10 ±0.05c ±0.07c ±0.06b ±0.09d ±0.09e ±0.19d ±0.05d 3.76 5.19 6.22 6.38 12.44 18.66 5.36 No.1700 Pp8 ±0.07d ±0.05b ±0.05b ±0.07b ±0.08b ±0.24f ±0.06e 4.24 5.13 6.79 6.15 13.49 15.84 4.72 No.1700 Pp9 ±0.06c ±0.05b ±0.06c ±0.07c ±0.09e ±0.12c ±0.05c 4.42 5.16 7.16 6.25 14.12 20.06 3.31 No.1700 Pp10 ±0.07d ±0.07d ±0.05b ±0.09f ±0.08f ±0.17d ±0.04b 3.40 4.54 6.16 5.30 11.98 23.79 3.81 No.1800 Pp8 ±0.04c ±0.04b ±0.07c ±0.04a ±0.05a ±0.19e ±0.04c 3.47 5.73 6.12 5.82 11.28 21.45 5.55 No.1800 Pp9 ±0.05b ±0.08d ±0.05b ±0.05b ±0.06b ±0.28f ±0.06b 3.64 5.37 5.95 5.67 11.18 18.42 6.99 No.1800 Pp10 ±0.06d ±0.08e ±0.04a ±0.07d ±0.08c ±0.19e ±0.07d 3.78 5.09 6.77 5.88 13.23 27.50 4.55 No.1702 Pp8 ±0.06c ±0.06c ±0.08c ±0.09e ±0.09b ±0.31e ±0.06c 3.63 4.52 6.27 5.28 12.36 30.56 5.98 No.1702 Pp9 ±0.05c ±0.05c ±0.06b ±0.07c ±0.08c ±0.27f ±0.05b 3.49 4.22 6.83 5.06 11.99 18.52 8.52 No.1702 Pp10 ±0.04b ±0.06b ±0.09c ±0.06c ±0.07d ±0.17d ±0.07c Solid state fermented samples 3.79 5.35 6.81 6.29 13.43 12.76 6.37 No.1701 Pp8 ±0.05c ±0.06b ±0.09e ±0.06b ±0.14d ±0.13b ±0.06bc 3.99 5.22 6.38 6.36 12.53 17.62 6.06 No.1701 Pp9 ±0.05d ±0.07c ±0.07d ±0.07c ±0.12c ±0.18c ±0.05b 4.60 5.32 6.31 6.76 12.95 12.32 4.51 No.1701 Pp10 ±0.07b ±0.06c ±0.06d ±0.08d ±0.15c ±0.13a ±0.04c 3.81 5.36 6.50 6.40 12.37 13.38 11.89 No.1700 Pp8 ±0.05b ±0.08d ±0.08e ±0.05b ±0.12b ±0.15b ±0.12d 3.98 5.26 6.92 6.29 13.96 17.23 11.95 No.1700 Pp9 ±0.05c ±0.07c ±0.07c ±0.05c ±0.15e ±0.19d ±0.14de 4.08 5.20 6.59 6.36 13.48 16.48 4.71 No.1700 Pp10 ±0.05d ±0.08d ±0.06b ±0.06b ±0.14d ±0.17c ±0.05b

Table 3.5.1.8 continued 1 2 3 4 5 6 7 8 3.96 4.73 6.42 5.57 12.39 18.75 7.54 No.1800 Pp8 ±0.07e ±0.05b ±0.09e ±0.08e ±0.11c ±0.21cd ±0.08c 3.65 4.55 5.60 5.68 11.15 19.99 3.12 No.1800 Pp9 ±0.06c ±0.06c ±0.04a ±0.07d ±0.10b ±0.20d ±0.04a 3.66 5.10 6.15 6.14 12.42 17.39 8.21 No.1800 Pp10 ±0.04b ±0.07d ±0.07c ±0.09f ±0.12c ±0.18e ±0.08e 3.68 4.52 6.84 5.36 13.05 15.86 6.94 No.1702 Pp8 ±0.05c ±0.08e ±0.08b ±0.06d ±0.14c ±0.16d ±0.06c 3.59 4.31 6.69 4.77 11.86 13.48 5.66 No.1702 Pp9 ±0.06d ±0.06c ±0.07d ±0.05c ±0.11b ±0.14c ±0.05b 3.88 4.13 7.42 5.94 6.37 17.29 10.24 No.1702 Pp10 ±0.04c ±0.07e ±0.09f ±0.06b ±0.08a ±0.17d ±0.12d TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; ALA – alanine; GLY – glycine; SER – serine; PRO – proline; ASP – asparagine; GLU – glutamine; TYR – tyramine; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10; a-f The mean values within a column with different letters are significantly different (p≤0.05).

3.5.2. The Free Amino Acids Content in Lupine Wholemeal Samples

Table 3.5.2.1. The free nonessential amino acids (FNAA) content (%) from total protein hydrolysed in seeds wholemeal nonfermented of lupine varieties Vilciai, Vilniai and hybrid lines No.1072, and No.1734 , SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains. FNAA content (%) from total protein hydrolysed Lupine ALA GLY SER PRO ASP GLU TYR samples 1 2 3 4 5 6 7 8 Nonfermented samples 2.93 3.79 5.11 4.56 10.64 25.88 8.78 Vilciai ±0.03a ±0.03a ±0.05a ±0.05b ±0.11b ±0.14b ±0.05e 3.14 3.83 5.02 4.35 10.43 26.58 10.61 Vilniai ±0.04a ±0.04a ±0.04a ±0.04a ±0.10a ±0.16c ±0.10f 3.37 4.06 5.53 4.67 11.34 27.34 5.57 No.1072 ±0.03b ±0.04b ±0.06b ±0.05b ±0.12d ±0.15d ±0.04b 3.99 4.59 6.11 4.70 10.39 26.20 5.64 No.1734 ±0.04d ±0.05d ±0.07e ±0.06c ±0.11a ±0.17b ±0.06b Submerged fermented samples 5.01 3.24 8.42 4.69 7.40 17.00 8.18 Vilciai Pp8 ±0.05b ±0.03c ±0.06a ±0.05d ±0.07d ±0.15c ±0.08h 4.76 2.75 11.44 4.13 5.91 19.25 12.32 Vilciai Pp9 ±0.04b ±0.03a ±0.12cd ±0.05b ±0.06a ±0.15f ±0.11j 5.36 2.99 11.14 4.07 6.36 18.38 13.04 Vilciai Pp10 ±0.06c ±0.03b ±0.09c ±0.06b ±0.08b ±0.09d ±0.12k

Table 3.5.2.1 continued 1 2 3 4 5 6 7 8 6.09 3.27 9.24 3.56 8.57 18.88 3.21 Vilniai Pp8 ±0.06d ±0.04c ±0.08b ±0.04a ±0.07e ±0.16e ±0.03a 6.78 3.53 8.56 3.93 9.74 17.46 4.86 Vilniai Pp9 ±0.08d ±0.04cd ±0.07a ±0.05b ±0.08f ±0.13c ±0.05e 7.23 3.38 10.80 3.41 9.64 15.80 7.52 Vilniai Pp10 ±0.09e ±0.05c ±0.09c ±0.04a ±0.07f ±0.12b ±0.08g No.1072 4.21 3.68 12.89 4.24 5.66 18.17 4.00 Pp8 ±0.05a ±0.04d ±0.12e ±0.05bc ±0.07a ±0.15d ±0.05c No.1072 4.14 3.06 13.52 4.20 5.72 19.34 8.54 Pp9 ±0.04a ±0.03b ±0.14f ±0.06bc ±0.04a ±0.17f ±0.08h No.1072 5.61 4.32 11.63 5.17 6.45 14.37 3.64 Pp10 ±0.05c ±0.05e ±0.11d ±0.05e ±0.06b ±0.14a ±0.05b No.1734 5.71 3.66 11.68 4.51 6.73 19.00 5.71 Pp8 ±0.06c ±0.04d ±0.11d ±0.04d ±0.07bc ±0.18e ±0.06f No.1734 6.78 4.43 11.96 5.24 6.14 15.85 4.36 Pp9 ±0.07d ±0.05e ±0.12d ±0.06e ±0.08ab ±0.14b ±0.05d No.1734 7.68 4.33 13.83 4.66 6.82 17.03 4.58 Pp10 ±0.09f ±0.04e ±0.14f ±0.05d ±0.09c ±0.15c ±0.06d Solid state fermented samples 3.31 4.11 7.52 4.76 10.89 28.05 3.68 Vilciai Pp8 ±0.03a ±0.05a ±0.06a ±0.05b ±0.09f ±0.16h ±0.05d 3.48 4.55 6.84 5.33 12.31 24.69 3.45 Vilciai Pp9 ±0.04b ±0.06b ±0.07a ±0.06c ±0.12h ±0.14g ±0.04c 3.43 4.32 7.15 5.15 11.56 24.03 3.34 Vilciai Pp10 ±0.04b ±0.05a ±0.09ab ±0.05c ±0.11g ±0.13g ±0.04c 6.58 5.23 21.22 6.85 7.00 13.18 4.38 Vilniai Pp8 ±0.09c ±0.06c ±0.18e ±0.08f ±0.06c ±0.11c ±0.05e 7.59 5.53 26.15 7.03 6.67 11.41 7.40 Vilniai Pp9 ±0.07de ±0.07cd ±0.21f ±0.07f ±0.05c ±0.09b ±0.08d 9.31 5.25 18.57 3.46 7.29 16.87 1.79 Vilniai Pp10 ±0.09f ±0.06c ±0.13d ±0.04a ±0.08cd ±0.15e ±0.02b No.1072 6.94 5.54 18.80 6.73 7.79 20.01 4.64 Pp8 ±0.07d ±0.07cd ±0.15d ±0.07f ±0.09e ±0.16f ±0.05e No.1072 7.19 4.90 25.36 5.25 5.52 21.07 3.82 Pp9 ±0.06d ±0.05b ±0.18f ±0.05c ±0.05b ±0.18f ±0.04d No.1072 7.79 5.26 31.67 6.38 6.40 15.73 1.20 Pp10 ±0.08e ±0.06c ±0.22h ±0.06e ±0.06c ±0.14d ±0.03a No.1734 7.46 4.12 13.81 5.32 7.90 16.72 3.24 Pp8 ±0.07d ±0.05a ±0.11c ±0.05c ±0.07e ±0.16e ±0.04c No.1734 6.90 5.44 29.35 7.05 5.70 11.57 7.19 Pp9 ±0.06d ±0.07c ±0.20g ±0.08f ±0.05b ±0.11b ±0.07f No.1734 6.48 4.32 22.17 5.56 5.15 7.76 7.45 Pp10 ±0.05c ±0.04a ±0.17e ±0.05cd ±0.05a ±0.06a ±0.08f TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation. ; ALA – alanine; GLY – glycine; SER – serine; PRO – proline; ASP – asparagine; GLU – glutamine; TYR – tyramine; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-gThe mean values within a column with different letters are significantly different (p≤0.05).

In all the cases, in SMF with P. pentosaceus No.8, No.9 and No.10 strains wholemeal the contents of ALA and SER amino acids was increased, in compare with nonfermented samples. The contents of GLY, ASP and

GLU in SMF with P. pentosaceus No.8 and No.9 strains varieties Vilciai, Vilniai and hybrid No.1072 and No.1734 wholemeal were decreased, except in sample No.1072, which was fermented with P. pentosaceus No.10 strain, where GLY content was increased, in compare with nonfermented one. In SSF wholemeal samples contents of ALA, GLY, SER and PRO (except in the samples fermented with P. pentosaceus No.10 variety Vilniai and with P. pentosaceus No.10 fermented hybrid No.1734 wholemeal) were increased, in compare with nonfermented samples. However, in SMF and SSF hybrid No.1072 and No.1734 wholemeal contents of ASP and GLU were decreased (except in SSF with P. pentosaceus No.8, No.9 and No.10 strains variety Vilciai wholemeal and in SMF with P. pentosaceus No.8 variety Vilciai wholemeal), in compare with nonfermented samples. The results of the ANOVA test have indicated that the amino acid profile of fermented lupine products was significantly affected by the selection of lupine variety, fermentation method, the types of microorganism applied for the fermentation and the interaction of these factors. The ALA, GLY, SER, PRO, ASP, GLU and TYR contents in lupine products were significantly affected by the selection of lupine variety (F(22.197)=5913.637, p≤0.0001; F(2.235)=287.577, p≤0.0001; F(232.288)=14629.009, p≤0.0001; F(1.977)= 653.402, p≤0.0001; F(18.766)=2912.717, p≤0.0001; F(109.977)=5347.975, p≤0.0001 and F(32.887)=8285.954, p≤0.0001, respectively), the fermentation method (F(6.301)=1678.735, p≤0.0001; F(30.772)=3959.634, p≤0.0001; F(1069.531)=67356.894, p≤0.0001; F(36.380)=12026.595, p≤ 0.0001; F(10.215)=1585.508, p≤0.0001; F(100.844)=25407.523, p≤0.0001, respectively, except GLU), the type of microorganism applied for the fermentation (F(5.660)=1507.987, p≤0.0001; F(0.269)=34.633, p≤0.0001; F(98.090)=6177.480, p≤0.0001; F(1.786)=590.496, p≤0.0001; F(1.680)= 260.826, p≤0.0001; F(41.504)=2018.267, p≤0.0001; F(21.208)= 5343.423, p≤0.0001, respectively) and their interaction (F(1.443)=384.444, p≤0.0001; F(0.190)=24.457, p≤0.0001; F(49.439)=3113.583, p≤0.0001; F(1.741)= 575.570, p≤0.0001; F(2.683)=416.388, p≤0.0001; F(17.409)=846.580, p≤0.0001; F(18.874)=4755.266, p≤0.0001, respectively). The free essential amino acids (FEAA) content (%) from total protein hydrolysed in lupine varieties Vilciai, Vilniai and hybrid lines No.1072 and No.1734 nonfermented wholemeal, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains is presented in Table 3.5.2.2. In all the cases, in SMF with P. pentosaceus No.8, No.9 and No.10 strains wholemeal samples contents of VAL, LEU, THR and PHE were increased (except in SMF with P. pentosaceus No.10 variety Vilciai samples and in SMF with P. pentosaceus No.9 and No.10 variety Vilniai samples, respectively), in compare with nonfermented wholemeal. In SMF wholemeal contents of 96

THR, LYS and HIS were increased (except in SMF with P. pentosaceus No.8 variety Vilniai samples, in which THR and LYS content was decreased), in compare with nonfermented lupine wholemeal. On average 13.22% higher content of THR in SSF with P. pentosaceus No.8, No.9 and No.10 strains lupine wholemeal was obtained, in compare to SMF. Also, in SSF wholemeal samples content of MET was increased, in compare to nonfermented samples. The content of ILE in SSF lupine wholemeal was decreased (except in SSF with P. pentosaceus No.9 and No.10 variety Vilciai samples, where ILE content was increased), in compare with nonfermented lupine wholemeal samples. The contents of LEU and PHE in SSF lupine wholemeal samples were decreased (except of PHE content in SSF with P. pentosaceus No.10 variety Vilciai samples), compare to nonfermented samples.

Table 3.5.2.2. The free essential amino acids (FEAA) content (%) from total protein hydrolysed in lupine varieties Vilciai, Vilniai and hybrid lines No.1072 and No.1734 nonfermented wholemeal, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains. FEAA content (%) from total protein hydrolysed Lupine VAL ILE LEU THR MET PHE LYS HIS samples 1 2 3 4 5 6 7 8 9 Nonfermented samples 4.35 4.99 8.34 3.37 0.16 5.52 6.42 5.17 Vilciai ±0.04a ±0.04a ±0.08b ±0.04a ±0.03b ±0.05c ±0.06b ±0.05b 4.32 4.83 7.05 3.45 0.15 4.75 7.16 4.34 Vilniai ±0.04a ±0.04a ±0.06a ±0.04a ±0.02a ±0.04a ±0.08c ±0.04a 4.56 5.10 7.39 3.75 0.15 5.10 7.66 4.40 No.1072 ±0.04ab ±0.05b ±0.07a ±0.05b ±0.02a ±0.05b ±0.08d ±0.04a 4.69 5.12 8.43 4.26 0.17 5.58 5.83 4.30 No.1734 ±0.05b ±0.05b ±0.08b ±0.06c ±0.02c ±0.06c ±0.05a ±0.03a Submerged fermented samples Vilciai 7.66 5.40 11.90 5.29 2.09 6.79 4.50 2.43 Pp8 ±0.06d ±0.06e ±0.09b ±0.05a ±0.04cd ±0.07e ±0.06c ±0.04b Vilciai 6.39 4.11 11.05 5.06 1.73 5.03 3.81 2.30 Pp9 ±0.05b ±0.05ab ±0.07a ±0.04a ±0.03a ±0.05c ±0.05a ±0.03b Vilciai 6.48 4.19 10.61 5.11 1.64 4.06 4.18 2.39 Pp10 ±0.06b ±0.05b ±0.06a ±0.05a ±0.04a ±0.04ab ±0.06b ±0.04b Vilniai 6.75 4.95 10.93 5.92 2.09 6.22 7.64 2.70 Pp8 ±0.08bc ±0.08cd ±0.07a ±0.06c ±0.05cd ±0.06d ±0.08g ±0.04d Vilniai 6.68 4.51 12.62 4.98 2.18 5.84 6.15 2.15 Pp9 ±0.06b ±0.06c ±0.12c ±0.04a ±0.04d ±0.05d ±0.06e ±0.03a Vilniai 5.98 4.09 11.00 5.42 1.95 3.82 7.03 3.02 Pp10 ±0.05a ±0.05a ±0.11a ±0.06b ±0.03bc ±0.04a ±0.09bc ±0.05b No.1072 7.32 4.90 11.98 6.37 1.66 6.90 5.10 2.92 Pp8 ±0.07d ±0.06cd ±0.12b ±0.08cd ±0.02a ±0.08e ±0.06d ±0.04c No.1072 6.43 3.89 11.68 5.50 1.77 5.90 3.82 2.31 Pp9 ±0.06b ±0.04a ±0.12b ±0.05b ±0.03ab ±0.06d ±0.04a ±0.03b

Table 3.5.2.2 continued 1 2 3 4 5 6 7 8 9 No.1072 8.22 5.11 13.62 6.64 1.90 5.29 4.71 3.34 Pp10 ±0.09e ±0.06d ±0.14d ±0.06e ±0.04b ±0.04c ±0.06c ±0.05c No.1734 6.80 4.71 11.32 5.78 1.81 6.24 4.22 2.13 Pp8 ±0.06bc ±0.04c ±0.12ab ±0.05bc ±0.0b ±0.07d ±0.05b ±0.04a No.1734 7.29 4.54 12.67 6.78 2.04 5.65 4.09 2.15 Pp9 ±0.07d ±0.05c ±0.13c ±0.07e ±0.04c ±0.06d ±0.05b ±0.04a No.1734 6.37 4.05 11.07 6.22 1.95 3.95 4.93 2.51 Pp10 ±0.06b ±0.04a ±0.11a ±0.06cd ±0.03bc ±0.04a ±0.05cd ±0.04bc Solid state fermented samples Vilciai 4.20 4.71 4.71 6.52 0.80 5.31 7.13 3.87 Pp8 ±0.04a ±0.06f ±0.05f ±0.06a ±0.02c ±0.05k ±0.08e ±0.04e Vilciai 4.81 5.53 5.53 6.84 0.78 5.43 6.25 3.94 Pp9 ±0.05c ±0.08h ±0.07h ±0.07a ±0.02c ±0.06k ±0.06d ±0.05e Vilciai 4.52 5.09 5.09 7.15 0.85 5.64 7.52 4.39 Pp10 ±0.03b ±0.06g ±0.06d ±0.09ab ±0.03d ±0.05kl ±0.07f ±0.06fg Vilniai 5.73 3.53 3.53 21.22 0.93 3.28 6.86 2.92 Pp8 ±0.06e ±0.05e ±0.04e ±0.16e ±0.04e ±0.04h ±0.06e ±0.03c Vilniai 4.86 2.59 2.59 26.15 0.87 2.43 3.02 3.23 Pp9 ±0.05c ±0.03c ±0.03c ±0.19f ±0.03d ±0.04f ±0.04b ±0.04d Vilniai 5.03 3.43 3.43 18.57 1.09 0.98 10.81 4.24 Pp10 ±0.04c ±0.05e ±0.05bc ±0.17d ±0.03f ±0.03c ±0.09g ±0.05f No.1072 5.21 3.17 3.17 18.80 0.81 2.95 1.63 4.04 Pp8 ±0.05cd ±0.04d ±0.04d ±0.18d ±0.02c ±0.04g ±0.03a ±0.06ef No.1072 4.10 2.26 2.26 25.36 0.87 1.61 5.68 2.54 Pp9 ±0.04a ±0.04b ±0.04b ±0.19f ±0.03d ±0.03e ±0.05c ±0.03b No.1072 4.03 2.15 2.15 31.67 0.54 0.64 5.98 3.76 Pp10 ±0.03a ±0.04b ±0.03b ±0.26h ±0.02a ±0.02a ±0.05c ±0.04e No.1734 6.94 4.85 4.85 13.81 1.30 4.44 6.42 2.27 Pp8 ±0.06f ±0.05b ±0.05b ±0.14c ±0.03g ±0.05j ±0.07d ±0.03a No.1734 4.51 1.97 1.97 29.35 0.70 1.49 5.86 3.14 Pp9 ±0.04b ±0.02a ±0.03a ±0.21g ±0.02b ±0.04d ±0.07c ±0.04d No.1734 4.03 2.25 2.25 22.17 0.68 0.82 23.35 2.45 Pp10 ±0.03a ±0.03b ±0.04b ±0.19e ±0.02b ±0.02b ±0.17h ±0.03b FEAA – total essential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; VAL – valine; ILE – isoleucine; LEU – leucine; THR – threonine; MET – methionine; PHE – phenylethylamine; LYS – lysine; HIS – histamine; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-lThe mean values within a column with different letters are significantly different (p≤0.05).

The contents of VAL, ILE, LEU, THR, MET, PHE, LYS and HIS in lupine products were significantly affected by the selection of lupine variety (F(0.339)=6.256, p≤0.0001; F(3.395)=1322.165, p≤0.0001; F(3.083)= 477.488, p≤0.0001; F(210.090)=15033.297, p≤0.0001; F(0.541)=603.068, p≤0.0001; F(7.961)=3244.840, p≤0.0001 and F(21.347)=4401.404, p≤0.0001, F(2.155)=1264.923, p≤0.0001, respectively), the fermentation method (F(78.542)=1451.286, p≤0.0001; F(20.866)=8125.764, p≤0.0001; F(1221.662)=189195.512, p≤0.0001; F(3141.866)=224820.497, p≤0.0001; F(19.814)=22102.723, p≤0.0001; F(117.581)=47922.433, p≤0.0001, F(114.989)=23708.992, p≤0.0001, F(13.546)=7951.538, p≤0.0001,

98 respectively), the type of microorganism applied for the fermentation (F(3.239)=59.858, p≤0.0001; F(5.111)=1990.317, p≤0.0001; F(0.983)= 152.298, p≤0.0001; F(69.524)=4974.860, p≤0.0001; F(0.076)=84.376, p≤0.0001; F(26.881)=10955.963, p≤0.0001; F(96.160)=19826.822, p≤0.0001, F(1.841)=1080.517, p≤0.0001, respectively) and their interaction (F(2.151)=39.737, p≤0.0001; F(2.019)=786.389, p≤0.0001; F(3.702)= 573.368, p≤0.0001; F(29.034)=2077.579, p≤0.0001; F(0.189)=210.600, p≤0.0001; F(1.815)=739.787, p≤0.0001; F(29.275)=6035.966, p≤0.0001, F(0.426)=250.016, p≤0.0001, respectively). The free nonessential amino acids (FNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.2.3. To compare nonfermented and SMF samples, the higher contents of ALA and SER and the lower content of GLU (on average 50% lower) were found in all SMF lupine wholemeal samples. Different tendencies of changes of GLY, PRO and TYR in SMF with L. sakei and P. acidilactici were discovered. In SMF and SSF lupine samples the ASP content has decreased; only in two samples (SMF with P. acidilactici and L. sakei variety Vilciai wholemeal samples, respectively) higher content of ASP was established, in compare to nonfermented samples. In all the cases, in SSF lupine wholemeal samples SER content has increased (on average by 20.82%) and was found to be higher, in compare to SMF samples, in which SER content has increased on average by 12.58%. The ALA, GLY, SER, PRO, ASP, GLU and TYR contents in lupine products were significantly affected by the selection of lupine variety (F(30.043)=4564.909, p≤0.0001; F(4.716)=1587.760, p≤0.0001; F(21.452)=725.332, p≤0.0001; F(0.207)=49.267, p≤0.0001; F(3.463)=514.941, p≤0.0001; F(15.277)=435.309, p≤0.0001 and F(25.536)= 6440.429, p≤0.0001, respectively), the fermentation method (F(23.081)= 3507.095, p≤0.0001; F(0.706)=237.601, p≤0.0001; F(816.008)=27591.130, p≤0.0001; F(26.686)=6353.790, p≤0.0001; F(35.656)=5301.973, p≤0.0001; F(0.642)=18.285, p≤0.0001, F(0.474)=119.551, p≤0.0001, respectively,) the type of microorganism applied for the fermentation (F(59.307)=9011.573, p≤0.0001; F(5.631)=1895.859, p≤0.0001; F(114.670)=3877.258, p≤0.0001; F(10.463)=2491.112, p≤0.0001; F(76.887)=2190.817, p≤0.0001; F(0.239)= 60.383, p≤0.0001, respectively, except ASP), and their interaction (F(4.881)=741.693, p≤0.0001; F(1.593)=536.439, p≤0.0001; F(19.663)= 664.846, p≤0.0001; F(2.303)=548.433, p≤0.0001; F(2.706)=402.352, p≤0.0001; F(17.232)=491.022, p≤0.0001; F(11.996)=3025.503, p≤0.0001, respectively).

Table 3.5.2.3. The free nonessential amino acids (FNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains. FNAA content (%) from total protein hydrolysed Lupine ALA GLY SER PRO ASP GLU TYR samples Nonfermented samples 3.26 4.19 5.72 4.72 11.77 24.86 8.40 No.1701 ±0.04b ±0.05b ±0.06b ±0.05b ±0.11d ±0.25c ±0.07b 3.64 4.38 5.90 5.08 12.16 26.25 3.58 No.1700 ±0.05b ±0.06c ±0.07a ±0.06c ±0.14b ±0.28b ±0.04a 3.27 3.99 5.63 4.53 11.31 27.80 7.00 No.1800 ±0.04b ±0.05b ±0.05d ±0.04b ±0.09c ±0.29c ±0.09b 3.22 4.05 5.38 4.69 11.04 27.54 6.74 No.1702 ±0.03a ±0.05c ±0.06c ±0.07d ±0.12c ±0.28d ±0.08c Submerged fermented samples 14.37 6.97 10.90 4.00 7.79 11.12 4.64 No.1701 Ls ±0.19ef ±0.06d ±0.12c ±0.04a ±0.08de ±0.12b ±0.06d 9.89 4.66 11.13 4.75 7.35 13.06 4.54 No.1701 Pa ±0.09c ±0.05c ±0.13b ±0.05b ±0.06 ±0.16c ±0.05c 7.71 4.14 14.80 5.67 5.92 16.37 4.14 No.1700 Ls ±0.08d ±0.04b ±0.17d ±0.07d ±0.07a ±0.18 ±0.06bc 7.58 4.26 15.49 5.93 6.08 16.12 3.42 No.1700 Pa ±0.06cd ±0.05c ±0.15e ±0.09d ±0.08b ±0.16e ±0.05b 8.20 4.37 13.74 4.99 6.74 14.42 2.69 No.1800 Ls ±0.09c ±0.04d ±0.12e ±0.06b ±0.09c ±0.19cd ±0.03a 5.31 3.21 10.68 4.38 6.79 19.57 6.35 No.1800 Pa ±0.05ab ±0.03c ±0.11c ±0.05c ±0.07cd ±0.21ef ±0.07ef 10.52 5.28 13.13 5.92 8.62 12.33 4.56 No.1702 Ls ±0.11d ±0.07d ±0.14de ±0.06d ±0.09e ±0.13b ±0.05d 6.14 4.08 10.73 4.75 6.46 15.48 6.85 No.1702 Pa ±0.06b ±0.05c ±0.12d ±0.05c ±0.07d ±0.16e ±0.09de Solid state fermented samples 10.51 6.53 21.39 8.58 5.14 17.43 1.74 No.1701 Ls ±0.11ef ±0.08b ±0.27cd ±0.09b ±0.07b ±0.18d ±0.02a 8.86 5.43 20.73 5.99 7.17 16.02 2.33 No.1701 Pa ±0.08e ±0.06c ±0.24b ±0.06b ±0.09c ±0.15c ±0.04b 7.86 4.45 14.72 6.15 6.18 16.49 3.59 No.1700 Ls ±0.07d ±0.05a ±0.19d ±0.07c ±0.06b ±0.17c ±0.05c 5.40 3.38 12.97 4.51 6.34 19.05 3.01 No.1700 Pa ±0.06c ±0.04ab ±0.16b ±0.06d ±0.07bc ±0.19d ±0.04c 5.30 5.21 24.86 6.64 4.56 9.50 9.12 No.1800 Ls ±0.05ab ±0.06c ±0.23ef ±0.08cd ±0.05ab ±0.09ab ±0.09e 5.37 6.23 20.99 6.91 3.77 10.08 4.74 No.1800 Pa ±0.06c ±0.08d ±0.21f ±0.09d ±0.04a ±0.11b ±0.05cd 8.61 3.73 32.41 8.14 3.93 11.61 6.94 No.1702 Ls ±0.09de ±0.04b ±0.31ef ±0.07e ±0.05b ±0.13c ±0.07f 6.73 3.95 18.50 5.40 4.87 20.14 7.31 No.1702 Pa ±0.07f ±0.05c ±0.22d ±0.05a ±0.07b ±0.17f ±0.09e FNAA – free nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; ALA – alanine; GLY – glycine; SER – serine; PRO – proline; ASP – asparagine; GLU – glutamine; TYR – tyramine. Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-f The mean values within a column with different letters are significantly different (p≤0.05). 100

The free nonessential amino acids (FNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine varieties Vilciai, Vilniai and hybrid lines No.1072 and No.1734, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.2.4. In all the cases, contents of ALA and SER were increased in all SMF and SSF samples, in compare to nonfermented. To compare GLY, ASP and GLU contents in nonfermented and fermented lupine samples, in SMF with L. sakei and P. acidilatici lupine samples contents of GLY, ASP and GLU were decreased. Amino acids ALA, GLY (except in SSF with P. acidilactici variety Vilciai wholemeal sample), SER and PRO contents in SSF wholemeal samples were increased, in compare to nonfermented samples. The amino acids contents obtained in SSF samples were on average 10.13% higher, in compare with nonfermented samples; however, in SMF samples amino acids content has increased on average by 2.00%, compared to nonfermented samples. The ALA, GLY, SER, PRO, ASP, GLU and TYR contents in lupine products were significantly affected by the selection of lupine variety (F(21.309)=5165.850, p≤0.0001; F(4.092)=1451.062, p≤0.0001; F(302.918)=8931.684, p≤0.0001; F(0.618)=186.049, p≤0.0001; F(24.251)= 2986.620, p≤0.0001; F(87.146)=2627.659, p≤0.0001 and F(3.837)= 1176.419, p≤0.0001, respectively), the fermentation method (F(3.967)= 961.818, p≤0.0001; F(37.224)=13200.007, p≤0.0001; F(1274.935)= 37592.059, p≤0.0001; F(16.673)=5022.114, p≤0.0001; F(0.581)=71.527, p≤0.0001; F(64.380)=1941.208, p≤0.0001; F(43.758)=13415.872, p≤ 0.0001, respectively), the type of microorganism applied for the fermentation (F(6.007)=1456.164, p≤0.0001; F(0.057)=20.113, p≤0.0001; F(113.467)=3345.644, p≤0.0001; F(0.105)=31.768, p≤0.0001; F(7.442)= 916.487, p≤0.0001; F(160.857)=4850.204, p≤0.0001; F(16.136)=4947.042, p≤0.0001, respectively) and their interaction (F(0.076)=18.442, p≤0.0001; F(0.229)=81.248, p≤0.0001; F(4.079)=120.280, p≤0.0001; F(0.254)= 76.602, p≤0.0001; F(2.077)=255.748, p≤0.0001; F(7.770)=234.293, p≤0.0001; F(9.318)=2856.700, p≤0.0001, respectively).

101

Table 3.5.2.4. The free nonessential amino acids (FNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of lupine variety Vilciai, Vilniai and hybrid lines No.1072 and No.1734, SMF and SSF with L. sakei and P. acidilactici strains. FNAA content (%) from total protein hydrolysed Lupine samples ALA GLY SER PRO ASP GLU TYR Nonfermented samples 2.93 3.79 5.11 4.56 10.64 25.88 8.78 Vilciai ±0.03a ±0.03a ±0.05a ±0.05b ±0.11b ±0.14b ±0.05e 3.14 3.83 5.02 4.35 10.43 26.58 10.61 Vilniai ±0.04a ±0.04a ±0.04a ±0.04a ±0.10a ±0.16c ±0.10f 3.37 4.06 5.53 4.67 11.34 27.34 5.57 No.1072 ±0.03b ±0.04b ±0.06b ±0.05b ±0.12d ±0.15d ±0.04b 3.99 4.59 6.11 4.70 10.39 26.20 5.64 No.1734 ±0.04d ±0.05d ±0.07e ±0.06c ±0.11a ±0.17b ±0.06b Submerged fermented samples 5.97 3.36 12.38 4.22 7.89 17.25 9.25 Vilciai Ls ±0.05c ±0.04a ±0.11c ±0.04ab ±0.09cd ±0.18d ±0.13e 5.98 3.52 11.94 5.05 6.90 18.31 3.62 Vilciai Pa ±0.07bc ±0.05ab ±0.10b ±0.06c ±0.07b ±0.21e ±0.04a 8.36 3.74 12.86 3.88 9.42 13.51 3.81 Vilniai Ls ±0.09e ±0.06b ±0.13c ±0.03a ±0.11d ±0.15ab ±0.05b 7.43 3.80 11.83 4.09 6.77 15.85 2.97 Vilniai Pa ±0.08d ±0.06c ±0.11b ±0.04ab ±0.06de ±0.17c ±0.03a 5.56 3.52 17.49 4.42 5.53 15.67 8.21 No.1072 Ls ±0.07b ±0.04b ±0.16de ±0.05b ±0.05a ±0.14c ±0.08d 4.50 3.55 14.55 4.49 5.67 17.63 5.01 No.1072 Pa ±0.06a ±0.05bc ±0.15d ±0.06b ±0.06ab ±0.19d ±0.05b 7.53 4.34 12.15 5.02 7.50 14.15 5.74 No.1734 Ls ±0.08ef ±0.07d ±0.12c ±0.06d ±0.08e ±0.15b ±0.06b 6.53 4.14 11.44 5.19 6.54 18.40 6.89 No.1734 Pa ±0.06d ±0.06d ±0.11b ±0.07e ±0.07c ±0.20d ±0.07c Solid state fermented samples 3.51 4.46 7.04 5.03 12.86 27.04 2.77 Vilciai Ls ±0.05ab ±0.05b ±0.07b ±0.05b ±0.18f ±0.27d ±0.04a 3.15 3.72 5.92 4.61 10.06 27.56 4.24 Vilciai Pa ±0.04a ±0.04a ±0.06a ±0.04a ±0.11de ±0.28d ±0.05b 8.71 6.06 25.75 7.08 7.38 12.57 2.63 Vilniai Ls ±0.08e ±0.06e ±0.24e ±0.08e ±0.09d ±0.15ab ±0.03a 7.85 6.56 21.98 6.05 5.88 21.84 1.32 Vilniai Pa ±0.06d ±0.07cd ±0.21d ±0.07bc ±0.07c ±0.21d ±0.02b 8.14 5.47 36.09 5.84 4.26 15.70 4.72 No.1072 Ls ±0.07de ±0.06c ±0.32c ±0.06b ±0.05ab ±0.17c ±0.05c 7.33 5.39 27.85 5.90 5.89 20.32 2.61 No.1072 Pa ±0.06c ±0.05c ±0.27d ±0.08ab ±0.06c ±0.19d ±0.04b 9.21 6.34 34.41 5.96 3.65 9.50 5.37 No.1734 Ls ±0.09d ±0.07e ±0.39f ±0.07d ±0.04a ±0.11c ±0.06d 8.56 6.06 28.06 5.32 4.48 14.77 6.42 No.1734 Pa ±0.08e ±0.06d ±0.32f ±0.06cd ±0.05b ±0.16d ±0.08d FNAA – free nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation.; ALA – alanine; GLY – glycine; SER – serine; PRO – proline; ASP – asparagine; GLU – glutamine; TYR – tyramine; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

102

The free essential amino acids (FEAA) content (%) from total protein hydrolysed in lupine seeds Vilciai, Vilniai, hybrid lines No.1072 and No.1734 nonfermented wholemeal, SMF and SSF with L. sakei and P. pentosaceus strains is presented in Table 3.5.2.5. In all the cases, higher contents of VAL and LEU in SMF samples, in compare to nonfermented samples, were established. However, in SMF with L. sakei and P. acidilactici strains lupine seeds wholemeal LEU content was increased. The THR content in all SMF lupine samples was higher. Also, higher THR content in SSF lupine wholemeal samples was found, except in SSF with P. Acidi- lactici Vilciai samples, in compare to nonfermented samples. In most of the cases, MET content in SMF lupine wholemeal samples was higher (in SMF with P. acidilactici variety Vilciai wholemeal 47.82% higher, however, in SMF with L. sakei variety Vilciai wholemeal 63.91% lower), in compare to nonfermented samples. Different tendencies of changes of PHE, LYS and HIS content in SMF lupine wholemeal samples were found, in compare to nonfermented samples. In most of the SSF samples, compared to SMF, the lower contents of VAL (in 14 samples out of 16 analysed), LEU (in 13 samples out of 16 analysed), ILE (in 12 samples out of 16 analysed), MET (in 13 samples out of 16 analysed) and PHE (in 12 samples out of 16 analysed) were established. In opposite, contents of LYS and HIS in most of the SSF samples, compared to SMF, were higher (in 10 and 9 samples, respectively, out of 16 analysed).

103

Table 3.5.2.5. The free essential amino acids (FEAA) content (%) from total protein hydrolysed in lupine seeds Vilciai, Vilniai, hybrid lines No.1072 and No.1734 nonfermented wholemeal, SMF and SSF with L. sakei and P. acidilactici strains. FEAA content (%) from total protein hydrolysed Lupine VAL ILE LEU THR MET PHE LYS HIS samples Nonfermented samples 4.35 4.99 8.34 3.37 0.16 5.52 6.42 5.17 Vilciai ±0.04a ±0.04a ±0.08b ±0.04a ±0.03b ±0.05c ±0.06b ±0.05b 4.32 4.83 7.05 3.45 0.15 4.75 7.16 4.34 Vilniai ±0.04a ±0.04a ±0.06a ±0.04a ±0.02a ±0.04a ±0.08c ±0.04a 4.56 5.10 7.39 3.75 0.15 5.10 7.66 4.40 No.1072 ±0.04ab ±0.05b ±0.07a ±0.05b ±0.02a ±0.05b ±0.08d ±0.04a 4.69 5.12 8.43 4.26 0.17 5.58 5.83 4.30 No.1734 ±0.05b ±0.05b ±0.08b ±0.06c ±0.02c ±0.06c ±0.05a ±0.03a Submerged fermented samples Vilciai 5.69 3.64 10.36 5.32 1.57 4.82 5.43 2.86 Ls ±0.06a ±0.04a ±0.11b ±0.06a ±0.03a ±0.05c ±0.06ab ±0.04c Vilciai 8.07 5.06 12.56 6.34 2.27 5.30 4.98 2.49 Pa ±0.09e ±0.07cd ±0.13c ±0.08b ±0.04d ±0.09f ±0.05b ±0.03b Vilniai 6.50 4.38 10.96 6.33 2.08 2.62 7.95 3.57 Ls ±0.07b ±0.05d ±0.11b ±0.07b ±0.03d ±0.03a ±0.09c ±0.05d Vilniai 6.83 4.51 12.23 6.85 2.19 5.00 7.35 2.33 Pa ±0.09bc ±0.06c ±0.14d ±0.07c ±0.05c ±0.06c ±0.07de ±0.04ab No.1072 6.49 3.84 9.97 6.14 1.58 2.99 5.19 3.39 Ls ±0.06b ±0.04b ±0.09a ±0.06bc ±0.03b ±0.03ab ±0.05d ±0.05c No.1072 7.10 4.31 12.53 6.57 1.82 5.86 4.96 2.17 Pa ±0.07e ±0.05c ±0.15e ±0.08e ±0.04c ±0.07c ±0.06bc ±0.03a No.1734 6.75 4.30 11.31 6.39 1.76 3.89 4.66 3.36 Ls ±0.06d ±0.05c ±0.12d ±0.07c ±0.05bc ±0.05b ±0.05b ±0.05d No.1734 7.09 4.46 12.73 6.43 1.94 5.87 3.61 2.37 Pa ±0.08de ±0.06d ±0.14de ±0.05d ±0.05b ±0.08d ±0.04a ±0.03b Solid state fermented samples Vilciai 4.22 4.82 9.44 3.88 0.72 5.03 5.31 3.86 Ls ±0.05b ±0.07c ±0.09d ±0.06b ±0.02b ±0.06c ±0.06c ±0.05d Vilciai 4.21 5.08 9.45 3.20 0.78 5.72 7.61 4.96 Pa ±0.05b ±0.09d ±0.11e ±0.05a ±0.04c ±0.08d ±0.09c ±0.06e Vilniai 5.23 2.81 3.99 6.54 0.56 0.88 6.10 3.72 Ls ±0.07c ±0.04b ±0.05b ±0.07d ±0.03b ±0.03b ±0.07d ±0.04d Vilniai 4.73 2.44 3.90 6.92 0.83 1.11 5.98 3.13 Pa ±0.06d ±0.04ab ±0.04c ±0.09ef ±0.04c ±0.04b ±0.09d ±0.05c No.1072 5.47 1.78 2.98 5.83 0.55 0.39 2.19 2.34 Ls ±0.08de ±0.03a ±0.05a ±0.06cd ±0.02a ±0.02a ±0.03a ±0.03b No.1072 5.39 1.90 3.04 5.55 0.59 0.62 5.71 3.43 Pa ±0.07e ±0.04d ±0.04a ±0.04c ±0.03a ±0.03a ±0.08d ±0.04d No.1734 4.79 2.33 3.99 6.31 0.90 1.05 3.97 1.48 Ls ±0.05a ±0.05c ±0.06b ±0.09e ±0.04c ±0.04ab ±0.05b ±0.02a No.1734 4.02 2.07 3.85 6.86 1.30 1.18 4.44 2.37 Pa ±0.07c ±0.04c ±0.05c ±0.11f ±0.04d ±0.04b ±0.06c ±0.03b FEAA – total essential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; VAL – valine; ILE – isoleucine; LEU – leucine; THR – threonine; MET – methionine; PHE – phenylethylamine; LYS – lysine; HIS – histamine. Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-fThe mean values within a column with different letters are significantly different (p≤0.05). 104

The VAL, ILE, LEU, THR, MET, PHE, LYS and HIS contents in lupine products were significantly affected by the selection of lupine variety (F(0.513)=124.078, p≤0.0001; F(3.636)=1351.642, p≤0.0001; F(19.265)=v 2127.508, p≤0.0001; F(6.966)=1531.028, p≤0.0001; F(0.161)=131.341, p≤0.0001; F(14.544)=5157.464, p≤0.0001, F(13.097)=3035.249 and p≤0.0001, F(2.849)=1675.766, p≤0.0001, respectively), the fermentation method (F(50.800)=12285.290, p≤0.0001; F(23.815)=8853.130, p≤0.0001; F(507.195)=56012.702, p≤0.0001; F(5.227)=1148.835, p≤0.0001; F(15.120)=12342.918, p≤0.0001; F(77.954)=27643.092, p≤0.0001, F(1.491)=345.556, p≤0.0001 and F(1.418)=834.099, p≤0.0001, respectively), the type of microorganism applied for the fermentation (F(0.992)= 239.873, p≤0.0001; F(0.698)=259.635, p≤0.0001; F(9.965)=110.444, p≤0.0001; F(0.735)=161.555, p≤0.0001; F(0.750)=612.245, p≤0.0001; F(15.086)=5349.794, p≤0.0001; F(2.765)=640.742, p≤0.0001, F(0.332)= 195.099, p≤0.0001, respectively) and their interaction (F(0.452)=109.377, p≤0.0001; F(0.105)=38.873, p≤0.0001; F(0.213)=23.503, p≤0.0001; F(0.658)=144.692, p≤0.0001; F(0.118)=96.061, p≤0.0001; F(1.175)= 416.762, p≤0.0001; F(1.526)=353.610, p≤0.0001, F(0.375)=220.570, p≤0.0001, respectively). The free nonessential amino acids (FNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.2.6. In all the cases, the higher contents of ALA and SER in SMF and SSF lupine wholemeal samples, compared to nonfermented, were found. However, contents of ASP and GLU in fermented samples were lower, compared to nonfermented samples. Desirable changes in SSF samples of GLY content were obtained (it has increased in all samples). In most of the cases, the lower contents of GLU (in 9 samples out of 13 analysed) and PRO (in 6 samples out of 13 analysed) in SMF samples were found. In both, SMF and SSF samples, the lower content of TYR (in 16 samples out of 24 analysed), compared to nonfermented samples, was established. The ALA, GLY, SER, PRO, ASP, GLU and TYR contents in lupine products were significantly affected by the selection of lupine variety (F(2.338)=404.429, p≤0.0001; F(0.417)=106.915, p≤0.0001; F(7.280)=234.047, p≤0.0001; F(2.062)=421.168, p≤0.0001; F(0.786)=140.861, p≤0.0001; F(9.813)=306.370, p≤0.0001 and F(1.688)= 243.607, p≤0.0001, respectively), the fermentation method (F(9.267)= 1602.609, p≤0.0001; F(65.723)=16867.527, p≤0.0001; F(2130.413)= 68494.154, p≤0.0001; F(118.734)=24249.113, p≤0.0001; F(9.968)= 178.714, p≤0.0001; F(172.608)=5389.194, p≤0.0001; F(8.799)=1269.961, p≤0.0001, respectively), the type of microorganism applied for the 105 fermentation (F(22.579)=3904.893, p≤0.0001; F(0.077)=19.694, p≤0.0001; F(28.991)=932.069, p≤0.0001; F(1.815)=370.712, p≤0.0001; F(8.698)= 1558.199, p≤0.0001; F(13.699)=427.713, p≤0.0001; F(58.410)=8430.333, p≤0.0001, respectively) and their interaction (F(2.962)=512.307, p≤0.0001; F(1.380)=354.236, p≤0.0001; F(30.110)=968.057, p≤0.0001; F(2.150)= 439.100, p≤0.0001; F(3.896)=698.024, p≤0.0001; F(32.489)=1014.386, p≤0.0001; F(38.723)=5588.958, p≤0.0001, respectively).

Table 3.5.2.6. The free nonessential amino acids (FNAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains. FNAA content (%) from total protein hydrolysed Lupine ALA GLY SER PRO ASP GLU TYR samples 1 2 3 4 5 6 7 8 Nonfermented samples 3.26 4.19 5.72 4.72 11.77 24.86 8.40 No.1701 ±0.04b ±0.05b ±0.06b ±0.05b ±0.11d ±0.25c ±0.07b 3.64 4.38 5.90 5.08 12.16 26.25 3.58 No.1700 ±0.05b ±0.06c ±0.07a ±0.06c ±0.14b ±0.28b ±0.04a 3.27 3.99 5.63 4.53 11.31 27.80 7.00 No.1800 ±0.04b ±0.05b ±0.05d ±0.04b ±0.09c ±0.29c ±0.09b 3.22 4.05 5.38 4.69 11.04 27.54 6.74 No.1702 ±0.03a ±0.05c ±0.06c ±0.07d ±0.12c ±0.28d ±0.08c Submerged fermented samples No.1701 7.05 4.32 9.31 5.28 7.02 12.76 6.37 Pp8 ±0.08c ±0.06c ±0.09b ±0.06d ±0.08e ±0.12c ±0.07d No.1701 7.31 3.58 10.49 4.60 6.81 17.62 6.06 Pp9 ±0.09d ±0.05b ±0.11bc ±0.05c ±0.06c ±0.17f ±0.05c No.1701 7.66 4.53 8.76 5.51 7.97 12.32 4.51 Pp10 ±0.08d ±0.07d ±0.07a ±0.07cd ±0.10f ±0.15b ±0.04a No.1700 6.94 4.49 12.67 5.97 4.58 13.38 11.89 Pp8 ±0.07cd ±0.05e ±0.12d ±0.08f ±0.05cd ±0.14c ±0.11f No.1700 6.01 3.76 15.11 5.21 4.97 17.23 11.58 Pp9 ±0.06c ±0.05cd ±0.15e ±0.06d ±0.06b ±0.19d ±0.10f No.1700 8.04 4.18 17.05 5.78 5.69 16.48 4.71 Pp10 ±0.11e ±0.07d ±0.17f ±0.07e ±0.07c ±0.17e ±0.05a No.1800 5.40 3.52 8.79 4.16 6.50 18.75 7.54 Pp8 ±0.06b ±0.06 ±0.08b ±0.05c ±0.06d ±0.21f ±0.08c No.1800 4.95 3.35 11.45 4.44 6.98 19.99 3.12 Pp9 ±0.05a ±0.05a ±0.13de ±0.04d ±0.09e ±0.24d ±0.04a No.1800 6.11 3.59 10.53 4.74 6.71 17.39 8.21 Pp10 ±0.07c ±0.05b ±0.11c ±0.06bc ±0.07d ±0.18ef ±0.09de No.1702 5.20 4.07 12.87 4.43 5.73 15.86 6.94 Pp8 ±0.06b ±0.06c ±0.16e ±0.07b ±0.08c ±0.15c ±0.07c No.1702 6.43 4.29 11.22 4.38 3.86 13.48 5.66 Pp9 ±0.06a ±0.07b ±0.12c ±0.05a ±0.04a ±0.12d ±0.05b No.1702 7.04 3.74 17.01 4.39 5.40 17.29 10.24 Pp10 ±0.09c ±0.05a ±0.18f ±0.06a ±0.06b ±0.19e ±0.11e

106

Table 3.5.2.6 continued 1 2 3 4 5 6 7 8 Solid state fermented samples No.1701 8.86 5.43 28.85 8.50 5.17 8.87 3.42 Pp8 ±0.09d ±0.06b ±0.32f ±0.09d ±0.06b ±0.09b ±0.03a No.1701 6.63 6.60 25.90 9.52 4.75 6.35 10.65 Pp9 ±0.07c ±0.08f ±0.23e ±0.12e ±0.04c ±0.07a ±0.12e No.1701 7.34 6.97 20.54 7.46 6.25 13.81 2.96 Pp10 ±0.08b ±0.10f ±0.19c ±0.08c ±0.07f ±0.14b ±0.04a No.1700 7.59 6.30 21.01 8.46 5.48 14.67 4.03 Pp8 ±0.09c ±0.07e ±0.22b ±0.10d ±0.06c ±0.16c ±0.05b No.1700 8.04 6.22 21.11 8.56 5.18 13.66 9.24 Pp9 ±0.08c ±0.06de ±0.19c ±0.09d ±0.04c ±0.12b ±0.09d No.1700 10.77 5.42 20.21 6.92 5.31 16.68 4.64 Pp10 ±0.13d ±0.05b ±0.23b ±0.07c ±0.06d ±0.18d ±0.06b No.1800 3.39 5.61 29.31 6.29 3.79 15.86 7.58 Pp8 ±0.04a ±0.06 ±0.29e ±0.05c ±0.03a ±0.15d ±0.08c No.1800 4.59 6.99 23.37 8.09 4.84 11.29 9.49 Pp9 ±0.06b ±0.08d ±0.24c ±0.09de ±0.05b ±0.13b ±0.11d No.1800 7.31 4.42 14.79 4.54 6.70 16.87 4.61 Pp10 ±0.08d ±0.04a ±0.15a ±0.05a ±0.08c ±0.19cd ±0.05c No.1702 5.10 5.57 24.39 7.45 4.43 9.29 19.49 Pp8 ±0.07b ±0.07c ±0.23d ±0.08c ±0.05ab ±0.12b ±0.18d No.1702 7.55 4.89 24.78 6.25 4.70 17.52 12.70 Pp9 ±0.09c ±0.06a ±0.26d ±0.06ab ±0.06c ±0.17e ±0.12cd No.1702 9.58 5.93 21.58 7.67 6.69 10.52 6.41 Pp10 ±0.11e ±0.08c ±0.21bc ±0.07c ±0.09e ±0.11d ±0.07c FNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation. Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-f The mean values within a column with different letters are significantly different (p≤0.05).

The free essential amino acids (FEAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.2.7. In all the cases, higher contents of VAL, LEU and MET in SMF wholemeal samples, compared to nonfermented samples, were found. However, in SSF and SMF samples contents of ILE and HIS (except in SMF with L. sakei hybrid No.1700 wholemeal samples) were lower, compared to nonfermented samples. In all SMF lupine wholemeal samples contents of MET and VAL were increased, in compare to nonfermented. In most of the cases, contents of THR and MET in SSF lupine wholemeal samples were higher (except in SSF with L. sakei hybrid No.1700 wholemeal samples), compared to nonfermented samples. Contents of PHE, LYS and HIS in SSF samples were decreased, compared to nonfermented samples, and were established to be on average 36.12%, 19.77%, and 49.12% lower, respectively. The LYS and HIS was significantly affected by the selection of lupine variety (F(3.891)=23.874, p≤0.0001; F(15.555)=9.463, p≤0.0001, respectively), however, the selection

107 of lupine variety had no significant influence on VAL, ILE, LEU, THR, MET and PHE contents. The VAL, ILE, LEU, MET, PHE and LYS was significantly affected by the fermentation method (F(71.151)=63.146, p≤0.0001; F(26.240)=41.993, p≤0.0001; F(373.972)=64.092, p≤0.0001; F(6.773)=100.584, p≤0.0001; F(73.359)=71.924, p≤0.0001; F(4.320)= 26.507, p≤0.0001, respectively), however, the fermentation method had no significant influence on THR and HIS contents, as well as the type of microorganisms applied for the fermentation had no signifacant influence on VAL, ILE, LEU, THR, MET, PHE, LYS and HIS contents. The free essential amino acids (FEAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains is presented in Table 3.5.2.8. In all the cases, in SMF lupine wholemeal samples contents of VAL, LEU, THR and MET were increased, in compare to nonfermented. In SMF lupine samples content LEU was increased, in compare to SSF wholemeal samples, in which LEU content was discivered to be on average 63.25% lower. The higher (on average by 24.69%) THR content in SSF samples, in compare to SMF samples, was established. The VAL, ILE, LEU, THR, MET, PHE, LYS and HIS contents in lupine products were significantly influenced by the selection of lupine variety (F(0.124)=25.429, p≤0.0001; F(0.574)=210.242, p≤0.0001; F(3.439)= 339.262, p≤0.0001; F(0.140)=31.267, p≤0.0001; F(0.407)=326.443, p≤0.0001; F(0.563)=163.968, p≤0.0001, F(5.330)=1554.499 and p≤0.0001, F(6.585)=3077.985, p≤0.0001, respectively), the fermentation method (F(80.836)=165533.215, p≤0.0001; F(79.506)=29138.343, p≤0.0001; F(1056.620)=104247.261, p≤0.0001; F(0.074)=16.575, p≤0.0001; F(14.231)=11417.511, p≤0.0001; F(233.172)=67867.114, p≤0.0001, F(23.188)=6763.181, p≤0.0001 and F(23.461)=10966.861, p≤0.0001, respectively), the type of microorganism applied for the fermentation (F(3.323)=679.616, p≤0.0001; F(1.898)=695.423, p≤0.0001; F(9.034)= 891.280, p≤0.0001; F(1.722)=385.120, p≤0.0001; F(0.143)=114.758, p≤0.0001; F(15.099)=4394.821, p≤0.0001; F(4.116)=1200.620, p≤0.0001, F(0.789)=368.926, p≤0.0001, respectively) and their interaction (F(1.213)= 248.042, p≤0.0001; F(0.416)=152.453, p≤0.0001; F(2.684)=264.820, p≤0.0001; F(3.248)=726.484, p≤0.0001; F(0.120)=96.619, p≤0.0001; F(1.912)=556.561, p≤0.0001; F(0.503)=146.763, p≤0.0001, F(0.148)= 69.414, p≤0.0001, respectively).

108

Table 3.5.2.7. The free essential amino acids (FEAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains. FEAA content % from total protein hydrolysed Lupine samples VAL ILE LEU THR MET PHE LYS HIS Nonfermented samples 4.74 5.05 7.36 3.87 0.15 5.06 6.40 4.47 No.1701 ±0.04d ±0.05c ±0.04b ±0.03d ±0.01a ±0.05c ±0.06c ±0.05c 6.07 6.04 9.34 4.36 0.38 6.41 3.63 9.27 No.1700 ±0.08e ±0.07e ±0.07f ±0.05d ±0.02d ±0.06f ±0.04a ±0.05f 4.50 4.89 7.32 3.76 0.17 4.85 6.61 4.37 No.1800 ±0.04b ±0.04a ±0.05b ±0.03c ±0.01c ±0.05b ±0.07e ±0.04b 4.54 5.09 7.47 3.69 0.15 5.17 6.47 4.78 No.1702 ±0.04b ±0.05c ±0.05d ±0.03c ±0.01a ±0.06d ±0.07d ±0.05d Submerged fermented samples 8.76 3.64 10.12 2.47 1.43 3.71 4.47 3.45 No.1701 Ls ±0.11ef ±0.04a ±0.11b ±0.03a ±0.04a ±0.05a ±0.07c ±0.04b 8.03 5.06 11.50 7.00 1.67 5.55 3.72 2.21 No.1701 Pa ±0.07e ±0.08d ±0.13c ±0.08d ±0.05b ±0.07d ±0.05ab ±0.03c 6.17 4.38 10.45 4.31 1.48 3.30 3.11 9.17 No.1700 Ls ±0.05a ±0.05c ±0.12c ±0.05c ±0.03a ±0.04a ±0.03a ±0.11d 6.63 4.51 11.72 4.98 1.57 4.59 3.92 3.35 No.1700 Pa ±0.05b ±0.06cb ±0.14c ±0.05c ±0.05ab ±0.06b ±0.05b ±0.05b 7.68 3.84 12.44 6.62 1.97 3.99 4.63 2.57 No.1800 Ls ±0.08d ±0.04b ±0.15d ±0.07d ±0.05c ±0.05ab ±0.07d ±0.04c 7.30 4.31 11.99 6.07 1.86 5.53 4.43 2.05 No.1800 Pa ±0.07c ±0.05cd ±0.13b ±0.06bc ±0.04cd ±0.07d ±0.06c ±0.03a 8.42 4.30 10.80 4.64 1.62 3.26 3.46 2.31 No.1702 Ls ±0.09e ±0.05d ±0.11c ±0.5d ±0.03c ±0.05c ±0.05c ±0.04ab 7.60 4.46 11.88 7.56 2.04 5.39 4.55 2.05 No.1702 Pa ±0.06c ±0.06d ±0.12cd ±0.08cd ±0.05d ±0.08d ±0.06d ±0.03a Solid state fermented samples 5.08 3.01 5.40 6.33 0.88 1.04 4.14 3.14 No.1701 Ls ±0.07d ±0.04c ±0.07de ±0.06d ±0.02b ±0.03c ±0.05c ±0.05c 5.96 3.34 5.98 7.04 1.76 1.78 5.85 2.31 No.1701 Pa ±0.09e ±0.05d ±0.06d ±0.09e ±0.04c ±0.04b ±0.07d ±0.04b 6.83 4.46 11.78 5.40 0.16 3.59 3.61 3.36 No.1700 Ls ±0.06f ±0.06e ±0.11e ±0.05d ±0.02a ±0.05c ±0.04a ±0.05d 7.59 4.70 12.07 6.51 1.54 5.67 4.63 2.41 No.1700 Pa ±0.07ef ±0.07d ±0.16f ±0.06c ±0.04c ±0.07d ±0.06b ±0.03b 4.12 2.07 3.15 5.51 0.64 1.17 5.30 2.85 No.1800 Ls ±0.05c ±0.04c ±0.04c ±0.06bc ±0.03b ±0.03ab ±0.07e ±0.04c 3.85 1.16 1.78 4.89 0.77 0.67 4.99 3.80 No.1800 Pa ±0.04b ±0.03a ±0.03a ±0.05b ±0.02a ±0.02a ±0.05cd ±0.05e 3.73 1.84 2.67 5.28 0.75 0.61 4.76 2.93 No.1702 Ls ±0.07a ±0.04b ±0.04b ±0.07c ±0.03a ±0.03a ±0.04d ±0.03c 3.95 2.09 3.41 5.24 1.13 1.01 3.81 1.70 No.1702 Pa ±0.06b ±0.04c ±0.05c ±0.08cd ±0.05c ±0.04b ±0.05ab ±0.04a FEAA – total essential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation;VAL – valine; ILE – isoleucine; LEU – leucine; THR – threonine; MET – methionine; PHE – phenylethylamine; LYS – lysine; HIS – histamine; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

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Table 3.5.2.8. The free essential amino acids (FEAA) content (%) from total protein hydrolysed in nonfermented seeds wholemeal of hybrid lines No.1701, No.1700, No.1800 and No.1702, SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains. FEAA content (%) from total protein hydrolysed Lupine VAL ILE LEU THR MET PHE LYS HIS samples 1 2 3 4 5 6 7 8 9 Nonfermented samples 4.74 5.05 7.36 3.87 0.15 5.06 6.40 4.47 No.1701 ±0.04d ±0.05c ±0.04b ±0.03d ±0.01a ±0.05c ±0.06c ±0.05c 6.07 6.04 9.34 4.36 1.38 6.41 3.63 9.27 No.1700 ±0.08e ±0.07e ±0.07f ±0.05d ±0.02d ±0.06f ±0.04a ±0.05f 4.50 4.89 7.32 3.76 0.17 4.85 6.61 4.37 No.1800 ±0.04b ±0.04a ±0.05b ±0.03c ±0.01c ±0.05b ±0.07e ±0.04b 4.54 5.09 7.47 3.69 0.15 5.17 6.47 4.78 No.1702 ±0.04b ±0.05c ±0.05d ±0.03c ±0.01a ±0.06d ±0.07d ±0.05d Submerged fermented samples No.1701 8.04 5.51 12.03 7.31 1.94 6.77 4.16 2.14 Pp8 ±0.09e ±0.07d ±0.12c ±0.08d ±0.04b ±0.09 ±0.05bc ±0.04a No.1701 6.89 4.46 12.35 5.62 1.87 6.19 4.07 2.07 Pp9 ±0.08b ±0.05bc ±0.14c ±0.05b ±0.03b ±0.07bc ±0.04b ±0.03b No.1701 7.93 5.22 14.28 6.26 2.19 6.61 3.62 2.64 Pp10 ±0.09c ±0.08d ±0.17e ±0.06de ±0.05c ±0.06d ±0.05b ±0.04b No.1700 7.74 5.27 12.85 6.53 1.79 7.01 3.05 2.41 Pp8 ±0.07c ±0.06d ±0.16d ±0.05b ±0.04b ±0.11d ±0.03a ±0.03c No.1700 5.89 3.78 10.39 4.46 1.40 4.43 3.65 2.91 Pp9 ±0.06d ±0.05b ±0.11b ±0.04a ±0.03a ±0.06b ±0.04b ±0.05c No.1700 6.42 4.19 11.39 4.80 1.38 3.49 3.55 2.83 Pp10 ±0.07a ±0.07b ±0.12ab ±0.07b ±0.04a ±0.04b ±0.04ab ±0.04b No.1800 7.38 4.83 11.73 6.19 1.70 6.42 4.70 2.37 Pp8 ±0.09de ±0.08c ±0.10d ±0.09d ±0.03d ±0.09de ±0.07c ±0.03d No.1800 7.39 4.56 12.50 6.16 2.03 6.29 4.60 2.18 Pp9 ±0.09c ±0.05bc ±0.14bc ±0.07cd ±0.06cd ±0.08d ±0.05cd ±0.04d No.1800 7.45 4.06 11.89 6.27 1.74 4.34 4.05 2.43 Pp10 ±0.09d ±0.05ab ±0.16b ±0.06b ±0.04d ±0.05b ±0.06c ±0.06b No.1702 7.26 4.67 11.00 7.24 1.83 6.08 5.14 1.80 Pp8 ±0.07cd ±0.07b ±0.13b ±0.09e ±0.05cb ±0.07c ±0.09d ±0.04c No.1702 8.38 5.24 15.87 7.53 2.43 8.09 2.16 1.99 Pp9 ±0.11f ±0.08d ±0.17d ±0.12ef ±0.06d ±0.10d ±0.03a ±0.03b No.1702 6.33 3.73 9.10 6.25 1.57 2.53 3.84 1.54 Pp10 ±0.08e ±0.04a ±0.11a ±0.07d ±0.04b ±0.04a ±0.04ab ±0.04a Solid state fermented samples No.1701 5.27 2.80 4.31 6.99 0.83 3.07 5.49 3.20 Pp8 ±0.06d ±0.04b ±0.05b ±0.08cd ±0.02a ±0.05d ±0.08d ±0.05bc No.1701 5.18 2.22 3.29 6.34 1.02 1.95 4.98 3.53 Pp9 ±0.05c ±0.03c ±0.03c ±0.06c ±0.03b ±0.03c ±0.07cb ±0.06c No.1701 6.07 3.26 4.95 7.22 1.13 1.50 4.90 2.78 Pp10 ±0.07e ±0.05d ±0.06d ±0.07d ±0.02b ±0.02bc ±0.06c ±0.04a No.1700 4.77 2.83 4.55 6.66 0.62 2.92 5.48 4.65 Pp8 ±0.06bc ±0.04b ±0.05b ±0.08c ±0.02b ±0.04c ±0.07d ±0.07d No.1700 4.46 2.20 3.55 5.80 0.56 1.83 5.31 4.32 Pp9 ±0.05b ±0.03a ±0.04b ±0.06b ±0.03ab ±0.03b ±0.05b ±0.05d

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Table 3.5.2.8 continued 1 2 3 4 5 6 7 8 9 No.1700 5.57 3.66 6.93 6.20 0.95 1.45 2.50 3.79 Pp10 ±0.07de ±0.04bc ±0.08bc ±0.07c ±0.04b ±0.02b ±0.04a ±0.04d No.1800 4.42 1.85 2.73 5.72 0.74 2.64 6.64 3.43 Pp8 ±0.05b ±0.03b ±0.04a ±0.05ab ±0.03c ±0.05d ±0.08e ±0.03c No.1800 5.62 1.74 3.20 6.70 0.94 2.40 6.13 4.61 Pp9 ±0.06d ±0.02ab ±0.05b ±0.08d ±0.04b ±0.04c ±0.07d ±0.08bc No.1800 7.27 4.05 10.07 6.91 1.62 3.04 5.45 2.56 Pp10 ±0.07f ±0.05c ±0.13c ±0.09cd ±0.03c ±0.06d ±0.05c ±0.03c No.1702 3.96 2.09 3.41 5.24 1.04 1.90 4.53 2.72 Pp8 ±0.05a ±0.04c ±0.05b ±0.06b ±0.05a ±0.03b ±0.06ab ±0.05a No.1702 3.25 1.81 3.21 5.13 0.63 1.05 4.05 2.64 Pp9 ±0.04a ±0.03b ±0.04b ±0.05b ±0.02a ±0.02a ±0.05a ±0.04b No.1702 5.83 1.79 3.24 4.94 1.12 1.31 4.75 2.78 Pp10 ±0.07d ±0.02a ±0.04ab ±0.04a ±0.02b ±0.03a ±0.07c ±0.05b FEAA – total essential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; VAL – valine; ILE – isoleucine; LEU – leucine; THR – threonine; MET – methionine; PHE – phenylethylamine; LYS – lysine; HIS – histamine; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

3.5.3. The Amino Acids Profile in Untreated and Biotreated Lupine Protein Isolates/Concentrates

The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented lupine wholemeal protein isolates/ concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.3.1. In compare with nonfermented, in SMF isolates/concentrates the higher contents of ALA, SER, PRO, GLU in SMF with P. pentosaceus No.8, No.9 and No.10 strains varieties Vilciai and Vilniai were found. In SSF samples the contents of ALA, GLY, SER and PRO in varieties Vilciai, Vilniai and hybrid No.1072 were increased, in compare with nonfermented samples. In SSF samples content of GLU was established to be on average 10.01% higher, in compare with SMF. Compared to nonfermented, in most of the SSF samples, the lower content of TYR (in 9 samples out of 16 analysed) was found. The ALA, GLY, SER, PRO, ASP, GLU and TYR contents in lupine protein isolates/concentrates were significantly affected by the selection of lupine variety (F(3.923)=930.060, p≤0.0001; F(1.768)=3233.111, p≤0.0001; F(97.713)=3896.841, p≤0.0001; F(7.676)=3980.304, p≤0.0001; F(30.885)=3665.879, p≤0.0001; F(187.549)= 4798.702, p≤0.0001 and F(4.753)=1995.224, p≤0.0001, respectively), the fermentation method (F(0.423)=100.335, p≤0.0001; F(7.703)=3643.222, p≤0.0001; F(374.422)=14932.068, p≤0.0001; F(3.328)=1725.733, p≤0.0001; F(3.063)=363.539, p≤0.0001; F(79.275)=

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2028.359, p≤0.0001; F(18.392)=7720.827, p≤0.0001, respectively), the type of microorganism applied for the fermentation (F(8.205)= 1945.250, p≤0.0001; F(1.424)= 673.543, p≤0.0001; F(97.092)=3872.055, p≤0.0001; F(3.649)=1892.158, p≤0.0001; F(19.366)=2298.619, p≤0.0001; F(32.743)= 837.785, p≤0.0001; F(9.413)=3951.406, p≤0.0001; F(9.413)= 3951.406, p≤0.0001, respectively) and their interaction (F(3.268)=774.745, p≤0.0001; F(2.018)=954.686, p≤0.0001; F(1.515)=11017.333, p≤0.0001; F(1.220)= 632.521, p≤0.0001; F(9.979)=1184.458, p≤0.0001; F(119.438)=3055.978, p≤0.0001; F(1.996)= 838.057, p≤0.0001, respectively).

Table 3.5.3.1. The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented lupine wholemeal protein isolates/ concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734, SMF and SSF with P. pentosaceus No. 8, N. 9, No. 10 strains. TNAA content (%) from total protein hydrolysed Lupine samples ALA GLY SER PRO ASP GLU TYR 1 2 3 4 5 6 7 8 Nonfermented samples 3.15 2.19 4.02 1.01 11.72 18.92 1.98 Vilciai ±0.04b ±0.03a ±0.05b ±0.02a ±0.09b ±0.15b ±0.04c 4.05 2.00 7.75 1.88 6.65 12.13 4.98 Vilniai ±0.06c ±0.02 ±0.09 ±0.03 ±0.07 ±0.12 ±0.06 4.87 3.02 17.61 2.07 9.75 19.45 4.71 No.1072 ±0.08d ±0.04 ±0.16 ±0.04 ±0.11 ±0.18 ±0.05 6.42 3.61 21.90 5.24 19.11 10.88 4.31 No.1734 ±0.09f ±0.05 ±0.19 ±0.06 ±0.18 ±0.11 ±0.04 Submerged fermented samples 3.22 2.15 7.02 2.47 9.00 19.98 4.88 Vilciai Pp8 ±0.04a ±0.03a ±0.08b ±0.04a ±0.09c ±0.19c ±0.05d 6.50 2.56 7.19 2.51 9.91 21.71 5.27 Vilciai Pp9 ±0.06de ±0.04b ±0.09c ±0.05b ±0.11cd ±0.21cd ±0.07de 7.11 3.15 13.89 2.99 9.29 20.62 2.88 Vilciai Pp10 ±0.08e ±0.05c ±0.13de ±0.05b ±0.09c ±0.20d ±0.04a 5.69 2.73 11.97 3.11 7.34 23.10 4.60 Vilniai Pp8 ±0.05d ±0.04b ±0.11d ±0.03c ±0.08bc ±0.23d ±0.05d 7.16 3.52 19.91 3.72 4.79 18.81 2.47 Vilniai Pp9 ±0.07e ±0.03bc ±0.19f ±0.04c ±0.06a ±0.19d ±0.03a 5.52 4.02 21.88 4.33 5.67 15.60 4.01 Vilniai Pp10 ±0.06c ±0.05d ±0.21f ±0.06b ±0.07b ±0.17b ±0.04c 4.33 2.85 9.91 2.96 6.01 21.74 4.95 No.1072 Pp8 ±0.04b ±0.04b ±0.10c ±0.04 ±0.08bc ±0.21d ±0.05cd 4.76 2.99 13.53 2.61 10.35 22.60 4.80 No.1072 Pp9 ±0.05b ±0.05b ±0.13d ±0.03bc ±0.12e ±0.22de ±0.05c 4.94 2.30 8.49 2.30 8.07 26.10 4.00 No.1072 Pp10 ±0.07bc ±0.03b ±0.08b ±0.04b ±0.08d ±0.25f ±0.04c 5.86 4.57 19.50 3.02 8.39 9.35 3.58 No.1734 Pp8 ±0.08d ±0.06d ±0.18e ±0.05d ±0.09d ±0.09a ±0.04b

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Table 3.5.3.1 continued 1 2 3 4 5 6 7 8 5.08 1.98 6.89 2.22 7.84 21.62 6.19 No.1734 Pp9 ±0.06c ±0.02a ±0.07a ±0.03a ±0.07b ±0.18d ±0.08d 5.38 4.55 21.30 4.72 6.34 21.64 3.47 No.1734 Pp10 ±0.07d ±0.06d ±0.21f ±0.05e ±0.06b ±0.19e ±0.05b Solid state fermented samples 3.47 3.57 17.56 2.58 6.85 13.53 4.46 Vilciai Pp8 ±0.04a ±0.05b ±0.18de ±0.03a ±0.07e ±0.13c ±0.06d 6.05 4.71 17.13 3.18 9.18 11.58 3.63 Vilciai Pp9 ±0.09d ±0.06d ±0.17d ±0.04d ±0.11f ±0.11a ±0.05c 5.75 3.76 21.95 3.78 6.10 25.98 2.34 Vilciai Pp10 ±0.06c ±0.04c ±0.21f ±0.05bc ±0.08d ±0.24de ±0.04b 4.17 3.28 15.30 3.47 6.01 32.76 3.76 Vilniai Pp8 ±0.05b ±0.04b ±0.16c ±0.04c ±0.06d ±0.31f ±0.05c 6.13 4.07 20.00 4.05 7.91 18.11 3.30 Vilniai Pp9 ±0.07 ±0.06 ±0.19d ±0.06d ±0.08cd ±0.17b ±0.05bc 5.58 4.89 22.02 4.06 3.09 24.64 3.20 Vilniai Pp10 ±0.06c ±0.07d ±0.21f ±0.05d ±0.05a ±0.22e ±0.04c 5.21 3.56 20.34 3.87 7.02 21.63 3.43 No.1072 Pp8 ±0.05c ±0.04c ±0.20 ±0.04c ±0.09d ±0.21d ±0.06d 6.20 4.22 19.71 4.11 7.86 18.62 3.32 No.1072 Pp9 ±0.07d ±0.05de ±0.18d ±0.06d ±0.11d ±0.17b ±0.05d 6.68 3.64 24.70 3.98 6.79 25.14 1.59 No.1072 Pp10 ±0.08d ±0.04c ±0.22e ±0.05c ±0.08b ±0.22e ±0.03a 7.07 3.17 11.37 2.84 10.67 19.11 3.38 No.1734 Pp8 ±0.08e ±0.06a ±0.13b ±0.04b ±0.12e ±0.19b ±0.04cd 4.15 2.98 5.26 2.42 9.18 36.84 3.81 No.1734 Pp9 ±0.05b ±0.04a ±0.08a ±0.03a ±0.09cd ±0.33f ±0.05d 6.93 3.37 20.87 3.79 7.39 20.15 2.75 No.1734 Pp10 ±0.07de ±0.05c ±0.19de ±0.05c ±0.08c ±0.19c ±0.03a TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; ALA – alanine; GLY – glycine; SER – serine; PRO – proline; ASP – asparagine; GLU – glutamine; TYR – tyramine; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented wholemeal protein isolates/concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734 , SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.3.2. In all the cases, in SMF with L. sakei and P. acidilactici lupine protein isolates/concentrates, the contents of ALA (except in SMF with P. acidilactici hybrids No.1072 and No.1734 isolates/concentrates), PRO (except in SMF with L. sakei and P. acidilactici hybrid No.1734 isolates/ concentrates), GLU and TYR (except in SMF with L. sakei and P. acidilactici hybrid No.1072 isolates/concentrates) were increased, in compare with nonfermented lupine isolates/concentrates. In SSF lupine protein

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Table 3.5.3.2. The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented wholemeal protein isolates/concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734, SMF and SSF with L. sakei and P. acidilactici strains. TNAA content (%) from total protein hydrolysed Lupine ALA GLY SER PRO ASP GLU TYR samples Nonfermented samples 3.15 2.19 4.02 1.01 11.72 18.92 1.98 Vilciai ±0.04b ±0.03a ±0.05b ±0.02a ±0.09b ±0.15b ±0.04c 4.05 2.00 7.75 1.88 6.65 12.13 4.98 Vilniai ±0.06c ±0.02 ±0.09 ±0.03 ±0.07 ±0.12 ±0.06 4.87 3.02 17.61 2.07 9.75 19.45 4.71 No.1072 ±0.08d ±0.04 ±0.16 ±0.04 ±0.11 ±0.18 ±0.05 6.42 3.61 21.90 5.24 19.11 10.88 4.31 No.1734 ±0.09f ±0.05 ±0.19 ±0.06 ±0.18 ±0.11 ±0.04 Submerged fermented samples 6.14 2.97 7.54 2.68 10.04 20.18 5.61 Vilciai Ls ±0.06de ±0.05e ±0.08d ±0.05d ±0.11d ±0.20d ±0.06c 6.50 2.76 7.19 2.61 9.91 21.71 5.27 Vilciai Pa ±0.08e ±0.04d ±0.06d ±0.04d ±0.09c ±0.21de ±0.05c 5.98 1.98 6.12 2.19 11.81 18.77 6.08 Vilniai Ls ±0.06d ±0.03a ±0.07c ±0.04b ±0.10d ±0.18b ±0.08d 5.34 2.11 5.62 2.06 12.26 19.56 5.84 Vilniai Pa ±0.05c ±0.04b ±0.06a ±0.03a ±0.12ef ±0.19c ±0.06cd 7.95 2.36 6.22 2.27 7.86 23.66 3.69 No.1072 Ls ±0.09f ±0.03b ±0.07e ±0.04c ±0.09d ±0.23d ±0.04a 4.59 2.56 11.53 2.76 7.22 21.22 4.47 No.1072 Pa ±0.05a ±0.04b ±0.11f ±0.05cd ±0.06b ±0.21cd ±0.05b 5.71 1.89 7.20 1.74 5.23 16.64 7.10 No.1734 Ls ±0.06c ±0.02a ±0.06d ±0.03a ±0.05a ±0.16a ±0.08e 4.99 2.58 6.38 2.51 10.16 21.61 5.56 No.1734 Pa ±0.05b ±0.04bc ±0.05b ±0.04c ±0.10d ±0.21e ±0.06d Solid state fermented samples 6.94 4.85 23.39 2.70 9.48 10.21 1.82 Vilciai Ls ±0.07b ±0.06f ±0.22f ±0.04b ±0.09f ±0.11b ±0.02a 5.21 3.78 16.89 2.20 7.63 13.43 3.21 Vilciai Pa ±0.06a ±0.04de ±0.16c ±0.03a ±0.07e ±0.14c ±0.05d 6.32 3.85 22.14 4.29 5.67 23.57 2.04 Vilniai Ls ±0.06b ±0.05e ±0.19e ±0.05de ±0.06b ±0.22d ±0.04b 5.90 3.35 16.28 2.99 6.49 28.10 2.51 Vilniai Pa ±0.08a ±0.04d ±0.18c ±0.03bc ±0.07d ±0.25ef ±0.04b 8.31 3.42 19.74 3.95 6.16 27.60 1.11 No.1072 Ls ±0.09e ±0.05bc ±0.19d ±0.04d ±0.06c ±0.28e ±0.02a 7.25 2.78 8.63 2.94 7.68 24.53 3.64 No.1072 Pa ±0.07c ±0.03a ±0.09b ±0.03b ±0.09ef ±0.22d ±0.05d 8.39 3.55 16.92 3.70 6.49 24.28 1.84 No.1734 Ls ±0.09f ±0.05c ±0.18cd ±0.05d ±0.07c ±0.19d ±0.03a 7.90 2.45 7.09 2.37 6.06 35.90 3.44 No.1734 Pa ±0.08d ±0.04a ±0.08a ±0.04a ±0.06b ±0.27f ±0.04cd TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; ALA – alanine; GLY – glycine; SER – serine; PRO – proline; ASP – asparagine; GLU – glutamine; TYR – tyramine; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-fThe mean values within a column with different letters are significantly different (p≤0.05). 114 isolates/concentrates contents of ALA, PRO (except in SSF with L. sakei and P. acidilactici hybrid No.1734 isolates/concentrates) and GLU (except in SSF with L. sakei and P. acidilactici hybrid variety Vilciai isolates/ concentrates) were increased, in compare with nonfermented lupine protein isolates/concentrates. To compare SMF and SSF lupine isolates/concentrates, in SMF samples content of ASP was on average 33.95% higher. The ALA content in lupine protein isolates/concentrates was significantly influenced by the fermentation method (F(15.255)=22.627, p≤0.0001), however, the selection of lupine variety and microorganism had no significant influence on ALA content. The GLY content in lupine protein isolates/concentrates was significantly affected by the fermentation method (F(14.586)=100.802, p≤0.0001), but the selection of lupine variety, and microorganism had no significant influence on GLY content in isolates/concentrates. The SER content in lupine protein isolates/concentrates was significantly affected by the selection of lupine variety (F(98.901)=14.638, p≤0.0001) and fermentation method (F(1007.142)=149.059, p≤0.0001), but the microorganism applied for fermentation had no significant influence on SER content. The PRO content in lupine protein isolates/concentrates was significantly affected by the selection of lupine variety (F(7.686)=70.653, p≤0.0001) and fermentation method (F(7.489)=68.844, p≤0.0001), however, the microorganism applied for fermentation had no significant influence on PRO content in isolates/concentrates. The TYR content in lupine protein isolates/concentrates was significantly affected by the selection of lupine variety (F(8.337)=37.537, p≤0.0001) and fermentation method (F(108.090)= 486.680, p≤0.0001), however, the microorganism applied for fermentation had no significant influence on TYR content. Finally, the interaction of analysed factors was not significant on ASP and GLU content in lupine protein isolates/concentrates. The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented wholemeal protein isolates/concentrates of lupine hybrid lines No.1700, No.1701, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.3.3. In all the cases, in SMF isolates/concentrates content of ASP (except in SMF with P. pentosaceus No.8, No.9 and No.10 strains hybrid No.1702 isolates/concentrates samples) and GLU was increased, in compare to nonfermented isolates/concentrates. Different tendencies of ALA (in 7 samples out of 16 analysed was found to be higher), GLY (in 5 samples out of 16 analysed was found to be higher) and SER (in 6 samples out of 16 analysed was found to be higher) contents in SMF lupine isolates/concentrates were found, compare to nonfermented samples. In compare with nonfermented, in all SSF protein isolates/concentrates the higher contents of GLY and 115

GLU (except in SSF with P. pentosaceus No.10 hybrid No.1800 protein isolates/concentrates) were established. Also, in SSF lupine protein isolates/concentrates the higher contents of ALA and SER (except both of them in SSF with P. pentosaceus No.8, No.9 and No.10 strains hybrid No.1800 protein isolates/concentrates) were found, in compare with nonfermented protein isolates/concentrates. However, content of TYR in SSF protein isolates/concentrates was on average 21.74% lower, compared to nonfermented samples. The analysed TNAA were significantly influenced by the analysed factors and their interaction was significant. The ALA, GLY, SER, PRO, ASP, GLU and TYR contents in lupine protein isolates/concentrates were significantly influenced by the selection of lupine variety (F(5.573)=1373.267, p≤0.0001; F(1.079)=522.510, p≤0.0001; F(30.828) 1972.970, p≤0.0001; F(0.261)=130.412, p≤0.0001; F(22.285)= 3598.440, p≤0.0001; F(33.261)=679.337, p≤0.0001; F(9.380)=2513.421, p≤0.0001, respectively), the fermentation method (F(0.202)=49.679, p≤0.0001; F(13.056)=6324.730, p≤0.0001; F(417.605)=26726.720, p≤ 0.0001; F(13.520)=6747.950, p≤0.0001; F(2.750)=443.981, p≤0.0001; F(0.845)= 17.259, p≤0.0001; F(23.564)=6313.829, p≤0.0001, respectively), the type of microorganism applied for the fermentation (F(0.135)=33.376, p≤0.0001; F(1.460)=707.079, p≤0.0001; F(31.281)=2001.999, p≤0.0001; F(4.151)=2071.557, p≤0.0001; F(31.422)=5073.942, p≤0.0001; F(17.186)= 351.018, p≤0.0001; F(0.878)=235.364, p≤0.0001, respectively) and the interaction of these factors was significant (F(4.658)=1147.733, p≤0.0001; F(1.382)=669.354, p≤0.0001; F(55.967)=3581.895, p≤0.0001; F(0.428)= 213.579, p≤0.0001; F(11.581)=1870.090, p≤0.0001; F(39.486)=806.476, p≤0.0001; F(2.935)=786.455, p≤0.0001, respectively).

Table 3.5.3.3. The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented wholemeal protein isolates/concentrates of lupine hybrid lines No.1700, No.1701, No.1800 and No.1702, SMF and SSF with P. pentosaceus No. 8, No. 9, No. 10 strains. TNAA content (%) from total protein hydrolysed Lupine samples ALA GLY SER PRO ASP GLU TYR 1 2 3 4 5 6 7 8 Nonfermented samples 5.84 1.78 5.31 3.47 3.67 12.67 8.08 No.1700 ±0.09d ±0.03b ±0.05b ±0.08b ±0.06c ±0.14c ±0.09b 5.77 2.46 7.04 3.24 3.58 12.36 8.00 No.1701 ±0.08b ±0.04b ±0.09c ±0.06b ±0.05b ±0.13b ±0.08c 5.79 2.48 11.50 4.18 4.95 15.52 6.75 No.1800 ±0.05c ±0.04c ±0.11d ±0.05c ±0.08b ±0.16b ±0.07c 4.70 2.73 8.85 3.18 8.90 19.20 5.14 No.1702 ±0.04b ±0.05d ±0.09c ±0.04b ±0.11d ±0.19c ±0.05b

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Table 3.5.3.3continued 1 2 3 4 5 6 7 8 Submerged fermented samples 7.34 2.65 11.44 3.21 8.28 18.91 4.01 No.1700 Pp8 ±0.09f ±0.05c ±0.12d ±0.04d ±0.09e ±0.19c ±0.05b 6.67 3.69 15.37 4.27 6.15 21.10 4.09 No.1700 Pp9 ±0.07d ±0.07d ±0.14e ±0.06e ±0.06b ±0.20b ±0.04b 6.35 2.39 10.06 2.27 7.91 24.34 5.37 No.1700 Pp10 ±0.07d ±0.06b ±0.11d ±0.04b ±0.08d ±0.22e ±0.06c 2.75 3.47 14.54 3.11 4.40 37.69 3.20 No.1701 Pp8 ±0.04a ±0.05d ±0.16f ±0.05d ±0.05a ±0.35f ±0.04a 6.61 1.92 5.59 1.93 7.95 21.34 7.67 No.1701 Pp9 ±0.06de ±0.03a ±0.06bc ±0.03c ±0.09d ±0.21d ±0.08e 6.93 2.03 4.75 1.66 9.45 20.78 7.57 No.1701 Pp10 ±0.08e ±0.04b ±0.05a ±0.02a ±0.11f ±0.20a ±0.06e 6.06 1.97 4.32 1.71 8.72 21.31 6.22 No.1800 Pp8 ±0.06e ±0.04a ±0.05a ±0.03a ±0.09de ±0.21b ±0.07d 4.46 2.71 10.09 3.02 7.81 22.19 5.07 No.1800 Pp9 ±0.05c ±0.05d ±0.12d ±0.02d ±0.08cd ±0.24c ±0.06c 4.89 2.41 10.50 2.63 8.05 23.50 5.01 No.1800 Pp10 ±0.06d ±0.06c ±0.13cd ±0.04b ±0.09d ±0.26d ±0.05cd 4.92 2.57 11.12 2.71 7.23 23.63 4.38 No.1702 Pp8 ±0.07c ±0.05b ±0.15de ±0.05b ±0.06c ±0.24d ±0.04b 3.99 2.17 7.80 2.05 7.63 23.21 7.41 No.1702 Pp9 ±0.05b ±0.03b ±0.08c ±0.04a ±0.07c ±0.23d ±0.08d 4.11 2.55 10.49 2.75 7.47 22.58 5.63 No.1702 Pp10 ±0.06b ±0.04bc ±0.11d ±0.06b ±0.08c ±0.21b ±0.06c Solid state fermented samples 7.34 2.65 11.44 3.21 8.28 18.91 4.01 No.1700 Pp8 ±0.09f ±0.03a ±0.11d ±0.05c ±0.07e ±0.17b ±0.05c 6.67 3.69 15.37 4.27 6.15 21.10 4.09 No.1700 Pp9 ±0.05e ±0.06d ±0.16c ±0.05d ±0.06c ±0.19c ±0.06c 6.35 2.39 10.06 2.27 7.91 24.34 5.37 No.1700 Pp10 ±0.04c ±0.04a ±0.12b ±0.03a ±0.08d ±0.22d ±0.08d 5.08 2.66 11.29 2.84 7.43 23.31 4.64 No.1701 Pp8 ±0.06d ±0.03a ±0.12b ±0.04a ±0.08d ±0.24d ±0.06cd 7.63 3.61 12.03 3.59 6.97 17.17 4.10 No.1701 Pp9 ±0.09f ±0.05b ±0.14cd ±0.05c ±0.05c ±0.19b ±0.05c 6.97 3.00 8.13 3.18 9.83 28.02 2.49 No.1701 Pp10 ±0.06e ±0.04a ±0.09a ±0.04c ±0.09ef ±0.22e ±0.03a 3.12 4.34 23.08 4.13 3.56 24.62 3.85 No.1800 Pp8 ±0.03a ±0.06e ±0.18f ±0.05d ±0.05a ±0.23de ±0.04c 3.18 4.18 14.10 3.53 4.61 36.24 3.74 No.1800 Pp9 ±0.03a ±0.05e ±0.14c ±0.04c ±0.04b ±0.35f ±0.04b 3.56 3.79 19.54 2.70 6.65 14.51 4.39 No.1800 Pp10 ±0.05b ±0.03c ±0.18e ±0.03a ±0.07c ±0.15a ±0.05d 6.52 3.72 16.54 4.32 7.09 20.05 7.10 No.1702 Pp8 ±0.08de ±0.03cd ±0.15d ±0.05e ±0.08de ±0.19d ±0.09e 5.06 3.62 19.47 4.44 5.52 29.20 3.93 No.1702 Pp9 ±0.06d ±0.05d ±0.19e ±0.05de ±0.06b ±0.29e ±0.05b 4.87 3.11 12.82 3.19 12.36 20.51 4.19 No.1702 Pp10 ±0.05b ±0.04c ±0.13b ±0.04c ±0.14f ±0.21d ±0.07d TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation. ALA – alanine; GLY – glycine; SER – serine; PRO – proline; ASP – asparagine; GLU – glutamine; TYR – tyramine; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

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The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented wholemeal protein isolates/concentrates of lupine hybrid lines No.1700, No.1701, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.3.4. In all the cases, in SMF protein isolates/concentrates contents of ALA (except in SMF with P. acidilactici hybrid No.1800 and No.1702 isolates/concentrates), ASP (except in SMF with L. sakei and P. acidilactici strains hybrid No.1702 isolates/concentrates) and GLU were increased, compared to nonfermented lupine isolates/concentrates. Different tendencies of PRO and TYR contents in SMF lupine isolates/concentrates were established (lower content in 6 samples out of 12 analysed and in 7 samples out of 12 analysed, respectively), compared to nonfermented samples. In SSF isolates/concentrates the contents of ALA (except in SSF with P. acidilactici hybrid No.1800 isolates/concentrates) and GLY (except in SSF with P. acidilactici hybrid No.1702 isolates/concentrates) were higher, in compare with nonfermented lupine isolates/concentrates. To compare SMF and SSF isolates/ concentrates, in SSF samples SER content was on average 55.78% higher. In SSF samples the content of GLU was on average 40.24% higher, in compare with nonfermented lupine isolates/concentrates. The most of analysed TNAA were significantly influenced by the analysed factors. The ALA, GLY, SER, PRO, ASP, GLU and TYR contents in lupine protein isolates/concentrates were significantly influenced by the selection of lupine variety (F(6.829)=1460.717, p≤0.0001; F(0.879)= 401.898, p≤0.0001; F(57.146)=2435.381, p≤0.0001; F(1.565)=703.180, p≤0.0001; F(10.611)=1642.535, p≤0.0001; F(40.015)=830.154, p≤0.0001; F(6.545)=1790.762, p≤0.0001, respectively), the fermentation method (F(21.644)=4629.687, p≤0.0001; F(7.971)=3645.137, p≤0.0001; F(1350.611)=57558.516, p≤0.0001; F(5.355)=2406.601, p≤0.0001; F(10.267)=1589.396, p≤0.0001; F(54.596)=1132.663, p≤0.0001; F(42.956)=11752.659, p≤0.0001, respectively) and the type of microorganism applied for the fermentation (F(1.115)=238.561, p≤0.0001; F(0.592)= 270.697, p≤0.0001; F(30.032)=1279.863, p≤0.0001; F(0.893)=401.393, p≤0.0001; F(2.147)=332.326, p≤0.0001; F(1.060)=21.992, p≤0.0001, except TYR, respectively).

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Table 3.5.3.4. The total nonessential amino acids (TNAA) content (%) from total protein hydrolysed in nonfermented wholemeal protein isolates/ concentrates of lupine hybrid lines No.1700, No.1701, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains. TNAA content (%) from total protein hydrolysed Lupine samples ALA GLY SER PRO ASP GLU TYR Nonfermented samples 5.84 1.78 5.31 3.47 3.67 12.67 8.08 No.1700 ±0.09d ±0.03b ±0.05b ±0.08b ±0.06c ±0.14c ±0.09b 5.77 2.46 7.04 3.24 3.58 12.36 8.00 No.1701 ±0.08b ±0.04b ±0.09c ±0.06b ±0.05b ±0.13b ±0.08c 5.79 2.48 11.50 4.18 4.95 15.52 6.75 No.1800 ±0.05c ±0.04c ±0.11d ±0.05c ±0.08b ±0.16b ±0.07c 4.70 2.73 8.85 3.18 8.90 19.20 5.14 No.1702 ±0.04b ±0.05d ±0.09c ±0.04b ±0.11d ±0.19c ±0.05b Submerged fermented samples 6.33 3.12 17.30 3.53 5.26 20.05 4.76 No.1700 Pa ±0.05cd ±0.05d ±0.15f ±0.05d ±0.06a ±0.23a ±0.04b 7.62 2.15 5.41 1.64 11.36 22.36 6.74 No.1701 Ls ±0.09e ±0.03c ±0.06b ±0.04a ±0.12f ±0.19d ±0.08e 9.16 2.34 7.43 2.46 5.15 21.10 6.25 No.1701 Pa ±0.11f ±0.05c ±0.08d ±0.05c ±0.04a ±0.17c ±0.07d 6.70 1.99 5.25 1.93 8.96 20.35 5.75 No.1700 Ls ±0.08c ±0.02b ±0.04b ±0.02b ±0.09d ±0.21b ±0.05c 6.50 2.71 9.97 2.81 7.42 20.83 4.73 No.1800 Ls ±0.06c ±0.04cd ±0.11d ±0.04c ±0.08c ±0.24b ±0.04b 5.73 1.80 4.79 1.42 7.69 23.21 7.48 No.1800 Pa ±0.05c ±0.03a ±0.05a ±0.02a ±0.09c ±0.27c ±0.09f 4.78 3.45 14.61 3.87 6.18 29.74 4.33 No.1702 Ls ±0.04b ±0.06e ±0.14e ±0.05e ±0.07b ±0.27e ±0.05a 4.46 2.62 8.10 1.56 8.48 23.92 5.03 No.1702 Pa ±0.04b ±0.04c ±0.09de ±0.03a ±0.11e ±0.23cd ±0.07e Solid state fermented samples 7.92 3.58 18.28 4.16 6.38 27.39 1.38 No.1700 Ls ±0.08ef ±0.05c ±0.18c ±0.06d ±0.06d ±0.26e ±0.03a 6.88 3.62 18.67 4.20 5.80 26.63 3.12 No.1700 Pa ±0.06d ±0.06c ±0.19d ±0.07de ±0.05c ±0.29e ±0.04d 6.99 3.47 16.82 3.46 8.15 21.27 2.56 No.1701 Ls ±0.07e ±0.05b ±0.17b ±0.04c ±0.09e ±0.21d ±0.04b 6.96 3.85 18.19 3.85 7.24 21.58 3.16 No.1701 Pa ±0.09e ±0.06de ±0.19c ±0.05b ±0.07de ±0.20d ±0.06e 7.72 3.86 15.65 2.86 8.31 19.79 2.48 No.1800 Ls ±0.07e ±0.07d ±0.14a ±0.04b ±0.09e ±0.19bc ±0.04c 4.93 4.61 31.31 4.41 3.46 22.29 2.26 No.1800 Pa ±0.05b ±0.06e ±0.32f ±0.05e ±0.05a ±0.22d ±0.03b 5.31 3.36 21.50 3.95 4.60 33.96 1.72 No.1702 Ls ±0.06c ±0.05b ±0.21de ±0.03d ±0.06b ±0.32f ±0.02a 4.77 1.99 24.28 1.47 10.21 27.12 7.10 No.1702 Pa ±0.05a ±0.04a ±0.25e ±0.02a ±0.11f ±0.25b ±0.09f TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; ALA – alanine; GLY – glycine; SER – serine; PRO – proline; ASP – asparagine; GLU – glutamine; TYR – tyramine; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

119

The total essential amino acids (TEAA) content (%) from total protein hydrolysed in nonfermented wholemeal protein isolates/concentrates of lupine hybrid lines No.1700, No.1701, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.3.5. In compare with nonfermented, in SMF isolates/concentrates higher contents of VAL, ILE (except in SMF with L. sakei hybrid No.1700 and in SMF with P. acidilactici hybrid No.1800 isolates/concentrates), MET and PHE (except in SMF with P. acidilactici hybrids No.1800 and No.1702 isolates/concentrates) were established. The contents of THR in SMF lupine isolates/ concentrates (except in SMF with P. acidilactici hybrid No.1700 isolates/ concentrates) and MET were decreased, in compare with nonfermented isolates/concentrates. In SSF lupine isolates/concentrates the contents of VAL (except in SSF with L. sakei hybrid No.1800 isolates/concentrates), ILE, LEU, THR (except in SSF with L. sakei and P. acidilactici strains hybrid No.1702 isolates/concentrates), PHE and TRP were decreased, in compare to nonfermented. Also, in SSF samples LYS content was on average 25.00% lower, in compare to nonfermented samples. The analysed TEAA were significantly influenced by the analysed factors. The VAL, ILE, LEU, THR, MET, PHE, LYS, HIS and TRP contents in lupine protein isolates/concentrates were significantly influenced by the selection of lupine variety (F(1.762)=448.889, p≤0.0001; F(4.114)= 1913.590, p≤0.0001; F(33.497)=2678.667, p≤0.0001; F(1.306)=552.034, p≤0.0001; F(0.233)=190.291, p≤0.0001; F(5.502)=1695.505, p≤0.0001; F(10.310)=831285.269, p≤0.0001; F(1.105)=437.678, p≤0.0001; F(11.281)=3766.546, p≤0.0001, respectively), the fermentation method (F(0.875)=222.879, p≤0.0001; F(16.078)=7477.988, p≤0.0001; F(82.687)= 6612.355, p≤0.0001; F(6.968)=2946.185, p≤0.0001; F(0.317)=258.673, p≤0.0001; F(26.659)=8215.434, p≤0.0001; F(39.683)=4947.015, p≤0.0001; F(3.301)=1307.407, p≤0.0001; F(4.421)=1476.256, p≤0.0001, respectively) and the type of microorganism applied for the fermentation (F(3.167)=807.006, p≤0.0001; F(2.978)=1384.930, p≤0.0001; F(15.462)= 1236.489, p≤0.0001; F(1.140)=481.850, p≤0.0001; F(0.097)=79.531, p≤0.0001; F(1.280)=394.307, p≤0.0001; F(10.545)=1314.567, p≤0.0001; F(0.448)=177.337, p≤0.0001; F(0.163)=54.324, p≤0.0001, respectively).

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Table 3.5.3.5. The total essential amino acids (TEAA) content (%) from total protein hydrolysed in nonfermented wholemeal protein isolates/concentrates of lupine hybrid lines No.1700, No.1701, No.1800 and No.1702, SMF and SSF with L. sakei and P. acidilactici strains. TEAA content (%) from total protein hydrolysed Lupine samples VAL ILE LEU THR MET PHE LYS HIS TRP Nonfermented samples 6.94 5.43 18.15 3.03 2.26 7.91 9.15 2.36 4.15 No.1700 ±0.06b ±0.05c ±0.17d ±0.03a ±0.04d ±0.08c ±0.11c ±0.04b ±0.05c 7.27 5.64 18.88 3.25 1.93 8.58 6.08 2.15 3.76 No.1701 ±0.08c ±0.06c ±0.18d ±0.04c ±0.03c ±0.09c ±0.08a ±0.03a ±0.04a 5.93 4.49 13.19 3.16 1.62 5.90 5.82 3.47 6.75 No.1800 ±0.07a ±0.06b ±0.14c ±0.04b ±0.03b ±0.06b ±0.07a ±0.06d ±0.07b 7.49 4.09 11.32 4.84 1.21 4.55 7.59 2.76 3.45 No.1702 ±0.09c ±0.04a ±0.12b ±0.06d ±0.02a ±0.04a ±0.08b ±0.04c ±0.05a Submerged fermented samples 6.00 5.54 12.73 1.84 1.57 4.77 8.94 2.78 5.41 No.1700 Ls ±0.07e ±0.05e ±0.13d ±0.03b ±0.03c ±0.06c ±0.09c ±0.03e ±0.05d 6.21 3.64 9.51 4.06 0.88 3.19 6.77 2.57 2.83 No.1700 Pa ±0.08f ±0.04c ±0.08b ±0.05e ±0.02a ±0.05c ±0.08b ±0.04c ±0.03b 4.87 3.65 10.86 1.30 1.46 5.26 11.36 2.36 5.58 No.1701 Ls ±0.02a ±0.04b ±0.11c ±0.03a ±0.04d ±0.06e ±0.13e ±0.04a ±0.06d 5.16 3.39 9.80 2.97 1.01 4.01 10.70 2.76 6.31 No.1701 Pa ±0.06d ±0.03b ±0.09b ±0.04d ±0.02b ±0.05c ±0.11d ±0.05d ±0.07e 5.63 3.34 10.49 2.98 1.43 3.29 11.84 3.19 2.84 No.1800 Ls ±0.06d ±0.06c ±0.12d ±0.04c ±0.04b ±0.04c ±0.12f ±0.05d ±0.04b 1.36 5.86 15.67 2.40 1.21 7.26 8.62 2.62 2.90 No.1800 Pa ±0.03b ±0.07f ±0.16d ±0.03c ±0.04c ±0.09e ±0.09c ±0.04b ±0.05c 4.59 2.53 5.16 4.04 0.66 2.39 6.54 2.80 4.34 No.1702 Ls ±0.04c ±0.03a ±0.06a ±0.06e ±0.02a ±0.04a ±0.07a ±0.04c ±0.06c 4.98 3.69 10.19 2.92 1.12 5.16 9.82 2.71 3.74 No.1702 Pa ±0.06cd ±0.07c ±0.13c ±0.04d ±0.03b ±0.07d ±0.11d ±0.05b ±0.04b Solid state fermented samples 5.09 2.87 5.81 3.94 1.53 1.50 6.16 3.05 2.95 No.1700 Ls ±0.06d ±0.05c ±0.06c ±0.05c ±0.04b ±0.04b ±0.08b ±0.07d ±0.05b 3.94 1.84 4.32 3.64 1.61 1.12 9.07 3.33 3.22 No.1700 Pa ±0.05b ±0.02b ±0.05a ±0.05c ±0.05c ±0.03b ±0.09e ±0.08d ±0.04c 5.86 3.33 7.99 4.92 1.03 2.02 6.09 2.59 3.45 No.1701 Ls ±0.09de ±0.03d ±0.09e ±0.06d ±0.03a ±0.05e ±0.09b ±0.05a ±0.06d 5.79 3.15 6.10 5.54 1.70 1.72 6.73 2.49 2.97 No.1701 Pa ±0.08d ±0.04d ±0.08d ±0.07e ±0.05c ±0.03c ±0.11c ±0.04a ±0.05b 6.03 3.67 7.27 5.28 0.98 1.70 9.13 2.99 2.29 No.1800 Ls ±0.07f ±0.04e ±0.08d ±0.08d ±0.02a ±0.05d ±0.10e ±0.06b ±0.03a 3.85 2.82 6.96 4.82 1.40 1.62 7.09 3.86 4.04 No.1800 Pa ±0.04b ±0.03c ±0.09d ±0.06c ±0.03b ±0.04c ±0.07d ±0.07e ±0.08e 4.10 1.50 4.44 2.93 1.46 0.90 5.79 2.90 2.59 No.1702 Ls ±0.05c ±0.02a ±0.06b ±0.04b ±0.04b ±0.02a ±0.05a ±0.05b ±0.04b 3.53 2.05 10.44 1.24 2.07 4.16 5.89 3.81 8.86 No.1702 Pa ±0.03a ±0.05b ±0.11f ±0.03a ±0.05d ±0.08f ±0.06a ±0.04e ±0.09f TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; VAL – valine; ILE – isoleucine; LEU – leucine; THR – threonine; MET – methionine; PHE – phenylethylamine; LYS – lysine; HIS – histamine; TRP – tryptophan; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

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The total essential amino acids (TEAA) content (%) from total protein hydrolysed in lupine hybrid lines No.1700, No.1701, No.1800 and No.1702 wholemeal protein isolates/concentrates nonfermented, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.3.6. In compare with nonfermented samples, in SMF lupine protein isolates/concentrates lower contents of LEU and PHE (except in SMF with P. pentosaceus No.9 and No.10 hybrid No.1702 samples) were established. Similar tendencies in SMF samples were found: lower content of ILE in 10 samples from 16 analysed and lower content of HIS in 8 samples from 16 analysed was found, in compare with nonfermented. In SSF protein isolates/concentrates contents of VAL (except in SSF with P. pentosaceus No.10 hybrid No.1701 protein isolates/concentrates) and HIS were increased, in compare with nonfermented protein isolates/concentrates. However, in SSF samples contents of ILE, LEU, MET (except in SSF with P. pentosaceus No.9 hybrid No.1702 protein isolates/concentrates, in which content was increased) and PHE were decreased. In compare SMF and SSF protein isolates/concentrates, in SSF samples content of LYS on average by 16.80% higher was established. The analysed TEAA were significantly influenced by the analysed factors and their interaction was significant. The VAL, ILE, LEU, THR, MET, PHE, LYS, HIS and TRP contents in lupine protein isolates/concentrates was significantly influenced by the selection of lupine variety (F(1.767)=398.925, p≤0.0001; F(2.727)=1324.732, p≤0.0001; F(47.506)=4280.762, p≤0.0001; F(3.849)=1668.404, p≤0.0001; F(2.122)= 2233.208, p≤0.0001; F(5.653)=2127.658, p≤0.0001; F(20.687)=2653.007, p≤0.0001; F(2.839)=1370.420, p≤0.0001; F(4.782)=1367.598, p≤0.0001, respectively), the fermentation method (F(48.807)=11020.981, p≤0.0001; F(27.876)=13542.780, p≤0.0001; F(592.999)=53434.831, p≤0.0001; F(11.454)=4964.783, p≤0.0001; F(2.543)=1102.018, p≤0.0001; F(7.354)= 7740.645, p≤0.0001; F(99.970)=37623.043, p≤0.0001; F(59.933)= 7686.064, p≤0.0001; F(3.877)=1871.878, p≤0.0001; F(27.491)=13271.572, p≤0.0001; F(24.640)=7047.248, p≤0.0001, respectively), the type of microorganism applied for the fermentation (F(8.126)=1834.802, p≤0.0001; F(0.486)=236.045, p≤0.0001; F(3.991)=359.660, p≤0.0001; F(2.202)= 2317.750, p≤0.0001; F(4.406)=1658.299, p≤0.0001; F(29.680)=3806.280, p≤0.0001; F(29.474)=8429.784, p≤0.0001) and the interaction of analysed factors was significant (F(14.460)=3265.158, p≤0.0001; F(4.074)= 1979.451, p≤0.0001; F(6.058)=545.897, p≤0.0001; F(2.965)=1285.318, p≤0.0001; F(2.275)=2394.504, p≤0.0001; F(1.868)=703.182, p≤0.0001; F(6.669)=855.220, p≤0.0001; F(3.501)=1690.132, p≤0.0001; F(20.324)= 5812.687, p≤0.0001, respectively).

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Table 3.5.3.6. The total essential amino acids (TEAA) content (%) from total protein hydrolysed in lupine hybrid lines No.1700, No.1701, No.1800 and No.1702 wholemeal protein isolates/concentrates nonfermented, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains. TEAA content (%) from total protein hydrolysed Lupine samples VAL ILE LEU THR MET PHE LYS HIS TRP 1 2 3 4 5 6 7 8 9 10 Nonfermented samples 6.94 5.43 18.15 3.03 2.26 7.91 9.15 2.36 4.15 No.1700 ±0.06b ±0.05c ±0.17d ±0.03a ±0.04d ±0.08c ±0.11c ±0.04b ±0.05c 7.27 5.64 18.88 3.25 1.93 8.58 6.08 2.15 3.76 No.1701 ±0.08c ±0.06c ±0.18d ±0.04c ±0.03c ±0.09c ±0.08a ±0.03a ±0.04a 5.93 4.49 13.19 3.16 1.62 5.90 5.82 3.47 6.75 No.1800 ±0.07a ±0.06b ±0.14c ±0.04a ±0.03b ±0.06b ±0.07a ±0.06d ±0.07b 7.49 4.09 11.32 4.84 1.21 4.55 7.59 2.76 3.45 No.1702 ±0.09c ±0.04a ±0.12b ±0.06d ±0.02a ±0.04a ±0.08b ±0.04c ±0.05a Submerged fermented samples 7.34 4.55 11.52 4.64 1.32 3.14 6.32 2.23 3.11 No.1700 Pp8 ±0.09e ±0.06d ±0.12c ±0.06d ±0.02c ±0.04b ±0.06c ±0.02b ±0.03c 6.68 3.99 10.73 4.16 1.07 3.58 4.07 2.39 2.00 No.1700 Pp9 ±0.07d ±0.05d ±0.10c ±0.04d ±0.04a ±0.05c ±0.05a ±0.05c ±0.02a 6.13 3.68 10.66 3.17 1.53 3.61 7.75 2.31 2.92 No.1700 Pp10 ±0.08d ±0.04c ±0.11c ±0.05c ±0.03d ±0.07bc ±0.08d ±0.04b ±0.03b 3.31 2.73 12.90 3.20 4.49 1.19 7.08 3.55 4.38 No.1701 Pp8 ±0.04a ±0.02a ±0.13e ±0.04c ±0.05d ±0.02a ±0.09d ±0.04d ±0.05e 5.05 3.86 11.36 2.62 1.34 6.02 9.30 2.29 5.15 No.1701 Pp9 ±0.06c ±0.05c ±0.12c ±0.03a ±0.02b ±0.08e ±0.11f ±0.02c ±0.06f 4.93 4.52 11.95 2.47 1.19 5.83 8.94 2.22 4.80 No.1701 Pp10 ±0.05b ±0.06d ±0.11d ±0.05a ±0.03b ±0.06d ±0.08ef ±0.03b ±0.04e 8.76 2.84 10.48 2.56 1.48 5.56 11.86 3.37 2.78 No.1800 Pp8 ±0.11f ±0.04b ±0.09c ±0.03b ±0.04c ±0.08d ±0.13 ±0.05c ±0.02b 7.41 4.57 11.20 5.02 1.22 5.02 5.08 1.95 3.19 No.1800 Pp9 ±0.08e ±0.06d ±0.13d ±0.06e ±0.02bc ±0.06cd ±0.06b ±0.03a ±0.05d 6.90 3.82 10.52 3.75 1.18 4.16 7.16 2.44 3.06 No.1800 Pp10 ±0.06d ±0.03c ±0.14c ±0.04c ±0.04b ±0.02e ±0.08d ±0.05b ±0.04d 7.42 3.98 9.73 5.25 1.26 3.61 7.66 2.32 2.21 No.1702 Pp8 ±0.09e ±0.04d ±0.09b ±0.07d ±0.05b ±0.05b ±0.06d ±0.04b ±0.03b 5.91 3.81 10.62 3.23 1.17 5.87 8.92 2.49 3.81 No.1702 Pp9 ±0.06c ±0.05c ±0.11c ±0.05c ±0.03b ±0.07f ±0.09e ±0.05b ±0.05d 6.80 4.25 10.68 4.38 1.31 5.32 5.59 1.99 4.11 No.1702 Pp10 ±0.08de ±0.06d ±0.12c ±0.04d ±0.02b ±0.06ef ±0.05b ±0.03a ±0.07e Solid state fermented samples 5.11 2.95 4.70 4.87 1.83 0.72 10.83 3.56 1.77 No.1700 Pp8 ±0.04d ±0.05d ±0.05d ±0.05e ±0.04d ±0.02a ±0.11d ±0.06d ±0.03a 3.93 1.83 3.41 4.49 1.46 1.47 5.36 3.41 3.72 No.1700 Pp9 ±0.05b ±0.04b ±0.03b ±0.04d ±0.04c ±0.02b ±0.06a ±0.04c ±0.05c 5.19 3.11 6.34 4.51 0.74 1.20 5.14 2.72 2.08 No.1700 Pp10 ±0.06d ±0.03e ±0.08e ±0.05d ±0.02a ±0.03b ±0.05a ±0.02b ±0.04b 7.34 4.11 10.03 5.99 1.36 3.77 6.39 2.49 2.97 No.1701 Pp8 ±0.09f ±0.05f ±0.11f ±0.09f ±0.03c ±0.05e ±0.07b ±0.04b ±0.06b

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Table 3.5.3.6 continued 1 2 3 4 5 6 7 8 9 10 7.48 4.37 11.65 5.15 1.26 3.34 6.77 2.41 2.48 No.1701 Pp9 ±0.06f ±0.07f ±0.13f ±0.06e ±0.02b ±0.04d ±0.09b ±0.03a ±0.05a 5.30 2.92 5.62 2.48 1.01 2.80 15.86 2.53 2.87 No.1701 Pp10 ±0.05e ±0.05d ±0.06e ±0.03b ±0.03c ±0.03c ±0.15f ±0.04a ±0.07b 4.49 1.34 2.81 4.81 2.09 9.83 3.62 4.26 No.1800 Pp8 - ±0.04c ±0.02b ±0.02a ±0.06d ±0.04c ±0.09c ±0.04d ±0.07d 3.83 0.81 3.38 3.70 1.41 8.11 4.05 5.14 No.1800 Pp9 - ±0.05b ±0.03a ±0.04c ±0.04b ±0.03b ±0.07b ±0.06e ±0.09e 3.18 1.97 3.72 3.26 0.88 2.67 11.83 5.13 12.22 No.1800 Pp10 ±0.04b ±0.02b ±0.05b ±0.03b ±0.02b ±0.05d ±0.13e ±0.07f ±0.13f 5.51 2.97 5.69d 4.97 0.78 1.85 8.52 3.24 4.25 No.1702 Pp8 ±0.06d ±0.03c ±0.08 ±0.05c ±0.03a ±0.03c ±0.10d ±0.05d ±0.05d 3.97 1.88 4.00 3.45 1.57 1.59 6.81 3.07 3.43 No.1702 Pp9 ±0.04b ±0.04c ±0.04c ±0.03b ±0.04c ±0.02b ±0.08b ±0.04c ±0.03c 1.55 1.39 2.12 1.28 1.72 12.38 9.15 10.37 No.1702 Pp10 - ±0.03a ±0.03b ±0.03a ±0.02a ±0.03b ±0.14e ±0.09f ±0.12f TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; VAL – valine; ILE – isoleucine; LEU – leucine; THR – threonine; MET – methionine; PHE – phenylethylamine; LYS – lysine; HIS – histamine; TRP – tryptophan; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

The total essential amino acids (TEAA) content (%) from total protein hydrolysed in nonfermented wholemeal protein isolates/concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains is presented in Table 3.5.3.7.

Table 3.5.3.7. The total essential amino acids (TEAA) content (%) from total protein hydrolysed in nonfermented wholemeal protein isolates/concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734, SMF and SSF with P. pentosaceus No.8, No.9, No.10 strains. TEAA content (%) from total protein hydrolysed Lupine samples VAL ILE LEU THR MET PHE LYS HIS TRP 1 2 3 4 5 6 7 8 9 10 Nonfermented samples 0.64 0.98 0.99 1.70 13.67 12.33 26.69 Vilciai - - ±0.02a ±0.02a ±0.02a ±0.03a ±0.13b ±0.11b ±0.24c 2.93 2.53 10.35 1.30 1.32 5.58 12.66 6.53 17.36 Vilniai ±0.04b ±0.04b ±0.09c ±0.04b ±0.03b ±0.05b ±0.11a ±0.06c ±0.16d 1.80 1.71 0.66 0.82 15.52 8.58 9.42 No.1072 - - ±0.03b ±0.03b ±0.03a ±0.02b ±0.15c ±0.09a ±0.06c 3.25 1.58 1.94 2.83 0.34 1.36 5.55 5.40 6.30 No.1734 ±0.05c ±0.02a ±0.04b ±0.05c ±0.01a ±0.04c ±0.06b ±0.05b ±0.05b Submerged fermented samples 4.99 4.04 9.00 3.52 1.15 3.98 6.25 2.02 4.30 Vilciai Pp8 ±0.05c ±0.04b ±0.09c ±0.05c ±0.03c ±0.05 ±0.08ab ±0.04a ±0.06c

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Table 3.5.3.7 continued 1 2 3 4 5 6 7 8 9 10 5.45 4.75 12.05 2.21 1.20 4.67 6.70 2.38 4.95 Vilciai Pp9 ±0.06d ±0.06c ±0.12d ±0.03a ±0.04c ±0.07 ±0.09b ±0.06b ±0.08d 6.24 3.87 9.52 4.61 1.22 2.49 6.34 2.40 3.39 Vilciai Pp10 ±0.08e ±0.04b ±0.10c ±0.04c ±0.02b ±0.03 ±0.07b ±0.04b ±0.04b 5.22 4.39 10.12 2.82 1.56 4.60 8.91 2.42 7.17 Vilniai Pp8 ±0.05d ±0.05c ±0.11d ±0.03b ±0.04d ±0.05 ±0.11c ±0.05c ±0.09c 5.93 3.88 10.37 3.51 1.17 3.45 7.37 2.60 3.19 Vilniai Pp9 ±0.07d ±0.05b ±0.12d ±0.04b ±0.03d ±0.04 ±0.09c ±0.03c ±0.05b 4.84 2.47 4.65 4.96 0.73 2.42 9.21 3.81 5.91 Vilniai Pp10 ±0.05c ±0.04a ±0.06b ±0.06 ±0.02a ±0.03 ±0.08d ±0.06d ±0.07d 8.06 5.11 11.86 5.35 1.16 5.36 5.54 2.35 2.46 No.1072 Pp8 ±0.09de ±0.06d ±0.13d ±0.07d ±0.03b ±0.07 ±0.06a ±0.05c ±0.04a 3.86 1.66 4.64 2.67 2.03 9.64 6.18 7.68 No.1072 Pp9 - ±0.04a ±0.03a ±0.05b ±0.04a ±0.04 ±0.11d ±0.09d ±0.08c 7.44 5.52 12.54 3.87 2.12 9.98 6.08 7.98 No.1072 Pp10 - ±0.08d ±0.07d ±0.13e ±0.03c ±0.05 ±0.13c ±0.07b ±0.09b 7.05 6.94 16.56 3.09 1.56 6.82 7.44 2.00 3.02 No.1734 Pp8 ±0.06d ±0.09d ±0.16e ±0.05b ±0.04c ±0.09 ±0.08d ±0.04a ±0.04a 7.35 6.73 15.50 4.08 1.09 6.18 4.48 1.51 2.28 No.1734 Pp9 ±0.07c ±0.08d ±0.14d ±0.06d ±0.02c ±0.07 ±0.05a ±0.03a ±0.03a 4.30 1.49 3.62 4.41 0.59 1.51 7.75 3.61 5.31 No.1734 Pp10 ±0.06b ±0.03a ±0.05a ±0.08d ±0.02a ±0.02 ±0.08c ±0.05b ±0.07b Solid state fermented samples 3.11 1.95 3.63 3.18 2.64 15.21 5.14 13.22 Vilciai Pp8 - ±0.03a ±0.02a ±0.04a ±0.04a ±0.03b ±0.15e ±0.06d ±0.13d 5.00 2.89 5.02 5.26 0.89 2.45 9.49 4.88 8.66 Vilciai Pp9 ±0.06c ±0.04b ±0.06c ±0.07d ±0.03c ±0.03b ±0.09d ±0.05c ±0.09e 4.42 2.08 3.97 3.99 0.42 0.78 5.98 3.53 5.64 Vilciai Pp10 ±0.05b ±0.03b ±0.04a ±0.05b ±0.02a ±0.02a ±0.06c ±0.03c ±0.07c 3.69 1.90 3.89 4.19 2.26 6.60 2.91 5.81 Vilniai Pp8 - ±0.03a ±0.04a ±0.07b ±0.06c ±0.04b ±0.07b ±0.02a ±0.08d 5.08 2.83 5.00 4.90 0.62 2.11 7.39 3.19 5.32 Vilniai Pp9 ±0.07c ±0.06c ±0.08 ±0.07c ±0.02 ±0.03b ±0.09b ±0.04b ±0.06cd 3.93 0.65 3.20 4.33 0.81 10.49 3.96 5.14 Vilniai Pp10 - ±0.05b ±0.02a ±0.04b ±0.05d ±0.02a ±0.12d ±0.05b ±0.05d 4.46 2.40 4.27 4.67 0.53 2.15 7.55 3.63 5.27 No.1072 Pp8 ±0.07d ±0.05b ±0.07b ±0.06c ±0.02b ±0.03b ±0.08c ±0.07c ±0.04d 5.03 2.78 4.93 4.75 0.63 2.16 6.87 3.12 5.69 No.1072 Pp9 ±0.06d ±0.06c ±0.09c ±0.08b ±0.03b ±0.03b ±0.06b ±0.06b ±0.07c 3.64 3.29 4.15 3.97 0.45 0.59 6.42 3.04 3.55 No.1072 Pp10 ±0.04b ±0.07d ±0.06c ±0.04b ±0.02a ±0.02a ±0.07b ±0.07d ±0.04a 6.54 4.42 10.39 4.51 1.29 3.00 5.74 2.31 4.18 No.1734 Pp8 ±0.09d ±0.06d ±0.12c ±0.05c ±0.04c ±0.02c ±0.05a ±0.03a ±0.05b 4.52 2.58 8.00 2.61 0.72 3.06 8.16 2.19 3.52 No.1734 Pp9 ±0.05c ±0.03c ±0.10d ±0.02a ±0.02b ±0.03c ±0.09d ±0.02a ±0.04b 5.38 3.07 5.40 4.49 0.61 1.43 7.62 2.78 3.98 No.1734 Pp10 ±0.07c ±0.04b ±0.09c ±0.06d ±0.02b ±0.02a ±0.08bc ±0.04b ±0.06a TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; VAL – valine; ILE – isoleucine; LEU – leucine; THR – threonine; MET – methionine; PHE – phenylethylamine; LYS – lysine; HIS – histamine; TRP – tryptophan; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

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In all the cases, in SMF samples the contents of VAL, ILE (except in SMF with P. pentosaceus No.10 variety Vilniai and hybrid No.1734 isolates/concentrates), LEU (except in SMF with P. pentosaceus No.10 variety Vilniai isolates/concentrates) and THR were increased, in compare with nonfermented lupine isolates/concentrates. However, contents of HIS and TRP in SMF isolates/concentrates were decreased, compared to nonfermented isolates/concentrates. The content of LYS (except in SMF with P. pentosaceus No.8 and No.10 hybrid No.1734 isolates/concentrates) in SSF isolates/concentrates was on average 8.70% lower, in compare with SMF samples. In all SSF isolates/concentrates the contents of VAL and THR were found to be higher, in compare with nonfermented lupine isolates/concentrates. The analysed TEAA were significantly influenced by the analysed factors and their interaction was significant. The VAL, ILE, LEU, THR, MET, PHE, LYS, HIS and TRP contents in lupine protein isolates/concentrates were significantly influenced by the selection of lupine variety (F(3.837)=1108.860, p≤0.0001; F(3.936)=1674.718, p≤0.0001; F(70.991)=8779.760, p≤0.0001; F(0.833)=313.040, p≤0.0001; F(1.423)= 2505.150, p≤0.0001; F(12.891)=7204.700, p≤0.0001; F(25.085)=2896.420, p≤0.0001; F(15.598)=4940.499, p≤0.0001; F(147.202)=20805.953, p≤ 0.0001, respectively), the fermentation method (F(31.721)=9165.915, p≤ 0.0001; F(50.050)=21297.878, p≤0.0001; F(429.392)=53104.957, p≤ 0.0001; F(4.176)=1569.522, p≤0.0001; F(2.116)=795.327, p≤0.0001; F(3.472)=6113.531, p≤0.0001; F(61.550)=34398.929, p≤0.0001; F(7.821)= 903.045, p≤0.0001; F(1.378)=436.407, p≤0.0001; F(19.034)=2690.382, p≤0.0001, respectively), the type of microorganism applied for the fermentation (F(0.845)=244.093, p≤0.0001; F(7.325)=3117.080, p≤0.0001; F(54.627)=6756.045, p≤0.0001; F(1.037)=1825.701, p≤0.0001; F(35.323)= 19741.369, p≤0.0001; F(1.486)=171.595, p≤0.0001; F(3.876)=1227.839, p≤0.0001; F(2.363)=334.034, p≤0.0001, respectively) and the interaction of these factors was significant (F(2.075)=599.514, p≤0.0001; F(2.464)= 1048.530, p≤0.0001; F(11.454)=1416.564, p≤0.0001; F(1.573)=591.325, p≤0.0001; F(0.693)=12.19.798, p≤0.0001; F(0.838)=468.185, p≤0.0001; F(23.387)=2700.406, p≤0.0001; F(5.403)=1711.380, p≤0.0001; F(17.290)= 2443.856, p≤0.0001, respectively). The total essential amino acids (TEAA) content (%) from total protein hydrolysed in nonfermented wholemeal protein isolates/concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734, SMF and SSF with L. sakei and P. acidilactici strains is presented in Table 3.5.3.8. In all SMF samples the higher contents of VAL, ILE, LEU (except in SMF with L. sakei variety Vilniai isolates/concentrates) and THR were established, in compare with nonfermented isolates/concentrates. However, 126 the contents of LYS (except in SMF with L. sakei and P. acidilactici in hybrid No.1734 isolates/concentrates, in which LYS content was increased), HIS and TRP were decreased, in compare with nonfermented lupine isolates/concentrates. In SSF isolates/concentrates the lower contents of HIS and TRP were established, in compare to nonfermented samples. In SSF isolates/concentrates the contents of VAL and THR were found to be higher, in compare with nonfermented isolates/concentrates. To compare SMF and SSF isolates/concentrates, in SSF samples TRP content was on average 26.87% higher. The VAL content in lupine protein isolates/ concentrates was significantly affected by the fermentation method (F(30.672)=19.153, p≤0.0001), however, the selection of lupine variety and the microorganism had no significant influence on VAL content in protein isolates/concentrates. The ILE content in lupine protein isolates/concentrates was significantly affected by the fermentation method (F(42.526)= 26.873, p≤0.0001), but the selection of lupine variety and the microorganism had no significant influence. The LEU content in lupine protein isolates/concentrates was significantly affected by the selection of lupine variety (F(58.605)=10.624, p≤0.0001) and the fermentation method (F(383.070)=69.446, p≤0.0001), however, the microorganism applied for the fermentation had no significant influence. The MET content was significantly affected by the fermentation method (F(4.763)=29.643, p≤0.0001), but the selection of lupine variety and the microorganism had no significant influence. The PHE content in lupine protein isolates/concentrates was significantly affected by the selection of lupine variety (F(13.340)=24.268, p≤0.0001) and the fermentation method (F(93.326)= 169.779, p≤0.0001), but the microorganism applied for the fermentation had no significant influence. The HIS content in lupine protein isolates/concentrates was significantly affected by the selection of lupine variety (F(29.341)=27.334, p≤0.0001) and the fermentation method (F(19.931)= 18.567, p≤0.0001), but the microorganism applied for the fermentation had no significant influence. The TRP content in lupine protein isolates/concentrates was significantly affected by the selection of lupine variety (F(192.946)=22.478, p≤0.0001) and the fermentation method (F(24.453)= 2.849, p≤0.0001), but the microorganism applied for the fermentation had no significant influence. Analysed factors had no significant influence on LYS and THR content in lupine protein isolates/concentrates.

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Table 3.5.3.8. The total essential amino acids (TEAA) content (%) from total protein hydrolysed in nonfermented wholemeal protein isolates/concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734, SMF and SSF with L. sakei and P. acidilactici strains. TEAA content (%) from total protein hydrolysed Lupine samples VAL ILE LEU THR MET PHE LYS HIS TRP Nonfermented samples 0.64 0.98 0.99 1.70 13.67 12.33 26.69 Vilciai - - ±0.02a ±0.02a ±0.02a ±0.03a ±0.13b ±0.11b ±0.24c 2.93 2.53 10.35 1.30 1.32 5.58 12.66 6.53 17.36 Vilniai ±0.04b ±0.04b ±0.09c ±0.04b ±0.03b ±0.05b ±0.11a ±0.06c ±0.16d 1.80 1.71 0.66 0.82 15.52 8.58 9.42 No.1072 - - ±0.03b ±0.03b ±0.03a ±0.02b ±0.15c ±0.09a ±0.06c 3.25 1.58 1.94 2.83 0.34 1.36 5.55 5.40 6.30 No.1734 ±0.05c ±0.02a ±0.04b ±0.05c ±0.01a ±0.04c ±0.06b ±0.05b ±0.05b Submerged fermented samples 6.85 4.58 11.37 4.68 1.48 2.77 7.95 2.65 4.41 Vilciai Ls ±0.08c ±0.06c ±0.13d ±0.06d ±0.02c ±0.04a ±0.09d ±0.05b ±0.05d 4.32 2.24 7.81 2.16 3.11 9.58 2.14 3.36 Vilciai Pa - ±0.06a ±0.02a ±0.08a ±0.03a ±0.05b ±0.11b ±0.03b ±0.03c 5.22 4.39 10.12 2.82 1.56 4.60 8.91 2.42 7.17 Vilniai Ls ±0.05b ±0.05c ±0.10c ±0.04b ±0.03d ±0.07d ±0.08c ±0.04a ±0.09e 5.93 3.88 10.37 3.51 1.17 3.45 7.37 2.60 3.19 Vilniai Pa ±0.07b ±0.06b ±0.12c ±0.05b ±0.02a ±0.04b ±0.09c ±0.05b ±0.05c 7.99 7.98 14.37 2.10 1.09 4.40 4.67 1.85 1.56 No.1072 Ls ±0.09d ±0.09d ±0.15d ±0.06a ±0.04b ±0.06c ±0.05b ±0.02a ±0.02a 6.92 3.53 10.54 4.23 1.53 3.84 9.50 3.08 2.49 No.1072 Pa ±0.06c ±0.05b ±0.12c ±0.05d ±0.05b ±0.03b ±0.09d ±0.06d ±0.04b 7.05 6.94 16.56 3.09 1.56 6.82 7.44 2.00 3.02 No.1734 Ls ±0.07d ±0.07c ±0.19de ±0.04c ±0.04c ±0.09d ±0.08c ±0.04b ±0.05c 5.08 2.92 9.70 3.24 1.22 3.93 11.25 2.94 5.94 No.1734 Pa ±0.05b ±0.04a ±0.09b ±0.06c ±0.03a ±0.05b ±0.12d ±0.06c ±0.08d Solid state fermented samples 3.30 2.64 1.82 3.86 0.70 0.63 11.52 6.42 9.72 Vilciai Ls ±0.05b ±0.04c ±0.04a ±0.05c ±0.02b ±0.03a ±0.13c ±0.07d ±0.09c 2.51 1.09 3.66 2.51 1.08 15.46 6.53 14.82 Vilciai Pa - ±0.03a ±0.03a ±0.05b ±0.04b ±0.04c ±0.16d ±0.07d ±0.15d 4.50 2.08 4.55 4.13 0.46 0.86 7.09 3.34 5.11 Vilniai Ls ±0.06c ±0.04b ±0.07c ±0.09e ±0.03a ±0.02b ±0.08b ±0.04b ±0.07d 3.87 1.78 4.04 3.83 0.91 0.84 12.00 3.05 4.06 Vilniai Pa ±0.04c ±0.02a ±0.05b ±0.04c ±0.02b ±0.02b ±0.15d ±0.03bc ±0.06bc 5.05 2.76 5.51 4.15 0.49 0.42 6.44 2.87 2.03 No.1072 Ls ±0.07d ±0.06c ±0.06d ±0.06d ±0.04a ±0.03a ±0.06b ±0.04c ±0.04b 8.39 6.42 13.36 1.44 1.30 3.98 3.83 2.11 1.73 No.1072 Pa ±0.09e ±0.09d ±0.14e ±0.02a ±0.05c ±0.06d ±0.05a ±0.03a ±0.03a 5.57 3.10 6.37 4.26 0.71 1.16 8.77 2.82 2.07 No.1734 Ls ±0.06d ±0.55c ±0.08de ±0.08e ±0.03b ±0.04c ±0.09c ±0.05b ±0.04b 3.38 1.53 5.62 1.61 1.64 15.15 2.85 3.02 No.1734 Pa - ±0.05b ±0.03a ±0.05d ±0.03a ±0.05bc ±0.18e ±0.06b ±0.05c TNAA – total nonessential amino acids; SMF – submerged fermentation; SSF – solid state fermentation. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; VAL – valine; ILE – isoleucine; LEU – leucine; THR – threonine; MET – methionine; PHE – phenylethylamine; LYS – lysine; HIS – histamine; TRP – tryptophan; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

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3.5.4. The Essential and Nonessential Amino Acids – Possibility to Modulate Their Profile in Lupine Seeds Wholemeal and Isolates/Concentrates

The amino acid (AA) profile is important, because it provides information about the quality of protein in fermented and non-fermented lupine, closely associated with its nutritional value. Fermentation with lactobacilli has been reported to increase the concentrations of free amino acids in legumes [312, 313]. AAs composition and digestibility are major determi- nants of nutritional value of proteins [314]. Legume crops, which represent the major food and feed sources for humans and livestock worldwide, possess limited levels of some of the essential AAs, particularly lysine and methionine [315]. In developed countries, these essential amino acids are generally provided from the dietary utilization of farm animals (particularly meat, eggs and milk) as well as from the variety of crop plants (particularly cereals and legumes) that together provide optimal levels of essential AAs [316]. Although AAs are critical for all forms of life, only proteogenic amino acids that humans and animals cannot synthesize de novo and therefore must acquire in their diets are classified as essential. Nine AAs - LYS, MET, THR, PHE, TRP, VAL, ILE, LEU, and HIS - fit this definition [317]. Legume proteins have been recognized to have a nutritional role far beyond providing essential AAs [318]. LYS and MET are the most limited essential AA in cereals and legumes, respectively [319]. LYS is generally the first limited AA in almost all practical diets, so if diets are formulated on a lysine basis, other amino acid requirements should be met [320]. The limited LYS and MET contents reduce the nutritional values of these crop plants to 50–75%, compared to those of a diet possessing balanced levels of essential amino acids. This limitation in EAA can lead to nonspecific signs of protein deficiencies in humans, such as lowered resistance to diseases, decreased blood proteins and retarded mental and physical development of children. This syndrome is referred to as protein-energy malnutrition (PEM), and the World Health Organization (WHO) estimates that around 30% of the populations in the developing world suffer from this syndrome [315]. In the past years, essential AA have been shown to be solely responsible for the AA induced stimulation of muscle protein anabolism [322]. Furthermore, essential AA are able to stimulate muscle protein synthesis to more than twice the extent as the same amount of a high quality whey protein [323]. The VAL, ILE, LEU, THR, LYS and HIS contents in lupine wholemeal were significantly influenced by the selection of lupine variety (F(10.920)=3.334, p≤0.0001; F(5.786)=4.060, p≤0.0001; F(3.498)= 10.493, p≤0.0001; F(4.771)=3.112, p≤0.0001; F(18.360)=9.285, p≤0.0001; 129

F(6.894)=3.932, p≤0.0001, respectively), except for MET and PHE, the fermentation method (F(31.005)=9.467, p≤0.0001; F(64.340)=45.141, p≤0.0001; F(19.413)=58.225, p≤0.0001; F(28.725)=18.737, p≤0.0001; F(76.151)=2.036, p≤0.0001; F(73.618)=93.822, p≤0.0001; F(78.982)= 39.940, p≤0.0001; F(120.361)=68.637, p≤0.0001, respectively), the type of microorganism applied for the fermentation (F(55.737)=17.018, p≤0.0001; F(22.463)=15.760, p≤0.0001; F(15.024)=45.060, p≤0.0001; F(41.145)= 1.100, p≤0.0001; F(10.451)=13.319, p≤0.0001; F(6.682)=3.379, p≤0.0001; F(6.670)=3.804, p≤0.0001, respectively), except for THR, and the interaction of these factors was significant (F(4.587)=1.400, p≤0.0001; F(7.580)=0.203, p≤0.0001; F(12.887)=6.517, p≤0.0001; F(4.475)=2.552, p≤0.0001, respectively), except for ILE, LEU, THR and PHE. The ALA, GLY, SER, PRO, ASP, GLU and TYR contents in lupine wholemeal were significantly influenced by the selection of lupine variety (F(27.149)=1460.717, p≤0.0001; F(10.067)=0.856, p≤0.0001; F(27.048)= 2.049, p≤0.0001; F(14.750)=1.549, p≤0.0001; F(34.930)=6.997, p≤0.0001; F(23.653)=170.237, p≤0.0001; F(188.677)=55.169, p≤0.0001, respectively), the fermentation method (F(56.022)=4.64, p≤0.0001; F(153.210)=16.088, p≤0.0001; F(90.458)=651.055, p≤0.0001, respectively), except for ALA, SER, ASP and TYR, and the type of microorganism applied for the fermentation (F(15.551)=0.430, p≤0.0001; F(7.081)=1.419, p≤0.0001; F(27.433)=8.021, p≤0.0001, respectively), except for GLY, SER, PRO nd GLU; and the interaction of these factors was significant (F(4.023)=0.119, p≤0.0001; F(7.040)=0.599, p≤0.0001; F(4.690)=0.355, p≤0.0001; F(8.495)= 0.892, p≤0.0001; (15.851)=3.175, p≤0.0001; F(5.475)=39.405, p≤0.0001; F(27.841)=8.141, p≤0.0001, respectively). Essential AAs play an important role in the body, for example HIS helps in the removal of excess metals from the body, stimulates the synthesis of collagen, which is the main extracellular structural protein in the various connective tissues of animals [324], and can be emphasized in the nutritional strategy for the prevention of loss of muscle mass following gastric bypass surgery [325]. THR and ILE are frequently found in the active centers of enzymes [326]. MET plays a special role in the protein biosynthesis [327]. TYR is converted into melanin via enzymatic oxidation [328]. The rest of the essential AAs are also involved in the maintenance and repair of all tissues. Thus, the enrichment in amino acids by fermentation of lupine seeds could constitute a valuable contribution to the diet, supporting the development and maintenance of structures in humans and animals. Not only proteins but also AA, the constituents that make up the proteins, is important part of nutrition which could not be neglected, especially when the resistance exercise is performed. Besides the supplementation of complex essential AAs, that the 130 body is not able to synthesize, the supplementation of branched chain AAs is important (LEU, ILE and VAL) [329]. According to literature sources, the lupine protein has a relatively good amino acid profile with high content of ARG (4.1–11.2%), LEU (7.5–9.4%), LYS (4.3–5.2%) and PHE (3.0– 6.8%). Among pulses, lupine is ranked third in protein quality, following soybean and chickpea [330]. The VAL, ILE, LEU, THR, MET, PHE, LYS, HIS and TRP contents in lupine protein isolates/concentrates was significantly influenced by the selection of lupine variety (F(6.043)=6.968, p≤0.0001; F(8.109)=5.836, p≤0.0001; F(29.108)=101.592, p≤0.0001; F(4.299)=2.421, p≤0.0001; F(25.926)=3.667, p≤0.0001; F(16.618)=18.366, p≤0.0001; F(48.769)=21.279, p≤0.0001; F(67.821)=78.196, p≤0.0001, except LYS, respectively), the fermentation method (F(67.821)=78.196, p≤0.0001; F(169.539)=122.009, p≤0.0001; F(351.155)=1225.608, p≤0.0001; F(84.419)=11.941, p≤0.0001; F(184.146)=203.518, p≤0.0001; F(71.312)=31.114, p≤0.0001; F(13.589)=44.025, p≤0.0001, except THR and LYS, respectively), the type of microorganism applied for the fermentation (F(7.799)=8.992, p≤0.0001; F(8.181)=5.888, p≤0.0001; F(18.085)=2.558, p≤0.0001; F(14.303)=15.808, p≤0.0001; F(26.114)= 11.394, p≤0.0001, except THR, LYS and TRP, respectively) and the interaction of these factors was significant (F(7.058)=8.138, p≤0.0001; F(6.020)=4.332, p≤0.0001; F(3.671)=12.813, p≤0.0001; F(5.316)=2.994, p≤0.0001; F(9.566)=1.353, p≤0.0001; F(3.420)=3.780, p≤0.0001; F(3.885)= 17.823, p≤0.0001; F(14.837)=6.474, p≤0.0001; F(8.040)=26.048, p≤0.0001, respectively). The ALA, GLY, SER, PRO, ASP, GLU and TYR contents in lupine protein isolates/concentrates were significantly influenced by the selection of lupine variety (F(6.722)=1.306, p≤0.0001; F(5.651)=70.777, p≤0.0001; F(14.640)=3.618, p≤0.0001; F(31.323)=48.753, p≤0.0001; F(13.142)= 126.523, p≤0.0001; F(24.829)=22.348, p≤0.0001, except ALA, respectively), the fermentation method (F(116.983)=22.730, p≤0.0001; F(104.161)=1304.632, p≤0.0001; F(65.690)=16.235, p≤0.0001; F(11.665)= 18.157, p≤0.0001; F(21.055)=202.708, p≤0.0001; F(69.625)=62.667, p≤0.0001, except ALA, respectively) and the type of microorganism applied for the fermentation (F(9.301)=116.495, p≤0.0001; F(7.631)=73.472, p≤0.0001, except ALA, GLY, PRO, ASP and TYR, respectively).

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3.6. The Influence of Technological Factors on Formation of Biogenic amines in Lupine Products

3.6.1. The Biogenic Amines Content in Untreated and Biotreated Lupine Wholemeal

The biogenic amines (BAs) content (mg/kg) in nonfermented lupine varieties Vilciai, Vilniai, hybrid lines No.1072, No.1734, No.1700, No.1701, No.1800 and No.1702 seeds wholemeal is presented in Table 3.6.1.1. In nonfermented lupine wholemeal PEA, SPRMD and SPRM were not found. TYM was established only in variety Vilciai (5.71 ± 0.09 mg/kg). HIS content in nonfermented samples has ranged from 0 mg/kg to 20.78 ± 1.12 mg/kg (in hybrid No.1734 and variety Vilniai wholemeal, respectively). PUT and CAD content in nonfermented wholemeal has ranged from 49.42 ± 2.51 mg/kg to 252.32 ± 17.14 mg/kg (in hybrids No.1702 and No.1701 samples, respectively) and from 21.81 ± 0.32 mg/kg to 115.36 ± 11.02 mg/kg (in variety Vilciai and hybrid No.1700 samples, respectively), respectively. Compared to CAD, 55.18% higher PUT content was found in nonfermented samples. The results of the ANOVA test have indicated that the PUT, CAD, HIS and TYM content in nonfermented lupine seeds was significantly affected by the selection of lupine variety (PUT (F(33309.626)=99.963, p≤0.0001; CAD (F(3243.835)=5.840, p≤0.0001; HIS (F(359.790)=12.694, p≤0.0001; TYM (F(21.396)=4525.278, p≤ 0.0001). Significant influence of the selected lupine variety on PEA, SPRMD and SPRM contents was not established. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains in wholemeal seeds of lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734 is presented in Table 3.6.1.2. In SMF samples PEA content has ranged from 11.65 ± 1.80 mg/kg to 103.15 ± 2.24 mg/kg (in SMF with P. pentosaceus No.10 hybrid No.1734 and in SMF with P. pentosaceus No.10 hybrid No.1072 samples, respectively). In most of the cases, in SMF samples CAD (in 7 samples out of 16 analysed) and HIS (in 10 samples out of 16 analysed) were found. To compare, in SMF samples TYM (in 2 samples out of 16 analysed) and SPRM (in 1 sample out of 16 analysed) were found. SPRMD content in SMF samples has ranged from 8.52 ± 0.86 mg/kg to 48.73 ± 1.32 mg/kg (in SMF with P. pentosaceus No.10 hybrid No.1734 and in SMF with P. pentosaceus No.10 hybrid No.1072 samples, respectively). In most of the SSF samples PUT and SPRMD were detected (in 12 samples out of 16 analysed).

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Table 3.6.1.1. The biogenic amines (BAs) content (mg/kg) in nonfermented lupine varieties Vilciai, Vilniai, hybrid lines No.1072, No.1734, No.1700, No.1701, No.1800 and No.1702 wholemeal seeds. Lupine PEA PUT CAD HIS TYM SPRMD SPRM samples Nonfermented samples Vilciai - 64.82±0.98b 21.81±0.32a 4.97±0.04a 5.71±0.09a - - Vilniai - 114.92±12.20d 86.64±3.24d 20.78±1.12b - - - No.1072 - 49.85±1.24a 23.84±0.87b 5.29±0.12a - - - No.1734 - 71.11±2.41c 45.20±2.12c - - - - No.1700 - 202.49±12.12k 115.36±11.02j 28.97±1.01m - - - No.1701 - 252.32±17.14j 31.62±2.02a 9.97±0.14a - - - No.1800 - 198.85±12.05m 49.20±3.14b 13.79±1.04b - - - No.1702 - 49.42±2.51a 76.22±3.01c 19.48±1.35c - - - Data values are expressed as means with the standard deviations (n = 3); PEA – phenylethylamine; PUT – putrescine; CAD – cadaverine; HIS – histamine; TYM – tyramine; SPRMD – spermidine; SPRM – spermine. a-jThe mean values within a column with different letters are significantly different (p≤0.05).

PEA content in SSF samples has ranged from 0 mg/kg to 58.42 ± 3.12 mg/kg (in SSF with P. pentosaceus No.8, No.9 and No.10 strains hybrid No.1734 samples and in SSF with P. pentosaceus No.10 variety Vilniai samples, respectively). TYM was established in 5 samples out of 16 SSF samples analysed, and SPRM was found in 6 samples out of 16 SSF samples analysed . CAD and HIS in half of the SSF samples were formed (in 8 samples out of 16 analysed). Most of the analysed BAs were significantly influenced by the analysed factors and their interaction (analysed factors were: lupine variety, type of microorganism and fermentation method). The PEA content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(853.411)=304.114, p≤0.0001), fermentation method (F(26649.324)=9496.531, p≤0.0001), the type of microorganism applied for the fermentation (F(314.533)=112.084, p≤0.0001) and the interaction of these factors was significant (interaction of lupine variety × type of microorganism × fermentation method F(3111.643)=1108.839, p≤0.0001).

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Table 3.6.1.2. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains in wholemeal seeds of lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734. Lupine samples PEA PUT CAD HIS TYM SPRMD SPRM Submerged fermented samples 16.12 100.68 115.34 34.57 8.46 12.10 Vilciai Pp8 - ±1.45b ±1.62k ±2.01b ±1.67b ±0.23b ±0.89b 40.59 33.96 284.49 97.97 28.99 Vilciai Pp9 - - ±1.78c ±0.59f ±4.81c ±1.02e ±1.02d 42.60 73.34 321.78 92.35 27.74 Vilciai Pp10 - - ±1.84c ±1.12h ±3.30d ±1.36d ±1.01d 62.85 520.78 495.87 123.01 35.93 Vilniai Pp8 - - ±2.01gh ±1.36m ±3.58g ±2.89h ±1.35f 53.64 20.03 386.49 113.10 35.77 Vilniai Pp9 - - ±1.85f ±1.41b ±2.90f ±1.68f ±0.36f 58.42 85.15 343.71 108.25 31.24 Vilniai Pp10 - - ±1.91g ±2.18j ±4.72e ±1.81f ±1.47e 43.02 21.25 293.02 88.54 27.52 No.1072 Pp8 - - ±1.69cd ±1.35c ±2.05c ±2.28c ±1.56d 49.07 23.21 308.15 91.19 25.73 No.1072 Pp9 - - ±1.77e ±1.47d ±2.21cd ±1.58d ±1.36c 103.15 42.83 523.65 170.07 48.73 No.1072Pp10 - - ±2.24k ±1.69g ±5.14h ±2.06j ±1.32g 60.30 101.74 398.51 115.43 36.91 No.1734 Pp8 - - ±1.14g ±1.78k ±3.21f ±2.57fg ±1.99f 90.84 24.54 340.31 109.81 31.23 No.1734 Pp9 - - ±2.97j ±1.19e ±4.03e ±1.98f ±1.57e 11.65 13.88 64.88 22.64 7.33 8.52 19.40 No.1734Pp10 ±1.80a ±2.01a ±1.08a ±2.84a ±0.14a ±0.86a ±1.41 Solid state fermented samples 41.58 26.87 341.01 34.05 1.97 Vilciai Pp8 - - ±2.63h ±1.56j ±3.37e ±1.01f ±0.14b 22.60 22.01 314.70 36.01 2.11 Vilciai Pp9 - - ±1.57f ±1.21h ±3.81d ±1.21g ±0.78c 23.87 22.98 342.47 36.69 2.39 Vilciai Pp10 - - ±1.62g ±1.53h ±4.14e ±0.97g ±0.41d 18.99 10.73 1.32 0.53 1.27 16.44 0.76 Vilniai Pp8 ±1.12e ±1.09g ±0.19c ±0.12b ±0.12d ±1.21k ±0.12b 1.44 4.62 0.79 4.05 6.33 Vilniai Pp9 - - ±0.21c ±0.82c ±0.08a ±0.62e ±0.84f 58.42 85.15 343.71 108.25 31.24 Vilniai Pp10 - - ±3.12j ±2.49k ±3.28e ±4.08h ±0.49m 0.89 28.20 0.72 0.11 10.70 0.94 No.1072 Pp8 - ±0.02a ±2.24j ±0.11c ±0.01a ±0.97j ±0.10c 1.02 5.95 0.26 3.53 0.24 No.1072 Pp9 - - ±0.23b ±0.90d ±0.04c ±0.18e ±0.04a 1.71 8.17 0.99 0.56 0.16 9.18 0.76 No.1072Pp10 ±0.35d ±1.11e ±0.11b ±0.09b ±0.03b ±0.21h ±0.05b 9.65 2.18 7.16 2.19 No.1734 Pp8 - - - ±1.16f ±0.15e ±0.31g ±0.37d 1.06 0.87 18.41 2.45 No.1734 Pp9 - - - ±0.21b ±0.11d ±0.73l ±0.45e 0.57 0.26 0.29 No.1734 Pp10 - - - - ±0.32a ±0.09a ±0.06a Data values are expressed as means with the standard deviations (n = 3); SMF – submerged samples; SSF – solid state fermented; PEA – phenylethylamine; PUT – putrescine; CAD – cadaverine; HIS – histamine; TYM – tyramine; SPRMD – spermidine; SPRM – spermine; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10; a-j The mean values within a column with different letters are significantly different (p≤0.05).

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The PUT content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(16827.527)=7982.682, p≤0.0001), the fermentation method (F(87301.401)=41414.245, p≤0.0001), the type of microorganism applied for the fermentation (F(46610.827)=22111.354, p≤0.0001) and the interaction of these factors was significant (F(28565.528)=13550.983, p≤0.0001). The HIS content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(4420.138)=1490.573, p≤0.0001), the fermentation method (F(111606.864)=37636.413, p≤0.0001), the type of microorganism applied for the fermentation (F(1923.356)=648.600, p≤0.0001) and the interaction of these factors was significant (F(6516.934)=2197.661, p≤0.0001). The CAD content was significantly affected by the selection of lupine variety (F(56264.198)=6799.935, p≤0.0001), the fermentation methods (F(800875.899)=96791.635, p≤0.0001), the type of microorganism applied for the fermentation (F(11349.317)=1371.647, p≤0.0001) and their interaction (F(143217.196)=17308.832, p≤0.0001). The TYM content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(9.985)=2139.664, p≤0.0001), the fermentation method (F(17.464)= 3742.296, p≤0.0001), the type of microorganism applied for the fermentation (F(13.173)=2822.756, p≤0.0001), and their interaction (F(22.181)= 4753.062, p≤0.0001). The SPRMD content was significantly influenced by the selection of lupine variety (F(9.985)=2139.664, p≤0.0001), the fermentation method (F(17.464)=3742.296, p≤0.0001), the type of microorganism applied for the fermentation (F(13.173)=2822.756, p≤0.0001), and the interaction of these factors was significant (F(22.181)=4753.062, p≤0.0001). The SPRM content was significantly affected by the selection of lupine variety (F(42.385)=431.665, p≤0.0001), the fermentation method (F(18.191)=185.260, p≤0.0001), the type of microorganisms applied for the fermentation (F(35.696)=363.540, p≤0.0001), and the interaction of these factors was significant (F(47.604)=484.825, p≤0.0001). Weak negative correlation between HIS and histamine (r=-0.3709; p=0.0005) and weak positive correlation between PEA and phenylethylamine (r=0.3914, p=0.0002) were established. However, correlations between TYM and tyramine and between LYS and cadaverine were not observed. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with L. sakei and P. acidilactici strains in hybrid lines No.1700, No.1701, No.1800 and No.1702 wholemeal is presented in Table 3.6.1.3. In SMF samples TYM and SPRM were not found. In SMF samples PUT content was established to be on average 2.41% lower, in compare with SSF samples. In SMF samples PEA content has ranged from 26.93 ± 0.64 mg/kg to 72.80 ± 0.89 mg/kg (in SMF with L. sakei hybrid No.1700 samples and in SMF with 135

P. acidilactici hybrid No.1800 samples, respectively). To compare SSF and SMF samples, in SMF samples CAD and HIS contents were established to be on average 31.48% and 45.96% higher, respectively. In most of the SSF samples PEA was not found (except in SSF with L. sakei hybrid No.1700 samples). PUT was found in 9 samples out of 12 SSF samples analysed. Also, in SSF samples CAD and HIS contents have ranged from 0 mg/kg to 224.92 ± 1.08 mg/kg (in SSF with L. sakei and P. acidilactici hybrid No.1701 samples and in SSF with P. acidilactici hybrid No.1701 samples, respectively) and from 0 mg/kg to 57.97 ± 0.98 mg/kg (in SSF with L. sakei hybrid No.1701 samples and in SSF with P. acidilactici hybrid No.1701 samples, respectively), respectively. In SSF samples TYM was found in 3 samples out of 12 analysed and SPRMD in 4 samples out of 12 analysed. Only in 1 SSF sample (from 12 analysed) SPRM was found. The PEA content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(1973.136)=12.404, p≤0.0001) and fermentation method (F(16146.169)=101.500, p≤0.0001), however, the microorganism applied for fermentation had no significant influence on PEA content. The CAD content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(118425.764)=14.293, p≤0.0001), however, the fermentation method and the microorganism applied for fermentation had no significant influence on CAD content. The HIS content in fermented lupine seeds was significantly affected by the fermentation method (F(22679.603)=49.440, p≤0.0001), however, the selection of lupine variety and the microorganism applied for fermentation had no significant influence on HIS content. The TYM content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(286.172)=37.061, p≤0.0001) and the fermentation method (F(464.074)=60.101, p≤0.0001), however, the microorganism applied for the fermentation had no significant influence on TYM content. The SPRMD content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(204.754)= 10.490, p≤0.0001) and the fermentation method (F(6600.941)=338.164, p≤0.0001), however, the microorganism applied for the fermentation had no significant influence on SPRMD content. However, the influence of analysed factors was not significant on PUT and SPRM content in fermented lupine samples.

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Table 3.6.1.2. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with L. sakei and P. acidilactici strains in hybrid lines No.1700, No.1701, No.1800 and No.1702 wholemeal seeds. Lupine samples PEA PUT CAD HIS TYM SPRMD SPRM Submerged fermented samples 26.93 192.94 158.11 47.96 15.87 No.1700 Ls - - ±0.64a ±3.80d ±1.98d ±0.74a ±0.72a 47.57 45.69 269.31 85.35 29.77 No.1700 Pa - - ±0.98d ±2.11b ±3.14f ±1.81d ±0.54b 32.85 232.74 192.19 56.05 22.31 No.1701 Ls - - ±0.78b ±4.99j ±4.71f ±0.95c ±0.95c 35.60 195.32 198.87 66.98 25.64 No.1701 Pa - - ±0.87c ±4.81e ±2.81e ±1.99d ±0.96d 68.80 37.34 399.79 120.69 35.72 No.1800 Ls - - ±0.69f ±1.69a ±4.15m ±1.89j ±0.92e 72.80 395.54 126.65 34.56 No.1800 Pa - - - ±0.89e ±3.14l ±3.08k ±0.91f 48.98 22.87 88.21 22.74 No.1702 Ls - - - ±0.49d ±0.69a ±1.87f ±0.97b 36.58 24.25 59.97 23.04 No.1702 Pa - - - ±0.42c ±0.79b ±2.52b ±0.90c Solid state fermented samples 27.68 18.01 199.89 10.01 7.99 No.1700 Ls - - ±0.62b ±1.59b ±0.89a ±0.90a ±0.90b 72.97 224.92 57.97 15.40 9.38 No.1700 Pa - - ±2.64d ±1.08f ±0.98g ±0.98b ±0.96c 10.03 5.97 No.1701 Ls - - - - - ±0.31a ±0.09b 73.00 29.98 No.1701 Pa - - - - - ±2.88f ±0.35b 86.35 194.15 48.19 12.86 4.65 No.1800 Ls - - ±2.69e ±1.18d ±0.64d ±0.89a ±0.18a 136.75 196.41 57.82 21.49 18.93 No.1800 Pa - - ±3.98g ±2.01e ±1.72e ±0.91c ±0.96e 26.47 145.83 44.58 No.1702 Ls - - - - ±1.45c ±1.31b ±1.31c 297.74 176.95 55.51 No.1702 Pa - - - - ±4.15d ±1.42c ±1.97f Data values are expressed as means with the standard deviations (n = 3). SMF – submerged samples; SSF – solid state fermented; PEA – phenylethylamine; PUT – putrescine; CAD – cadaverine; HIS – histamine; TYM – tyramine; SPRMD – spermidine; SPRM – spermine; Ls – Lactobacillus sakei; Pa – Pediococus acidilactici. a-jThe mean values within a column with different letters are significantly different (p≤0.05).

The biogenic amines (BAs) content (mg/kg) in SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains in hybrid lines No.1700, No.1701, No.1800 and No.1702 wholemeal seeds is presented in Table 3.6.1.3. PEA, SPRMD (except in SMF with P. pentosaceus No.8 hybrid 137

No.1700 samples, in which SPRMD was not found) and HIS in all SMF lupine samples were formed. Also, in all SMF samples CAD and HIS were found and their content has ranged from 22.87 ± 0.69 mg/kg to 399.54 ± 3.14 mg/kg (in SSF with L. sakei hybrids No.1702 and No.1800 samples, respectively) and from 47.96 ± 0.74 mg/kg to 126.65 ± 3.08 mg/kg (in SMF with L. sakei hybrid No.1700 samples and in SMF with P. acidilactici hybrid No.1800 samples, respectively), respectively. In SMF with P. pentosaceus No.8, No.9 and No.10 hybrids No.1800 and No.1702 samples PUT was not found. However, in SMF hybrids No.1700 and No.1701 wholemeal samples PUT was formed. TYM and SPRM were established only in few SMF samples (in SMF with P. pentosaceus No.9 hybrid No.1701 samples and in SMF with P. pentosaceus No.10 hybrid No.1700 samples, respectively). In SMF samples PEA and SPRMD content has ranged from 0 mg/kg to 66.38 ± 7.48 mg/kg (in SMF with P. pentosaceus No.8 hybrid No.1700 samples and in SMF with P. pentosaceus No.8 hybrid No.1800 samples, respectively) and from 0 mg/kg to 36.02 ± 9.01 mg/kg (in SMF with P. pentosaceus No.8 hybrid No.1700 samples and in SMF with P. pentosaceus No.8 hybrid No.1800 samples, respectively), respectively. PUT and CAD contents have ranged from 9.52 ± 2.03 mg/kg to 178.36 ± 22.05 mg/kg (in SMF with P. pentosaceus No.8 hybrid No.1701 samples and in SMF with P. pentosaceus No.9 hybrid No.1702 samples, respectively) and from 0 mg/kg to 221.50 ± 35.28 mg/kg (in SMF with P. pentosaceus No.8 and No.9 hybrid No.1701 samples and in SMF with P. pentosaceus No.8 hybrid No.1800 samples, respectively), respectively. In SSF samples HIS content has ranged from 9.68 ± 1.56 mg/kg to 78.12 ± 6.31 mg/kg (in SSF with P. pentosaceus No.10 hybrid No.1701 samples and in SSF with P. pentosaceus No.9 hybrid No.1702 samples, respectively). SPRM was established only in SSF with P. pentosaceus No.9 hybrid No.1700 samples. The TYM content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(178.063)=9.264, p≤0.0001), however, the fermentation method and the microorganism applied for the fermentation had no significant influence on TYM content. The PEA content - by the fermentation method (F(5796.785)=18.843, p≤0.001), however, the selection of lupine variety and the microorganism applied for the fermentation had no significant influence on PEA content. The HIS content - by the fermentation method (F(90935.042)=192.785, p≤0.0001), however, the selection of lupine variety and the microorganism applied for the fermentation had no significant influence on HIS content.

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Table 3.6.1.3. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains in hybrid lines No.1700, No.1701, No.1800 and No.1702 wholemeal seeds. Lupine samples PEA PUT CAD HIS TYM SPRMD SPRM Submerged fermented samples 252.32 31.62 9.97 No.1700 Pp8 - - - - ±37.14f ±2.02b ±0.14b 39.28 37.09 234.67 75.23 25.49 No.1700 Pp9 - - ±11.02d ±6.71d ±108.17e ±9.04e ±6.38d 15.62 16.91 84.17 25.75 10.82 6.55 No.1700 Pp10 - ±2.98b ±3.17c ±11.08c ±4.21d ±1.01a ±0.47b 35.69 112.05 213.41 65.50 22.78 No.1701 Pp8 - - ±7.82f ±14.56e ±14.33d ±9.01d ±2.14d 23.90 178.56 138.00 43.01 15.60 13.14 No.1701 Pp9 - ±2.34d ±16.22f ±14.84c ±8.25c ±1.99c ±1.74b 30.95 95.43 171.78 90.53 17.52 No.1701 Pp10 - - ±3.11e ±11.05d ±16.89d ±17.01f ±2.27b 66.38 387.69 118.25 36.02 No.1800 Pp8 - - - ±7.48e ±98.57f ±27.87f ±9.01f 37.65 501.25 109.64 20.36 No.1800 Pp9 - - - ±1.98d ±144.41f ±14.85f ±0.78e 39.14 27.41 67.29 24.01 No.1800 Pp10 - - - ±1.75d ±1.04b ±3.14e ±1.28e 41.05 23.45 57.09 29.87 No.1702 Pp8 - - - ±2.89f ±0.99b ±4.75c ±1.31ef 24.58 19.95 50.15 20.56 No.1702 Pp9 - - - ±1.14c ±0.81a ±4.02b ±0.87c 41.28 18.39 45.91± 29.14 No.1702 Pp10 - - ±2.91f ±0.79a 3.51b ±1.29c Solid state fermented samples 70.45 186.18 47.34 19.89 13.98 No.1700 Pp8 - - ±7.01c ±31.04d ±12.25d ±2.04e ±1.41f 96.78 161.69 43.04 12.73 10.01 50.70 No.1700 Pp9 - ±10.14d ±27.14d ±11.58d ±2.04d ±1.47e ±13.05d 132.48 119.83 28.18 12.61 7.93 No.1700 Pp10 - - ±20.03e ±18.89c ±5.27c ±2.02d ±1.16c 9.52 15.83 No.1701 Pp8 - - - - - ±2.06b ±3.04b 38.01 20.25 8.54 2.47 0.26 No.1701 Pp9 - - ±5.18e ±4.05b ±1.58b ±0.89b ±0.02b 37.18 22.14 14.25 9.68 2.98 10.50 No.1701 Pp10 - ±5.05d ±4.25bc ±2.89b ±1.56b ±0.47c ±2.18d 163.45 221.50 55.77 No.1800 Pp8 - - - - ±25.87f ±35.28f ±12.87e 54.62 198.70 50.78 No.1800 Pp9 - - - - ±8.98d ±29.82d ±11.38e 155.51 128.66 39.65 No.1800 Pp10 - - - - ±24.62f ±19.36d ±7.21d 48.45 191.89 48.28 No.1702 Pp8 - - - - ±11.02b ±24.58f ±8.60e 178.36 145.58 78.12 No.1702 Pp9 - - - - ±22.05f ±22.01e ±6.31f 47.20 197.30 29.99 No.1702 Pp10 - - - - ±11.99e ±34.81f ±2.33d Data values are expressed as means with the standard deviations (n = 3). SMF – submerged samples; SSF – solid state fermented; PEA – phenylethylamine; PUT – putrescine; CAD – cadaverine; HIS – histamine; TYM – tyramine; SPRMD – spermidine; SPRM – spermine; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

139

The SPRMD content - by the selection of lupine variety (F(676.346)= 16.361, p≤0.0001) and the fermentation method (F(1366.720)=33.061, p≤0.0001), however, the microorganism applied for the fermentation had no significant influence on SPRMD content. The SPRM content - by the fermentation method (F(42.413)=17.032, p≤0.0001), however, the selection of lupine variety and the microorganism applied for the fermentation had no significant influence on SPRM content. Analysed factors had no significant influence on PUT and CAD contents in fermented lupine samples. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with L. sakei and P. acidilactici strains in lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734 wholemeal seeds is presented in Table 3.6.1.4. In SMF samples PUT content has ranged from 19.97 ± 1.68 mg/kg to 553.68 ± 5.35 mg/kg (in SMF with P. acidilactici hybrid No.1734 samples and in SMF with L. sakei Vilciai samples, respectively). TYM was found only in one SMF with P. acidilactici variety Vilniai sample(14.81 ± 1.01 mg/kg). In SMF samples SPRM was not established, however, CAD and HIS content in SMF samples has ranged from 68.75 ± 1.91 mg/kg to 346.08 ± 2.08 mg/kg (in SMF with P. acidilactici variety Vilniai samples and in SMF with L. sakei hybrid No.1072 samples, respectively) and from 51.23 ± 1.47 mg/kg to 138.20 ± 1.05 mg/kg (in SMF with P. acidilactici Vilniai samples and in SMF with L. sakei hybrid No.1734 samples, respectively), respectively. To compare SMF and SSF samples, PUT content in SSF samples was established to be on average 30.75% lower. PHE in 8 samples out of 12 analysed, HIS in 5 samples out of 12 analysed and SPRMD in 8 samples out of 12 analysed were found. To compare SMF and SSF samples, in SMF samples CAD content was on average 56.41% higher. The results of the ANOVA test indicated that the PEA content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(538.272)=37.998, p≤0.0001) and fermentation method (F(12826.147)= 905.434, p≤0.0001), and the interaction of lupine variety × type of microorganism × fermentation method F(1184.494)=83.617, p≤0.0001) was significant, however, the type of microorganism applied for the fermentation havd no significant influence on PEA content. The PUT content in fermented samples was significantly affected by the fermentation method (F(11769.615)=59.707, p≤0.0001), the type of microorganism applied for the fermentation (F(3264.199)=16.559, p≤0.0001) and the interaction of lupine variety × type of microorganism × fermentation method was significant (F(34453.375)=174.781, p≤0.0001), however, the selection of lupine variety had no significant influence on PUT content. The HIS content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(3081.937)=30.966, p≤0.0001), the fermentation method 140

(F(11485.217)=115.398, p≤0.0001), the type of microorganism applied for the fermentation (F(1436.080)=14.429, p≤0.0001), and the interaction of lupine varieties × type of microorganism × fermentation method was significant (F(3057.993)=30.725, p≤0.0001). The CAD content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(30876.858)=14.825, p≤0.0001), the type of microorganism applied for the fermentation (F(41991.201)=20.161, p≤0.0001) and the interaction of lupine variety × type of microorganism × fermentation method was significant (F(65331.322)=31.368, p≤0.0001), however, the fermentation method had no significant influence on CAD content. The TYM content in fermented lupine seeds was significantly affected by the selection of lupine variety (F(68.258)=94.275, p≤0.0001), the fermentation method (F(153.826)=212.458, p≤0.0001), the type of microorganism applied for the fermentation (F(23.054)=31.841, p≤0.0001), and their interaction was significant (F(162.965)=225.081, p≤0.0001). The SPRMD content was significantly affected by the selection of lupine variety (F(72.003)=11.347, p≤0.0001), the fermentation method (F(5357.678)=844.305, p≤0.0001) and the interaction of lupine variety × type of microorganism × fermentation method was significant (F(464.345)=73.175, p≤0.0001), however, the type of microorganism applied for the fermentation had no significant influence on SPRMD content. The SPRM content was significantly affected by the selection of lupine variety (F(299.000)=42.082, p≤0.0001), the fermentation method (F(243.653)=34.292, p≤0.0001) and the type of microorganism applied for the fermentation (F(285.163)=40.135, p≤0.0001), and their interaction was signifinant (F(312.837)=44.030, p≤0.0001). Weak negative correlation between HIS and histamine (r=-0.3709; p=0.0005) was found. Also, weak positive correlation between PEA and phenylethylamine (r=0.3914, p=0.0002) was established. However, no correlation between TYR and tyramine and between LYS and cadaverine was found.

141

Table 3.6.1.4. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with L. sakei and P. acidilactici strains in lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734 wholemeal seeds. Lupine samples PEA PUT CAD HIS TYM SPRMD SPRM Submerged fermented samples 26.52 553.68 239.46 101.90 13.87 Vilciai Ls - - ±0.91d ±5.35e ±3.16b ±1.99d ±0.39b 38.19 24.30 282.65 98.81 27.60 Vilciai Pa - - ±0.95e ±1.68b ±4.09c ±1.87c ±0.72c 32.86 130.42 230.07 75.26 24.62 Vilniai Ls - - ±0.54c ±3.86f ±1.02b ±2.58d ±0.55d 6.87 44.73 68.75 51.23 14.81 1.46 Vilniai Pa - ±0.46b ±1.54c ±1.91a ±1.47b ±1.01b ±0.01a 62.33 24.80 346.08 111.93 30.90 No.1072 Ls - - ±0.62d ±1.88d ±2.02e ±1.75d ±0.84e 78.75 30.32 414.57 135.96 40.13 No.1072 Pa - - ±0.95c ±1.53d ±3.28f ±3.05e ±0.89d 37.62 26.48 237.63 138.20 13.06 No.1734 Ls - - ±0.90d ±2.89b ±4.79d ±1.05f ±0.64b 43.48 19.97 327.61 107.93 25.68 No.1734 Pa - - ±0.92e ±1.68a ±2.08e ±2.08c ±0.57f Solid state fermented samples 26.04 23.69 328.36 52.98 7.58 28.31 Vilciai Ls - ±0.41c ±1.45e ±2.19e ±0.98d ±0.96d ±0.88f 36.58 19.14 298.36 24.17 5.19 2.01 Vilciai Pa - ±0.51c ±1.39c ±3.08f ±0.59f ±0.94b ±0.02a 39.47 25.80 299.87 34.99 2.64 Vilniai Ls - - ±0.51e ±1.52f ±2.03f ±0.84d ±0.26a 31.00 21.23 280.54 0.40 0.26 11.15 0.99 Vilniai Pa ±0.49d ±1.21d ±3.01e ±0.01a ±0.01a ±0.85d ±0.01a 17.31 15.93 2.47 4.69 23.62 23.62 2.11 No.1072 Ls ±0.44b ±1.69c ±0.04c ±0.65c ±0.92c ±0.92e ±0.04b 0.39 4.65 1.44 2.87 4.68 No.1072 Pa - - ±0.01a ±0.38b ±0.01b ±0.04b ±0.42b 43.55 2.09 2.01 3.06 6.47 No.1734 Ls - - ±1.43d ±0.04b ±0.03b ±0.03b ±0.09c 1.01 2.61 12.48 5.47 No.1734 Pa - - - ±0.01a ±0.05b ±0.83c ±0.08c Data values are expressed as means with the standard deviations (n = 3). SMF – submerged samples; SSF – solid state fermented; PEA – phenylethylamine; PUT – putrescine; CAD – cadaverine; HIS – histamine; TYM – tyramine; SPRMD – spermidine; SPRM - spermine. SMF – submerged fermentation; SSF – solid state fermentation; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

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3.6.2. The Influence of Fermentation on the Biogenic Amines Content in Lupine Protein Isolates/Concentrates

The biogenic amines (BAs) content (mg/kg) in nonfermented lupine seeds protein isolates/concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072, No.1734, No.1700, No.1701, No.1800 and No.1702 is presented in Table 3.6.2.1. In most of the nonfermented protein isolates/ concentrates PEA was detected (except in hybrids No.1700 and No.1701 protein isolate). Also, in all of the nonfermented wholemeal protein isolates/concentrates PUT was found, and its content has ranged from 16.45 ± 0.84 mg/kg to 52.59 ± 1.92 mg/kg, in varieties Vilciai and Vilniai samples, respectively. CAD was found in 3 samples out of 8 analysed samples (in Vilciai and hybrids No.1700, and No.1701 samples). HIS and SPRMD contents have ranged from 0 mg/kg to 58.05 ± 2.15 mg/kg, and from 0 mg/kg to 30.41 ± 1.29 mg/kg (in No.1701 and Vilciai samples and in No.1701 and Vilniai samples, respectively), respectively. TYM was established in 2 samples out of 8 analysed. SPRM content was found to be on average 31.50% lower, in compare with SPRMD content in nonfermented protein isolates. The results of the ANOVA test have indicated that the PEA, PUT, CAD, HIS, TYM, SPRMD and SPRM content in nonfermented lupine protein isolates was significantly affected by the selection of lupine variety (on PEA (F(1124.232)=1099.916, p≤0.0001); on PUT (F(745.428)= 703.068, p≤0.0001; on CAD (F(2066.167)=3540.208, p≤0.0001; on HIS (F(1781.299)=2611.164, p≤0.0001; on TYM (F(44.302)=21240.856, p≤0.0001; on SPRMD (F(560.362)=1105.313, p≤0.0001; on SPRM (F(404.357)=2100.946, p≤0.0001). The biogenic amines (BAs) content (mg/kg) in SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains in lupine seeds protein isolates/ concentrates of varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734 is presented in Table 3.6.2.2. In SMF samples TYM and SPRM were not established. PEA content in SMF samples has ranged from 0 mg/kg to 13.99 ± 0.15 mg/kg (in SMF with P. pentosaceus No.10 hybrid No.1072 protein isolates and in SMF with P. pentosaceus No.8 hybrid No.1734 isolates, respectively). PUT was found in 8 samples out of 12 analysed. In SSF samples CAD content was on average 80.69% lower, in compare with SMF samples. HIS content in SSF samples was on average 74.72% higher, in compare with SMF samples. In SSF samples SPRMD and SPRM were not established. TYM was found in 2 SSF samples out of 12 analysed. PUT content in SSF samples has ranged from 2.08 ± 0.03 mg/kg to 19.99 ± 0.19 mg/kg (in SSF with P. pentosaceus No.9 hybrid No.1734 isolates and in SSF with P. pentosaceus No.10 Vilniai isolates, respectively). 143

Table 3.6.2.1. The biogenic amines (BAs) content (mg/kg) in nonfermented lupine seeds protein isolates/concentrates of lupine varieties Vilciai, Vilniai, hybrid lines No.1072, No.1734, No.1700, No.1701, No.1800 and No.1702. Lupine samples PEA PUT CAD HIS TYM SPRMD SPRM Nonfermented samples 17.81 52.59 54.60 58.05 5.18 Vilciai - - ±0.55b ±1.92e ±2.02e ±2.15e ±0.08c 24.15 16.45 7.28 30.41 23.25 Vilniai - - ±0.47d ±0.84c ±0.11c ±1.29e ±0.95e 38.25 21.44 12.58 27.60 19.97 No.1072 - - ±1.51e ±0.98d ±0.16d ±1.08f ±0.64d 41.21 25.89 18.77 7.91 14.80 8.31 No.1734 - ±1.88f ±0.86f ±0.28d ±0.12c ±0.82d ±0.11c 33.98 5.73 0.54 0.40 15.33 1.92 No.1700 - ±0.92e ±0.08b ±0.02a ±0.02a ±0.22c ±0.04a 31.02 5.05 No.1701 - - - - - ±0.87d ±0.06a 44.01 31.17 16.39 12.16 14.66 No.1800 - - ±1.24c ±1.01c ±0.21c ±0.15b ±0.18c 19.22 16.98 12.44 5.24 7.69 No.1702 - - ±0.31b ±0.19b ±0.17b ±0.09a ±0.11b Data values are expressed as means with the standard deviations (n = 3). PEA – phenylethylamine; PUT – putrescine; CAD – cadaverine; HIS – histamine; TYM – tyramine; SPRMD – spermidine; SPRM – spermine. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

However, in compare SMF and SSF samples, PUT content in SSF protein isolates on average by 43.11% lower was established. Most of the analysed BAs were significantly influenced by the analysed factors and their interaction (analysed factors: lupine variety, type of microorganism, fermentation method). The PEA content in lupine protein isolates/concentrates was significantly affected by the selection of lupine variety (F(35.837)= 5319.060, p≤0.0001), the fermentation method (F(360.193)=53460.898, p≤0.0001), the type of microorganism applied for the fermentation (F(60.944)=9045.564, p≤0.0001) and the interaction of these factors was significant (interaction of lupine variety × type of microorganism × fermentation method F(33.826)=5020.605, p≤0.0001). The PUT content in lupine protein isolates was significantly affected by the selection of lupine variety (F(87.974)=1734.761, p≤0.0001), the fermentation method (F(813.254)=16036.568, p≤0.0001), the type of microorganism applied for the fermentation (F(251.867)=4966.573, p≤0.0001) and the interaction of these factors was significant (F(954.916)=18829.988, p≤0.0001). The CAD content in lupine protein isolates was significantly affected by the selection of lupine variety (F(75.138)=12102.749, p≤0.0001), the fermentation method (F(929.236)=149675.605, p≤0.0001), the type of microorganism applied for the fermentation (F(3.678)=592.438, p≤0.0001) and their

144

Table 3.6.2.2. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains in lupine seeds protein isolates/ concentrates of varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734. Lupine samples PEA PUT CAD HIS TYM SPRMD SPRM Submerged fermented samples 9.66 7.25 21.64 5.62 4.03 Vilciai Pp8 - - ±0.09c ±0.08b ±0.12d ±0.07d ±0.05d 8.76 20.31 4.22 5.00 Vilciai Pp9 - - - ±0.07b ±0.11c ±0.05c ±0.06e 10.32 16.33 1.99 5.52 Vilciai Pp10 - - - ±0.11d ±0.19b ±0.04a ±0.08f 9.64 12.66 4.44 Vilniai Pp8 - - - - ±0.09c ±0.12b ±0.05d 10.07 17.01 5.30 Vilniai Pp9 - - - - ±0.12d ±0.15b ±0.07e 5.07 5.46 12.64 3.66 2.29 Vilniai Pp10 - - ±0.06a ±0.06a ±0.17b ±0.05b ±0.04b 9.88 20.09 4.58 2.98 No.1072 Pp8 - - - ±0.09d ±0.14c ±0.06c ±0.06c 11.88 26.71 6.77 2.08 No.1072 Pp9 - - - ±0.13c ±0.18d ±0.08e ±0.04b 45.76 1.88 0.69 No.1072Pp10 - - - - ±0.88f ±0.02a ±0.02a 13.99 22.06 2.33 2.55 No.1734 Pp8 - - - ±0.15d ±0.18cd ±0.04b ±0.04c 9.20 19.14 4.88 1.95 No.1734 Pp9 - - - ±0.08c ±0.15b ±0.06c ±0.02b 9.99 33.88 6.32 7.32 2.06 No.1734Pp10 - - ±0.11d ±0.42e ±0.08a ±0.09f ±0.04b Solid state fermented samples 4.45 12.36 3.66 1.02 0.99 Vilciai Pp8 - - ±0.06b ±0.14e ±0.04a ±0.03c ±0.03b 6.99 10.33 0.85 Vilciai Pp9 - - - - ±0.09e ±0.11d ±0.02b 5.02 11.01 Vilciai Pp10 - - - - - ±0.07d ±0.13d 7.55 12.55 4.25 0.78 Vilniai Pp8 - - - ±0.08f ±0.16e ±0.06c ±0.02 13.90 3.82 0.42 Vilniai Pp9 - - - - ±0.15e ±0.05a ±0.01a 19.99 5.05 1.30 0.79 Vilniai Pp10 - - - ±0.19f ±0.07d ±0.04c ±0.02a 3.77 3.04 3.91 0.66 No.1072 Pp8 - - - ±0.04a ±0.05b ±0.04b ±0.02a 5.01 4.12 0.88 No.1072 Pp9 - - - - ±0.07c ±0.06c ±0.03b 4.97 5.44 1.12 No.1072Pp10 - - - - ±0.05c ±0.08d ±0.05c 5.87 4.15 0.55 No.1734 Pp8 - - - - ±0.06d ±0.04c ±0.03a 6.33 2.08 1.44 No.1734 Pp9 - - - - ±0.08e ±0.03a ±0.03d 4.82 3.67 1.91 No.1734Pp10 - - - - ±0.05b ±0.04b ±0.04d Data values are expressed as means with the standard deviations (n = 3); PEA – phenylethylamine; PUT – putrescine; CAD – cadaverine; HIS – histamine; TYM – tyramine; SPRMD – spermidine; SPRM – spermine; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-f The mean values within a column with different letters are significantly different (p≤0.05).

145 interaction (F(159.224)=25646.779, p≤0.0001). The HIS content in lupine protein isolates was significantly affected by the selection of lupine variety (F(4.914)=2574.821, p≤0.0001), the fermentation method (F(130.573)= 68422.428, p≤0.0001), the type of microorganism applied for the fermentation (F(1.793)=939.773, p≤0.0001) and the interaction of these factors was significant (F(10.949)=5737.441, p≤0.0001). The TYM content in lupine protein isolates was significantly affected by the selection of lupine variety (F(0.135)=2498.769, p≤0.0001), the fermentation method (F(0.396)= 7311.692, p≤0.0001), the type of microorganism applied for the fermentation (F(0.103)=1897.154, p≤0.0001) and their interaction (F(0.233)= 4303.615, p≤0.0001). The SPRMD content in lupine protein isolates was significantly influenced by the selection of lupine variety (F(0.323)= 252.362, p≤0.0001), the fermentation method (F(189.054)=147794.668, p≤0.0001), the type of microorganism applied for the fermentation (F(1.635)=1277.951, p≤0.0001) and the interaction of these factors was significant (F(6.032)=4715.814, p≤0.0001). However, analysed factors have not significant influence on SPRM content in lupine protein isolate samples. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with L. sakei and P. acidilactici strains hybrid lines No.1700, No.1701, No.1800 and No.1702 lupine seeds protein isolates/concentrates is presented in Table 3.6.2.3. In all SMF samples, CAD and TYM were not found. PEA and PUT in half of the SMF samples were found. In SMF hybrids No.1700, No.1800 and No.1702 protein isolates HIS, SPRMD and SPRM were not found. However, in SMF with L. sakei and P. acidilactici strains hybrid No.1701 isolates the contents of HIS, SPRMD and SPRM were ranged from 0.88 ± 0.01 mg/kg till 1.17 ± 0.02 mg/kg (in SMF with P. acidilactici hybrid No.1701 isolates and in SMF with L. sakei hybrid No.1701 isolates, respectively), from 0.19 ± 0.01 mg/kg till 0.36 ± 0.02 mg/kg (in SMF with L. sakei hybrid No.1701 isolates and in SMF with P. acidilactici hybrid No.1701 isolates respectively) and from 0.42 ± 0.02 mg/kg till 0.81 ± 0.03 mg/kg (in SMF with L. sakei hybrid No.1701 isolates and in SMF with P. acidilactici hybrid No.1701 isolates respectively), respectively. TYM just in 1 SSF sample from 12 analysed was found (0.09 ± 0.01 mg/kg). In SSF samples, PEA, HIS and SPRMD in all hybrids No.1701, No. 1800 and No.1702 protein isolates were found, except in hybrid No.1700 isolate. CAD only in SSF with L. sakei and P. acidilactici hybrid No.1800 protein isolate samples was found. SPRM in 3 SSF samples from 12 analysed was found. The PEA content in lupine protein isolates was significantly affected by the selection of lupine variety (F(41.820)=27.451, p≤0.0001), however, the fermentation method and the microorganism applied for fermentation have not significant influence on PEA content in isolates. The PUT content 146 in lupine protein isolates was significantly affected by the selection of lupine variety (F(43.043)=50.705, p≤0.0001), however, the fermentation method and the microorganism applied for fermentation have not significant influence on PUT content. The HIS content in isolates was significantly affected by the selection of lupine variety (F(1.933)=19.351, p≤0.0001), however, the fermentation method and the microorganism applied for fermentation have not significant influence on HIS content. The SPRM content in lupine protein isolates was significantly affected by the selection of lupine variety (F(0.615)=14.323, p≤0.0001), however, the fermentation method and the microorganism applied for fermentation have not significant influence on SPRM content. However, the influence of analysed factors interactions was not significant on PEA, PUT, CAD, HIS, TYM, SPRMD and SPRM content. The analysed factors do not influenced CAD, TYM and SPRMD content in lupine protein isolate samples.

Table 3.6.2.3. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with L. sakei and P. acidilactici strains hybrid lines No.1700, No.1701, No.1800 and No.1702 lupine seeds protein isolates/concentrates. Lupine samples PEA PUT CAD HIS TYM SPRMD SPRM Submerged fermented samples No.1700 Ls ------No.1700 Pa ------3.14 4.62 1.17 0.19 0.42 No.1701 Ls - - ±0.05b ±0.07c ±0.02b ±0.01a ±0.02a 2.23 2.32 0.88 0.36 0.81 No.1701 Pa - - ±0.02a ±0.03a ±0.01a ±0.02b ±0.03b No.1800 Ls ------No.1800 Pa ------0.76 3.86 No.1702 Ls - - - - - ±0.02a ±0.07c 7.81 3.55 No.1702 Pa - - - - - ±0.09c ±0.05b Solid state fermented samples No.1700 Ls ------No.1700 Pa ------3.01 4.51 1.14 0.18 0.30 No.1701 Ls - - ±0.06e ±0.07e ±0.04e ±0.02a ±0.02a 2.07 2.24 0.67 0.34 0.51 No.1701 Pa - - ±0.03c ±0.05d ±0.03c ±0.03b ±0.02b 1.34 0.54 0.26 0.49 0.11 No.1800 Ls - - ±0.02a ±0.03b ±0.03a ±0.02a ±0.02a 1.28 0.48 0.22 0.47 0.99 No.1800 Pa - - ±0.02a ±0.02a ±0.02a ±0.02a ±0.03c 2.51 0.64 0.94 1.00 0.52 No.1702 Ls - - ±0.04d ±0.03c ±0.03d ±0.04c ±0.03b 1.73 0.56 0.09 0.22 No.1702 Pa - - - ±0.02b ±0.02b ±0.01a ±0.03a Data value are expressed as means with the standard deviations (n = 3). PEA – phenylethylamine; PUT – putrescine; CAD – cadaverine; HIS – histamine; TYM – tyramine; SPRMD – spermidine; SPRM – spermine; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici. a-eThe mean values within a column with different letters are significantly different (p≤0.05).

147

The biogenic amines (BAs) content (mg/kg) in SMF and SSF with L. sakei and P. acidilactici strains in lupine seeds protein isolates/concentrates of varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734 is presented in Table 3.6.2.4. In all SMF samples TYM was not established. PEA and PUT were established in 6 samples out of 8 analysed. In SMF lupine isolates HIS content has ranged from 0.52 ± 0.02 mg/kg to 9.25 ± 0.09 mg/kg (in SMF with L. sakei hybrid No.1072 isolates and in SMF with L. sakei Vilniai isolates, respectively). SPRMD content in SMF samples has ranged from 0.67 ± 0.02 mg/kg to 25.23 ± 0.58 mg/kg (in SMF with P. acidilactici hybrid No.1734 isolates and in SMF with P. acidilactici Vilniai isolates, respectively). In SMF samples SPRM content was on average 2.52% lower, in compare with SSF samples. PEA was established in 3 SSF samples out of 8 analysed. PUT content in SSF isolates has ranged from 0 mg/kg to 44.47 ± 0.41 mg/kg (in SSF with P. acidilactici Vilciai and hybrid No.1734 isolates, and in SSF with L. sakei Vilniai protein isolates, respectively). CAD content has ranged from 0 mg/kg to 15.40 ± 0.19 mg/kg (in SSF with L. sakei and P. acidilactici strains Vilniai isolates and in SSF with P. acidilactici strain Vilciai isolates, respectively). HIS was established in 2 SSF samples out of 8 analysed. SPRMD content in SSF samples has ranged from 0 mg/kg to 15.21 ± 0.14 mg/kg (in SSF with L. sakei varieties Vilciai and Vilniai isolates, and in SSF with P. acidilactici Vilniai isolates, respectively). The PEA content in lupine protein isolates was highy affected by the selection of the microorganism applied for the fermentation (F(341.867)=25.001, p≤0.0001), however, the selection of lupine variety and the fermentation method had no significant influence on PEA content in protein isolates. The HIS content in lupine protein isolates was significantly affected by the fermentation method (F(202.089)=70.675, p≤0.0001), but the selection of lupine variety and the microorganism applied for the fermentation had no significant influence on HIS content. The TYM content in lupine protein isolates was significantly affected by the fermentation method (F(369.297)=36.647, p≤0.0001), however, the fermentation method and the microorganism applied for fermentation had no significant influence on TYM content. Analysed factors did not have significant influence on PUT, CAD, SPRMD and SPRM content in lupine protein isolate samples. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains in lupine protein isolates/concentrates of hybrid lines No.1701, No.1700, No.1800 and No.1702 is presented in Table 3.6.2.5. The PEA was only found in SMF with P. Pento- saceus No.8 hybrid No.1800 isolates (28.42 ± 0.44 mg/kg). In SMF samples PUT and SPRMD content has ranged from 0 mg/kg to 42.61 ± 0.58 mg/kg

148

Table 3.6.2.4. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with L. sakei and P. acidilactici strains in lupine seeds protein isolates/ concentrates of varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734. Lupine PEA PUT CAD HIS TYM SPRMD SPRM samples Submerged fermented samples 11.87 9.95 4.66 4.65 Vilciai Ls - - - ±0.12d ±0.09c ±0.05c ±0.06c 11.74 5.06 4.57 6.58 Vilciai Pa - - - ±0.11d ±0.06a ±0.04b ±0.08d 2.00 9.25 2.09 2.99 Vilniai Ls - - - ±0.02a ±0.09d ±0.02a ±0.03b 9.01 8.21 6.41 25.23 19.87 Vilniai Pa - - ±0.09b ±0.07b ±0.06c ±0.58d ±0.19d 7.62 0.52 12.18 1.40 No.1072 Ls - - - ±0.06b ±0.02a ±0.13c ±0.02a 7.85 6.52 5.75 9.02 No.1072 Pa - - - ±0.06c ±0.05b ±0.06d ±0.09c 8.72 8.45 18.58 3.79 3.77 No.1734 Ls - - ±0.08d ±0.09c ±0.16b ±0.04a ±0.04b 10.05 0.62 4.85 0.67 0.43 No.1734 Pa - - ±0.10c ±0.02a ±0.06c ±0.02a ±0.01a Solid state fermented samples 0.93 42.75 2.07 2.75 10.99 1.52 Vilciai Ls - ±0.02a ±0.38d ±0.03a ±0.04a ±0.11b ±0.04b 15.40 3.41 2.66 Vilciai Pa - - - - ±0.19d ±0.04b ±0.06c 9.89 44.47 Vilniai Ls - - - - - ±0.11c ±0.41d 5.72c 7.88 4.23 15.21 7.22 Vilniai Pa - - ±0.06b ±0.08b ±0.07b ±0.14d ±0.08e 5.01 2.92 10.30 1.38 3.45 No.1072 Ls - - ±0.06b ±0.03a ±0.09a ±0.02a ±0.05d 5.89 4.95 3.46 2.96 No.1072 Pa - - - ±0.07c ±0.05b ±0.05c ±0.06c 5.47 4.83 10.67 3.18 4.25 No.1734 Ls - - ±0.06b ±0.04b ±0.19c ±0.04b ±0.06d 4.87 12.42 6.33 3.26 No.1734 Pa - - - ±0.07c ±0.22d ±0.08c ±0.04c Data values are expressed as means with the standard deviations (n = 3). PEA – phenylethylamine; PUT – putrescine; CAD – cadaverine; HIS – histamine; TYM – tyramine; SPRMD – spermidine; SPRM – spermine; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici; a-d The mean values within a column with different letters are significantly different (p≤0.05). and from 0 mg/kg to 31.20 ± 0.41 mg/kg, respectively (in SMF with P. pentosaceus No.8 and No.9 hybrid No.1701 isolates and in SMF with P. pentosaceus No.9 hybrid No.1700 isolates, respectively). CAD was established in 3 SMF samples out of 16 analysed.

149

Table 3.6.2.5. The biogenic amines (BAs) content (mg/kg) in SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains in lupine seeds protein isolates/concentrates of hybrid lines No.1701, No.1700, No.1800 and No.1702. Lupine samples PEA PUT CAD HIS TYM SPRMD SPRM Submerged fermented samples No.1701 Pp8 ------No.1701 Pp9 ------33.18 13.88 No.1701 Pp10 - - - - - ±0.52e ±0.16b 26.59 1.77 10.51 No.1700 Pp8 - - - - ±0.41d ±0.04c ±0.12a 42.61 2.63 13.34 No.1700 Pp9 - - - - ±0.58f ±0.04c ±0.15b 30.04 0.39 14.01 1.19 No.1700 Pp10 - - - ±0.49d ±0.02a ±0.18c ±0.02a 28.42 1.94 27.28 4.04 No.1800 Pp8 - - - ±0.44b ±0.03b ±0.29e ±0.06e 17.12 0.47 2.23 19.19 1.63 No.1800 Pp9 - - ±0.28 ±0.03b ±0.03b ±0.19d ±0.03c 10.86 1.10 3.16 31.20 3.64 No.1800 Pp10 - - ±0.12c ±0.02a ±0.05d ±0.41f ±0.05d 7.28 0.66 3.09 14.11 No.1702 Pp8 - - - ±0.08a ±0.03b ±0.04d ±0.14c 31.37 2.00 2.78 19.82 1.40 No.1702 Pp9 - - ±0.46d ±0.04d ±0.03c ±0.18d ±0.04b 7.92 0.62 2.03 19.97 1.30 No.1702 Pp10 - - ±0.09c ±0.02a ±0.02a ±0.21d ±0.02b Solid state fermented samples No.1701 Pp8 ------7.64 0.55 0.44 15.09 1.42 No.1701 Pp9 - - ±0.08b ±0.02a ±0.03a ±0.15b ±0.03 32.06 2.48 15.00 0.92 No.1701 Pp10 - - - ±0.37e ±0.04b ±0.18b ±0.02b 5.32 2.07 13.77 0.59 No.1700 Pp8 - - - ±0.05 ±0.04b ±0.12a ±0.01a 5.85 1.46 16.22 0.97 No.1700 Pp9 - - - ±0.07c ±0.02a ±0.17c ±0.03b No.1700 Pp10 ------3.85 11.81 No.1800 Pp8 - - - - - ±0.06c ±0.13f 8.69 3.00 13.98 1.05 No.1800 Pp9 - - - ±0.09c ±0.05c ±0.12a ±0.02c 8.45 8.25 20.31 1.14 No.1800 Pp10 - - - ±0.07c ±0.09d ±0.22d ±0.02c 9.72 1.59 18.62 1.21 No.1702 Pp8 - - - ±0.12d ±0.04b ±0.19d ±0.03d 6.10 0.59 24.65 1.82 No.1702 Pp9 - - - ±0.06 ±0.02a ±0.25f ±0.03e 12.79 1.12 22.23 1.27 No.1702 Pp10 - - - ±0.14c ±0.03b ±0.23e ±0.04d Data values are expressed as means with the standard deviations (n = 3). PEA – phenylethylamine; PUT – putrescine; CAD – cadaverine; HIS – histamine; TYM – tyramine; SPRMD – spermidine; SPRM – spermine; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-dThe mean values within a column with different letters are significantly different (p≤0.05).

150

In SMF samples HIS and SPRMD content was found to be on average 24.26% and 12.83% lower, in compare with SSF samples. In SSF samples PEA was not found, and TYM was established only in SSF with P. pentosaceus No.9 hybrid No.1702 isolate (0.59 ± 0.02 mg/kg). The PUT was established in 9 SSF samples out of 12 analysed. In SSF samples SPRM content was found to be on average 38.89% higher, in compare with SMF samples. CAD was established only in SSF with P. pentosaceus No.9 hybrid No.1701 isolates (0.55 ± 0.02 mg/kg). The analysed BAs were renarkably influenced by the analysed factors and their interaction was significant. The PEA content in lupine protein isolates was significantly affected by the selection of lupine variety (F(100.962)=12515.957, p≤0.0001), the fermentation method (F(100.962)=12515.957, p≤0.0001), the type of microorganism applied for the fermentation (F(100.992)=15125.759, p≤0.0001) and the interaction of these factors was significant (interaction of lupine variety × type of microorganism × fermentation method F(100.958)= 12551.001, p≤0.0001). The PUT content in lupine protein isolates was notably affected by the selection of lupine variety (F(206.811)=3233.111, p≤0.0001), the fermentation method (F(1526.005)=23856.253, p≤0.0001), the type of microorganism applied for the fermentation (F(790.881)= 12363.951, p≤0.0001) and the interaction of these factors was significant (F(539.868)=8439.828, p≤0.0001). The CAD content in lupine protein isolates was remarkably affected by the selection of lupine variety (F(1.384)=10068.970, p≤0.0001), the fermentation method (F(3.277)= 23831.273, p≤0.0001), the type of microorganism applied for the fermentation (F(0.411)=2986.182, p≤0.0001) and their interaction (F(1.515)= 11017.333, p≤0.0001). The HIS content in lupine protein isolates was significantly affected by the selection of lupine variety (F(4.584)=4074.767, p≤0.0001), the fermentation method (F(42.090)=37413.611, p≤0.0001), the type of microorganism applied for the fermentation (F(2.829)=2515.078, p≤0.0001) and the interaction of these factors was not found (F(13.045)= 11.595, p≤0.0001). The TYM content in lupine protein isolates was significantly affected by the selection of lupine variety (F(0.146)=522.149, p≤0.0001), the fermentation method (F(20.161)=72219.403, p≤0.0001), the type of microorganism applied for the fermentation (F(0.680)=2435.418, p≤0.0001) and their interaction (F(4.675)=16746.403, p≤0.0001). The SPRMD content in isolates was outstandingly influenced by the selection of lupine variety (F(168.114)=5095.656, p≤0.0001), the fermentation method (F(68.679)=2081.714, p≤0.0001), the type of microorganism applied for the fermentation (F(274.070)=8307.235, p≤0.0001) and the interaction of these factors was significant (F(239.220)=7250.931, p≤0.0001). The SPRM content in isolates was significantly influenced by the selection of lupine 151 variety (F(28.012)=20496.415, p≤0.0001), the fermentation method (F(10.125)=7408.537, p≤0.0001), the type of microorganism applied for the fermentation (F(9.753)=7136.643, p≤0.0001) and the interaction of these factors was remarkable (F(6.421)=4698.198, p≤0.0001).

3.6.3. The Formation of Biogenic Amines in Plant Based Substrates and Factors Influencing Their Formation

The BAs can occure in the proteinaceous substrate during its processing or storage, primarily due to the release of specific amino acids via the activity of decarboxylases produced by LAB and by other numerous factors [331, 332]. The BAs are present in all animal feeds that contain proteins or free amino acids, and they also exist in fermented feed [333]. BAs are biologically active substances with numerous physiological or pathophysiological functions in humans and animals. High level of BAs is frequently observed in silages prepared from high-protein forage. Some BAs have significant biological characteristics, as they are tissue hormones (HIS), protoalkaloids and the building blocks for biosynthesis of other hormones in animals (PEA), phytohormones, auxins, alkaloids and other secondary metabolites of plants [334]. HIS, TYR and PEA have been observed to have negative effects, influencing the nervous system, affecting blood pressure, and provoking skin manifestations [335]. The results of the ANOVA test have indicated that the BAs content in lupine wholemeal products was notably affected by the selection of lupine variety (on PHE (F(1253.566)=13.833, p≤0.0001); on PUT (F(17482.130)=5.161, p≤0.0001); on CAD (F(42207.521)=7.947, p≤0.0001; on HIS (F(2417.926)=12.022, p≤0.0001; on TYR (F(80.708)=15.592, p≤0.0001; on SPRMD (F(256.898)=19.804, p≤0.0001); on SPRM (F(81.022)=34.920, p≤0.0001), the fermentation method (on PHE (F(58871.763)=649.662, p≤0.0001); on PUT (F(53104.908)= 15.678, p≤0.0001); on CAD (F(750790.753)=141.361, p≤0.0001); on HIS (F(194257.816)=965.853, p≤0.0001); on TYR (F(325.091)=62.806, p≤0.0001); on SPRMD (F(19512.906)=1504.232, p≤0.0001); on SPRM (F(105.542)=45.488, p≤0.0001), the type of microorganism applied for the fermentation (on PUT (F(28226.403)=8.333, p≤0.0001); on TYR (F(34.434)=6.652, p≤0.0001); on SPRM (F(75.904)=32.714, p≤0.0001), and the interaction of lupine variety × type of microorganism × fermentation method was significant (on PHE (F(1660.689)=18.326, p≤0.0001), on PUT (F(22888.701)=6.758, p≤0.0001); on CAD (F(73313.407)=13.804, p≤0.0001); on HIS (F(3166.822)=15.745, p≤0.0001); on TYR (F(117.183)= 22.639, p≤0.0001); on SPRMD (F(341.518)=26.327, p≤0.0001) and on SPRM (F(151.352)=65.232, p≤0.0001). Overall, BAs play an important role

152 in brain activity, regulation of body temperature and stomach pH, as well as gastric acid secretion, the immune response, and cell growth and diffe- rentiation [336]. The BAs in feeds are mostly synthesized by microorganisms and are usually considered to be potential toxins. However, polyamines PUT, SPRM and SPRMD are cationic compounds, which have hormone-like properties of anabolic compounds , and it has been proposed that exogenous dietary polyamines may play an important role in promoting animal growth and maintaining their health. Although all polyamines promote the intestinal tract development, only PUT may promote chicken body growth. Smith et al. [337] have reported that the PUT was proven to overcome the toxicity of raw legumes when brought into chicks diets. Also, laying hens diets supplemented with PUT had a decrease in egg shell deformations and an increase in egg shell thickness. Nevertheless, BAs should not always be considered as potential toxicants, but can also be considered to be nonhormonal growth promotants [337]. During fermentation, the enzymatic activity of the raw material and the metabolic activity of microorganisms can change the nutritive and bioactive properties of food/feed matrices in a manner that has beneficial consequences for human/animal health [338]. However, LAB involved in fermentation can produce BAs [165]. The BAs are organic, basic, nitrogenous compounds of low molecular weight, and they are formed by the removal of the α-carboxyl group from amino acids and they are usually named after corresponding precursor amino acid, e.g. histidine is decar- boxylated to produce histamine tryptophan to tryptamine, tyrosine to tyramine and lysine to cadaverine. However, putrescine can be produced from three amino acids: glutamine, arginine and agmatine. Arginine, once formed is easily converted to agmatine or under effect of bacterial activity can be degraded to ornithine from which putrescine is formed by decarboxylation [339–341]. According to their chemical structure, biogenic amines can be classified as heterocyclic (histamine and tryptamine), aliphatic (putrescine and cadaverine) or aromatic (tyramine and phenylethylamine) [336]. Also, BAs production can be associated with product contamination. However, the use of LAB producing bacteriocins can have an important potential in limiting BAs accumulation [342]. Some LAB have proteolytic capacities active in different types of foods that can result in increased concentrations of bioactive peptides and polyamines such as PUT, SPRM and SPRMD [332]. The BAs accumulation in fermented foods is a complex process affected by multiple factors and their interactions, the combinations of which are numerous, variable and product-specific [165]. Low concentrations of BAs occur naturally in all animals, plants and microorganisms, where they perform many important functions such as being the stabilisers 153 of macromolecules, precursors of hormones or local tissue hormones etc. [343]. Although the BAs have been studied for more than thirty years, the interest in their study is more relevant since food/feed quality requirements became higher [344]. In general, there is no legal regulation of the BA content in feed. Adverse health effects can occur both in case of high intake of these amines or when the ability to metabolize them is compromised by different causes (including enzymatic deficiencies due to genetic or physiological circumstances or enzymatic blockage). Under these circumstances they accumulate in plasma and exert bioactive effects [345, 346]. According to the results of our analysis, the safer technology for proteinaceous stock preparation should include the isolation/concentration processes. To compare all lupine protein isolates/concentrates (nonfermented and fermented), the results of the ANOVA test have indicated that the BAs content in lupine protein isolates was significantly affected by the selection of lupine variety (on PEA (F(1018.934)=326.199, p≤0.0001); on PUT (F(429.919)=18.074, p≤0.0001); on CAD (F(962.804)=295.191, p≤0.0001; on HIS (F(583.502)= 864.961, p≤0.0001; on TYM (F(19.275)=9.753, p≤0.0001; on SPRMD (F(235.672)=41.491, p≤0.0001); on SPRM (F(139.992)=44.150, p≤0.0001) and the fermentation method (on PEA (F(607.331)=194.430, p≤0.0001); on PUT (F(766.206)=32.211, p≤0.0001); on CAD (F(249.728)=76.565, p≤ 0.0001); on HIS (F(69.961)=103.707, p≤0.0001); on TYM (F(34.374)= 17.393, p≤0.0001); on SPRMD (F(316.913)=55.793, p≤0.0001), however, significant influence of the selection of lupine variety on SPRM content was not established; also it was affected by the type of microorganism applied for the fermentation (on PEA (F(87.031)=27.862, p≤0.0001); on PUT (F(1103.722)=46.400, p≤0.0001); on TYM (F(21.928)=11.095, p≤0.0001); on SPRMD (F(443.426)=78.066, p≤0.0001); on SPRMD (F(30.107)=9.495, p≤0.0001), however, significant influence of the type of microorganism applied for fermentation on CAD and HIS content in isolates was not establishedThe interaction of lupine variety × type of microorganism × fermentation method was significant (on PEA (F(53.638)=17.172, p≤0.0001), on PUT (F(508.481)=21.376, p≤ 0.0001); on CAD (F(85.310)= 26.156, p≤0.0001); on HIS (F(8.754)=12.977, p≤0.0001) and on TYM (F(9.101)=4.605, p≤0.0001); on SPRMD (F(90.583)=15.947, p≤0.0001), however, significant influence of the interaction on SPRM content was not found. Although BA contents in fermented products vary from undetected to more than 1000 mg/kg, the available data indicated that it is possible to produce fermented products without or with low contents of BA. Therefore, the implementation of specific technological measures should mainly aim to control aminogenic microorganisms participating in the fermentation process [165]. 154

3.7. The Total Phenolic Compounds Content in Lupine Wholemeal and Protein Isolates/Concentrates and Their Antioxidant Properties

Results of the TPC content and free radical (DPPH) scavenging activity in nonfermented wholemeal and protein isolates/concentrates of varieties Vilciai, Vilniai and hybrid lines No.1072, No.1734 in SMF and SSF with L. sakei and P. acidilactici strains are presented in Table 3.7.1. The TPC content in lupine products has ranged from 310.61 ± 3.49 GAE 100/g d.w. to 699.47 ± 6.69 GAE 100/g d.w. (in SMF with L. sakei Vilniai and in SSF with L. sakei hybrid No.1734 samples, respectively).

Table 3.7.1. The TPC content and free radical (DPPH) scavenging activity in nonfermented wholemeal and protein isolates/concentrates of varieties Vilciai, Vilniai and hybrid lines No.1072, No.1734in SMF and SSF with L. sakei and P. acidilactici strains. Lupine Vilciai Vilniai No.1072 No.1734 samples BI AI BI AI BI AI BI AI Total phenolic compounds content, GAE 100/g d.w. 643.97 628.21 500.38 527.82 318.21 422.38 466.31 489.29 NF ±6.12d ±6.52d ±4.89b ±5.22c ±3.54b ±4.25b ±4.58a ±4.62a 542.90 567.92 310.61 441.32 469.69 552.14 513.05 502.23 Ls SMF ±5.18b ±5.64c ±3.49a ±4.99a ±4.82c ±5.09c ±4.01b ±4.99b 459.55 447.32 576.96 452.21 257.40 312.27 520.94 500.62 Pa SMF ±4.85a ±4.99a ±5.29c ±5.01b ±3.17a ±3.99a ±4.63b ±4.92b 543.18 479.20 586.54 641.63 525.72 621.41 648.20 699.47 Ls SSF ±5.28b ±5.11b ±5.32e ±6.77e ±5.16d ±4.35d ±6.15d ±6.69d 614.97 652.17 588.23 622.63 554.44 632.64 642.57 687.26 Pa SSF ±6.01c ±5.18e ±5.33d ±6.23d ±5.20e ±4.39d ±6.13c ±6.79c Free radical (DPPH) scavening activity, % 66.30 69.99 59.28 65.72 61.04 65.84 66.85 73.42 NF ±1.02a ±1.11a ±0.98a ±1.09a ±0.97b ±1.32b ±1.04a ±1.46a 71.47 75.52 65.65 72.25 62.98 69.82 73.78 79.52 Ls SMF ±1.10b ±1.29c ±1.01c ±1.21c ±0.98c ±1.41c ±1.15d ±1.52c 72.21 74.69 63.16 66.47 63.81 72.25 73.22 81.09 Pa SMF ±1.12c ±1.32b ±0.99b ±1.10b ±0.99c ±1.45d ±1.14d ±1.62d 73.22 82.36 88.18 95.62 35.37 42.62 71.84 77.65 Ls SSF ±1.14d ±1.47e ±1.41e ±1.47e ±0.52a ±0.61a ±1.11b ±1.56b 71.38 81.98 71.28 82.52 72.95 75.69 72.95 82.85 Pa SSF ±1.08b ±1.44d ±1.07d ±1.29d ±1.11d ±1.46d ±1.12c ±1.65d Data expressed as means (n = 3) ± SD; SD: standard deviation; p significant, when p ≤ 0.05. GAE 100/g d.w. - gallic acid equivalent per 100/g of dry weight; BI – before protein isolation; AI – after protein isolation; TPC – total phenolic compounds content; NF - nonfermented samples; Ls – Lactobacillus sakei, Pa – Pediococcus acidilactici. a-dThe mean values within a column with different letters are significantly different (p≤0.05).

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Most of the fermented lupine samples had a higher TPC content, in compare with nonfermented samples. The results of free radical (DPPH) scavenging activity of fermented lupine samples have shown that after 48 h of fermentation the DPPH has increased, in comparison with nonfermented samples (except hybrid lines No.1734, No.1072 and No.1800 seeds) and has ranged from 35.37% to 88.18% (in SSF with L. sakei hybrid No.1072 and in SSF with L. sakei Vilniai samples, respectively). The results of the ANOVA test have indicated that the TPC content and free radical (DPPH) scavenging activity of lupine products and lupine protein isolates/concentrates were notably affected by the selection of lupine variety (on TPC (F(33587.649)= 118.895, p≤0.0001); on DPPH (F(142.571)=6.371, p≤0.0001), the fermentation method (on TPC (F(106281.093)=376.218, p≤0.0001); on DPPH (F(142.571)=6.371, p≤0.0001), the type of microorganism applied for the fermentation (on TPC content F(2131.595)=7.545, p=0.0001; on DPPH F(529.680)=23.670, p≤0.0001), the protein isolation process (on DPPH F(1887.750)=84.358, p≤0.0001), however, analysed factors had no significant influence on TPC content. Correlation between TPC content and free radical scavening (DPPH) activity of fermented lupine was not found. The TPC content and free radical (DPPH) scavenging activity in nonfermented wholemeal and protein isolates/concentrates of hybrid lines No.1700, No.1701, No.1800 and No.1702, in SMF and SSF with L. sakei and P. acidilactici strains are presented in Table 3.7.2. In compare to lupine protein isolates/concentrates, in all the cases higher TPC content in lupine wholemeal samples was obtained, ranging from 414.61 ± 4.11 GAE 100/g d.w. to 625.77 ± 6.82 GAE 100/g d.w. (in SMF with L. sakei hybrid No.1701 and in SSF with P. acidilactici hybrid No.1702). In all the cases, higher antioxidant activity of the lupine protein isolates/concentrates was found, in compare to lupine seeds wholemeal (in wholemeal samples on average by 79.09%, and in protein isolates on average by 87.56%). The TPC content and free radical (DPPH) scavenging activity of lupine products and lupine protein isolates/concentrates were significantly affected by the selection of lupine variety (on DPPH (F(579.656)=82.301, p≤0.0001), the fermentation method (on TPC (F(106281.093)=376.218, p≤0.0001); on DPPH (F(4709.037)=668.605, p≤0.0001), however, analysed factors had no significant influence on TPC content, the type of microorganism applied for the fermentation (on TPC content F(6906.062)= 11.973, p≤0.0001; on DPPH F(160.531)=22.793, p≤0.0001) and the protein isolation process (on TPC F(22440.943)=38.907, p≤0.0001; on DPPH F(206.105)=29.264, p≤0.0001).

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Table 3.7.2. The TPC content and free radical (DPPH) scavenging activity in nonfermented wholemeal and protein isolates/concentrates of hybrid lines No.1700, No.1701, No.1800 and No.1072, in SMF and SSF with L. sakei and P. acidilactici strains. Lupine No. 1700 No. 1701 No. 1800 No. 1702 samples BI AI BI AI BI AI BI AI Total phenolic compounds content, GAE 100/g d.w. 419.29 436.87 408.78 400.14 557.82 529.14 552.47 501.28 NF ±3.86a ±4.25a ±4.18a ±4.02a ±5.22a ±5.02a ±5.16a ±5.09a 447.67 458.74 414.61 442.34 589.63 591.53 566.50 574.52 Ls SMF ±4.22b ±4.16b ±4.11a ±4.32b ±5.02b ±5.75c ±5.89b ±5.65b 449.59 478.67 424.72 455.62 587.51 589.64 584.35 587.99 Pa SMF ±4.36b ±4.39c ±4.19c ±4.38d ±4.98b ±5.61b ±5.72c ±5.76c 457.42 499.53 421.33 461.57 599.63 602.32 597.58 599.78 Ls SSF ±4.52c ±4.56e ±4.14b ±4.53e ±5.08d ±6.52d ±5.86d ±5.93d 468.87 487.64 442.21 453.42 593.42 614.84 599.72 625.77 Pa SSF ±4.48d ±4.51d ±4.29d ±4.49c ±5.02c ±6.76e ±5.99e ±6.82e Free radical (DPPH) scavening activity, % 70.27 85.20 64.54 84.96 74.24 83.69 75.62 81.02 NF ±1.09a ±1.64a ±0.97a ±1.58a ±1.09a ±1.52a ±1.14a ±1.62a 72.54 91.41 79.62 85.64 79.22 85.20 80.18 86.83 Ls SMF ±1.25b ±1.48e ±1.15b ±1.32b ±1.08b ±1.54c ±1.35b ±1.52b 76.81 89.56 80.50 88.51 80.36 84.52 80.87 89.67 Pa SMF ±1.31c ±1.43c ±1.26d ±1.37c ±1.11c ±1.53b ±1.38b ±1.59d 82.56 88.84 79.89 89.64 80.97 89.51 85.40 88.51 Ls SSF ±1.38d ±1.41b ±1.16b ±1.42d ±1.12c ±1.58e ±1.44c ±1.52c 88.95 90.41 80.23 89.99 82.54 88.82 86.58 89.24 Pa SSF ±1.42e ±1.49d ±1.25c ±1.43d ±1.14d ±1.56d ±1.48d ±1.58e Data expressed as means (n = 3) ± SD; SD: standard deviation; p significant, when p ≤ 0.05. GAE 100/g d.w. - gallic acid equivalent per 100/g of dry weight; BI – before protein isolation; AI – after protein isolation; TPC – total phenolic compounds content; NF - nonfermented samples; Ls – Lactobacillus sakei, Pa – Pediococcus acidilactici. a-eThe mean values within a column with different letters are significantly different (p≤0.05).

The TPC content and free radical (DPPH) scavenging activity of nonfermented wholemeal and protein isolates/concentrates of varieties Vilciai, Vilniai, hybrid lines No.1072, and No.1734 in SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains are presented in Table 3.7.3. The highest TPC content was found in SMF with P. pentosaceus No.8 and P. pentosaceus No.9 Vilciai lupine wholemeal (402.90 ± 5.18 GAE 100/g d.w. and 406.27 ± 3.14 GAE 100/g d.w., respectively). The comparison of wholemeal samples and protein isolates has shown that in all the cases, lower TPC content and DPPH free radical scavenging activity of lupine isolates/concentrates were found. In all the cases, fermentation has increased TPC content and DPPH free radical scavenging activity of lupine products, while in different fermentation conditions (SMF and SSF), in most of the 157 cases, higher TPC content in SSF samples was established. The results have shown that in most of the cases, the higher TPC content can be obtained in fermented lupine wholemeal, in compare to lupine protein isolates/concentrates.

Table 3.7.3. The TPC content and free radical (DPPH) scavenging activity in nonfermented wholemeal and protein isolates/concentrates of varieties Vilciai, Vilniai and hybrid lines No.1072, No.1734 in SMF and SSF with P. pentosaceus No.8, No.9, and No.10 strains. Lupine Vilciai Vilniai No. 1072 No. 1734 samples BI AI BI AI BI AI BI AI Total phenolic compounds content, GAE 100/g d.w. 243.97 207.42 260.38 211.25 218.21 201.74 216.31 191.47 NF ±6.12a ±5.01a ±4.89a ±4.77a ±3.54a ±2.43a ±4.58a ±4.15a 402.90 321.44 352.14 277.41 259.08 214.47 260.93 203.74 Pp8 SMF ±5.18d ±5.42e ±6.04c ±3.01c ±3.19b ±4.69b ±4.63c ±6.21b 406.27 348.36 350.73 299.87 244.95 199.55 293.34 247.52 Pp9 SMF ±3.14d ±4.98f ±6.02c ±3.41e ±3.01b ±4.01a ±4.19e ±4.03e 329.79 307.01 296.90 241.58 236.56 205.87 282.78 235.21 Pp10 SMF ±3.01c ±4.84d ±4.20b ±2.99b ±2.85b ±3.69a ±4.18d ±4.14c 316.66 276.17 381.33 311.01 290.24 240.14 265.24 221.10 Pp8 SSF ±6.02b ±5.03c ±5.30d ±4.70f ±4.16d ±4.59c ±6.18c ±5.35c 317.79 256.26 299.08 289.54 270.82 212.25 241.29 200.14 Pp9 SSF ±6.03b ±4.81b ±5.28b ±3.15d ±4.79c ±3.13b ±6.16b ±2.51b 311.60 261.70 296.97 273.25 273.63 243.19 282.13 236.40 Pp10 SSF ±5.99b ±5.11b ±5.23b ±4.24c ±4.85c ±3.67c ±6.22d ±2.12d Free radical (DPPH) scavening activity, % 66.30 69.99 59.28 65.72 61.04 65.84 66.85 73.42 NF ±1.02a ±1.11a ±0.98a ±1.09a ±0.97a ±1.32a ±1.04a ±1.46a 67.69 71.22 62.24 68.14 63.64 69.67 72.65 78.68 Pp8 SMF ±1.05b ±1.12b ±1.01b ±1.08b ±1.04b ±1.13b ±1.17b ±1.31b 68.55 73.57 67.51 67.25 63.99 71.41 73.84 74.99 Pp9 SMF ±1.07b ±1.19c ±1.06c ±1.05a ±1.07b ±1.16b ±1.19b ±1.22a 75.38 73.98 71.25 69.82 65.58 73.54 75.17 79.37 Pp10 SMF ±1.29c ±1.22c ±1.11d ±1.12c ±1.11c ±1.18c ±1.22c ±1.32b 74.99 81.47 74.34 72.36 72.27 82.41 84.24 85.97 Pp8 SSF ±1.28d ±1.31d ±1.19e ±1.16d ±1.15d ±1.33d ±1.31d ±1.41c 82.25 82.54 77.82 85.78 76.98 85.24 92.41 91.54 Pp9 SSF ±1.39e ±1.35d ±1.29f ±1.47f ±1.19e ±1.40e ±1.53f ±1.49e 83.47 85.67 79.71 84.24 79.54 81.37 90.71 89.72 Pp10 SSF ±1.44f ±1.48e ±1.30f ±1.41e ±1.31f ±1.38d ±1.50e ±1.48d Data expressed as means (n = 3) ± SD; SD: standard deviation; p significant, when p ≤ 0.05; BI – before protein isolation; AI – after protein isolation; GAE 100/g d.w. – gallic acid equivalent per 100 g of dry weight; TPC – total phenolic compounds content; NF – nonfermented lupine wholemeal samples. Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

The TPC content and free radical (DPPH) scavenging activity of lupine products and lupine protein isolates/concentrates were significantly affected

158 by the selection of lupine variety (on TPC F(72264.776)=2677.906, p≤0.0001; on DPPH (F(878.926)=588.324, p≤0.0001), the fermentation method (on TPC (F(501615.975)=18588.316, p≤0.0001); on DPPH (F(154.305)=103.287, p≤0.0001), the type of microorganism applied for the fermentation (on TPC content F(1419.036)=52.585, p≤0.0001; on DPPH F(154.915)=103.695, p≤0.0001), the protein isolation process (on TPC F(26255.645)=972.952, p≤0.0001; on DPPH F(1167.819)=781.699, p≤ 0.0001) and their interaction (on TPC content F(4637.576)=171.854, p≤0.0001; on DPPH F(11.464)=7.674, p≤0.0001). The TPC content and free radical (DPPH) scavenging activity in nonfermented wholemeal and protein isolates/concentrates of hybrid lines No.1700, No.1701, No.1800 and No.1702in SMF and SSF with P. pentosaceus No.8, No.9 and No.10 strains are presented in Table 3.7.4. In all the cases, higher TPC content was found in lupine wholemeal samples, compared to lupine protein isolates, while the highest content was observed in SSF with P. pentosaceus No.9 hybrid No.1701 isolate samples (588.74 ± 5.49 GAE 100/g d.w.). In all the cases, the higher antioxidant activity of the lupine isolates/concentrates, compared to lupine seeds wholemeal (in wholemeal samples on average 74.56 %, and in protein isolates/concentrates on average 84.07 % higher) was found. The TPC content and free radical (DPPH) scavenging activity of lupine products and lupine protein isolates/ concentrates were significantly affected by the selection of lupine variety (on TPC F(173563.401)=6851.225, p≤0.0001; on DPPH (F(23.837)=12.626, p≤0.0001), the fermentation method (on TPC (F(7424.739)=293.083, p≤0.0001); on DPPH (F(240.413)=127.340, p≤0.0001), the type of microorganism applied for the fermentation (on TPC content F(1333.849)= 52.652, p≤0.0001; on DPPH F(43.740)=23.168, p≤0.0001), the protein isolation process (on TPC F(1433.045)=56.568, p≤0.0001; on DPPH F(2263.488)=1198.901, p≤0.0001) and their interaction (on TPC F(1.138)= 0.603, p≤0.0001), however, analysed factors had no notable influence on antioxidant activity.

159

Table 3.7.4. The TPC content and free radical (DPPH) scavenging activity in nonfermented wholemeal and protein isolates/concentrates of hybrid lines No.1700, No.1701, No.1800 and No.1702 in SMF and SSF with P. pentosaceus No.8, No.9, and No.10 strains. Lupine No. 1700 No. 1701 No. 1800 No. 1702 samples BI AI BI AI BI AI BI AI Total phenolic compounds content, GAE 100/g d.w. 419.29 436.87 408.78 400.14 557.82 529.14 552.47 501.28 NF ±3.86c ±4.25c ±4.18b ±4.02c ±5.22d ±5.02e ±5.16e ±5.09c 387.76 399.25 417.04 399.81 462.93 499.82 472.51 521.36 Pp8 SMF ±4.00b ±4.14b ±3.82b ±3.88b ±5.02b ±4.75c ±4.71c ±5.29d 361.01 354.24 391.14 402.69 459.84 489.69 478.98 522.39 Pp9 SMF ±3.88a ±4.18a ±3.61a ±3.99c ±5.00b ±4.69b ±4.89c ±5.35d 363.54a 382.39 385.50 344.72 473.35 524.35 482.92 500.02 Pp10 SMF ± 3.91 ±3.88a ±3.69a ±3.69a ±4.80c ±4.89e ±4.92d ±5.07c 473.91 447.82 554.44 569.29 445.19 487.55 419.85 498.41 Pp8 SSF ±4.14d ±4.12d ±5.22e ±5.29e ±4.87a ±4.92a ±4.17a ±4.99b 465.19 502.35 557.25 588.74 447.45 496.32 410.56 452.25 Pp9 SSF ±4.57d ±4.89e ±5.22e ±5.49e ±4.89a ±4.96b ±4.11a ±4.82a 522.90 539.52 500.38 529.41 476.45 502.41 428.86 457.28 Pp10 SSF ±5.23e ±4.77f ±4.90d ±4.83d ±4.90c ±5.03d ±4.25b ±4.93a Free radical (DPPH) scavening activity, % 70.27 85.20 64.54 84.96 74.24 83.69 75.62 81.02 NF ±1.09a ±1.64d ±0.97b ±1.58c ±1.09a ±1.52b ±1.14b ±1.62b 80.52 87.25 77.56 86.39 84.39 87.82 80.88 82.20 Pp8 SMF ±1.27d ±1.72c ±1.19c ±1.61d ±1.36d ±1.58c ±1.32d ±1.57b 80.24 85.15 79.59 87.04 82.73 89.69 77.19 86.39 Pp9 SMF ±1.24d ±1.62c ±1.21e ±1.63e ±1.31c ±1.59d ±1.28c ±1.78d 79.84 84.36 77.10 83.36 82.36 91.25 76.92 85.10 Pp10 SMF ±1.19c ±1.58b ±1.19d ±1.49b ±1.32c ±1.67e ±1.21b ±1.73c 73.78 81.20 61.96 79.64 74.42 82.25 81.16 87.30 Pp8 SSF ±1.16b ±1.42a ±0.97b ±1.33b ±1.18b ±1.50a ±1.19d ±1.77e 71.84 82.00 53.00 76.91 73.96 83.28 63.16 76.99 Pp9 SSF ±1.14a ±1.39a ±0.47a ±1.41a ±1.14a ±1.53a ±1.01a ±1.29a 71.38 83.63 77.47 88.42 74.15 84.35 67.50 77.22 Pp10 SSF ±1.12a ±1.51b ±1.18c ±1.49d ±1.15a ±1.47b ±1.02a ±1.37a Data expressed as means (n = 3) ± SD; SD: standard deviation; p significant, when p ≤ 0.05; BI – before protein isolation; AI – after protein isolation; GAE 100/g d.w. - gallic acid equivalent per 100/g of dry weight; TPC – total phenolic compounds content; NF - nonfermented lupine wholemeal samples. Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

3.7.1. The Formation of Antioxidant Properties Showing Compounds and Their Modulation by Using Fermentation

Phenolic compounds are classified by their chemical structure. Phenolic compounds or phenolics are substances that possess an aromatic ring with hydroxyl group [347], and they are one of the biggest and most widely distributed groups of secondary metabolites in plants. They play a role of

160 protection against insects and other plant stress elicitors and one of their main roles are protection and signaling. They are involved in many functions in plants, such as sensorial properties, processes of seed, development, and reproduction [348, 349]. The AA PHE, TYR and TRP are precursors for phenolic acids [350]. Bioactive compounds such as polyphenols have a wide therapeutic potential as they can contribute towards the antioxidant activities [351]. Phenolic compounds have been widely studied, due to their protection against diseases that may be associated with the powerful antioxidant and free radical scavenging properties of these compounds [352]. Phenolic acids and their derivatives flavanols, anthocyanins, condensed tannins are the main polyphenol categories present in legume seeds [353]. Dehulling of legumes results in removal of large amounts of polyphenols as these are concentrated in the seed coat portions. Girish et al. [354] have reported that about one-fourth of the black gram grain was removed as a byproduct after milling process which includes the seed coat rich in polyphenols. The presence of LAB in controlled fermentation contributes to the simple phenolic conversion and the depolymerization of high molecular weight phenolic compounds [355]. Fermentation increases the TPC content in legumes, and these changes could be associated with bio-modification between soluble phenolic compounds and the release of bound phenolic compounds by microorganisms involved in the fermentation process [356]. Finally, after comparing all lupine samples (nonfermented and fermented), the results of the ANOVA test have indicated that the TPC content and free radical (DPPH) scavenging activity of lupine products were significantly affected by the selection of lupine variety (on TPC F(317112.720)=175.838, p≤ 0.0001; on DPPH (F(1673.546)=169.838, p≤0.0001), the fermentation method (on TPC (F(280799.902)=155.703, p≤0.0001); on DPPH (F(794.091)=80.588, p≤0.0001), the type of microorganism applied for the fermentation (on TPC content F(731496.755)=405.613, p≤0.0001; on DPPH F(390.805)=39.661, p≤0.0001) and the protein isolation process (on TPC F(43000.631)=23.844, p≤0.0001; on DPPH F(4404.501)=446.987, p≤ 0.0001). Additionally, during fermentation microorganisms produce phenolic compounds as secondary metabolites [357]. In recent years, much attention has been paid to researching a number of substances with antioxidant properties, which include vitamins, microelements, and many other compounds naturally occurring in plants and producing positive effects both in human and animal nutrition [358–360]. It is considered that phenolic structures play a crucial role in bioactive activities [361]. Antioxidative action is one of the prime physiological functions that protect living organisms from oxidative damage caused by reactive oxygen species [362]. The free radicals attack various biomolecules, cellular machinery, 161 cell membrane, lipids, proteins, enzymes and DNA causing oxidative stress and ultimately cell death [363]. Antioxidant activity is closely related to phenolic content in legumes [364].

3.8. Influence of Technological Factors on Isoflavones Content in Lupine Bioproducts

The genistein concentration in nonfermented seeds wholemeal and protein isolates/concentrates of lupine varieties Vilciai, Vilniai and hybrid lines No.1072, and No.1734 in SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.8, No.9 and No.10 strains is presented in Table 3.8.1. Among all the isoflavones analysed (daidzein, genistein, formononetin, and biochanin-A), only genistein in seeds wholemeal and protein isolates/concentrates was present. In all the cases, the genistein concentration in lupine protein isolates/concentrates was found to be lower, compared to wholemeal samples, and in lupine wholemeal genistein concentration has ranged from 0 µg/g to 35.60 ± 0.41 µg/g (in SMF with P. acidilactici variety Vilniai, and with L. sakei variety Vilniai samples, respectively). In lupine protein isolates/concentrates genistein concentration has ranged from 0 µg/g to 52.33 µg/g (in SMF with P. acidilactici variety Vilniai, and in SMF with P. pentosaceus No.9 variety Vilciai samples, respectively). On average 19.11% higher concentration of genistein in lupine wholemeal samples was found, compared to lupine protein isolates/concentrates. The results of the ANOVA test have indicated that the genistein concentration in lupine wholemeal and protein isolates/concentrates was significantly affected by the selection of lupine variety (F(1349.540)=1318.630, p≤0.0001), the fermentation method (F(75.172)=73.451, p≤0.0001), the type of microorganism applied for the fermentation (F(672.254)=656.856, p≤0.0001), the protein isolation process (F(260.534)=254.566, p≤0.0001) and the interaction of these factors (F(4.000)=3.908, p≤0.0001). The genistein concentration in nonfermented seeds wholemeal and protein isolates/concentrates in lupine hybrid lines No.1700, No.1701, No.1800 and No.1702 in SMF and SSF with L. sakei and P. acidilactici and P. pentosaceus No.8, No.9 and No.10 strains is presented in Table 3.8.2. In all the cases, the genistein concentration in lupine protein isolates/concentrates was found to be lower, compared to wholemeal samples, and in wholemeal samples genistein concentration has ranged from 0 µg/g to 42.55 ± 0.36 µg/g (in SMF with L. sakei and P. acidilactici hybrid No.1702, and in SSF with L. sakei hybrid No.1800 samples, respectively). On average, 18.31% higher concentration of genistein in lupine wholemeal samples was found, compared to lupine protein isolates/concentrates. In lupine protein 162

Table 3.8.1. The genistein concentration in nonfermented seeds wholemeal and protein isolates/concentrates of lupine varieties Vilciai, Vilniai and hybrid lines No.1072, and No.1734 in SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.8, No.9 and No.10 strains. Genistein concentration, µg/g Lupine Vilciai Vilniai No.1072 No.1734 samples BI AI BI AI BI AI BI AI 11.79 9.94 11.60 9.82 9.33 7.54 7.49 5.35 NF ±0.13c ±0.08b ±0.12c ±0.09c ±0.09d ±0.08d ±0.09b ±0.05b 6.42 4.58 35.60 30.93 20.41 16.20 19.24 15.51 SMF Ls ±0.07a ±0.05a ±0.41d ±0.39d ±0.23d ±0.17d ±0.24e ±0.21e 24.88 20.16 6.89 4.60 6.87 4.74 SMF Pa - - ±0.22c ±0.19c ±0.07a ±0.05b ±0.07b ±0.06b 1.25 29.32 27.11 3.49 2.87 51.20 48.43 SMF Pp8 - ±0.02a ±0.27e ±0.32e ±0.04a ±0.03a ±0.47f ±0.49f 52.33 48.17 5.02 2.68 10.23 9.78 34.41 33.34 SMF Pp9 ±0.45e ±0.42f ±0.06a ±0.03a ±0.12d ±0.09e ±0.33e ±0.38e 26.30 22.98 7.60 5.30 4.99 3.39 9.93 8.39 SMF Pp10 ±0.29d ±0.24c ±0.09c ±0.06c ±0.06b ±0.04b ±0.09c ±0.08c 34.20 29.22 10.77 8.19 7.02 4.51 7.69 4.22 SSF Ls ±0.38e ±0.26d ±0.12b ±0.09c ±0.09b ±0.07a ±0.09c ±0.05b 29.32 27.75 6.21 4.22 24.39 20.20 4.83 3.62 SSF Pa ±0.27d ±0.24d ±0.08a ±0.05a ±0.23e ±0.19e ±0.05a ±0.03a 35.21 30.79 6.81 4.90 7.58 6.00 17.88 15.93 SSF Pp8 ±0.35c ±0.28d ±0.07b ±0.04b ±0.09c ±0.08d ±0.17d ±0.19f 39.98 36.81 15.77 13.45 14.38 12.45 9.64 6.95 SSF Pp9 ±0.39d ±0.37e ±0.16d ±0.14d ±0.16e ±0.13f ±0.08c ±0.07c 6.79 4.74 49.44 47.77 6.31 4.93 5.71 3.69 SSF Pp10 ±0.07b ±0.05a ±0.42f ±0.40f ±0.09c ±0.05c ±0.04a ±0.05a The data expressed as mean values (n = 3) ± SD; SD – standard deviation. a-f The mean values within a column with different letters are significantly different (p≤0.05). BI – before isolation; AI – after isolation; NF – nonfermented samples; SMF – submerged fermentation; SSF – solid state fermentation; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-fThe mean values within a column with different letters are significantly different (p≤0.05). isolates/concentrates genistein concentration has ranged from 2.94 µg/g to 37.37 µg/g (in SMF with L. sakei hybrid No.1800, and in SMF with P. acidilactici hybrid No.1700 samples, respectively). The results of the ANOVA have test indicated that the genistein concentration in lupine wholemeal and protein isolates/concentrates was significantly affected by the selection of lupine variety (F(1742.100)=12086.822, p≤0.0001), the fermentation method (F(9.186)=63.733, p≤0.0001), the type of microorganism applied for the fermentation (F(159.181)=1104.410, p≤0.0001), the protein isolation process (F(477.468)=3312.710, p≤0.0001) and the interaction of these factors (F(6.885)=47.766, p≤0.0001).

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Table 3.8.2. The genistein concentration in nonfermented seeds wholemeal and protein isolates/concentrates of lupine hybrid lines No.1700, No.1701, No.1800 and No.1702 in SMF and SSF with L. sakei and P. acidilactici and P. pentosaceus No.8, No.9 and No.10 strains. Genistein concentration, µg/g Lupine No.1700 No.1701 No.1800 No.1702 samples BI AI BI AI BI AI BI AI 4.37 2.49 17.19 13.02 17.12 14.36 6.38 5.27 NF ±0.11b ±0.15a ±0.72f ±0.16e ±0.68b ±0.22b ±0.14a ±0.09a 23.55 20.86 24.69 20.87 4.82 2.94 SMF Ls - - ±0.26d ±0.21c ±0.27f ±0.22f ±0.05a ±0.03a 41.27 37.37 9.98 7.49 25.70 21.60 SMF Pa - - ±0.39f ±0.44f ±0.09b ±0.08b ±0.28c ±0.20d SMF 4.38 3.92 31.03 25.63 21.57 18.46 7.05 6.39 Pp8 ±0.12b ±0.09b ±0.67g ±0.41f ±0.79c ±0.57d ±0.15b ±0.21b SMF 25.45 19.11 11.25 7.36 3.81 2.75 8.46 7.02 Pp9 ±0.84d ±0.35d ±0.19d ±0.21c ±0.08a ±0.12a ±0.18c ±0.11c SMF 3.16 2.35 9.48 8.81 41.68 36.46 24.07 16.96 Pp10 ±0.43a ±0.21a ±0.15c ±0.09d ±1.02e ±0.23f ±0.92e ±0.28e 26.40 23.93 9.32 7.78 42.55 38.14 SSF Ls - - ±0.24e ±0.24d ±0.07b ±0.06b ±0.36d ±0.34e 17.50 14.31 14.35 10.66 18.63 14.84 SSF Pa - - ±0.17c ±0.18b ±0.13d ±0.12d ±0.21b ±0.16c SSF 17.72 14.62 3.77 2.59 29.12 21.24 7.04 5.91 Pp8 ±0.81c ±0.39c ±0.06a ±0.10a ±0.98d ±0.35e ±0.17b ±0.09a SSF 28.03 16.29 4.88 3.26 18.95 16.01 8.31 6.93 Pp9 ±0.93d ±0.28e ±0.13b ±0.15b ±0.82b ±0.18c ±0.21c ±0.31b SSF 18.60 13.42 14.31 9.89 19.82 16.41 17.30 14.16 Pp10 ±0.73c ±0.13c ±0.62e ±0.27d ±0.79b ±0.23c ±0.60d ±0.20d The data expressed as mean values (n = 3) ± SD; SD – standard deviation. NF– nonfermented samples; SMF – submerged fermentation; SSF – solid state fermentation, BI – before isolation; AI – after isolation; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

3.8.1. Isoflavones in Lupine Seeds and Their Possible Changes During Technological Processes

Isoflavones are a class of plant secondary metabolites [365]. Isoflavones are similar to estradiol-17 beta molecules and they can induce similar effects to those of estrogens, avoiding the risks associated with the treatments with these drugs [366]. They are most widely known bioactive compounds of legumes, which together with phenolic acids and procyanidins constitute the major phenolic compounds present in their seeds [367]. Lupine seeds contain various bioactive compounds, such as isoflavones and phytosterols [368], and in lupine and soybean seeds genistein is the main isoflavone

164

[369]. Numerous studies have provided a wealth of information on the anti- inflammatory and anti-carcinogenic properties effects and the underlying mechanisms of isoflavones, however, some studies have raised concerns about negative effects induced by isoflavones [370, 371]. Also, anti- flammatory properties of isoflavones occure in various animal models through increased antioxidative activities, NF-κB regulation, and reduced pro-inflammatory enzymes activities as well as cytokine levels, thereby encouraging the application of isoflavones in a range of inflammatory diseases. The potential carcinogenic and immunosuppressive effects of isoflavone raise concerns about the risks of its consumption noticeably. However, extensive evaluations are still warranted to explore the exact mechanisms and answer the safety questions [371]. There are studies claiming that genistein is capable of normalizing the increased superoxide anion production and nitrotyrosine formation in streptozotocin-induced type 1 diabetic mice [372]. Isoflavones are usually found in free form as aglycones, but glycosides can be formed with some types of monosaccharides, O‐glycosides being the most common [373]. The highest content of isoflavones has been found in soybeans (Glycine max) [374] and red clover (Trifolium pratense) [375] but lupine seeds (Lupinus spp.) can also be a good source of these compounds [376]. It has been reported that the isoflavones content is reduced during the processing of soybeans, and the loss of isoflavones in the water used for soaking raw soybeans can be as high as 31% [377]. The genistein concentration in lupine wholemeal was significantly affected by the selection of lupine variety (F(479.754)= 646.967, p≤0.0001), the type of microorganism applied for the fermentation (F(46.791)=63.099, p≤0.0001) and the interaction of these factors (F(557.277)=751.509, p≤0.0001), however, the fermentation method had no notable influence on genestein concentration. Furthermore, the amount of isoflavones can be decreased during coagulation of proteins (by 44%) and during alkaline extraction of protein isolate (by 53%) [378]. The genistein concentration in lupine protein isolates/concentrates was significantly affected by the selection of lupine variety (F(484.754)=246.967, p≤0.0001), the type of microorganism applied for the fermentation (F(40.701)=23.121, p≤0.0001), the protein isolation process (F(509.129)=302.289, p≤0.0001) and the interaction of these factors (F(2,886)=4.861, p≤0.0001), however, the fermentation method had no significant influence on genistein concentration.

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3.9. The Trypsin Inhibitors Activity in Lupine Products

The trypsin inhibitors activity (TIA) (%) in nonfermented, SMF and SSF with L. sakei and P. acidilactici and P. pentosaceus No.8, No.9 and No.10 strains in lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734 seeds wholemeal is presented in Table 3.9.1. In all the cases, in SMF and SSF with L. sakei, P. acidilactici and P. pentosaceus No.8, No.9 and No.10 strains lupine wholemeal samples TIA was on average 7.16% lower, in compare with nonfermented samples. In SMF samples TIA has ranged from 19.40 ± 0.48% to 26.74±0.62% (in SMF with L. sakei Vilniai and in SMF with P. pentosaceus hybrid No.1734 samples, respectively), and in SSF samples TIA has ranged from 18.42±0.38% to 26.72±0.61% (in SSF with P. acidilactici Vilciai and in SSF with P. acidilactici hybrid No.1072 samples, respectively). To compare SMF and SSF samples, TIA in SSF samples was on average 1.41% lower. The results of the ANOVA test have indicated that the TIA in lupine wholemeal was significantly affected by the selection of lupine variety (F(19.114)=68.271, p≤0.0001), the fermentation method (F(59.714)=213.283, p≤0.0001), the type of microorganism applied for the fermentation (F(13.971)=49.902, p≤0.0001) and the interaction of these factors (F(9.223)=32.944, p≤0.0001). The TIA (%) in nonfermented, SMF and SSF with L. sakei and P. acidilactici and P. pentosaceus No.8, No.9 and No.10 strains in lupine hybrid lines No.1700, No.1701, No.1800 and No.1702 seeds wholemeal is presented in Table 3.9.2. In most of the fermented lupine seeds wholemeal samples TIA (except in SMF with P. pentosaceus No.8 hybrid No.1701, in which it was increased) was decreased. In SMF and SSF wholemeal TIA was on average 5.02% lower, in compare with nonfermented samples. In SMF samples TIA has ranged from 18.40±0.38% to 27.30 ± 0.65% (in SMF with P. acidilactici hybrid No.1701 and in SMF with P. pentosaceus No.8 hybrid No.1701, respectively). In SSF samples TIA was slightly lower (on average by 0.53%), compared to SMF, and has ranged from 19.29 ± 0.32% to 25.90±0.59% (in SSF with P. pentosaceus No.9 and in SSF with L. sakei hybrid No.1702 wholemeal samples, respectively). The TIA in lupine seeds wholemeal was significantly affected by the selection of lupine variety (F(19.118)=68.272, p≤0.0001), the fermentation method (F(8.395)= 31.134, p≤0.0001), the type of microorganism applied for the fermentation (F(8.042)=29.824, p≤0.0001) and the interaction of these factors (F(2.541)= 9.425, p≤0.0001).

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Table 3.9.1. The trypsin inhibitors activity (TIA) (%) in nonfermented, SMF and SSF with L. sakei and P. acidilactici and P. pentosaceus No.8, No.9 and No.10 strains in lupine varieties Vilciai, Vilniai, hybrid lines No.1072 and No.1734 seeds wholemeal. Trypsin inhibitors activity, % Lupine samples Vilciai Vilniai No.1072 No.1734 NF 29.30±0.62e 28.80±0.60e 28.90±0.61d 29.80±0.63d SMF Ls 20.10±0.51c 19.40±0.48a 19.60±0.46a 20.80±0.52a SMF Pa 21.12±0.56d 20.52±0.49b 20.28±0.53b 22.41±0.57b SMF Pp8 23.52±0.69d 25.47±0.57d 26.43±0.69d 26.74±0.62d SMF Pp9 21.08±0.52c 22.55±0.48c 25.44±0.62c 24.99±0.55c SMF Pp10 22.31±0.54d 23.77±0.51c 23.61±0.58c 24.62±0.59c SSF Ls 19.00±0.45b 22.80±0.51d 24.00±0.54c 22.70±0.50b SSF Pa 18.42±0.38a 21.57±0.56c 26.72±0.61c 24.44±0.66c SSF Pp8 20.89±0.50b 21.23±0.52b 21.08±0.45b 20.05±0.43a SSF Pp9 20.44±0.47a 20.09±0.46a 20.49±0.38a 19.69±0.29a SSF Pp10 20.19±0.45a 20.11±0.41a 22.26±0.48b 20.39±0.35b TIA – trypsin inhibitors activity; NF – nonfermented samples; SMF – submerged fermented; SSF – solid state fermented. The data are expressed as mean values (n = 3) ± SD; SD – standard deviation. Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-eThe mean values within a column with different letters are significantly different (p≤0.05).

Table 3.9.2. The TIA (%) in nonfermented, SMF and SSF with L. sakei and P. acidilactici and P. pentosaceus No.8, No.9 and No.10 strains in lupine hybrid lines No.1700, No.1701, No.1800 and No.1702 seeds wholemeal. Trypsin inhibitors activity, % Lupine samples No.1700 No.1701 No.1800 No.1702 NF 28.20±0.58d 26.80±0.55d 28.60±0.59e 28.00±0.58d SMF Ls 22.70±0.56a 19.80±0.49b 20.80±0.51b 20.50±0.52a SMF Pa 23.66±0.59b 18.40±0.38a 20.75±0.49a 21.87±0.56b SMF Pp8 24.51±0.57d 27.30±0.65e 24.64±0.61c 25.13±0.58d SMF Pp9 23.78±0.51c 24.71±0.61d 25.99±0.52d 23.77±0.55c SMF Pp10 23.11±0.47c 24.97±0.57d 23.44±0.48c 23.01±0.49c SSF Ls 24.20±0.55c 25.37±0.58c 24.30±0.56d 25.90±0.59c SSF Pa 25.14±0.61c 24.01±0.57c 22.32±0.53c 24.14±0.58c SSF Pp8 21.44±0.54b 23.81±0.48c 22.31±0.41b 21.62±0.38b SSF Pp9 19.29±0.32a 22.66±0.53b 21.07±0.39a 20.18±0.27a SSF Pp10 20.73±0.46b 21.52±0.41a 21.53±0.44a 20.74±0.32a TIA - trypsin inhibitors activity; NF– nonfermented samples; SMF – submerged fermented; SSF – solid state fermented. The data are expressed as mean values (n = 3) ± SD; SD – standard deviation. Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-eThe mean values within a column with different letters are significantly different (p≤0.05).

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3.9.1. The Perspectives of Reduction of Trypsin Inhibitors in Plant Based Material

Trypsin inhibitors (TIs) are one of the most relevant anti-nutritional factors because they reduce digestion and absorption of dietary proteins [379]. Trypsin, a water soluble globular protein, is a proteolytic enzyme that cleaves peptide bonds at the carboxylic groups of arginine and lysine. TIs are classified as small proteins or polypeptides that exhibit inhibitory activity against trypsin and can lead to certain diseases in animals and humans [380, 381]. The inhibitory role of trypsin inhibitors comes from their bindings to trypsin and other proteins, causing major protein structural changes [382, 383]. TIs strongly inhibit the activity of key pancreatic enzymes, particularly trypsin and chymotrypsin, thereby reducing digestion and absorption of dietary proteins by the formation of complexes that are indigestible even in the presence of high amounts of digestive enzymes [384]. In order to improve the nutritional quality of legumes, different processing methods have been developed to inactivate or to diminish TIs below threshold limit [385]. The TIA in lupine seeds wholemeal was significantly affected by the selection of lupine variety (F(20.124)=54.257, p≤0.0001), the fermentation method (F(7.694)=28.945, p≤0.0001), the type of microorganism applied for the fermentation (F(7.882)=27.554, p≤0.0001) and the interaction of these factors (F(1.997)=8.447, p≤0.0001). Legume TIs are classiﬁed into 2 families according to their molecular size: Kunitz with molecular weights around 20 kDa and Bowman-Birk of approximately 8 kDa [379]. Fermentation is an anaerobic and catabolic process where complex molecules are transformed into simple samples by microorganisms. For example, bacterial and yeast fermentation involves proteolytic activity that increases AA bioavailability by degrading unwanted substances, such as proteinase inhibitors. Likewise, Adeyemo and Onilude [386] have concluded that fermentation with L. plantarum inactivated 99.17% of the TIA, and Chi and Cho [387] have reported that by SSF with L. acidophilus, L. plantarum, B. amyloliquefaciens and S. cerevisiae separately, over a period of 2 days, TIA was reduced by 82.6%, 88.9%, 85.9%, and 73.8%, respectively.

3.10. The Digestibility In Vitro of Lupine Wholemeal Protein and Protein Isolates/Concentrates

The in vitro protein digestibility (%) of nonfermented, SMF and SSF lupine varieties Vilciai, Vilciai and hybrid lines No.1072, No.1734 wholemeal and seeds protein isolates/concentrates is presented in Table 3.10.1. 168

After comparison of nonfermented wholemeal and isolates/concentrates samples in vitro, protein digestibility in isolates/concentrates was discovered to be 2.63% higher. In all the cases, higher protein digestibility was established in lupine protein isolates/concentrates (except in SSF with L. sakei hybrid No.1072 sample) and it has ranged from 86.15±0.74% to 97.88± 1.16% (in SSF with L. sakei Vilciai isolates and in SMF with P. acidilactici Vilciai isolates, respectively), compared to wholemeal.

Table 3.10.1. The in vitro protein digestibility (%) of nonfermented, SMF and SSF lupine varieties Vilciai, Vilciai and hybrid lines No.1072, No.1734 wholemeal and seeds protein isolates/concentrates. In vitro protein digestibility, % Lupine Vilciai Vilniai No.1072 No.1734 samples BI AI BI AI BI AI BI AI 81.75 83.43 86.63 89.55 84.83 86.71 84.29 88.34 NF ±0.77a ±0.71a ±0.79d ±0.80b ±0.74a ±0.77b ±0.69a ±0.81a 94.60 97.60 79.94 88.47 86.27 92.02 88.45 92.07 SMF Ls ±1.04c ±1.15d ±0.69a ±0.81a ±0.77c ±0.98d ±0.86c ±0.97c 96.78 97.88 89.53 92.74 84.82 87.92 93.70 96.29 SMF Pa ±1.14d ±1.16d ±0.84c ±0.97 ±0.68a ±0.82c ±0.94d ±1.02d 79.94 85.24 94.06 97.58 90.62 93.45 85.55 89.63 SMF Pp8 ±0.88b ±0.92b ±1.03d ±1.05d ±0.93d ±0.93d ±0.81d ±0.80d 83.38 88.41 85.73 91.09 83.92 89.55 87.54 91.40 SMF Pp9 ±0.93d ±0.99c ±0.90c ±1.01cd ±0.73a ±0.75d ±0.69d ±0.79d 81.20 85.69 93.69 95.47 86.09 91.99 80.12 84.96 SMF Pp10 ±0.77a ±0.95b ±0.94d ±0.98d ±0.79d ±0.81d ±0.75a ±0.86a 83.01 86.15 83.38 88.74 85.91 82.14 86.09 91.52 SSF Ls ±0.79b ±0.74b ±0.78b ±0.83a ±0.62b ±0.73a ±0.84c ±0.99b 83.56 86.74 83.74 89.84 86.63 87.00 85.91 88.42 SSF Pa ±0.77b ±0.76c ±0.72b ±0.88b ±0.65d ±0.82c ±0.78b ±0.81a 81.85 84.98 84.28 85.47 85.19 89.40 83.92 91.05 SSF Pp8 ±0.78c ±0.96b ±0.82b ±0.89b ±0.76c ±0.82c ±0.66b ±1.03d 81.50 86.47 83.92 87.11 83.92 88.41 83.74 88.41 SSF Pp9 ±0.85a ±1.01d ±0.80a ±0.92b ±0.72b ±0.93c ±0.71b ±0.91b 83.74 87.52 83.01 84.12 83.74 84.91 83.92 89.54 SSF Pp10 ±0.73d ±0.97d ±0.77a ±0.79a ±0.89b ±0.94a ±0.74c ±1.01c SMF – submerged fermentation; SSF – solid state fermentation; NF – nonfermented samples. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; . Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-dThe mean values within a column with different letters are significantly different (p≤0.05).

After comparison of varieties Vilciai and Vilniai and hybrids No.1072 and No.1734 samples, the highest in vitro protein digestibility was established in SMF with P. acidilactici variety Vilciai isolates (97.88 ±

169

1.16%). In vitro protein digestibility of SMF lupine protein isolates was established to be on average 3.82% higher, in compare to SSF samples. In SMF wholemeal in vitro protein digestibility was 2.64% higher, compared to nonfermented samples. The lowest in vitro protein digestibility was found to be of SMF with L. sakei variety Vilniai wholemeal (79.94 ± 0.69%). The results of the ANOVA test have indicated that the in vitro protein digestibility of both lupine products (wholemeal and protein isolates/concentrates) was significantly affected by the selection of lupine variety (F(47.242)=53.448, p≤0.0001), the fermentation method (F(922.432)= 1043.619, p≤0.0001), the type of microorganism applied for the fermentation (F(86.620)=98.000, p≤0.0001), the protein isolation process (F(687.943)=778.323, p≤0.0001) and the interaction of these factors (F(4.408)=4.988, p≤0.0001). The in vitro protein digestibility (%) of nonfermented, SMF and SSF lupine hybrid lines No.1700, No.1701, No.1800 and No.1702 wholemeal and seeds protein isolates/concentrates is presented in Table 3.10.2 After comparison of in vitro protein digestibility of nonfermented wholemeal and nonfermented protein isolates/concentrates, 2.15% higher digestibility of isolates/concentrates was established. In all the cases, higher in vitro protein digestibility of lupine protein isolates/concentrates was found and it has ranged from 82.11 ± 0.88% to 97.85 ± 0.99% (in SMF with P. pentosaceus hybrid No.1700 isolates and in SMF with P. pentosaceus No.9 hybrid No.1800 isolates, respectively), compared to wholemeal. After comparison of in vitro protein digestibility of hybrid lines No.1700, No.1701, No.1800 and No.1702 wholemeal protein and protein isolates/concentrates, the highest in vitro protein digestibility was established to be of SMF with P. pentosaceus No.9 hybrid No.1700 protein isolates (95.50 ± 1.11%), while the lowest (21.90% lower) in vitro digestibility of SMF with P. pentosaceus No.9 hybrid No.1800 wholemeal samples was found. The in vitro protein digestibility of both lupine products (wholemeal and protein isolates/ concentrates) was significantly affected by the selection of lupine variety (F(172.954)=210.564, p≤0.0001), the fermentation method (F(332.280)= 404.539, p≤0.0001), the type of microorganism applied for the fermentation (F(40.960)=49.867, p≤0.0001) and the protein isolation process (F(0.139)= 0.169, p≤0.0001).

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Table 3.10.2. The in vitro protein digestibility (%) of nonfermented, SMF and SSF lupine hybrid lines No.1700, No.1701, No.1800 and No.1702 wholemeal and seeds protein isolates/concentrates. In vitro protein digestibility, % Lupine No.1700 No.1701 No.1800 No.1702 samples BI AI BI AI BI AI BI AI 85.55 88.14 83.92 85.02 83.56 85.24 85.73 88.96 NF ±0.87b ±0.90b ±0.82b ±0.85b ±0.81a ±0.83a ±0.85b ±0.92b 87.72 92.54 78.49 83.69 89.77 94.24 82.65 87.69 SMF Ls ±0.95c ±0.99bc ±0.68a ±0.82a ±0.92c ±0.94d ±0.78a ±0.71a 80.84 85.24 83.92 87.63 88.62 92.25 85.73 89.47 SMF Pa ±0.74a ±0.78a ±0.78c ±0.93c ±0.85b ±0.92c ±0.87b ±0.78c SMF 86.09 89.97 81.57 86.41 92.97 97.54 90.44 93.41 Pp8 ±0.95a ±0.88b ±0.78a ±0.91b ±0.98f ±1.03e ±0.90c ±0.91c SMF 95.50 97.85 87.00 92.04 73.60 82.11 94.06 96.88 Pp9 ±1.11f ±0.99 ±0.84d ±0.95d ±0.72a ±0.88a ±0.94e ±0.97e SMF 91.52 94.36 93.69 96.57 81.57 85.34 84.46 88.67 Pp10 ±1.02e ±1.07d ±0.94e ±0.98e ±0.88b ±0.92c ±0.77a ±0.86b 89.26 93.55 83.97 89.54 90.62 91.65 91.70 93.62 SSF Ls ±0.93d ±0.91c ±0.67d ±0.97d ±0.93d ±0.84b ±0.91d ±0.92d 89.35 94.45 85.37 89.62 91.34 93.54 90.80 93.79 SSF Pa ±0.87d ±0.94d ±0.79c ±0.99d ±0.92d ±0.96cd ±0.88c ±0.96d 88.99 91.25 84.10 87.44 90.62 93.00 93.69 95.62 SSF Pp8 ±0.88c ±1.01c ±0.79b ±0.85c ±0.93d ±0.97d ±0.99d ±0.91d 87.00 89.41 88.00 92.01 92.43 95.40 94.42 96.11 SSF Pp9 ±0.82b ±0.91b ±0.82d ±0.93d ±0.98e ±1.01d ±1.05f ±1.04e SSF 92.07 95.57 83.92 87.64 89.53 92.51 91.52 94.90 Pp10 ±0.94d ±0.93e ±0.76b ±0.83c ±0.84d ±0.94c ±0.95c ±0.98c SMF – submerged fermentation; SSF – solid state fermentation; NF – nonfermented lupine samples. Data expressed as mean values (n = 3) ± SD; SD – standard deviation; Ls – Lactobacillus sakei; Pa – Pediococcus acidilactici; Pp8 – P. pentosaceus No.8; Pp9 – P. pentosaceus No.9; Pp10 – P. pentosaceus No.10. a-fThe mean values within a column with different letters are significantly different (p≤0.05).

3.10.1. The Digestibility of Lupine Seed Protein and Technologies to Improve It

The quality of a protein source encompasses both the amino acid composition to be digested, absorbed, and used, and the ability of that protein to be digested, absorbed, and used for metabolic functions. Protein isolates/ concentrates manufacturing can increase protein content and digestibility and this is related to the removal of other components, such as starch. In addition, the methods of manufacturing the isolates/concentrates may affect protein content and digestibility [388]. The in vitro protein digestibility of lupine wholemeal was significantly affected by the selection of lupine

171 variety (F(39.338)=210.564, p≤0.0001), the fermentation method (F(10.473)=27.164, p≤0.0001), the type of microorganism applied for the fermentation (F(15.787)=40.947, p≤0.0001) and the interaction of these factors (F(24.710)=64.093, p≤0.0001). There is an opinion stating that poor bioavailability and limited essential amino acid content in plant-based protein (legumes and cereals) represent a major issue for the level of their consumption. β-sheets are the main components in legume proteins, and these elements play a major role in decreasing protein digestibility [389]. The majority of dietary proteins are fully degraded and absorbed in the small intestine: after a meal, proteins are denatured by acid and hydrolysed by gastric pepsin in the stomach, further hydrolysed by pancreatic proteases, subsequently degraded by small intestinal enterocyte membrane exopeptida- ses and absorbed across the small intestinal enterocytes into the bloodstream as individual AA for use in the body. However, proteins are not always digested in this way. Some proteins are not easily digested (either inherently based on their source or due to processing conditions) and can survive intact or partially intact in the colon [390]. The in vitro protein digestibility of lupine protein isolates/concentrates was significantly affected by the selection of lupine variety (F(24.853)=73.239, p≤0.0001), the fermentation method (F(10.684)=31.484, p≤0.0001), the type of microorganism applied for the fermentation (F(19.204)=56.592, p≤0.0001), the protein isolation process (F(186.815)=519.425, p≤0.0001) and the interaction of these factors (F(26.100)=76.915, p≤0.0001). Also, protein digestibility varies depending on the source, and the digestibility of animal proteins (meat and dairy proteins) is higher (typically >90%) than that of plant proteins (70–90%). In early weaned-piglets, plant sources of protein such as soybean meal were lower in digestibility compared with animal proteins such as whey and fish [391].

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CONCLUSIONS

1. The analysed newly bred in Lithuania lupine seed varieties (Vilciai, Vilniai and hybrid lines Nos.1072, 1734, 1700, 1701, 1800 and 1702) according to their chemical composition are useful for the preparation of the high value proteinaceous stock/products, and lupine variety has a significant influence on seeds chemical composition (p≤0.0001): 1.1. The highest protein content was established in Vilciai variety seeds (40.8%); in hybrid lines seed protein content was from 20.1% to 25.7% lower (in Nos.1700 and 1800, respectively). 1.2. Carbohydrates content in lupine seeds has ranged from 41.6% to 50.8%, in Vilciai and No.1800 seeds, respectively. 1.3. The lowest fat content was obtained in Vilciai seeds (4.4%), and the main fatty acids in lupine seeds were the following: unsaturated C18:1 and C18:2, saturated - C16:0, monounsaturated - C15:1 and C20:1. 1.4. The highest ash content was discovered in Vilciai seeds (4.3%). 1.5. The alkaloid content in Vilciai and Vilniai seeds was 0.030% and 0.021% respectively, and it was higher than the one in hybrid lines seed. 1.6. Macro elements content in lupine seeds was ranged in the following order: K > Mg > Ca > Na (13.230 > 2.446 > 2.021 > 1.099 g/kg d.w.) 1.7. Micro elements content in lupine seeds was ranged in the following order: Mn > Fe > Zn > Sr > Cu > Al > Ni > Cr > Se > Co > Pb > Cd > As > Ag (95.96 > 60.08 > 43.92 > 17.45 > 6.273 > 3.114 > 1.659 > 1.281 > 0.1063 > 0.1050 > 0.076 > 0.038 > 0.023 mg/kg d.w.)

2. In most of the cases, the fermentation with selected LAB strains is usefull technology for reducing TIA and increasing lupine seeds protein digestibility in vitro: 2.1. In most of the fermented samples (except SMF with P. pentosaceus Nr.8 No.1701) TIA was lower than in NF samples. TIA in lupine seeds was remarkably influenced by lupine variety (p≤0.0001), fermentation technology (p≤0.0001) and LAB used for fermentation (p≤0.0001). 2.2. Treatment by using SMF and SSF has increased lupine seeds wholemeal protein digestibility in vitro, and it has ranged from 85.3% to 92.6% (SMF No.1800 and SSF No.1702, respectively) on average, in compare to NF samples. It was discovered that the significant influence on protein isolates/concentrates digestibility in vitro has lupine variety, fermentation technology, LAB used for fermentation and protein isolation/concentration process, and interaction of analysed factors was not found (p≤0.0001).

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3. The influence of technological factors on the changes of TPC and genistein content, as well as on antioxidant properties of the treated lupine seeds wholemeal and protein isolates/concentrates has varied according to the technology used: 3.1. In most of the cases, higher TPC content was found in lupine wholemel (on average TPC content has ranged from 318.2% to 644.0%), compared to TPC content in isolates/concentrates (on average TPC content has ranged from 236.6% to 525.1%). 3.2. In most of the cases, higher antioxidant activity was established in fermented lupine samples and it has ranged from 4.4% to 17.2%, in compare to nonfermented samples. The significant influence on antioxidant activity and TPC content in lupine wholemeal and isolates/concentrates have lupine variety, fermentation technology and LAB used for fermentation. Interaction of analysed factors was significant (p≤0.0001), however, protein isolation process had no significant influence on above mentioned parameters. 3.3. The fermentation has increased genistein content in lupine samples, and in NF samples genistein content has ranged on average from 4.37 μg/g to 17.19 μg/g, while in SMF samples it was established to be on average from 47.0% to 77.7% higher , compared to NF. Protein isolation process reduces the genistein content, and significant influence on genistein content of lupine variety, fermentation technology, LAB used for fermentation, protein isolation/concentration process and interaction of analysed factors was found (p≤0.0001).

4. The highest protein yield was obtained at pH 10.0, and significant influence on protein solubility (at pH 10.0) have both the fermentation technology and LAB used for fermentation (p≤0.0001). Significant influence on the protein content in isolates/concentrates have lupine variety, fermentation technology, LAB used for fermentation and interaction of analysed factors (p≤0.0001).

5. Microbial hydrolysis is a suitable technology for both the lupine seeds protein molecular weight and amino acids profile biomodification: 5.1. After comparison of amino acids profile in fermented and NF seeds wholemeal, in most of the cases higher content of VAL, THR and MET was established in SMF with L. sakei and P. acidilactici samples. In SSF samples contents of LEU, THR and MET were increased, compared to NF. 5.2. In compare amino acids profile in fermented and NF lupine seeds protein isolates/concentrates, the higher contents of ALA, SER, PRO and 174

GLU were found in SMF with P. pentosaceus Nr.8, Nr.9 and Nr.10 varieties Vilciai and Vilniai samples. 5.3. The processes of SMF and SSF treatment have influence on protein molecular weight, and in biodegraded samples the higher content of low molecular weight protein was established (from 10 kDa to 20 kDa), compared to NF. The main protein fractions in SMF and SSF isolates/ concentrates had molecular weight ranging from 120 kDa to 200 kDa. The protein fractions were decreased from 38 kDa to 47 kDa in fermented samples, compared to NF.

6. Lupine seeds protein isolation/concentration process can be used for high biological value and safe stock/products preparation: 6.1. By using protein isolation process, the D(-) lactic acid does not remainn in products, however, the biological value of the products is significantly improved after the biodegradation. 6.2. After comparison of BAs content in lupine protein isolates/concentrates, in most of the cases the higher content of BA was found in NF samples, compared to SMF and SSF. In SMF and SSF protein isolates/ concentrates HIS content was on average 83.6% and 93.2% lower, compared to NF samples.

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PRACTICAL RECOMMENDATIONS

The analysed newly bred in Lithuania narrow-leafed (Lupinus angustifolius) lupine seed varieties (Vilciai, Vilniai and hybrid lines Nos.1072, 1734, 1700, 1701, 1800 and 1702), especially Vilciai seeds (because, in above mentioned variety seeds, the highest content of protein was established) can be recommended for preparation of the proteinaceous stock/ products. For the lupine seeds wholemeal and protein isolates/concentrates treatment fermentation with selected technological microorganisms (LAB) can be recommended, and fermentation conditions (SMF and/or SSF) according to the preferred substrate characteristics should be selected, because optimised technological process reduces activity of antinutritional factors, increases protein digestibility, TPC content, as well as free genistein concentration, and these changes have positive influence on antioxidant properties of the substrate. The preparation of the lupine seeds protein isolates/concentrates at pH 10.0 can be recommended, because at these conditions the highest protein yield can be obtained. Microbial hydrolysis for the lupine seeds protein molecular weight and amino acids profile biomodification can be recommended, to improve characteristics of the lupine seeds protein, especially biological value.

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PUBLICATIONS

Publications based on the results of the dissertation were published in journals with an impact factor refereed by the „Clarivate Analytics Web of Science“ 1. Starkutė V. Bartkienė E. Bartkevics V. Rusko J. Žadeikė D. Juodeikienė G. Amino acids profile and antioxidant activity of different Lupinus angustifolius seeds after solid state and submerged fermentations. Journal of food science & technology. New Delhi: Springer. 2016; 53(12):4141- 4148. [Citav. rod.: 1.262 (2017)][Indėlis: 0.167; indeksas: 0.211] 2. Bartkienė E. Bartkevics V. Rusko J. Starkutė V. Žadeikė D. Juodeikienė G. Changes in the free amino acids and the biogenic amine contents during lactic acid fermentation of different lupin species. International journal of food science & technology. Oxford: Wiley. 2016; 51(9):2049- 2056. [Citav. rod.: 1.64 (2016)][Indėlis: 0.167; indeksas: 0.274] 3. Bartkienė E. Bartkevics V. Starkutė V. Krunglevičiūtė V. Čižeikienė D. Žadeikė D. Juodeikienė G. Maknickienė Z. Chemical composition and nutritional value of seeds of Lupinus luteus L. L. angustifolius L. and new hybrid lines of L. angustifolius L. Žemdirbystė = Agriculture. 2016; 103(1):107-114. [Citav. rod.: 0.644 (2016)][Indėlis: 0.125; indeksas: 0.081] 4. Bartkienė E. Bartkevics V. Rusko J. Starkutė V. Bendoraitienė EA. Žadeikė D. Juodeikienė G. The Effect of Pediococcus acidilactici and Lactobacillus sakei on biogenic amines formation and free amino acid profile in different lupin during fermentation. LWT-Food science and technology. Amsterdam: Elsevier. 2016; 74: 40-47. [Citav. rod.: 2.329 (2016)][Indėlis: 0.143; indeksas: 0.333] 5. Bartkienė E. Bartkevics V. Starkutė V. Žadeikė D Juodeikienė G. The Nutritional and safety challenges associated with lupin lactofermentation. Frontiers in plant science. Lausanne: Frontiers Research Foundation. 2016; 7:1-5. [Citav. rod.: 4.291 (2016)][Indėlis: 0.2; indeksas: 0.858] 6. Bartkienė E. Šakienė V. Bartkevics V. Juodeikienė G. Lėlė V. Wiacek C. Braun P.G. Modulation of the nutritional value of lupine wholemeal and protein isolates using submerged and solid-state fermentation with Pediococcus pentosaceus strains. International journal of food science & technology. Oxford: Wiley. 2018; 00: 00-00. [Citav. rod.: 2.383 (2017)] [Indėlis: 0.143; indeksas: 0.235] 7. Bartkienė E. Šakienė V. Bartkevics V. Rusko J. Lėlė V. Juodeikienė G. Wiacek C. Braun P.G. Lupinus angustifolius L. lactofermentation and

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protein isolation: effects on phenolic compounds and genistein, antioxidant properties, trypsin inhibitor activity, and protein digestibility. European food research and technology = Zeitschrift für Lebensmittel- Untersuchung und -Forschung. A. Berlin: Springer Verlag. AGRICOLA; Scopus; CAB Abstracts]. 2018; 00: 00-00. [Citav. rod.: 1.919 (2017)] [Indėlis: 0.125; indeksas: 0.208] 8. Bartkienė E. Šakienė V. Bartkevics V. Rusko J. Lėlė V. Ruzauskas M. Bernatoniene J. Jakstas V. Juodeikiene G. Wiacek C. Braun P.G. Nutra- ceuticals in gummy candies form prepared from lacto-fermented lupine protein concentrates, as high-quality protein source, incorporated with Citrus paradise L. essential oil and xylitol. International Journal of Food Science & Technology. ISSN 0950-5423. 2018, vol. 00, p. 00-00. [Citav. rod.: 2.383 (2017)]

Publications based on the results of the dissertation were published in peer – reviewed journals refereed by other data bases

1. Krunglevičiūtė V. Starkutė V. Bartkienė E. Bartkevics V. Juodeikienė G. Vidmantienė D. Maknickienė Z. Design of lupin seeds lactic acid fermentation – changes of digestibility. amino acid profile and antioxidant activity. Veterinarija ir zootechnika. Kaunas: Lietuvos sveikatos mokslų universiteto Veterinarijos akademija. 2016; 73(95):47-53. [In- dėlis: 0.143] 2. Starkutė V. Bartkienė E. Krunglevičiūtė V. Juodeikienė G. Maknickienė Z. Lubinų baltymų izoliatai ir koncentratai. jų taikymo maisto bei pašarų pramonėje apžvalga. Maisto chemija ir technologija. Kaunas: KTU Mais- to institutas. 2015; 49(1):1-9. [Indėlis: 0.2] 3. Starkutė V. Andrulionytė D. Bartkienė E. Krunglevičiūtė V. Mozūrienė E. Juodeikienė G. Maknickienė Z. Lubinų baltymų izoliatų panaudojimas makaronų gamybai. Maisto chemija ir technologija. Kaunas: KTU Maisto institutas. 2015; 49(1:1-14. [Indėlis: 0.143]

Abstracts of international conferences based on the results of the dissertation

1. Bartkienė E. Zavistanavičiūtė P. Lėlė V. Šakienė V. Ružauskas M. Bernatonienė J. Juodeikienė G. Viškelis P. Jakštas V. Formulation of multifunctional nutraceuticals with addition of bovine colostrum, essential oils and probiotics. 1st International meeting on I&D [Innovation & Development] in the food sector. 3° workshop de I&D no setor agroalimentar : 05 June 2018, Viseu, Portugal. Viseu: Instituto 204

Politécnico de Viseu, 2018. (Session I. Keynote speeches.). ISBN 9789899693746. p. 11-12. 2. Šakienė V. Bartkevics V. Lėlė V. Juodeikienė G. Žadeikė D. Čižeikienė D. Wiacek C. Braun PG. Bartkienė E. Alternative technologies for plant proteins, in natural matrices and isolates, nutritional value and functionality increasing. The Vital Nature Sign : 11th International scientific conference "The Vital Nature Sign" : 19-20 October, 2017, Vilnius, Lithuania : abstract book / Vytautas Magnus University. Instrumental Analysis Open Access Center ; Editors: Dr. Nicola Tiso, dr. Vilma Kaškonienė. Kaunas: Vytautas Magnus University. (Poster presentation. 2nd day, 20 October, 2017.). ISSN 2335-8653. 2017, p. 59-59. 3. Šakienė V. Bartkienė E. Bartkevics V. Rusko J. Lėlė V. Ružauskas M. Bernatonienė J. Jakštas V. Juodeikienė G. Wiacek C. Braun P.G. Lacto- fermented lupine protein isolates, as a high quality protein and genistein source, Citrus paradise L. essential oil and xylitol, as an antimicrobial agents, for gummy candies preparation. 1st International doctoral students’ conference “Science for Health” : book of abstracts : April 13, 2018, Kaunas, Lithuania / Lithuanian university of health sciences. LSMU Department of Research Affairs. Council of LSMU Doctoral Students ; [Edited by Indrė Šveikauskaitė]. Kaunas: Lietuvos sveikatos mokslų universiteto Leidybos namai, 2018. [Indėlis: 0.091] 4. Sakiene V. Bartkevics V. Lele V. Juodeikiene G. Zadeike D. Cizeikiene D. Wiacek C. Braun PG. Bartkiene E. Alternative technologies for plant proteins. in natural matrices and isolates. nutritional value and functionality increasing. The Vital Nature Sign : 11th International scientific conference ”The Vital Nature Sign“. Kaunas: Vytautas Magnus University. 2017. 19-20 October. P. 59-59. 5. Starkute V. Krungleviciute V. Bartkiene E. Bartkevics V. Zadeike D. Juodeikiene G. Cereal by - products conversion to stock with high content of P. Acidilactici by using enzymatic treatment combined with solid state and submerged fermentation. Foodbalt 2017 : 11th Baltic Conference on Food Science and Technology ”Food science and technology in a changing world“. Latvia University of Agriculture Faculty of Food Technology. 2017. 27-28 April. P. 89. 6. Sakiene V. Lele V. Zavistanaviciute P. Bartkevics V. Rusko J. Cizeikiene D. Zadeike D. Juodeikiene G. Bartkiene E. Challenges associated with the lupine seeds fermentation by using selected antimicrobial properties showing lactic acid bacteria strains. Tarptautinė mokslinė konferencija „Gyvūnų fiziologijos ir patologijos aktualijos“ : programa ir tezės : Kaunas. 2017 rugsėjo 28-29 d. = International 205

scientific conference ”Actualities in animal physiology and pathology“ : programme and abstracts : Kaunas. 28-29 September. 2017 / Lietuvos sveikatos mokslų universitetas. Veterinarijos akademija. 2017. P. 60- 60. 7. Zavistanavičiūtė P. Lėlė V. Šakienė V. Antanaitis R. Televičius M. Ružauskas M. Juodeikienė G. Žadeikė D. Bernatonienė J. Viškelis P. Bartkienė E. Characterisation. including in vivo studies. and encapsulation of Lactobacillus plantarum and Lactobacillus paracasei through spray drying using enriched whey as a wall material. Tarptautinė mokslinė konferencija „Gyvūnų fiziologijos ir patologijos aktualijos“ : programa ir tezės : Kaunas. 2017 rugsėjo 28-29 d. = International scientific conference ”Actualities in animal physiology and pathology“. Kaunas. 28-29 September. 2017. P. 72-72. 8. Zavistanavičiūtė P. Krunglevičiūtė V. Starkutė V. Bartkienė E. Fer- mented and immobilized hemp seed products for the higher value biscuits production / P. Zavistanaviciute. V. Krungleviciute. V. Star- kute. E. Bartkiene // Foodbalt 2017 : 11th Baltic Conference on Food Science and Technology ”Food science and technology in a changing world“. Jelgava. 2017. April 27-28. P. 72-72. 9. Starkutė V. Bartkienė E. Bartkevics V. Krunglevičiūtė V. Zavistana- vičiūtė P. Juodeikienė G. The influence of fermentation technology on the amino acids profile and antioxidant properties of lupin seeds. Foodbalt 2017 : 11th Baltic Conference on Food Science and Techno- logy "Food science and technology in a changing world". Jelgava. April 27-28. 2017. P. 75-75. 10. Bartkienė E. Bartkevics V. Starkutė V. Krunglevičiūtė V. Čižeikienė D. Žadeikė D. Maknickienė Z. Juodeikienė G. Chemical and nutritional characterization of Lupinus luteus L. L. angustifolius L. and new hybrid lines from L. angustifolius L. seeds. The Food Factor I Barcelona Con- ference ”Emerging and exploratory food science and technology“ : 2-4 November 2016. Barcelona (Spain). 2016. P. 184-184. 11. Bartkienė E. Bartkevics V. Rusko J. Starkutė V. Juodeikienė G. The Influence of Pediococcus acidilactici strain KTU05-7 and Lactobacillus sakei strain KTU05-6 on biogenic amine formation and free amino acid profile in lupin seeds. The Food Factor I Barcelona Conference ”Emerging and exploratory food science and technology“ : 2-4 Novem- ber 2016. P. 308-308. 12. Krunglevičiūtė V. Starkutė V. Bartkienė E. Bartkevics V. Juodeikienė G. Vidmantienė D. Maknickienė Z. Design of lupin seeds lactic acid fermentation - changes of digestibility. amino acid profile and antioxidant activity. Tarptautinė mokslinė konferencija „Vietinių pašarinių 206

žaliavų panaudojimas gyvūnų mitybai: nauda ir apribojimai. susiję su virškinimo fiziologija bei jų įtaka produkcijos kokybei ir gyvūnų sveikatai“ : 2016 m. spalio 6 d. Kaunas = International scientific conference "The Use of local raw material in the animal nutrition: benefits and limitations regarding digestive physiology. products quality and animal health. 2016. P. 39-39. 13. Starkutė V. Bartkienė E. Bartkevics V. Rusko J. Maknickienė Z. Žadeikė D. Juodeikienė G. Free Amino acid profile and biogenic amines in fermented L. angustifolius and L. luteus Lupin seeds. The Vital Nature Sign : 10th International Scientific Conference ”The Vital Nature Sign“ : May 19-20. 2016. Vilnius. Lithuania. P. 99-99. 14. Starkute V. Bartkiene E. Bartkevics V. Zadeike D. Juodeikiene G. The Influence of solid state and submerged fermentation with Pediococcus pentosaceus KTU05-8. KTU05-9. and KTU05-10 strains on the free amino acids profile in lupine seeds. Tagungsband: 15. BOKU-Sym- posium Tierernährung ”Verarbeitung von Futtermitteln für die Misch- futterherstellung“; Universität für Bodenkultur Wien. 2016. 7 April. P. 124-128. 15. Starkutė V. Bartkienė E. Bartkevics V. Maknickienė Z. Chemical composition of a new varieties of the lupin seeds cultivated in Lithuania. IV International Conference of PhD Students ”Multi- directional research in agriculture and forestry“ : Cracow. 21-22 March 2015. P. 102-102. 16. Krunglevičiūtė V. Bartkienė E. Starkutė V. Juodeikienė G. Vid- mantienė D. Maknickienė Z. The Influence of lactic acid fermentation on proteins digestibility and biogenic amines formation in lupine and soybean. 17th International Veterinary medicine students Scientific Research Congress; Istanbul. Turkey = 17. 2015. 28-30 April. P. 182- 183. 17. Starkutė V. Bartkienė E. Bartkevics V. Maknickienė Z. Juodeikienė G. Analysis of micro- and macroelements of Lupine Seeds bred In Lithaunia by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The Vital Nature Sign : 9th International Scientific Con- ference ”The Vital Nature Sign“. May 14-16. 2015 Kaunas. Lithuania. P. 127-127. 18. Starkutė V. Andrulionytė D. Bartkienė E. Krunglevičiūtė V. Juo- deikienė G. The Use of lupine protein isolates for pasta production. 10th Baltic Conference on Food Science and Technology ”Future Food: Innovations. Science and Technology“ – FoodBalt – 2015. May 21-22. 2015. Kaunas. Lithuania P. 45-45.

207

Abstracts of national conferences based on the results of the dissertation

1. Starkutė V. Krunglevičiūtė V. Bartkienė E. Kietafazės fermentacijos Lactobacillus Sakei įtaka aminorūgščių ir antioksidacinių savybių po- kyčiams lubinuose. X nacionalinė doktorantų mokslinė konferencija „Mokslas – sveikatai“ : konferencijos tezių knyga : 2017 balandžio 7 d. Kaunas / Lietuvos sveikatos mokslų universitetas ; 2017. P. 49-50. 2. Starkutė V. Bartkienė E. Bartkevics V. Lietuvoje išvestų naujų lubinų hibridų sėklų detali cheminės sudėties analizė. IX nacionalinė doktorantų mokslinė konferencija „Mokslas – sveikatai” : konferencijos tezių knyga : 2016 balandžio 13 d. Kaunas / Lietuvos sveikatos mokslų universitetas. P. 48-49. 3. Starkutė V. Bartkienė E. Bartkevics V. Krunglevičiūtė V. Čižeikienė D. Žadeikė D. Juodeikienė G. Maknickienė Z. Kokybinis alkaloidų tyrimas lubinų sėklose „Vilčiai“ ir „Vilniai“ bei naujai Lietuvoje išvestose hib- ridų linijų sėklose. IV Jaunųjų mokslininkų konferencijos „Jaunieji mokslininkai – žemės ūkio pažangai“ pranešimų tezės. 2015 m. lapkričio 5 d. Vilnius. P. 22-22. 4. Starkutė V. Andrulionytė D. Bartkienė E. Krunglevičiūtė V. Mozūrienė E. Juodeikienė G. Biotechnologinių sprendimų taikymas lubinų baltymų izoliatų ekstrakcijai ir jų panaudojimas makaronų gamybai. VIII nacionalinė doktorantų mokslinė konferencija „Mokslas – sveikatai“ : konferencijos pranešimų tezės [skirta] Pasaulinei sveikatos dienai paminėti. kuri minima balandžio 7-ąją. Šių metų tema – “Maisto sauga” : 2015 m. balandžio 10 d. Kaunas / Lietuvos sveikatos mokslų universitetas. P. 125-126. 5. Krunglevičiūtė V. Bartkienė E. Starkutė V. Kantautaitė J. Juodeikienė G. Kietafazės pienarūgštės fermentacijos (KF) įtaka skirtingų rūšių lubinų ir sojų sėklų saugos rodikliams. III Jaunųjų mokslininkų konferencijos „Jaunieji mokslininkai – žemės ūkio pažangai“. pranešimų tezės : Vil- nius. 2014 m. lapkričio 6 d. P. 54.

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SUMMARY

SANTRUMPOS

AA – antioksidacinis aktyvumas AR – aminorūgštys BA – biogeniniai aminai BTR – bendras titruojamasis rūgštingumas BFJ – bendras fenolinių junginių kiekis ALA – alaninas GLY – glicinas SER – serinas PRO – prolinas ASP – asparaginas GLU – glutaminas TYR – tiraminas PHE – feniletilaminas CAD – kadaverinas VAL – valinas ILE – izoleucinas THR – treoninas MET – metioninas LYS – lizinas HIS – histaminas PEA – feniletilaminas TYM – tiraminas KF – kietafazė fermentacija Ls – Lactobacillus sakei Pa – Pediococcus acidilactici Pp – Pediococcus pentosaceus PRB – pieno rūgšties bakterijos PUT – putrescinas SPRM – sperminas SPRMD – spermidinas TF – tradicinė fermentacija TRP – triptaminas TI – tripsino inhibitoriai TIA – tripsino inhbitorių aktyvumas NF – nefermentuoti mėginiai

301

ĮVADAS

Vis didėjantis augalinės kilmės baltymingų žaliavų poreikis pašarų/mais- to pramonei yra glaudžiai susijęs ne tik su vartotojų poreikių patenkinimu, bet ir su gyvūninių produktų gamybos neigiamo poveikio aplinkai bei, tuo pačiu, klimatos kaitos mažinimu. Ankštiniai augalai yra perspektyvus alter- natyvių baltymų šaltinis, kuris galėtų būti efektyviai taikomas naujos kartos maisto/pašarų gamyboje [12]. Prognozuojama, kad iki 2050 m. gyventojų skaičius pasaulyje pasieks 9,5 mlrd. ribą, todėl poreikis alternatyviems baltymų šaltiniams sparčiai augs. Didėjant pigių ir genetiškai nemodifikuotų augalinių baltymų poreikiui, maisto/pašarų pramonė ieško alternatyvių gyvūniniams, augalinių baltymų šaltinių [6]. Gyvūniai baltymai maistiniu aspektu yra pranašesni už augalinius baltymus, tačiau globaliai susiduriama su pakankamo jų kiekio užtikrinamo vartotojams problema bei sąlyginai nedideliu jų tvarumu. Siekiant kompensuoti gyvūninių baltymų trūkumą, visame pasaulyje populiariausiu baltymų šaltiniu tapo soja ir Europa tapo labai priklausoma nuo sojų pupelių importo, apimančio prekybos susitari- mus ir kokybės standartus, kurie tik dalinai atitinka europiečių lūkesčius. Lubinai galėtų būti alternatyva sojai Europoje, nes juose didelis kiekis aukš- tos kokybės baltymų (iki 44 proc.), taip pat lubinai genetiškai nemodifikuoti [15]. Lubinai gerai dera įvairaus klimato regionuose, jie dirvą papildo azotu bei fosforu, tinkami sėjomainai, priešsėliams, gerai auga sąlyginai neder- lingose žemėse, sudaro simbiozę su gumbelinių bakterijų rūšimis ir sukau- pia atmosferos azotą ant augalo šaknų [16]. Lubinų pasėliai, tai vertingas indėlis į tvarų bei saugų nederlingų dirvų kokybės gerinimą. Australijoje lubinai ir jų produktai (pagrinde baltymai) sėkmingai dominuoja rinkoje, o lubinų baltymų gamybos pramonė yra labai išvystyta. Tačiau Europoje ši tendencija tik pradeda populiarėti ir viena iš priežasčių, dėl ko neplečiami lubinų auginimo plotai, tai nepakankamai išvystytos technologijos jų perdirbimui į paklausius bei konkurencingus produktus ir/ar žaliavas. Todėl labai svarbiu uždaviniu išlieka efektyvus lubinų panaudojimas. Pastaruoju metu lubinai sulaukė nemažo susidomėjimo visame pasaulyje, dėl savo puikių maistinių savybių ir galimybės jais praturtinti pašarus [10]. Lubinų sėklose didžiausią dalį sudaro baltymai, mažiau yra skaidulinių medžiagų ir nedidelis kiekis riebalų. Šis cheminės sudėties savitumas yra labai svarbus aukštos kokybės augalinių baltymų išgavimui. Visi šie veiksniai nulėmė lu- binų baltymų gamybos augimą, kartu ir lubinų baltymų išgavimo techno- logijų efektyvumo didinimo poreikį [15]. Lubinų baltymų fermentacija pieno rūgšties bakterijomis (PRB) gali būti naudojama, siekiant pagerinti juslines savybes bei baltymų bioprieinamumą [20]. Lubinų panaudojimas pašarų pramonėje ilgą laiką buvo ribojamas, dėl juose esančių antimitybinių 302 faktorių. Pagrindiniai antimitybiniai komponentai lubinuose yra kvino- lizidino grupės alkaloidai [19]. Daugelio lubinų veislių sėklų sudėtyje yra didelis šių junginių kiekis ir tai mažina jų, kaip pažarų žaliavos, integra- vimą, dėl kartaus skonio bei toksiškumo. Lubinų baltymų izoliatų/kon- centratų gamybos technologijos taikymas galėtų išspręsti lubinuose esančių alkaloidų problemą, nes alkaloidai yra tirpūs vandenyje, todėl būtų pašalinti baltymingų žaliavų gamybos metu [20]. Šiai problemai spręsti siūlomi ir kiti technologiniai sprendimai: sėklų terminis apdorojimas, sėklų mirkymas, fermentacija ir kt. [21]. Šiuo metu sukurtos lubinų veislės gali būti nau- dojamos gyvūnų šėrimui be papildomo apdorojimo, nes jose alkaloidų kiekis yra labai mažas [16]. Daugiausiai lubinų perdirbama Australijoje (1,6 milijonų tonų kasmet) ir tai sudaro 80 proc. visos pasaulinės produkcijos, kitos, didelį kiekį lubinų perdirbančios šalys, yra Rusija ir Lenkija [16]. Lubinų veislių selekcijos srityje pažangos Europoje iki II pasaulinio karo buvo nedaug. Vėliau šioje srityje tyrimai buvo vykdomi, kuriant naujus, mažu alkaloidų kiekiu pasižyminčius, genotipus Vokietijoje, Prancūzijoje, Ispanijoje, Lenkijoje [400]. Lubinų selekcijos programa Lietuvoje buvo pradėta 1995 metais. Darbas buvo vykdomas trimis pagrindinėmis kryptimis: (I) lubinų su mažu alkaloidų kiekiu, skirtų maisto pramonei, selekcija; (II) lubinų su mažu alkaloidų kiekiu, skirtų pašarų pramonei, selekcija; (III) lubinų, skirtų žaliajai masei, veislių selekcija. Pastaruoju metu vyraujančios pasaulinės tendencijos, ieškant ne tik patrauklių, bet ir draugiškų aplinkai augalų bei jų perdirbimo technologijų, išgaunant itin vertingas žaliavas yra labai perspektyvi ir savalaikė. Dėl šių priežasčių lubinų poreikis turėtų augti, o jų perdirbimo technologijų ir naujų žaliavų kūrimas bei taikymas maisto/pa- šarų pramonėje augti.

1. DISERTACINIO DARBO TIKSLAS IR UŽDAVINIAI

Darbo tikslas: atlikti išsamią Lietuvoje naujai išvestų lubinų veislių sėklų cheminės sudėties analizę ir, naudojant biotechnologinius sprendimus nelukštentų sėklų (beatliekinė technologija) ir baltymų izoliatų/koncentratų (mažaatliekinė technologija) apdorojimui, sukurti didesnės vertės, tvarias ir saugias augalinių baltymų žaliavas/produktus.

Uždaviniai: 1. Atrinkti Lietuvoje populiarinamas lubinų veislių sėklas, pasižyminčias mažais alkaloidų kiekiais ir detaliai įvertinti jų cheminę sudėtį. 2. Taikant tradicinę ir/ar kietafazę fermentaciją bakteriocinus produkuojan- čiomis pieno rūgšties bakterijomis, parinkti tinkamiausią būdą lubinų 303

baltymų virškinamumo padidinimui bei tripsino inhibitorių inaktyva- vimui. 3. Įvertinti technologinių veiksnių įtaką baltymingos žaliavos pokyčiams: bendram fenolinių junginių ir izoflavonų (genisteino) kiekiui bei antioksidaciniam aktyvumui. 4. Atlikti lubinų baltymų izoliatų/koncentratų gamybą ir įvertinti juose esančių baltymų molekulinę masę, tirpumą bei išeigą. 5. Nustatyti mikrobinės hidrolizės poveikį aminorūgščių profiliui izoliatuose/koncentratuose bei smulkintose lubinų sėklose. 6. Įvertinti technologinių veiksnių įtaką biogeninių aminų bei D(-) pieno rūgšties formavimuisi žaliavoje.

Darbo aktualumas ir naujumas

Šiame disertaciniame darbe pristatomas inovatyvių ir tvarių procesų taikymas bei naujai Lietuvoje išvestų lubinų sėklų ir mikroorganizmų atranka įgalins sukurti aukštos pridėtinės vertės novatoriškas baltymingas žaliavas/produktus, pasižyminčius dideliu biofunkcionalumu. Augant gyventojų populiacijai ir iškylant būtinumui agrarinius resursus panaudoti išskirtinai efektyviai, tvarių ir saugių žemės ūkio technologijų kūrimas atitinka šiuolaikinius maisto/pašarų pramonės poreikius ne tik Europos, bet ir pasauliniu mastu. Šiame disertaciniame darbe pristatyti rezultatai sudaro prielaidas didelės biologinės vertės maisto/pašarų žaliavų technologijų pro- totipų sukūrimui. Sukurtos aukštos pridėtinės vertės lubinų sėklų žaliavos ir jų perdirbimo technologijos yra labai savalaikės, nes pastaruoju metu auganti vartotojų, kurie atsisako rinktis genetiškai modifikuotus ir/ar nena- tūralius produktus, poreikis įpareigoja pramonę perorientuoti gamybą šia linkme. Pagrindinis šio darbo mokslinis naujumas ir praktinė nauda yra ta, kad apibendrinant gautus tyrimų rezultatus galima teigti, kad didesnės vertės maisto/pašarų žaliavos gali būti gaminamos ekologiniu, socialiniu ir eko- nominiu aspektu tvariu būdu, užtikrinant jų saugą bei skatinant bendrą visuomenės tvarumą.

2. MEDŽIAGOS IR METODAI

2.1. Tyrimų vieta ir laikas

Eksperimentai atlikti 2014–2018 metais Lietuvos sveikatos mokslų universitete Veterinarijos akademijoje (LSMU VA) Maisto saugos ir kokybės katedroje, Latvijos maisto saugos, gyvūnų gerovės ir aplinkosaugos institute – BIOR (Ryga, Latvija), Leipcigo universiteto Maisto higienos institute (Leipcigas, Vokietija). 304

2.2. Medžiagos

Augalinė žaliava. Siauralapių lubinų sėklų veislės Vilčiai ir Vilniai ir hibridinių linijų Nr. 1072, Nr. 1734, Nr. 1700, Nr. 1701, Nr. 1800 ir Nr. 1702 (Lupinus angustifolius L.). Lubinų sėklos (2014 metų derliaus) gautos iš Lietuvos agrarinių ir miškų mokslo centro (Trakų Vokė, Lietuva). Mikroorganizmai. Lactobacillus sakei KTU05-6, Pediococcus acidilactici KTU05-7 ir Pediococcus pentosaceus KTU05-8, KTU05-9, KTU05-10 pasižymintys antimikrobinėmis savybėmis [218] buvo gauti iš KTU Maisto mokslo ir technologijos katedros, Grūdai ir grūdų produktai mokslo grupės kolekcijos.

2.3. Lubinų žaliavos fermentacija

Lubinų sėklų fermentacija atlikta L. sakei KTU05-06, P. acidilactici KTU05-07, P. pentosaceus KTU05-8, KTU05-9, KTU05-10, atitinkamai, 30 °C; 32 °C; 35 °C temperatūroje 48 val., taikant TF ir KF (KF ≤ 45 proc.; TF ≥ 65 proc.).

2.4. Lubinų sėklų izoliatų/koncentratų gamybos technologija

Lubinų baltymų izoliavimas/koncentravimas atliktas pagal Muranyi ir kt. [156] metodiką.

2.5. Analizės metodai

Mikrobiologinė analizė atlikta naudojant dešimtainį praskiedimo metodą ir sėjimo į Petri lėkšteles metodą. Rūgštingumo rodiklių nustatymas. pH išmatuotas pH-metru (PP – 15, Sartorius, Goettingen, Vokietija). Bendras titruojamasis rūgštingumas (BTR) nustatytas vandeniniuose tirpaluose mėginių tirpaluose. juos tit- ruojant 0,1 N NaOH tirpalu iki terpės pH = 8,5. L(+)- ir D(–)- pieno rūgšties izomerų kiekis nustatytas taikant fermentinį testą „Megazyme“ spektro- fotometriniu metodu (International Ireland, Wicklow, Airija). Drėgmės kiekis nustatytas džiovinant termostate (UNB400, Memmert, Vokietija) 103 ± 2 °C temperatūroje iki pastovios masės. Maistinės vertės analizė. Baltymų kiekis nustatytas Kjeldalio metodu (AOAC, 2000). Riebalų kiekis nustatytas Soksleto metodu pagal Khalil (1990) metodiką. Angliavandenių kiekis pagal AOAC (1990). Pelenų kiekis nustatytas ICC 104/1:1990 metodu, bendras mineralinių medžiagų kiekis nustatytas pagal AOAC (1990). 305

Riebalų rūgščių sudėties analizė atlikta dujų chromatografijos metodu su liepsnos jonizacijos detektoriumi (metodika detaliai aprašyta 2.4.2 skyriuje). Mikro ir makrokomponentų analizė atlikta induktyviai susietos plazmos su masių spektrometru (metodika detaliai aprašyta 2.4.3 skyriuje). Alkaloidų analizė atlikta dujų chromatografijos masių spektrometrijos metodu pagal Harris ir Wilson (1988) metodiką. In vitro baltymų virškinamumas nustatytas spektrofotometriškai pagal Lqari ir kt. (2002) metodiką. Antioksidacinis aktyvumas nustatytas spektrofotometriškai pagal Zhu ir kt. (2011) metodiką. Biogeninių aminų analizė atlikta efektyvaus slėgio skysčių chromatografijos metodu pagal Ben-Gigirey ir kt. (1999) metodiką. Aminorūgščių sudėties analizė įvertinta dujų chromatografijos su liepsnos jonizacijos detektoriumi (metodika detaliai aprašyta 2.4.7 skyriuje). Izoflavonų analizė atlikta skysčių chromatografijos metodu su masių spektrometrijos detektoriumi (metodika detaliai aprašyta 2.4.13 skyriuje). Bendro fenolinių junginių kiekio analizė atlikta pagal Vaher ir kt. (2010) metodiką. Laisvųjų radikalų surišimo geba įvertinta spektrofotometriškai pagal Zhu ir kt. (2011) metodiką. Izoliatų/koncentratų liofilizavimas atliktas pagal Magni ir kt. (2005) metodiką. Baltymų tirpumo analizė atlikta pagal King ir kt. (1985) metodiką. Bal- tymų tirpumas apskaičiuotas pagal Bradford (1976) metodiką. Baltymų kiekio analizė atlikta Kjeldalio metodu pagal AACC (1999) su ,,Nitrogen Analyzer“ (Vapodestas, Gerhardt GmbH Co. KG, Königswin- teris, Vokietija). Apskaičiuojant baltymų kiekį naudotas faktorius 5.7 (ICC 105/2). Baltymų molekulinės masės analizė atlikta pagal Muranyi ir kt. [198] metodiką.

2.6. Statistinė analizė

Visi fizikiniai cheminiai tyrimai kartoti tris kartus, tiriant du parelelinius mėginius, mikrobiologiniai tyrimai kartoti penkis kartus, tiriant du para- lelinius mėginius. Siekiant įvertinti technologinių veiksnių ir jų sąveikos įtaką analizuojamiems rodikliams buvo atlika daugiafaktorinė dispersinė analizė (R 3.2.1 R, 2015), taip pat taikant Tjukio-HSD testą (R 3.2.1, Core Team, 2015). Rezultatai patikimi (p), kai p ≤ 0,05.

306

3. TYRIMŲ REZULTATAI IR JŲ APTARIMAS

3.1. Lubinų sėklų cheminė sudėtis

Lubinų veislių Vilčiai, Vilniai ir hibridų linijų Nr. 1072, Nr. 1734, Nr. 1700, Nr. 1701, Nr. 1800 ir Nr. 1702 sėklų maistinė vertė ir alkaloidų kiekis pavaizduotas 3.1.1 a, b paveiksle. Didžiausias baltymų kiekis nustatytas Vilčiai veislės sėklose – 40,80 ± 0,10 proc., o hibridų linijų Nr. 1700 ir Nr. 1800 sėklose baltymų kiekis nustatytas nuo 20,10 proc. iki 25,70 proc. mažesnis. a)

3.1.1 pav. Lubinų veislių Vilčiai, Vilniai ir hibridų linijų Nr. 1072, Nr. 1734, Nr. 1700, Nr. 1701, Nr. 1800 ir Nr. 1702 sėklų maistinė vertė (a), drėgnis, pelenų ir alkaloidų kiekis (b)

307

Baltymų kiekis lubinų sėklose varijuoja priklausomai nuo rūšies, augimo sąlygų bei dirvožemio [394]. Angliavandenių kiekis lubinų sėklose kito nuo 41,60 ± 0,10 proc. iki 50,80 ± 0,30 proc., atitinkamai, Vilčiai ir Nr. 1800 sėklose. Mažiausias riebalų kiekis nustatytas Vilčiai veislės sėklose (4,40 ± 0,10 proc.). Riebalų kiekis lubinų sėklose yra sąlyginai didelis, panašus kiekis yra žemės riešutuose ir sojų sėklose [230]. Didžiausias pelenų kiekis – Vilčių veislės sėklose (4,30 ± 0,20 proc.). Alkaloidų kiekis Vilčiai ir Vilniai veislių sėklose nustatytas, atitinkamai, 0,0300 proc. ir 0,0210 proc., ir jis buvo didesnis nei hibridų linijų sėklose. Lubinų sėklų cheminė sudėtis reikšmingai priklausė nuo lubinų veislės (baltymų kiekis (F(483,139) = 244,136, p = 0,0001), riebalų kiekis (F(138,794) = 14,816, p ≤ 0,0001), angliavandenių kiekis (F(686,995) = 174,986, p ≤ 0,0001), mineralinių medžiagų kiekis (F(23,574) = 3,956, p ≤ 0,0001), alkaloidų kiekis (F(148,527) = 0,001, p ≤ 0,0001). Vidutinė riebalų rūgščių sudėtis (proc. nuo bendro riebalų kiekio) lubinų sėklose pateikta 3.1.2 paveiksle. Lubinų hibridų sėklose vyravo nesočiosios oleino (C18:1) ir linolo (C18:2) riebalų rūgštys (vidutiniškai 33,20 ± 3,90 ir 38,40 ± 4,50 proc.). Sočiosios palmitino rūgšties (C16:0) kiekis sėklose vidutiniškai nustatytas 10,80 ± 2,70 proc. Mononesočiųjų riebalų rūgščių C15:1 ir C20:1 nustatyta tik Vilčių veislės sėklose (1,30 ± 0,09 proc.).

3.1.2 pav. Vidutinė riebalų rūgščių sudėtis (proc. nuo bendro riebalų kiekio) lubinų sėklose

Makroelementų kiekis lubinų sėklose pavaizduotas 3.1.3 paveiksle. Didžiausias Na kiekis nustatytas hibridų sėklose Nr. 1072, Nr. 1734, Nr. 1700, Nr. 1701, Nr. 1800 ir Nr. 1702, ir jis kito nuo 1,07 ± 0,04 mg/g iki 1,19 ± 0,04 mg/g (atitinkamai, mėginiuose Nr. 1701 ir Nr. 1800). Didžiausias Mg kiekis nustatytas Vilčiai veislės sėklose (3,44 ± 0,09 mg/g). Panašius Mg ir K kiekius L. angustifolius sėklose nustatė ir Porres ir kt.

308

[234]. Mažiausiu Ca ir K kiekiu pasižymėjo Vilniai veislės sėklos, atitinkamai, 12,60 ± 0,09 mg/g and 1,46 ± 0,14 mg/kg.

3.1.3 pav. Makroelementų kiekis (mg/kg s.m.) lubinų sėklose

Mikroelementų: Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, Se ir Sr kiekiui lubinų sėklose reikšmingos įtakos turėjo lubinų veislė (Al (F(363,070) = 93,559, p = 0,0001), Cr (F(22,357)=2,422, p≤ 0,0001), Mn (F(90216,207) = 34772,709, p ≤ 0,0001), Fe (F(4508,221) = 1164,079, p = 0,0001), Co (F(10,218) = 0,046, p = 0,0001), Ni (F(26,274) = 2,506, p = 0,0001), Cu (F(118,689) = 15,931, p = 0,0001), Zn (F(7794,620) = 1553,663, p = 0,001), Se (F(10,759) = 0,014, p = 0,001) ir Sr (F(6341,740) = 1044,511, p ≤ 0,0001), tačiau As, Cd ir Pb kiekiui sėklose veislė įtakos neturėjo. Didžiausia Cr koncentracija nustatyta hibrido Nr. 1702 sėklose (1,81 ± 0,03 μg/g), mažiausia – Vilčiai veislės sėklose (0,73 ± 0,01 μg/g). Didžiausia Fe ir Zn koncentracija nustatyta Vilčiai veislės sėklose, atitinkamai, 59,84 ± 3,22 μg/g ir 73,52 ± 4,77 μg/g, o Ni koncentracija kito nuo 1,25 ± 0,04 μg/g (Nr. 1701 sėklose) iki 2,31 ± 0,02 μg/g (Nr.1702 sėklose). Sr koncentracija kito nuo 9,09 ± 0,09 μg/g (Nr. 1701 sėklose) iki 28,49 ± 0,21 μg/g (Nr. 1072 sėklose). Ag tirtuose mėginiuose nenustatyta, o Co koncentracija kito nuo 0,06 ± 0,02 μg/g (Nr. 1800 sėklose) iki 0,18 ± 0,03 μg/g (Nr. 1702 sėklose). Didžiausias Se kiekis nustatytas Nr.1702 ir Vilčiai veislės sėklose (0,13 ± 0,01 μg/g), o Cd koncentracija kito nuo 0,026 ± 0,01 μg/g (Nr. 1701 sėklose) iki 0,049 ± 0,02 μg/g (Nr. 1734 sėklose). Pb koncentracija kito nuo 0,06 ± 0,02 μg/g (Vilniai veislės sėklose) iki 0,11 ± 0,03 mg/kg (Nr. 1072 sėklose), o As – nuo 0,015 ± 0,01 μg/g (Nr. 1701 309 sėklose) iki 0,033 ± 0,01 μg/g (Vilčiai veislės sėklose). Apie mikroelementų kiekius lubinų sėklose publikuotų rezultatų yra labai nedaug, o kai kurie mikroelementai (pvz.: Cr) iš esmės apibūdinami labai menkai [235]. Labai svarbu žinoti mikroelementų kiekius pagrindinėse žaliavose, nes, pvz., keturių mikroelementų (kobalto, kaip vitamino B12, jodo, geležies ir cinko) trūkumas juntamas daugelyje regionų ir jis asocijuojasi su sveikatos proble- momis [236]. Seleno trūkumas siejamas su onkologinėmis ir širdies krauja- gyslių ligomis, antro diabeto rizika [238]. Todėl duomenų bazės kaupimas apie šiuos komponentus yra labai reikšmingas.

3.2. Lubinų sėklų fermentacijos efektyvumo rodikliai

Fermentuotų lubinų sėklų rūgštingumo rodikliai ir PRB KSV skaičius grame mėginio pavaizduoti 3.2.1 paveiksle. Lubinų sėklų pH vertės kito nuo 3,62 iki 5,00 (atitinkamai, mėginio Vilčiai TF su P. pentosaceus Nr. 9 ir hibridų Nr. 1734 KF su P. pentosaceus Nr. 8 mėginio) ir reikšmingai pri- klausė nuo lubinų veislės (F(146,151) = 0,403, p ≤ 0,0001), fermentacijos technologija (6463,010) = 17,833, p ≤ 0,0001), fermentacijai naudotos PRB (F(94,132) = 0,260, p ≤ 0,0001) ir šių faktorių sąveika buvo reikšminga (F(35,572) = 0,08, p ≤ 0,0001).

3.2.1 pav. Fermentuotų lubinų sėklų rūgštingumo rodikliai (pH ir BTR) bei PRB KSV/g mėginio NF – nefermentuoti mėginiai; TF – tradicinė fermentacija; KF – kietafazė fermententacija. 310

Didžiausias bendras titruojamasis rūgštingumas nustatytas Vilniai veislės sėklose (12,34 ± 0,05) ir šiam rodikliui reikšmingos įtakos turėjo lubinų veislė (F(114,392) = 3,600, p ≤ 0,0001), fermentacijos technologija (5469,105) = 172,098, p ≤ 0,0001), fermentacijai naudotos PRB (F(163,958) = 5,159, p ≤ 0,0001) ir šių veiksnių tarpusavio sąveika buvo reikšminga (F(71,304) = 2,244, p ≤ 0,0001). Nustatyta silpna koreliacija tarp pH ir PRB kiekio (r = 0,2142; p = 0,0361, tačiau tarp BTR ir PRB koreliacija nenustatyta. Pagrindiniai rodikliai apibūdinantys fermentacijos pro- cesą yra pH, BTR ir technologinių mikroorganizmų kiekis [243]. PRB koncentracija fermentuojamame substrate reikšmingiau priklauso nuo pH nei nuo BTR [244]. L(+) ir D(–) pieno rūgšties izomerų kiekis fermentuotuose produktuose reikšmingai priklausė nuo lubinų veislės (F(73,380) = 1,986, p ≤ 0,0001; F(10,880) = 0,454, p ≤ 0,0001); fermentacijos technologijos (1920,232) = 51,973, p ≤ 0,0001; F(970,818) = 40,486, p ≤ 0,0001); fermentacijai naudotų PRB (F(84,787) = 2,295, p ≤ 0,0001; F(23,126) = 0,964, p ≤ 0,0001) ir analizuotų veiksnių tarpusavio sąveika buvo reikš- minga (F(14,018) = 0,212, p ≤ 0,0001; F(21,911) = 0,914, p ≤ 0,0001). D(–) pieno rūgšties kiekis žinduolių plazmoje ir urine tiriamas kaip žymuo iden- tifikuojant įvairias ligas [166]. Vidutinis PRB KSV skaičius grame produkto nustatytas 10,09 ± 0,05 log10 KSV/g ir PRB kiekiui fermentuotuose produktuose reikšmingos įtakos turėjo lubinų veislė (F(53,650) = 0,813, p ≤ 0,0001), fermentacijos technologija (2,202) = 0,033, p≤ 0,0001), fermentacijai naudotos PRB (F(1073,521) = 16,258, p ≤ 0,0001) ir šių veiksnių są- veika buvo reikšminga (F(21,911) = 0,914, p ≤ 0,0001).

3.3. Baltymų kiekis lubinų baltymų izoliatuose/koncentratuose bei jų tirpumas

Baltymų kiekis (proc.) NF, TF ir KF apdorotuose lubinų sėklų izoliatuose/koncentratuose pavaizduotas 3.3.1 a paveiksle. NF mėginiuose baltymų kiekis kito nuo 82,39 ± 0,79 iki 88,24 ± 0,97 proc. (atitinkamai, NF Nr. 1734 ir Vilčiai mėginiuose). TF ir KF apdorotuose izoliatuose/koncentratuose baltymų kiekis varijavo, atitinkamai nuo 77,78 iki 82,89 proc. ir nuo 79,32 iki 86,45 proc. (atitinkamai, Nr. 1701 ir Vilniai mėginiuose bei Vilčiai ir Nr. 1072 mėginiuo- se). Lubinų izoliatuose/koncentratuose baltymų kiekiui reikšmingos įtakos turėjo lubinų veislė (F(35,355) = 28.041, p ≤ 0,0001), fermentacijos technologija (F(73,836) = 58,560, p ≤ 0,0001), fermentacijai naudotos PRB (F(38,526) = 30,555, p ≤ 0,0001) ir analizuotų faktorių sąveika (F(33,006) = 26,177, p ≤ 0,0001). Baltymų, gaunamų iš lubinų sėklų išeiga, priklauso nuo

311 a)

3.3.1 pav. Baltymų kiekis (proc.) lubinų baltymų izoliatuose/koncentratuose (a) bei jų tirpumas (b) esant pH 10 NF – nefermentuoti mėginiai; TF – tradicinė fermentacija; KF – kietafazė fermententacija. apdorojimo proceso ir techninių galimybių, kurios taip pat turi įtakos išga- naumų baltymų savybėms [266]. Lubinų baltymų izoliatų/koncentratų tirpumas (proc.) pavaizduotas 3.3.1 b paveiksle. Didžiausias TF ir KF apdorotų baltymų tirpumas nustatytas esant pH 10,0, ir, atitinkamai, kito nuo 70,82 ± 0,82 proc. iki 92,75 ± 1,52 proc. (atitinkamai, TF P. pentosaceus Nr. 10 apdorotų Vilčiai mėginių ir TF L. sakei Nr. 1072 mėginių). Baltymų tirpumas turi sąsajų su technologinėmis ir funkcionaliosiomis savybėmis (tokiomis kaip emulsijų, putų ir gelių

312 formavimo ir t. t.) [256]. Mikro ir makrokomponentai (pvz.: polifenoliniai junginiai), esantys lubinų sėklose gali turėti įtakos lubinų tirpumo profiliui [258]. Lubinų baltymų izoliatų/koncentratų tirpumui (esant pH 10,0) reikšmingos įtakos turėjo fermentacijos technologija (F(150,088) = 28,291, p ≤ 0,0001) ir fermentacijai naudotos PRB (F(80,499) = 15,174, p ≤ 0,0001).

3.4. Technologinių veiksnių įtaka izoliatų/koncentratų baltymų molekulinei masei

TF ir KF technologija turėjo įtakos baltymų molekulinei masei ir daugiau mažos molekulinės masės baltymų (nuo 10 kDa iki 20 kDa) nustatyta biodegraduotuose mėginiuose, lyginant su NF. NF, TF ir KF Nr. 1072 mėginio baltymų molekulinės masės pokyčiai pateikti 3.4.1 lentelėje. TF ir KF izoliatuose/koncentratuose vyravo baltymai, kurių molekulinė masė nuo 120 kDa iki 200 kDa. TF ir KF mėginiuose 38 kDa iki 47 kDa baltymų frakcijos nebuvo nustatytos, tačiau minėtos frakcijos buvo nustatytos NF mėginiuose.

3.4.1 lentelė. Nr. 1072 mėginio baltymų molekulinės masės pokyčiai prieš ir po biodegradavimo Nr. 1072 Nr. 1072 TF Nr. 1072 KF Molekulinė masė, kDa NF Pp Pp 220 160 120 100 90 80 70 60 50 40 30 20 10 NF – nefermentuoti mėginiai; TF – tradicinė fermentacija; KF – kietafazė fermententacija.

313

3.5. Fermentacijos įtaka bendram aminorūgščių kiekiui lubinų sėklose bei jų baltymų izoliatuose/koncentratuose

Pakeičiamųjų ir nepakeičiamųjų aminorūgščių (AR) vidutinis kiekis (proc.) NF, TF ir KF būdu apdorotose lubinų sėklose pavaizduotas 3.5.1 a, b paveiksle. Lyginant su NF, daugeliu atvejų TF L. sakei ir P. acidilactici apdorotuose lubinų sėklų mėginiuose VAL (išskyrus TF su P. acidilactici hibridų Nr. 1734 mėginiuose), THR ir MET kiekis nustatytas didesnis. KF apdorotuose mėginiuose LEU, THR (išskyrus KF P. acidilactici Vilčiai ir KF L. sakei ir P. acidilactici hibridų Nr. 1734 mėginius) ir MET kiekis padidėjo, lyginant su NF mėginiais. KF apdorotuose mėginiuose nustatyta vidutiniškai 62,40 ± 0,75 proc. mažesnis kiekis MET, lyginant su TF mėginiais. Lyginant TF ir KF mėginius, GLU kiekis, atitinkamai, 17,06 ± 0,18 proc. ir 35,7 ± 0,35 proc., nustatytas didesnis, lyginant su NF mėginiais. TF mėginiuose GLY, PRO ir ASP kiekis, atitinkamai, 20,92 ± 0,32 proc., 24,46 ± 0,38 proc. ir 17,76 ± 0,21 proc. nustatytas didesnis, lyginant su NF mėginiais. Fermentacija lactobacilli technologiniais mikroorganizmais padidina laisvųjų aminorūgščių kiekį ankštinių javų sėklose [313]. Aminorūgščių kompozicija ir virškinamumas yra pagrindiniai balty- mų kokybės rodikliai [314]. Įvertinus technologinių veiksnių įtaką AR kiekiui smulkintų lubinų sėklų mėginiuose, reikšmingos įtakos pakeičiamųjų AR kiekiui turėjo lubinų veis- lė (GLY (F(6,722) = 1,306, p ≤ 0,0001; SER (F(5,651) = 70,777, p ≤ 0,0001; PRO (F(14,640) = 3,618, p ≤ 0,0001; ASP (F(31,323) = 48,753, p ≤ 0,0001; GLU (F(13,142) = 126,523, p ≤ 0,0001; TYR (F(24,829) = 22,348, p ≤ 0,0001, išskyrus ALA), fermentacijos technologija (GLY (F(116,983) = 22,730, p ≤ 0,0001; SER (F(104,161) = 1304,632, p ≤ 0,0001; PRO (F(65,690) = 16,235, p ≤ 0,0001; ASP (F(11,665) = 18,157, p = 0,0001; GLU (F(21,055) = 202,708, p ≤ 0,0001; TYR (F(69,625) = 62,667, p ≤ 0,0001, išskyrus ALA), fermentacijai naudotos PRB (SER (F(9,301) = 116,495, p ≤ 0,0001; GLU (F(7,631) = 73,472, p = 0,0001, iš- skyrus ALA, GLY, PRO, ASP, TYR) bei analizuotų veiksnių sąveika (ALA (F(3,197) = 3,792, p ≤ 0,0001; GLY (F(7,117) = 1,383, p ≤ 0,0001; SER (F(6,198) = 77,630, p ≤ 0,0001; PRO (F(4,390) = 1,085, p ≤ 0,0001; ASP (F(8,419) = 13,104, p ≤ 0,0001; GLU (F(8,802) = 84,737, p ≤ 0,0001; TYR (F(9,797) = 8,817, p ≤ 0,0001). Įvertinus technologinių veiksnių įtaką nepa- keičiamųjų AR kiekiui smulkintų lubinų sėklų mėginiuose, reikšmingos įtakos turėjo lubinų veislė (LYS (F(8,562) = 4,330, p ≤ 0,0001, išskyrus VAL, ILE, LEU, THR, MET, PHE, HIS), fermentacijos technologija (VAL (F(31,005) = 9,467, p ≤ 0,0001; ILE (F(64,340) = 45,141, p ≤ 0,0001; LEU

314 a)

3.5.1 pav. Nepakeičiamųjų (a) ir pakeičiamųjų (b) aminorūgščių kiekis (proc.) smulkintose lubinų sėklose NF – nefermentuoti mėginiai; TF – tradicinė fermentacija; KF – kietafazė fermententacija.

(F(19,413) = 58,225, p ≤ 0,0001; THR (28,725) = 18,737, p = 0,0001; MET (76,151) = 2,036, p≤ 0,0001; PHE (F(73,618) = 93,822, p ≤ 0,0001; LYS (F(78,982) = 39,3940, p ≤ 0,0001; HIS (F(120,361) = 68,637, p ≤ 0,0001), fermentacijai naudotos PRB (VAL (F(55,737) = 17,018, p ≤ 0,0001; ILE (F(22,463) = 15,760, p ≤ 0,0001; LEU (F(15,024) = 45,060, p ≤ 0,0001; MET (F(41,145) = 1,100, p ≤ 0,0001; PHE (F(10,451) = 13,319, p ≤ 0,0001; LYS (F(6,682) = 3,379, p ≤ 0,0001; HIS (F(6,670) = 3,804, p ≤ 0,0001; iš- skyrus THR) bei analizuotų veiksnių sąveika (VAL (F(4,587)=1,400, p ≤ 0,0001; MET (F(7,580) = 0,203, p ≤ 0,0001; LYS (F(12,887) = 6,517,

315 p ≤ 0,0001; HIS (F(4,475) = 2,552, p ≤ 0,0001, iškyrus ILE, LEU, THR ir PHE). Pakeičiamųjų ir nepakeičiamųjų AR vidutinis kiekis (proc.) NF, TF ir KF būdu apdorotuose lubinų baltymų izoliatuose/koncentratuose pavaizduotas 3.5.2 a, b paveiksluose. Lygininant NF ir TF mėginius, TF P. pentosaceus Nr. 8, Nr. 9 ir Nr. 10 veislių Vilčiai ir Vilniai izoliatai/koncentratai pasižymėjo didesniu ALA, SER, PRO ir GLU kiekiu. Lyginant NF ir KF veislių Vilčiai, Vilniai ir hibridų Nr. 1072 mėginius, KF mėginiuose nustatytas didesnis ALA, GLY, SER ir PRO kiekis. a)

3.5.2 pav. Nepakeičiamųjų (a) ir pakeičiamųjų (b) aminorūgščių kiekis (proc.) lubinų baltymų izoliatuose/koncentratuose NF – nefermentuoti mėginiai; TF – tradicinė fermentacija; KF – kietafazė fermententacija.

316

KF mėginiuose, lyginant su TF, GLU kiekis nustatytas vidutiniškai 10,01 proc. didesnis. Lyginant NF ir KF mėginius, daugeliu atvejų KF mėginiuose TYR kiekis nustatytas mažesnis. KF mėginiuose LEU ir PHE kiekis, atitinkamai, 39,60 ± 0,44 proc. ir 60,57 ± 0,57 proc. nustatytas didesnis kiekis, lyginant su NF mėginiais. TF mėginiuose HIS kiekis vidutiniškai 50,83 ± 0,49 proc. nustatytas didenis, lyginant su NF. Įvertinus technologinių veiks- nių įtaką AR kiekiui lubinų sėklų baltymų izoliatuose/koncentratuose, reikš- mingos įtakos pakeičiamųjų AR kiekiui turėjo lubinų veislė (GLY (F(6,722) = 1,306, p ≤ 0,0001; SER (F(5,651) = 70,777, p ≤ 0,0001; PRO (F(14,640) = 3,618, p ≤ 0,0001; ASP (F(31,323) = 48,753, p ≤ 0,0001; GLU (F(13,142) = 126,523, p ≤ 0,0001; TYR (F(24,829) = 22,348, p ≤ 0,0001, išskyrus ALA), fermentacijos technologija (GLY (F(116,983) = 22,730, p ≤ 0,0001; SER (F(104,161) = 1304,632, p ≤ 0,0001; PRO (F(65,690) = 16,235, p ≤ 0,0001; ASP (F(11,665) = 18,157, p = 0,0001; GLU (F(21,055) = 202,708, p ≤ 0,0001; TYR (F(69,625) = 62,667, p ≤ 0,0001, išskyrus ALA), fermentacijai naudotos PRB (SER (F(9,301) = 116,495, p ≤ 0,0001; GLU (F(7,631) = 73,472, p = 0,0001, išskyrus ALA, GLY, PRO, ASP, TYR), baltymų izoliavimo/koncentravimo procesas (ALA (F(101,110) = 87,277, p ≤ 0,0001; GLY (F(121,009) = 188,701, p ≤ 0,0001; SER (F(105,651) = 51,702, p ≤ 0,0001; PRO (F(104,214) = 89,445, p ≤ 0,0001; ASP (F(121,325) = 148,741, p ≤ 0,0001; GLU (F(103,122) = 127,053, p ≤ 0,0001; TYR (F(104,009) = 102,348, p ≤ 0,0001) bei anali- zuotų veiksnių sąveika (ALA (F(3,197)=3,792, p ≤ 0,0001; GLY (F(7,117) = 1,383, p ≤ 0,0001; SER (F(6,198) = 77,630, p ≤ 0,0001; PRO (F(4,390) = 1,085, p ≤ 0,0001; ASP (F(8,419) = 13,104, p ≤ 0,0001; GLU (F(8,802) = 84,737, p≤ 0,0001; TYR (F(9,797) = 8,817, p ≤ 0,0001). Įvertinus techno- loginių veiksnių įtaką nepakeičiamųjų AR kiekiui lubinų sėklų baltymų izoliatuose/koncentratuose, pastarajam reikšmingos įtakos turėjo lubinų veislė (VAL (F(6,043) = 6,968, p ≤ 0,0001); ILE (F(8,109) = 5,836, p ≤ 0,0001; LEU (F(29,108) = 101,592, p ≤ 0,0001; THR (F(4,299) = 2,421, p ≤ 0,0001; MET (F(25,926) = 3,667, p ≤ 0,0001); PHE (F(16,618) = 18,366, p ≤ 0,0001), MET (F(25,926) = 3,667, p ≤ 0,0001); PHE (F(16,618) = 18,366, p ≤ 0,0001, išskyrus LYS), fermentacijos technologija (VAL (F(67,821) = 78,196, p ≤ 0,0001; ILE (F(169,539) = 122,009, p ≤ 0,0001; LEU (F(351,155) = 1225,608, p ≤ 0,000; MET (F(84,419) = 11,941, p = 0,0001; PHE (F(184,146) = 203,518, p ≤ 0,0001; HIS (F(71,312) = 31,114, p ≤ 0,0001; TRP (F(13,589) = 44,025, p ≤ 0,0001, išskyrus ALA ir LYS), fermentacijai naudotos PRB (SER (F(9,301) = 116,495, p ≤ 0,0001; GLU (F(7,631) = 73,472, p = 0,0001, išskyrus ALA, GLY, PRO, ASP ir TYR), baltymų izoliavimo/koncentravimo procesas (ALA (F(101,110) = 87,277, p ≤ 0,0001; GLY (F(121,009) = 188,701, p ≤ 0,0001; SER (F(105,651) = 317

51,702, p ≤ 0,0001; PRO (F(104,214) = 89,445, p ≤ 0,0001; ASP (F(121,325) = 148,741, p ≤ 0,0001; GLU (F(103,122) = 127,053, p ≤ 0,0001; TYR (F(104,009) = 102,348, p ≤ 0,0001) bei analizuotų veiksnių sąveika (VAL (F(7,058) = 8,138, p ≤ 0,0001; ILE (F(6,020) = 4,332, p ≤ 0,0001; LEU (F(3,671) = 12,813, p ≤ 0,0001; THR (F(5,316) = 2,994, p ≤ 0,0001; MET (F(9,566) = 1,353 , p ≤ 0,0001; PHE (F(3,420) = 3,780, p ≤ 0,0001; LYS (F(3,855) = 17,823, p ≤ 0,0001; HIS (F(14,837) = 6,474, p ≤ 0,0001; TRP (F(8,040) = 26,048, p ≤ 0,0001).

3.6. Fermentacijos įtaka biogeninių aminų kiekiui smulkintose lubinų sėklose ir jų baltymų izoliatuose/koncentratuose

Vidutinis biogeninių aminų kiekis (mg/kg) NF, TF ir KF būdu apdorotose lubinų sėklose bei jų izoliatuose/koncentratuose pavaizduotas 3.6.1 a, b paveiksle. NF smulkintų lubinų sėklų mėginiuose PEA, SPRM ir SPRM – nenustatyta, o TYM vidutinis kiekis nustatytas 0,71 ± 0,07 mg/kg. Lygininant BA kiekius NF, TF ir KF mėginiuose, vidutiniškai 23,00 ± 0,32; 86,70 ± 0,84 ir 91,01 ± 0,85 proc. didesnis, atitinkamai, PUT, CAD ir HIS kiekis nustatytas fermentuotuose mėginiuose. SPRMD kiekis KF mėginiuose nustatytas vidutiniškai 54,62 ± 0,51 proc. didesnis, lyginant su šio biogeninio amino kiekiu TF apdorotuose mėginiuose. Lyginant BA kiekius lubinų sėklų baltymų izoliatuose/koncentratuose, daugeliu atvejų NF lubinų baltymų izoliatuose/koncentratuose nustatytas didesnis BA kiekis, lyginant su TF ir KF apdorotais mėginiais. Lyginant TF ir KF apdorotus mėginius, PEA ir PUT kiekis TF apdorotuose mėginiuose nustatytas vidutiniškai, atitinkamai, 35,80 ± 0,52 proc. ir 29,17 ± 0,44 proc. didesnis. Lyginant su NF mėginiais, TF ir KF apdorotuose izoliatuose/koncentratuose HIS kiekis nustatytas vidutiniškai, atitinkamai, 83,62 ± 0,72 proc., ir 93,15 ± 0,87 proc. mažesnis. BA dideliais kiekiais gali susidaryti didelį kiekį baltymų turinčiuose produktuose ir/ar žaliavose laikymo metu, skylant atitinkamoms aminorūgštims, kurių degradavimo procesą gali inicijuoti dekarboksilazes išskiriančios PRB bei daugelis kitų veiksnių [331, 333]. Jų fiziologinis poveikis organizmui gali būti įvairus: hormoninis, inhibicinis ir kt. [334]. Įvertinus technologinių veiksnių įtaką BA kiekiui smulkintų lubinų sėklų mėginiuose, reikšmingos įtakos BA formavimuisi turėjo lubinų veislė (PEA F(1253,566) = 13,833, p ≤ 0,0001; PUT F(17482,130) = 5,161, p ≤ 0,0001; CAD F(42207,521) = 7,947, p ≤ 0,0001; HIS F(2417,926) = 12,022, p ≤ 0,0001; TYR F(80,708) = 15,592, p ≤ 0,0001; SPRMD F(256,898) = 19,804, p ≤ 0,0001; SPRM F(81,022) = 34,920, p ≤ 0,0001), fermentacijos technologija (PEA (F(58871,763) = 649,662, p ≤ 0,0001; PUT 318

(F(53104,908) = 15,678, p ≤ 0,0001; CAD (F(750790,753) = 141,361, p ≤ 0,0001; HIS F(194257,816) = 965,853, p ≤ 0,0001; TYR (F(325,091) = 62,806, p ≤ 0,0001; SPRMD F(19512,906) = 1504,232, p ≤ 0,0001; SPRM F(105,542) = 45,488, p ≤ 0,0001), fermentacijai naudotos PRB (PUT F(28226,403) = 8,333, p ≤ 0,0001; TYR F(34,434) = 6,652, p ≤ 0,0001; SPRM F(75,904) = 32,714, p ≤ 0,0001) ir analizuotų veiksnių sąveika buvo reikšminga (PEA F(1660,689) = 18,326, p ≤ 0,0001; PUT F(22888,701) = 6,758, p ≤ 0,0001; CAD (F(73313,407) = 13,804, p ≤ 0,0001; HIS F(3166,822) = 15,745, p ≤ 0,0001; TYR F(117,183) = 22,639, p ≤ 0,0001; SPRMD F(341,518) = 26,327, p ≤ 0,0001; SPRM F(151,352) = 65,232, p ≤ 0 ,0001). a)

3.6.1 pav. Biogeninių aminų kiekis (mg/kg) smulkintose lubinų sėklose (a) ir jų baltymų izoliatuose/koncentratuose (b) NF – nefermentuoti mėginiai; TF – tradicinė fermentacija; KF – kietafazė fermententacija. 319

Įvertinus technologinių veiksnių įtaką BA kiekiui lubinų sėklų baltymų izoliatuose/koncentratuose, reikšmingos įtakos BA kiekiui turėjo lubinų veislė (PEA (F(1018,934) = 326,199, p ≤ 0,0001); PUT (F(429,919) = 18,074, p ≤ 0,0001); CAD (F(962,804) = 295,191, p ≤ 0,0001; HIS (F(583,502) = 864,961, p ≤ 0,0001; TYM (F(19,275) = 9,753, p ≤ 0,0001; SPRMD (F(235,672) = 41,491, p ≤ 0,0001); SPRM (F(139,992) = 44,150, p ≤ 0,0001), fermentacijos technologija (PEA (F(607,331) = 194,430, p ≤ 0,0001); PUT (F(766,206) = 32,211, p ≤ 0,0001); CAD (F(249,728) = 76,565, p ≤ 0,0001); HIS (F(69,961) = 103,707, p ≤ 0,0001); TYM (F(34,374) = 17,393, p ≤ 0,0001); SPRMD (F(316,913) = 55,793, p ≤ 0,0001, išskyrus SPRM), fermentacijai naudotos PRB (PEA (F(87,031) = 27,862, p ≤ 0,0001); PUT (F(1103,722) = 46,400, p ≤ 0,0001); TYM (F(21,928) = 11,095, p ≤ 0,0001); SPRMD (F(443,426) = 78,066, p ≤ 0,0001); SPRMD (F(30,107) = 9,495, p ≤ 0,0001, išskyrus CAD ir HIS), baltymų izoliavimo/koncentravimo procesas (PEA (F(199,471)=13064,800, p ≤ 0,0001); PUT (F(239,297) = 401868,846, p≤0,0001); CAD (F(881,931) = 2030274,789, p ≤ 0,0001; HIS (F(1409,803) = 153579,001, p ≤ 0,0001; SPRMD (F(31,069) = 1093,939, p ≤ 0,0001); SPRM (F(33,065) = 423,759, p ≤ 0,0001, išskyrus TYM) bei analizuotų veiksnių sąveika (PEA (F(5,872) = 384,589, p ≤ 0,0001), PUT (F(6,207) = 10424,714, p ≤ 0,0001); CAD (F(5,062) = 11652,106, p ≤ 0,0001); HIS (F(11,398) = 1241,618, p ≤ 0,0001), SPRMD (F(4,447) = 156,587, p ≤ 0,0001), SPRM (F(4,150) = 53,181, p ≤ 0,0001, išskyrus TYM). Fermentacijos metu technologiniai mikroorganizmai modifikuoja fermentuojamo substrato maistinę ir biologinę vertę, susidarant nebūdingiems natūraliai tam substratui komponentams, pvz.: fenolinėms rūgštims bei biogeniniams aminams [338].

3.7. Bendras fenolinių junginių kiekis smulkintose lubinų sėklose ir jų baltymų izoliatuose/koncentratuose bei jų antioksidacinis aktyvumas (DPPH)

Vidutinis bendras fenolinių junginių kiekis (BFJ) (proc.) NF, TF ir KF būdu apdorotose lubinų sėklose bei jų izoliatuose/koncentratuose pavaizduotas 3.7.1 a, b paveiksle. Daugeliu atvejų, didesnis BFJ kiekis nustatytas smulkintose lubinų sėklose, lyginant su jų kiekiu lubinų baltymų izoliatuose/koncentratuose. Smulkintose lubinų sėklose BFJ kiekis vidutiniškai kito nuo 318,21 ± 4,52 proc. iki 643,97 ± 5,71 proc., atitinkamai, NF hibridų Nr. 1072 ir Vilčiai veislės sėklose. TF ir KF apdorotuose lubinų baltymų izoliatuose/koncentratuose BFJ kiekis vidutiniškai varijavo nuo 236,56 ± 3,34 proc. iki 525,14 ± 5,12 proc. (atitinkamai, TF hibridų Nr. 1072 ir KF hibridų Nr. 1800 mėginiuose). TF ir KF Vilčiai veislės mėginiuose BFJ 320 kiekis nustatytas vidutiniškai, atitinkamai, 48,79 ± 0,81 proc. ir 27,81 ± 0,48 proc. mažesnis. Daugelis fenolinių junginių pasižymi prevenciniu poveikiu daugeliui ligų [352]. PRB buvimas fermentuojamame substrate gali iniciuoti įvairius pokyčius, pvz.: paprastų fenolinių junginių konversiją ir depoli- merizaciją, naujų junginių susidarymą [355, 356]. a)

3.7.1 pav. Bendras fenolinių junginių kiekis (proc.) smulkintose lubinų sėklose (a) ir jų baltymų izoliatuose/koncentratuose (b) NF – nefermentuoti mėginiai; TF – tradicinė fermentacija; KF – kietafazė fermententacija.

321

Vidutinis antioksidacinis aktyvumas (AA) (DPPH) (proc.) NF, TF ir KF būdu apdorotose lubinų sėklose bei jų izoliatuose/koncentratuose pavaizduotas 3.7.2 a, b paveiksle. Daugeliu atvejų TF ir KF smulkintų lubinų sėklų mėginių antioksidacinis aktyvumas nustatytas mažesnis ir kito nuo 4,37 ± 0,11 proc. iki 17,19 ± 0,24 proc. (atitinkamai, NF hibridų Nr. 1700 ir Nr. 1701 sėklų), lyginant su NF. TF hibridų Nr. 1700 ir Vilčiai veislės mėginių a)

3.7.2 pav. Smulkintų lubinų sėklų (a) ir jų baltymų izoliatų/koncentratų (b) antioksidacinis aktyvumas (DPPH) (proc.) NF – nefermentuoti mėginiai; TF – tradicinė fermentacija; KF – kietafazė fermententacija.

322 antioksidacinis aktyvumas nustatytas vidutiniškai 46,99 ± 0,78 proc. ir 22,34 ± 0,33 proc. didesnis (atitinkamai, Vilčiai ir hibridų Nr. 1700 mėgi- niuose), nei NF mėginiuose. Lyginant su NF lubinų baltymų izoliatų/kon- centratų antioksidacinis aktyvumas nustatytas vidutiniškai didesnis TF ir KF mėginių ir varijavo nuo 6,07 ± 0,14 proc. iki 25,86 ± 0,42 proc. (atitinkamai, TF hibridų Nr.1800 ir KF Vilčiai veislės mėginių). TF ir KF hibridų Nr. 1701 mėginių antioksidacinis aktyvumas nustatytas, atitinkamai 5,52 ± 0,09 proc. ir 10,66 ± 0,17 proc. mažesnis, nei NF mėginiuose. Dažniausiai bendras fenolinių junginių kiekis koreliuoja su antioksidaciniu aktyvumu, tačiau yra nuomonių, kad antioksidacinis aktyvumas gali priklausyti nuo fenolinių junginių struktūros [361]. Įvertinus technologinių veiksnių įtaką BFJ kiekiui smulkintų lubinų sėklų mėginiuose ir jų antioksidaciniam aktyvumui, šiems rodikliams reikšmingos įtakos turėjo lubinų veislė (BFJ F(1253,566)=13,833, p≤0,0001; AA (DPPH) F(17482,130) = 5,161, p ≤ 0,0001), fermentacijos technologija (BFJ F(1253,566)=13,833, p ≤ 0,0001; AA (DPPH) F(17482,130)=5,161, p ≤ 0,0001), fermentacijai naudotos PRB (BFJ F(28226,403) = 8,333, p ≤ 0,0001; AA (DPPH) F(34,434) = 6,652, p ≤ 0,0001) ir analizuotų veiks- nių sąveika buvo reikšminga (BFJ F(1660,689) = 18,326, p ≤ 0,0001; AA (DPPH) F(22888,701) = 6,758, p ≤ 0,0001). Įvertinus technologinių veiksnių įtaką BFJ kiekiui lubinų sėklų baltymų izoliatuose/koncentratuose ir jų antioksidaciniam aktyvumui, šiems rodikliams reikšmingos įtakos turėjo lubinų veislė (BFJ F(1253,566) = 13,833, p ≤ 0,0001; AA (DPPH) F(17482,130) = 5,161, p ≤ 0,0001), fermentacijos technologija (BFJ F(1253,566) = 13,833, p ≤ 0,0001; AA (DPPH) F(17482,130) = 5,161, p ≤ 0,0001), fermentacijai naudotos PRB (BFJ F(28226,403) = 8,333, p ≤ 0,0001; AA (DPPH) F(34,434) = 6,652, p ≤ 0,0001) ir šių analizuotų veiksnių sąveika buvo reikšminga (BFJ F(1660,689) = 18,326, p ≤ 0,0001; AA (DPPH) F(22888,701) = 6,758, p ≤ 0,0001), tačiau baltymų izoliavimo/koncentravimo procesas reikšmingos įtakos neturėjo.

3.8. Technologinių veiksnių įtaka izoflavonų kiekiui smulkintose lubinų sėklose ir jų baltymų izoliatuose/koncentratuose

Vidutinė genisteino koncentracija (μg/g) NF, TF ir KF būdu apdorotose lubinų sėklose bei jų izoliatuose/koncentratuose pavaizduota 3.8.1 a, b paveiksle. NF smulkintose lubinų sėklose genisteino koncentracija viduti- niškai kito nuo 4,37 ± 0,07 μg/g iki 17,19 ± 0,19 μg/g, atitinkamai hibridų Nr. 1700 ir Nr. 1701 mėginiuose. TF Vilčiai ir hibridų Nr. 1700 mėginiuose

323 a)

3.8.1 pav. Genisteino kiekis (μg/g) smulkintose lubinų sėklose (a) ir jų baltymų izoliatuose/koncentratuose (b) NF – nefermentuoti mėginiai; TF – tradicinė fermentacija; KF – kietafazė fermententacija. vidutiniškai genisteino kiekis nustatytas 46,99 ± 0,43 proc. ir 77,66 ± 0,75 proc. didesnis, lyginant su NF mėginiais. Vidutiniškai genisteino koncentracija nustatyta 60,38 ± 0,31 ir 52,28 ± 0,48 proc. didesnė, atitinkamai, KF hibridų linijų Nr. 1700 ir Nr. 1702 mėginiuose. Daugeliu atvejų mažesnė genisteino koncentracija nustatyta lubinų baltymų izoliatuose/koncentratuose, lyginant su fermentuotomis smulkintų lubinų sėklomis. TF ir KF apdorotuose Vilčiai veislės sėklų mėginiuose vidutiniškai nustatytas, atitinkamai, 51,82 ± 0,57 proc. ir 61,56 ± 0,64 proc., didesnis genisteino kiekis. Įvertinus technologinių veiksnių įtaką genisteino kiekiui smulkintose sėklose ir baltymų izoliatuose/koncentratuose, reikšmingos įtakos jam turėjo lubinų veislė (F(930,701) = 1475,906, p ≤ 0,0001), fermentacijos 324 technologija (F(17,301) = 27,435, p ≤ 0,0001), fermentacijai naudotos PRB (F(153,483) = 243,393, p ≤ 0,0001), baltymų izoliavimo/koncentravimo procesas (F(356,728) = 565,699, p ≤ 0,0001) ir šių analizuotų faktorių są- veika buvo reikšminga (F(3,065) = 4,86, p ≤ 0,0001). Apie izoflavonų fiziologinį poveikį literatūroje pateikiamos prieštaringos nuomonės [371], tačiau biodegradavimas technologiniais mikroorganizmais padidina šių junginių kiekį substrate [377]. TI slopina tripsino ir chimotripsino akty- vumą, o tai tiesiogiai veikia virškinimo procesą [104]. Dėl šios priežasties ieškoma įvairių būdų tripsino inhibitorių aktyvumui žaliavoje mažinti [103].

3.9. Fermentacijos įtaka tripsino inhibitorių aktyvumui lubinų sėklose

Tripsino inhibitorių aktyvumas lubinų sėklose (proc.) NF, TF ir KF būdu apdorotose lubinų sėklose pavaizduotas 3.9.1 paveiksle. Daugeliu atvejų TF ir KF smulkintuose lubinų sėklų mėginiuose tripsino inhibitorių aktyvumas (TIA, proc.) nustatytas mažesnis (išskyrus, TF P. pentosaceus Nr. 8 apdorotuose hibridų Nr. 1701 mėginiuose), lyginant su NF mėginiais. Lyginant su NF, TF ir KF mėginius, vidutiniškai mažesnis TIA (atitinkamai, 16,65 ± 0,22 proc. ir 23,05 ± 0,28 proc. mažesnis) nustatytas, KF mėginiuose. TIA vidutiniškai kito nuo 19,79 ± 0,19 proc. iki 23,47 ± 0,24 proc., atitinkamai, Vilčiai ir hibridų Nr. 1701 mėginiuose. Įvertinus technologinių veiksnių įtaką TIA smulkintose lubinų sėklose, TIA reikšmingos įtakos turėjo lubinų veislė (F(13,863) = 4,179, p ≤ 0,0001), fermentacijos technologija (F(5,405) = 13,844, p ≤ 0,0001), fermentacijai naudotos PRB (F(6,058) = 15,518, p ≤ 0,0001).

3.9.1 pav. Tripsino inhibitorių aktyvumas (proc.) lubinų sėklose TIA – tripsino inhibitorių aktyvumas; NF – nefermentuoti mėginiai; TF – tradicinė fermentacija; KF – kietafazė fermententacija. 325

3.10. Fermentacijos įtaka smulkintų lubinų sėklų ir jų baltymų izoliatų/koncentratų baltymų virškinamumui in vitro

Vidutinis lubinų baltymų in vitro virškinamumas (proc.) NF, TF ir KF būdu apdorotose lubinų sėklose bei jų izoliatuose/koncentratuose pavaizduotas 3.10.1 a, b paveiksle. Daugeliu atvejų TF ir KF smulkintų lubinų sėklų mėginių baltymų virškinamumas in vitro nustatytas vidutiniškai didesnis (išskyrus, KF Vilčiai ir Vilniai veislių sėklose) ir varijavo nuo 85,31 ± 0,84 proc. iki 92,61 ± 0,94 proc. (atitinkamai, TF hibridų Nr. 1800 ir KF Nr. 1702 sėklų), lyginant su NF. Didžiausias baltymų virškinamumas in vitro vidutiniškai nustatytas KF hibridų linijų Nr. 1800 ir Nr. 1702 sėklų (atitinkamai, 90,98 ± 0,95 proc. ir 92,61 ± 0,98 proc.), mažiausias – KF Vilčiai ir Vilniai veislės sėklų mėginių (atitinkamai, 82,66 ± 0,73 proc. Ir 83,74 ± 0,76 proc.). Įvertinus technologinių veiksnių įtaką baltymų virškinamumui in vitro, smulkintų lubinų sėklų mėginių baltymų virškinamumui reikšmingos įtakos turėjo lubinų veislė (F(45,302) = 128,470, p ≤ 0,0001), fermentacijos technologija (F(18,539) = 52,573, p ≤ 0,0001), fermentacijai naudotos PRB (F(13,819) = 39,189, p ≤ 0,0001) ir analizuotų veiksnių sąveika buvo reikšminga (F(84,640) = 29,846, p ≤ 0,0001). Lyginant izoliatų/koncentratų baltymų virškinamumą in vitro, nustatyta, kad TF ir KF apdoroti izoliatai/koncentratai, lyginant su NF, pasižymėjo didesniu virškinamumu ir TF bei KF apdoruotuose mėginiuose virškinamumas in vitro vidutiniškai varijavo nuo 89,27 ± 0,76 proc. iki 93,07 ± 0,87 proc. (atitinkamai, TF hibridų Nr. 1701 ir Vilniai veislės sėklų) ir nuo 86,37 ± 0,84 proc. iki 94,81 ± 0,93 proc. (atitinkamai, KF hibridų Nr. 1072 iki hibridų Nr. 1702 mėginių). Įvertinus technologinių veiksnių įtaką lubinų sėklų baltymų izoliatų/koncentratų baltymų virškinamumui in vitro reikšmingos įtakos šiam rodikliui turėjo lubinų veislė (F(30,668) = 567,783, p ≤ 0,0001), fermentacijos technologija (F(5,748) = 106,424, p ≤ 0,0001), fermentacijai naudotos PRB (F(4,921) = 91,102, p ≤ 0,0001), baltymų izoliavimo/koncentravimo procesas (F(178,543) = 506,319, p ≤ 0,0001) ir veiksnių sąveika buvo reikšminga (F(29,846) = 84,640, p ≤ 0,0001). Baltymų kokybę pagrinde apsprendžia aminorūgščių profilis, tačiau taip nemažiau svarbus yra ir jų virškinamumas. Augalinių baltymų virškinamumas yra šiek tiek mažesnis nei gyvulinės kilmės baltymų, tačiau taikant technologinius procesus būtų galima padidinti jų virškinamumą [221].

326 a)

3.10.1 pav. Smulkintų lubinų sėklų (a) ir jų baltymų izoliatų/koncentratų (b) baltymų in vitro virškinamumas (proc.) NF – nefermentuoti mėginiai; TF – tradicinė fermentacija; KF – kietafazė fermententacija.

IŠVADOS

1. Išanalizuotos naujai išvestų Lietuvoje lubinų veislių (Vilčiai, Vilniai ir hibridų linijų Nr. 1072, 1734, 1700, 1701, 1800 ir 1702) sėklos, pagal cheminę sudėtį yra tinkama žaliava baltymingų žaliavų/produktų gamybai ir reikšmingos įtakos sėklų cheminei sudėčiai turi lubinų veislė (p ≤ 0,0001): 1.1. Didžiausias baltymų kiekis nustatytas Vilčiai veislės sėklose (40,8 proc.), o hibridų linijų Nr. 1700 ir 1800 sėklose baltymų kiekis nustatytas nuo 20,1 iki 25,7 proc. mažesnis.

327

1.2. Angliavandenių kiekis lubinų sėklose kito nuo 41,6 iki 50,8 proc., atitinkamai, Vilčiai ir Nr. 1800 sėklose. 1.3. Mažiausias riebalų kiekis nustatytas Vilčiai veislės sėklose (4,4 proc.), o vyraujančios NRR (C18:1) ir (C18:2), SRR – (C16:0), MNRR (C15:1 ir C20:1). 1.4. Didžiausias pelenų kiekis nustatytas Vilčiai veislės sėklose (4,30 proc.). 1.5. Alkaloidų kiekis Vilčiai ir Vilniai veislių sėklose (atitinkamai, 0,0300 proc. ir 0,0210 proc.) nustatytas didesnis, nei naujai išvestų hib- ridų linijų sėklose. 1.6. Makroelementų vidutinis kiekis lubinų sėklose kito tokiu eiliškumu: K > Mg > Ca > Na (13,230 > 2,446 > 2,021 > 1,099 g/kg s.m.). 1.7. Mikroelementų vidutinis kiekis lubinų sėklose kito tokiu eiliškumu: Mn > Fe > Zn > Sr > Cu > Al > Ni > Cr > Se > Co > Pb > Cd > As > Ag (95,96 > 60,08 > 43,92 > 17,45 > 6,273 > 3,114 > 1,659 > 1,281 > 0,1063 > 0,1050 > 0,076 > 0,038 > 0,023 mg/kg s.m.)

2. Daugeliu atvejų fermentacija parinktomis PRB yra tinkama technologija TIA lubinų sėklų žaliavoje sumažinti ir lubinų baltymų virškinamumui in vitro padidinti: 2.1. Daugelyje fermentuotų mėginių (išskyrus, TF P. pentosaceus Nr. 8 apdorotus Nr. 1701 mėginius) TIA nustatytas mažesnis, lyginant su NF mėginiais. TIA mėginiuose reikšmingos įtakos turėjo lubinų veislė, fermentacijos technologija ir fermentacijai naudotos PRB (p ≤ 0,0001). 2.2. Apdorojimas TF ir KF daugeliu atvejų padidino smulkintų lubinų sėklų baltymų virškinamumą in vitro ir jis kito nuo 85,3 iki 92,6 proc. (atitinkamai, TF hibridų Nr. 1800 ir KF Nr. 1702 mėginių), lyginant su NF. Baltymų izoliatų/koncentratų virškinamumui in vitro reikšmingos įtakos turėjo lubinų veislė, fermentacijos technologija, fermentacijai naudotos PRB, baltymų izoliavimo/koncentravimo procesas ir analizuotų veiksnių sąveika buvo reikšminga (p ≤ 0,0001).

3. Technologinių veiksnių įtaka BFJ ir genisteino kiekiui bei antioksida- cinėms lubinų sėklų ir jų baltymų izoliatų/koncentratų savybėms kito priklausomai nuo taikytos technologijos: 3.1. Daugeliu atvejų, didesnis BFJ kiekis nustatytas smulkintose lubinų sėklose (vidutiniškai kito nuo 318,2 iki 644,0 proc.), lyginant su jų kiekiu lubinų baltymų izoliatuose/koncentratuose (vidutiniškai varijavo nuo 236,6 iki 525,1 proc.). 3.2. Daugeliu atvejų TF ir KF apdorotų smulkintų lubinų sėklų mėginių antioksidacinis aktyvumas nustatytas mažesnis ir kito nuo 4,4 iki 328

17,2 proc. lyginant su NF. BFJ kiekiui smulkintų lubinų sėklų ir kon- centratų/izoliatų mėginiuose ir jų antioksidaciniam aktyvumui reikš- mingos įtakos turėjo lubinų veislė, fermentacijos technologija, fermentacijai naudotos PRB ir analizuotų veiksnių sąveika buvo reikšminga (p ≤ 0,0001), baltymų izoliavimo/koncentravimo procesas reikšmingos įtakos neturėjo. 3.3. Daugeliu atvejų fermentacija padidino genisteino kiekį mėginiuose ir NF smulkintose lubinų sėklose genisteino koncentracija vidutiniškai kito nuo 4,37 iki 17,19 μg/g, o TF mėginiuose vidutiniškai genisteino kiekis nustatytas nuo 47,0 iki 77,7 proc. didesnis, lyginant su NF mėginiais. Baltymų izoliavimo/koncentravimo procesas sumažino genisteino kiekį mėginiuose ir įvertinus technologinių veiksnių įtaką genisteino kiekiui, reikšmingos įtakos jam turėjo lubinų veislė, fermentacijos technologija, fermentacijai naudotos PRB, baltymų izoliavimo/koncentravimo procesas ir šių veiksnių sąveika buvo reikšminga (p ≤ 0,0001).

4. Didžiausia baltymų išeiga gaunama esant pH 10,0, o lubinų baltymų tirpumui (esant pH 10,0) reikšmingos įtakos turėjo fermentacijos technologija ir fermentacijai naudotos PRB (p ≤ 0,0001). Baltymų kiekiui lubinų izoliatuose/koncentratuose reikšmingos įtakos turėjo lubinų veislė, fermentacijos technologija, fermentacijai naudotos PRB ir analizuotų veiksnių sąveika (p ≤ 0,0001).

5. Mikrobinė hidrolizė yra tinkama priemonė lubinų sėklų baltymų mole- kulinės masės ir aminorūgščių profilio biomodifikavimui: 5.1. Lyginant AR kiekį NF ir fermentuotuose smulkintų sėklų mėgi- niuose, daugeliu atvejų TF L. sakei ir P. acidilactici apdorotuose lubinų sėklų mėginiuose VAL, THR ir MET kiekis nustatytas didesnis. KF apdorotuose mėginiuose LEU, THR ir MET kiekis padidėjo, lyginant su NF mėginiais. 5.2. Lyginant AR kiekį NF ir fermentuotuose lubinų sėklų baltymų izoliatuose/koncentratuose, TF mėginiai (TF P. pentosaceus Nr. 8, Nr. 9 ir Nr. 10 veislių Vilčiai ir Vilniai izoliatai/koncentratai) pasižymėjo didesniu ALA, SER, PRO ir GLU kiekiu. Lyginant NF ir KF apdorotus veislių Vilčiai, Vilniai ir hibridų Nr. 1072 mėginius, KF mėginiuose nustatytas didesnis ALA, GLY, SER ir PRO kiekis. 5.3. TF ir KF technologija turėjo įtakos baltymų molekulinei masei ir daugiau mažos molekulinės masės baltymų frakcijų (nuo 10 kDa iki 20 kDa) nustatyta biodegraduotuose mėginiuose, lyginant su NF. TF ir KF izoliatuose/koncentratuose vyravo nuo 120 kDa iki 200 kDa mole- kulinės masės baltymų frakcijos, o baltymų frakcijų, kurių molekulinė 329

masė nuo 38 kDa iki 47 kDa, nebuvo nustatyta, tačiau minėtos frakcijos buvo nustatytos NF mėginiuose.

6. Lubinų sėklų baltymų izoliavimo/koncentravimo procesas gali būti taikomas aukštos biologinės vertės saugių žaliavų/produktų gamybai, nes: 6.1. Taikant baltymų izoliavimo procesą fermentuotuose produktuose nelieka D(–) pieno rūgšties, o žaliavos/produkto biologinė vertė po biodegradavimo reikšmingai pagerėja. 6.1. Lyginant BA kiekį lubinų sėklų baltymų izoliatuose/koncentratuose, daugeliu atvejų NF lubinų baltymų izoliatuose/koncentratuose nustatytas didesnis BA kiekis, lyginant su TF ir KF apdorotais mėginiais. Lyginant TF ir KF apdorotus mėginius, PEA ir PUT kiekis TF apdorotuose mė- giniuose nustatytas vidutiniškai, atitinkamai, 35,8 ir 29,2 proc. didesnis. Lyginant su NF mėginiais, TF ir KF apdorotuose izoliatuose/koncentratuose, HIS kiekis nustatytas vidutiniškai, atitinkamai, 83,6 ir 93,2 proc. mažesnis.

PRAKTINĖS REKOMENDACIJOS

Naujai Lietuvoje išvestos siauralapių lubinų (Lupinus angustifolius) veis- lių ir jų hibridų (Vilčiai, Vilniai ir hibridų linijų Nr. 1072, 1734, 1700, 1701, 1800 ir 1702 sėklos) sėklos (ypač Vilčiai, nes juose nustatytas didžiausias baltymų kiekis) gali būti rekomenduojamos baltymingų žaliavų/produktų gamybai. Lubinų sėklų ir jų baltymų izoliatų/koncentratų apdorojimui galima rekomenduoti fermentaciją (priklausomai nuo lubinų veislės bei naudojamo mikroorganizmo ir norimų gauti substrato savybių, kietafazę arba tradicinę) atrinktais technologiniais mikroorganizmais (PRB), nes pastarieji, daugeliu atvejų, sumažina antimitybinių veiksnių aktyvumą, pagerina baltymų virš- kinamumą, padidina BFJ kiekį bei laisvo genisteino kiekį, o tai turi įtakos didesniam substrato antioksidaciniam aktyvumui. Lubinų baltymų izoliatus/koncentratus galima rekomenduoti gaminti, esant pH 10,0, nes šiomis sąlygomis gaunama didžiausia baltymų išeiga. Mikrobinė hidrolizė gali būti rekomenduojama lubinų sėklų baltymų mo- lekulinės masės ir aminorūgščių profilio biomodifikavimui, siekiant pagerinti baltymingo substrato savybes, ypač biologinę vertę.

330

ANNEX

Tyrimų sąlygos. Lubinų selekciniai bandymų pasėliai buvo auginami LAMMC Vokės filialo šešialaukėje sėjomainoje, priešsėlis – vasariniai javai; dirvožemis – paprastasis išplautžemis ( IDp ) vidutinio rūgštingumo, mažo humusingumo (2,0–2,1), azoto 0,096–0,117 proc., fosforo 113,2– 147,3 mg/kg, kalio 126,4–153,3 mg/kg dirvožemio. Dirva lubinams buvo paruošta pagal įprastą intensyvią technologiją: gilus rudeninis arimas, kultivavimas (pagal galimybes ir atsižvelgiant į meteorologines sąlygas kultivuota 2 kartus).

1 lentelė. Trakų Vokės meteorologinės stoties oro temperatūros (°C) ir kritulių duomenys (mm). Mėnuo Daugiametis vidurkis 2014 m. Oro temperatūra, °C Gegužė 12,5 13,4 Birželis 15,7 14,5 Liepa 16,9 20,1 Rugpjūtis 16,3 17,8 Kritulių kiekis, mm Gegužė 60 71 Birželis 77 48 Liepa 78 68 Rugpjūtis 68 117

Meteorologiniai duomenys (2014 m.) Balandžio mėn. Vyravo šilti orai. Trečiąjį balandžio dešimtadienį vidu- tinė dirvožemio temperatūra po natūralia danga iki 20 cm. buvo 9–13 °C, kai kur atskiromis dienomis 5 cm gylyje pasiekdavo 22–25 °C. Trečią balandžio mėn. dešimtadienį ariamasis sluoksnis buvo labai sausas, saulės spindėjimo trukmė 110–130 val., maždaug dvigubai viršijo dešimtadienio standartinę klimato normą. Gegužės mėn. Orai buvo permainingi. Mėnesio pradžioje užregistruotos šalnos iki –5 °C. Lubinai sudygo gegužės 11 dieną, vasariniai kviečiai ge- gužės 9 dieną, dobilai gegužės 14 dieną. Pirmą dešimtadienį ariamasis sluoksnis buvo sausas. Nuo gegužės 9 dienos drėgmės perteklius, kuris buvo jaučiamas iki gegužės 23 dienos, nuo 27 dienos drėgmės trūkumas, lubinai bei dobilai vystosi lėtai oro temperatūra svyruoja nuo 25 iki 29 °C. Birželio mėn. 1,2 °C vėsesnis nei įprasta. Šilčiausias buvo pirmasis de- šimtadienis. Vėliau vyravo vėsūs drėgni orai. Pagal Selianinovo hidrotermi- nį koeficientą rytinėje šalies dalyje vyravo drėgmės perteklius (HTK > 1,5) pirmame, antrame ir trečiame dešimtadieniuose. Saulė birželio mėnesį

331 spindėjo 175–220 val. (70–100 val. trumpiau nei SKN). Bandyminiame lauke vyravo drėgmės perteklius visą birželio mėnesį iki liepos 3 dienos. Liepos mėn. Liepą vyravo karšti ir sausi orai, temperatūra buvo 3,2 °C aukštesnė nei SKN. Aukščiausia oro temperatūra kilo iki 30–35 °C. Liepos mėnesio vidutinė oro temperatūra buvo 20,1 °C. Aukščiausia pirmo dešim- tadienio 29 °C, antro – 28 °C, trečio dešimtadienio – 33 °C. Bandyminiame lauke pirmo dešimtadienio dienomis įsivyravus sausiems ir karštiems orams dešimtadienio pabaigoje sumažėjo dirvožemio drėgmė, antrąjį dešimtadienį 20 cm gylyje dirvožemiai sausi, trečiąjį dešimtadienį 50 cm gylyje dirvo- žemis buvo perdžiūvęs, liepos 24 dieną išdegė dobilų ir motiejukų mišinys. Lubinai pradėjo mesti lapus, kviečiai – gelsta. Rugpjūčio mėn. Orai permainingi. Pirmąjį dešimtadienį užregistruota stichinė kaitra – 6–7 dienas iš eilės aukščiausia oro temperatūra 30 °C ir daugiau. HTK < 0,5 Nuo antrojo dešimtadienio pabaigos orai atvėso 1,0– 1,5. Ypač lietingas rugpjūčio trečias dešimtadienis, dėl liūčių kritulių kiekis šį dešimtadienį viršijo mėnesio kritulių normą. Lubinai subrendo liepos tre- čio dešimtadienio pabaigoje.

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CURRICULUM VITAE

Name, Surname: Vytautė Šakienė (Starkutė) Address: Department of Food Safety and Quality, Lithuanian University of Health Sciences, Tilžės 18, LT-47181 Kaunas, Lithuania

Phone: +370 646 30033 E-mail: [email protected]

Education: 2014–2018 PhD studies in Zootechics, Lithuanian University of Health Sciences, Kaunas. 2012–2014 Master degree in Public Health, Lithuanian University of Health Sciences, Kaunas. 2008–2012 Bachelor in Public Health, Lithuanian Veterinary Academy, Kaunas

Professional from 2016–until now Activity: Assistant at the Department of Food Safety and Quality, Lithuanian University of Health Sciences

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ACKNOWLEDGEMENTS

I would like to thank my supervisor Prof. Dr. Elena Bartkienė for her kind support and useful advices during the whole PhD process. I would like to thank Assoc. Prof. Dr. Vadims Bartkevics (University of Latvia (LU)), Prof. Dr. Peggy G. Braun and Dr. Claudia Wiacek (University of Leipzig (LU)) for sharing their scientific knowledge and experience. Finally, I am thankful to my family for being very supportive and encouraging.

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