Alternative Brewing Organisms in Wort Fermentation for Novel Non-To-Low Alcoholic Beverages with Unique Flavour Profiles

Alternative brewing organisms in wort fermentation for novel non-to-low alcoholic beverages with unique flavour profiles

MASTER THESIS

Mariana Machado Pires Canoso Integrated Master on Bioengineering – Biological Engineering Faculty of Engineering of University of Porto (FEUP), Portugal

Supervisor (FEUP) Prof. F. Xavier Malcata

Supervisor (Carlsberg Research Laboratory) Marta Mikš Gemma Buron-Moles

June 2017

Never be afraid of going through the hot sand to reach the sea

Mom

Acknowledgments

First of all, I would like to say the biggest thank you I can to Gemma and Marta. For Gemma, thanks for the HUGE support and teaching, for all your dedication and patient for my questions and mistakes, for always be ready to help, for spending so much time with me even when a bunch of other things were around… THANKS for caring so much about me and for being my non-Danish mum in Denmark for the past 4/5 months! For Marta, thanks for the advices, guidance and support, for the discussions and great learnings, for always giving your best and getting time for me in your super busy agenda. Without you both, for sure, this project would not be possible! Thanks to Kathrine for your constant good mood and friendship during the long days in the laboratory, for always helping me, for making my days funnier and more relaxed. For Jonas and Rocío, thanks for such a nice environment at the office, for all the laughs and chitchats, but also for the productive discussions and support. For Rosa and Jochen, I feel quite grateful for the chance of developing this project at Carlsberg Research Laboratory. Thanks to all those that made lunch time, cake club and Friday bar always so enjoyable and amusing. I would also like to express my gratitude to Prof. Malcata for accepting me under his academic guidance in this project - and in particular his careful review of this thesis, and also on behalf the MIB’s Direction for allowing me to take this great journey. For Catarina, João and Rita, also enormous thanks for all the excellent moments during the last years, for sure you contributed a lot to make it much better. Last but not least, thanks to Augusto for being the input of this great adventure and for being always present during this 5 year journey. Thanks to my family and friends for always believing in me and giving me their best support on everything.

O Prof. Francisco Xavier Malcata, orientador desta dissertação, é membro integrado do LEPABE – Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia, financiado pelos Projetos (i) POCI-01-0145-FEDER- 006939 (Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia, UID/EQU/00511/2013) financiado pelo Fundo Europeu de Desenvolvimento Regional (FEDER), através do COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) e por fundos nacionais através da Fundação para a Ciência e a Tecnologia I.P., (ii) NORTE-01-0145-FEDER-000005 – LEPABE-2-ECO-INNOVATION, financiado pelo Fundo Europeu de Desenvolvimento Regional (FEDER), através do COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) e Programa Operacional Regional do Norte (NORTE2020)

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Abstract

The non-alcoholic and clean label beverages are getting more and more important in the market, due to an increased interest in terms of health and nutrition. Food grade lactic acid bacteria (LAB) strains and non-conventional yeast were tested in attempts to find strains with high productivity of volatile organic compounds (VOCs), associated to a fresh and sour profile (without off-flavours) and a low or inexistent level of alcohol. All strains were inoculated in high fan glucose wort (HFGW); after preliminary sensory evaluation, the best candidates were selected, and also tested in maltose wort (MW). In the first screening, optical density (OD), pH, alcohol by volume (ABV) and VOCs concentration were measured; an aroma evaluation was also performed. In order to explain the correlation between all this data and the strains tested, as well as shed light onto their relative influence, cluster analysis (CA), principal component analysis (PCA) and partial least squares analysis (PLS) were performed. For the candidates, growth parameters (OD, pH and ABV) were measured, and a flavour (aroma and taste) evaluation was performed. In terms of results encompassing LAB, PCA explained 93% of the variance obtained in the VOCs and growth parameters (OD, pH and ABV) data, being acetaldehyde and 4-vinylguaiacol the principal components identified (83% and 10%, respectively). Comparisons between PCA results and the candidates for a role in flavour evaluation indicated that acetaldehyde might be the chief responsible VOC for the fresh and sour profile obtained. For non-conventional yeast, PCA explained 94% variance obtained in the VOCs and growth parameters data, being isoamyl alcohol and acetaldehyde the principal components identified (accounting for 63% and 31% of variability, respectively). Comparisons between the PCA results and the candidates to flavour evaluation showed that isoamyl alcohol and acetaldehyde were probably associated with the fresh and sour profile; however, more studies are suggested to take a more educated decision. Apart from Lactobacillus pentosus (Lb.25), Lactobacillus salivarius (Lb.56) and confidential yeast 2 (Y2) that presented a sweeter profile in MW, almost no sensory differences were noticed between the yeast and bacteria candidates, when fermented in HFGW and MW. For downstream processing of fermented MW, pasteurization was performed, yet no sensory differences were perceived between pasteurized and non-pasteurized samples. Different fermentation times (24 h and 7 d) were also tested for the MW fermented by non-conventional yeast, and differences in ABV level and aroma profile were noticed.

Contents

1. Introduction ...... 1 2. State of Art ...... 2 2.1. Non-to-low alcoholic beer ...... 2 2.1.1. Background ...... 2 2.1.2. Production processes ...... 3 2.2. Lactic acid bacteria ...... 3 2.2.1. Taxonomy ...... 4 2.2.1.1. Lactobacillus ...... 5 2.2.1.2. Pediococcus ...... 5 2.2.1.3. Leuconostoc ...... 5 2.2.1.4. Lactococcus ...... 5 2.2.2. Growth and metabolism ...... 6 2.2.2.1. Alternative pathways ...... 8 2.2.2.2. Impact of media characteristics ...... 8 2.2.3. Flavour ...... 9 2.3. Non-conventional yeasts ...... 12 2.3.1. Overview ...... 12 2.3.2. Growth and metabolism ...... 13 2.3.3. Flavour ...... 14 2.4. Aims of the project ...... 15 3. Material and Methods ...... 16 3.1. Media ...... 16 3.1.1. MRS medium ...... 16 3.1.2. GM17 medium ...... 16 3.1.3. YPD medium ...... 16 3.1.4. High fan glucose wort ...... 16 3.1.5. Maltose wort ...... 17 3.2. Microorganisms ...... 18 3.2.1. Lactic acid bacteria ...... 18 3.2.2. Non-conventional yeast ...... 19 3.3. Fermentation and samples processing ...... 20 3.4. Analysis ...... 21 3.4.1. Bacterial growth monitoring - optical density ...... 21 3.4.2. Acidifying activity - pH ...... 22 3.4.3. Ethanol content - alcohol by volume ...... 22 3.4.4. Sensory evaluation ...... 22

3.4.5. Aroma profiles - volatile organic compounds analysis ...... 22 3.4.6. Fermentation rate - carbon dioxide release ...... 23 3.5. Statistical analysis ...... 23 3.5.1. Cluster analysis ...... 23 3.5.2. Principal component analysis ...... 24 3.5.3. Partial least squares regression ...... 24 4. Results and Discussion ...... 25 4.1. Lactic acid bacteria ...... 25 4.1.1. Cluster analysis ...... 25 4.1.2. Principal component analysis ...... 27 4.1.3. Partial least squares regression ...... 28 4.2. Non-conventional yeast ...... 31 4.2.1. Cluster analysis ...... 31 4.2.2. Principal component analysis ...... 33 4.2.3. Partial least squares regression ...... 34 4.3. Candidates ...... 36 4.3.1. Lactic acid bacteria ...... 36 4.3.2. Non-conventional yeasts ...... 37 4.3.2.1. Yeast fermentation rate ...... 39 5. Conclusions ...... 41 6. Future Work ...... 42 References ...... 43 Annexes ...... I Annex A: Tapping wort out from a barrel ...... I Annex B: Sterile filtration of wort ...... II Annex C: ABV measurement ...... III Annex D: Sensory analysis ...... IV Annex D.1: Aroma evaluation ...... IV Annex D.2: Taste evaluation ...... V Annex E: Lactic acid bacteria and non-conventional yeast data ...... VI Annex E.1: Volatile organic compounds ...... VI Annex E.2: Sensory analysis ...... IX Annex E.3: Growth parameters ...... XV Annex F: PCA analysis ...... XXI Annex F.1: Lactic acid bacteria ...... XXI Annex F.2: Non-conventional yeast ...... XXII Annex G: Growth parameters data for candidates ...... XXIV

List of Figures

Figure 1- Most common production methods of alcohol-free beer (Brányik et al. 2012) ...... 3 Figure 2- Homofermentative and heterofermentative pathways of LAB (Reis et al. 2012) ...... 7 Figure 3- Pathways for the alternative fates of pyruvate (Lahtinen et al. 2012)...... 8 Figure 4- Functional metabolic pathways for the generation of aroma compounds from existing precursors, during lactic acid fermentation of malt wort (Nsogning Dongmo et al. 2016)...... 10 Figure 5- Kefir grains (Kniesel 2017) ...... 12 Figure 6- Respiration (left) and fermentation (right) pathways of yeast (Walker 1998) ...... 13 Figure 7- Cluster analysis plot for LAB, based on VOCs concentration ...... 25 Figure 8- Principal component analysis bi-plot obtained for LAB, based on VOCs and growth parameters (PC-1 (83%) vs PC-2 (10%)) ...... 27 Figure 9- Principal component analysis plot obtained for LAB metabolism (PC-1 (83%) vs. PC-2 (10%)) ...... 27 Figure 10- Partial least squares regression plot obtained for LAB, based on VOCs and sensory analysis (Factor-1 (85%,1%) vs Factor-2 (10%,1%) ...... 29 Figure 11- Cluster analysis plot for non-conventional yeast, based on VOCs concentration ...... 31 Figure 12- Principal component analysis plot obtained for non-conventional yeast, based on VOCs and growth parameters (PC-1 (63%) vs PC-2 (31%)) ...... 33 Figure 13- Partial least squares regression plot obtained for non-conventional yeast, based on VOCs and sensory analysis (Factor-1 (40%,11%) vs Factor-2 (54%,5%) ...... 35

Figure 14- CO2 release plot for non-conventional yeast candidates in HFGW ...... 39

Figure 15- CO2 release plot for non-conventional yeast candidates in MW ...... 40

List of Figures in Annexes :

Figure F.1. 1- Principal component analysis plot obtained for LAB, based on VOCs and growth parameters (PC-3 (3%) vs PC-4 (1%))...... XXI Figure F.1. 2- Principal component analysis 3D-plot obtained for LAB, based on VOCs and growth parameters (PC-1 (83%) vs PC-3 (3%) vs PC-4(1%)) ...... XXI Figure F.2. 1- Principal component analysis plot obtained for non-conventional yeast, based on VOCs and growth parameters (PC-3 (3%) vs PC-4 (2%))...... XXII Figure F.2. 2- Principal component analysis plot obtained for non-conventional yeast, based on VOCs and growth parameters (PC-4 (2%) vs PC-5 (1%))...... XXII Figure F.2. 1- Principal component analysis plot obtained for non-conventional yeast, based on VOCs and growth parameters (PC-3 (3%) vs PC-4 (2%))...... XXII Figure F.2. 2- Principal component analysis plot obtained for non-conventional yeast, based on VOCs and growth parameters (PC-4 (2%) vs PC-5 (1%))...... XXII

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List of Tables

Table 1- LAB families and respective genus ...... 4 Table 2- Common aroma active compounds produced by LAB fermentation ...... 11 Table 3- Common aroma active compounds produced by non-conventional yeasts fermentation ...... 14 Table 4- Chemical composition of the high fan glucose wort ...... 17 Table 5- Code and taxonomic name of the LAB tested ...... 18 Table 6- Code and taxonomic name of the non-conventional yeasts tested ...... 20 Table 7- Volatile compounds screened in all samples ...... 22 Table 8- LAB candidates selected from the sensory analysis ...... 36 Table 9- Non-conventional yeasts candidates selected from the sensory analysis ...... 38

List of Tables in Annexes:

Table D. 1- Aroma evaluation table ...... IV Table D. 2- Taste evaluation table ...... V Table E.1. 1- Volatile organic compounds data for LAB (part I) ...... VI Table E.1. 2- Volatile organic compounds data for LAB (part II) ...... VII Table E.1. 3- Volatile organic compounds data for non-conventional yeast ...... VIII Table E.2. 1- Sensory analysis data for LAB (part I) ...... IX Table E.2. 2- Sensory analysis data for LAB (part II) ...... X Table E.2. 3- Sensory analysis data for LAB (part III) ...... XI Table E.2. 4- Sensory analysis data for LAB (part IV) ...... XII Table E.2. 5- Sensory analysis data for LAB (part V) ...... XIII Table E.2. 6- Sensory analysis data for non-conventional yeasts ...... XIV Table E.3. 1- Growth parameters data for LAB (part I) ...... XV Table E.3. 2- Growth parameters data for LAB (part II) ...... XVI Table E.3. 3- Growth parameters data for LAB (part III) ...... XVII Table E.3. 4- Growth parameters data for LAB (part IV) ...... XVIII Table E.3. 5- Growth parameters data for LAB (part V) ...... XIX Table E.3. 6- Growth parameters data for LAB (part VI) ...... XX Table E.3. 7- Growth parameters data for non-conventional yeast ...... XXI Table G. 1- Growth parameters data for LAB candidates ...... XXIV Table G. 2- Growth parameters data for non-conventional yeast candidates ...... XXIV

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List of Abbreviations

ABV: Alcohol by Volume AFB: Alcohol Free Beer ATP: Adenosine Tri-Phosphate CA: Cluster Analysis DoE: Design of Experiments Fr.: Fructobacillus GC: Gas Chromatography GM17: Glucose Media 17 GRAS: Generally Recognized as Safe HFGW: High Fan Glucose Wort LA: Low-Alcoholic LAB: Lactic Acid Bacteria Lb.: Lactobacillus Lc.: Lactococcus Leuc: Leuconostoc MR: Multiple Regression MRS: Man, Rogosa and Sharpe MVA: Multi-Variate Analysis MW: Maltose Wort OD: Optical Density P.: Pediococcus PC: Principal Component PCA: Principal Components Analysis PLS: Partial Least Squares QPS: Qualified Presumption of Safety ssp: subspecies Strep.: Streptococcus

T0: Time 0 h after inoculation

T24: Time 24 h after inoculation

T7days: Time 7 days after inoculation VOC: Volatile Organic Compound YPD: Yeast Extract Peptone Dextrose

1. Introduction

The Carlsberg group was founded in 1847 by Jacob Christian Jacobsen and, some years later, in 1875, he also founded the Carlsberg Laboratory driven by the need of understanding the beer chemistry and the physiology of the organisms involved. In 1979, the brewery was moved to Frederica (centre of Denmark), leaving the laboratory and all business offices in Copenhagen. J. C. Jacobsen spent his whole life trying to get the latest knowledge available, attending many lectures during his youth that allowed him to realize how scientific research is important to increase the product quality and the industrial productivity. A few years later, he visited many brewing colleges in Europe in order to share his knowledge and experience; and, at the same time, to bring new ideas and insights back to Carlsberg. Since the Laboratory was established, the research major goal has been the development of beer as close to perfection as possible, providing a brewing model in Denmark and for the rest of the World. Based on these principles, the Carlsberg Laboratory has been presenting innovative and pioneering results, and revolutionised modern brewing. Some of the most important scientific highlights coming from Carlsberg Laboratory are: creation of the first method for culturing pure yeasts (Emil Christian Hansen, 1883), development of a method for quantifying nitrogen in organic compounds and raw materials called Kjeldahl method (Johan Kjeldahl, 1883), introduction of the pH concept for measuring the level of acidity or alkalinity of a solution and development of its scale (Søren Sørensen, 1909), discovery of sexual reproduction by yeast cells and first genetic manipulation of this microorganism (Øjvind Winge, 1935), discovery of the enzyme subtilisin as responsible for degradation of egg albumin (Martin Ottesen, 1970) and, more recently, discovery of new barley sorts able to convey to beer an increased freshness sensation, a longer shelf life and a better head (superficial foam on top of beer) stability (Birgitte Skadhauge, 2000’s) (Carlsberg Foundation 2016; Carlsberg Group 2016; Skadhauge, Haldrup, and Olsen 2016). Nowadays, the renamed Carlsberg Research Laboratory is working towards development of new opportunities in terms of brewing and biotechnology, with its research divided in four areas: brewing science and technology, yeast and fermentation, raw materials and new ingredients (Carlsberg Foundation 2016). The current project was developed in the Yeast and Fermentation Department.

2. State of Art

A bibliographic review of the main topics supporting the goals of the project is presented in this chapter, starting with a general overview about non-to-low alcoholic beer and its manufacture processes. A characterization of LAB ensues, from the taxonomy of the bacteria screened in this project to their growth and metabolism, as well as flavour characteristics. At the end, a similar description is presented for non- conventional yeasts that can positively contribute to non-to-low alcoholic wort beverages.

2.1. Non-to-low alcoholic beer Non-to-low alcoholic beers are getting more and more popular in recent years. In the majority of the European countries, low alcohol content beers are divided between alcohol-free beers (AFBs) with ≤0.5% alcohol by volume (ABV) and low-alcoholic (LA) beers with a maximum of 1.2% ABV (Michel et al. 2016). In the United States of America, there are stricter rules, so AFBs do not have any alcohol present in their composition and the upper limit for LA beers is 0.5% ABV. In contrast, in the countries where drinking of alcoholic beverages is prohibited by religion, LA beers in general must not exceed 0.05% ABV (Brányik et al. 2012).

2.1.1. Background The creation of non-to-low alcoholic beers can be justified by several historical reasons in the past century. During both the 1st and the 2nd World Wars (1914-1918 and 1939-1945), the production of beers with non-alcoholic content arose due to the lack of raw materials (water, barley, hops and yeast) necessary to produce standard beer. However, even during peace time (1919-1933), the production process of this type of beers was developed because in the USA authorities did not allow manufacture, sale and consumption of alcohol. Late in the 20th century, after both wars, the production of AFBs was expanded due to different reasons, such as to provide alternatives to beer consumers during activities/conditions not compatible with alcohol consumption (driving, sports practicing, pregnancy, medication), but also to try to enter the market where one is not allowed to consume alcohol due to religious reasons (Brányik et al. 2012) . Nowadays, apart from a number of incompatible activities and religious reasons, the market of non- alcoholic brews has widely expanded also due to health issues, because taking care of eating habits and personal weight are getting quite trendy topics. There are still many consumers that prioritize flavour over nutritional benefits; since many of the available non-alcoholic beverages present a poor flavour profile that is not accepted by them, production techniques of these type of beverages have been improved in order to adjust the flavour of non-alcoholic beverages to the characteristics of standard beers (Catarino and Mendes 2011).

2.1.2. Production processes The methods to produce AFBs can be split in physical and biological processes, being included some specific strategies in each category (Figure 1). The physical methods are more related to removal of alcohol from regular beers, requiring some costly special equipment that, after optimisation, produce AFBs with trace alcohol levels. In terms of biological methods, many of them rely on modification of the normal brewing process, in order to limit the fermentation and have lower production of ethanol. Since some of these methods are performed during beer fermentation, there is no requirement of extra equipment or investment, only an accurate and controlled process, as well as a careful selection and propagation of the microorganism(s) involved. However, when following continuous fermentation, it is also based on limited alcohol formation, but requires some special equipment such as continuously operating bioreactors or carriers for cell immobilization, being also profitable due to the high productivity attained. Another possibility, also related to fermentation but not with the microorganisms, is taking a part of the mashing product obtained during the brewing process, heating it up and then putting it back with the rest of the mixture; this allows inactivation of some enzymes, which prevents buildup of normal concentrations of fermentable sugars, that is later reflected in lower ethanol production during fermentation (Brányik et al. 2012; Bamforth 2009; Preedy 2009). As mentioned before, many consumers complain about the lack of beer flavour in non- or low-alcoholic beers. To solve this problem, the biological methods are claimed as advantageous because they avoid the unwanted extraction of flavour compounds, a common problem when ethanol extraction is performed after fermentation (physical processes) (Basso, Alcarde, and Portugal 2016).

Figure 1- Most common production methods of alcohol-free beer (Brányik et al. 2012)

2.2. Lactic acid bacteria LAB are characterized as gram-positive bacteria, non-sporulating, aerotolerant, catalase-negative; in terms of morphology, they can be categorized as cocci or bacilli. In general, LAB are considered beneficial for health, but some genera include species identified as human or animal pathogens (Lahtinen et al. 2012).

In terms of brewing, LAB are well known as the main spoilage agent, having some studies showed that can be responsible for 60% to 70% of all cases of beer spoilage (Garofalo et al. 2015). However, the brewing industry has been improving and applying a number of inventions, including the use of LAB as a player to improve wort characteristics, as well as aroma profiles and flavour stability (Lowe and Arendt 2004). Some well-known beers as German Berliner Weisse and several Belgian styles are made with a mixture of yeast and LAB, resulting in a tasty and characteristic final product (Vriesekoop et al. 2012).

2.2.1. Taxonomy The basis of LAB classification was published by Sigurg Orla-Jensen in 1919 (Orla-Jensen 1919), and the four genera recognized at that time were Lactobacillus, Leuconostoc, Pediococcus and Streptococcus. The criteria used at that time for classification of LAB included cellular morphology, glucose fermentation, ranges of growth temperature and patterns of sugar consumption. As time passed by, more advanced tools have been used, with a special focus on molecular biology methods that allow a more accurate classification. In this way, LAB are now known to belong to the phylum Firmicutes, class Bacilli and order Lactobacillales; in Table 1, it is possible to observe the existing LAB families and the corresponding genera (Lahtinen et al. 2012).

Table 1- LAB families and respective genus

Family Genera Aerococcaceae Aerococcus Carnobacteriaceae Carnobacterium Enterococcus Enterococcaceae Tetrageonococcus Vagococcus Lactobacillus Lactobacillaceae Pediococcus Leuconostoc Leuconostocaecae Oenococcus Weissella Lactococcus Streptococcaceae Streptococcus

From the four genera originally described in 1919, it is now possible to observe that are more genera included in the different LAB families. For this project, the four main LAB genera tested were Lactobacillus, Pediococcus, Leuconostoc and Lactococcus, so a brief description of them will be presented below.

2.2.1.1. Lactobacillus Bacteria from the genus Lactobacillus can be found in many different microbial-heavy host habitats (i.e. human mucosa surfaces), dairy environments rich in nutrients or natural ecological niches as plants and soil. The wide range of conditions where this genus can live is reflected on its diversity because it comprises over a hundred different species. Some of the most well characterized and relevant species, both scientifically and industrially, are Lb. acidophilus, Lb. casei, Lb. delbrueckii ssp. bulgaricus, Lb. plantarum, Lb. rhamnosus, and Lb. salivarius (Lahtinen et al. 2012). In terms of food industry applications, the genus Lactobacillus is often used in bread, cheese, cured ham, soy sauce, yogurt and wine (Vuyst and Vandamme 1994).

2.2.1.2. Pediococcus During many decades, only five species were considered the taxonomic core of this LAB genus, i.e. P. damnosus, P. acidilactici, P. pentosaceus (including the subspecies pentosaceus and intermedius), P. parvulus and P. inopinatus. However, seven more species have been added to this genus in recent years, encompassing also P. claussenii, P. cellicola, P. stilesii, P. ethanolidurans, P. siamensis, P. argentinicus and P. lolii. In general, these species can be found in raw or processed foods, and in the intestinal tract of animals and humans (Batt and Tortorello 2014; Lahtinen et al. 2012). This bacteria genus has many applications in the food industry due to its recognized capacities as a probiotic (live microorganisms that confer health benefits to the host when administered in adequate amounts); for example, they stimulate gut microbiota balance due to their resistance to low pH and to bile salts present in the intestinal tract. Usually, Pediococcus species can be used for example, in milk, bread, sausages and pork meat (Porto et al. 2017) .

2.2.1.3. Leuconostoc There are many species included in the Leuconostoc bacteria genera, such as Leuc. carnosum, Leuc. citreum, Leuc. fallax, Leuc. gasicomitatum, Leuc. gelidum, Leuc. holzapfelii, Leuc. inhae, Leuc. kimchi, Leuc. lactis, Leuc. mesenteroides, Leuc. palmae, Leuc. garlicum and Leuc. pseudomesenteroides. The natural habitats for these bacteria are vegetables, fruit and animal products as meat, fish and milk (and its derivatives), not being spontaneously present in healthy warm-blooded animals, including humans (Batt and Tortorello 2014; Lahtinen et al. 2012). The species Leuc. carnosum, Leuc. gasicomitatum and Leuc. gelidum are often associated with food spoilage, but they can also contribute positively to fermented foodstuff (sauerkraut, pickles, processed meat, etc.), dairy products and play an important role in functional food (Hemme and Foucaud-Scheunemann 2004).

2.2.1.4. Lactococcus Bacteria from the genus Lactococcus comprise seven different species: Lc. lactis (including the subspecies cremoris, lactis, and hordniae), Lc. garvieae, Lc. piscium, Lc. plantarum, Lc. raffinolactis, Lc. chungangensis and Lc. fujiensis. The typical ecological niches of Lactococci are vegetables, milk (or other animal sources as, for example, human gut), but they can also be found in the foam of activated

5 sludge, in forage or in fish (Lahtinen et al. 2012). This is a LAB genus widely used in the food industry, being applied for example in the production of butter, cheese, sour cream (Vuyst and Vandamme 1994).

2.2.2. Growth and metabolism LAB do not possess a functional respiratory system, so they need to obtain energy through substrate phosphorylation. As shown in Figure 2, there are two basic fermentative pathways: homofermentation, which is based on glycolysis (Embden-Meyerhof-Parnas pathway), resulting only in lactic acid production, and heterofermentation (pentose phosphate pathway, hexose monophosphate shunt or 6- phospho-gliconate pathway), that (besides lactic acid) also produces considerably amounts of CO2 and ethanol or acetate, requiring the presence of hexoses in the media as starter sugars. If the hexose that enter the pathways is other than glucose (mannose, galactose, fructose), different isomerization and phosphorylation steps are taken. In terms of pentose fermentation, they can only be fermented heterofermentatively by entering the pathway as either ribulose-5-phosphate or xylulose-5-phosphate, without CO2 production. In terms of energy yield, the homolactic fermentation produces 2 moles of adenosine triphosphate (ATP) per mole of glucose consumed, but the heterolactic fermentation only results in 2 moles of ATP if acetate is produced because the production of ethanol only generates 1 mole of ATP. Bacteria form the genus Lactobacillus can be divided in three groups: obligately homofermentative (i.e. Lb. acidophilus, Lb. delbrueckii, Lb. salivarius), facultatively heterofermentative (i.e. Lb. casei, Lb. curvatus, Lb. plantarum, Lb. sakei) or obligately heterofermentative (i.e. Lb. brevis, Lb. buchneri, Lb. fermentum, Lb. reuteri), respectively. The other three main genera used in this project do not present fermentation groups, being Lactococcus and Pediococcus characterized as homofermentative and Leuconostoc characterized as obligately heterofermentative, which may change due to medium conditions (Lahtinen et al. 2012; Priest and Campbell 1996).

Homofermentative Heterofermentative

Figure 2- Homofermentative and heterofermentative pathways of LAB (Reis et al. 2012)

*Homofermentative: 1. glucokinase, 2. fructose-1,6-diphosphate aldolase, 3. glyceraldehyde-3-phosphate dehydrogenase, 4. pyruvate kinase, 5. lactate dehydrogenase; Heterofermentative: 1. glucokinase, 2. glucose-6-phosphate dehydrogenase, 3. 6-phosphogluconate dehydrogenase; 4. phosphoketolase; 5. glycer-aldehyde-3-phosphate dehydrogenase; 6. pyruvate kinase; 7. lactate dehydrogenase; 8. acetaldehyde dehydrogenase; 9. alcohol dehydrogenase;

2.2.2.1. Alternative pathways In Figure 2 is possible to observe that pyruvate occupies a central role in both pathways (homo- and heterofermentative), being responsible for acceptance of electrons in order to obtain lactic acid and keep the oxidation-reduction balance in the cell. However, there are alternative pathways for the pyruvate, depending on the LAB strain selected and on particular growth conditions, that result in the formation of uncommon fermentation products such as diacetyl, acetoin, 2,3-butanediol and acetaldehyde (Figure 3). In the case of diacetyl, it can contribute positively to some dairy products (butter, cottage cheese, etc), but in products like fermented sausages, wine and beer it is usually considered unpleasant (Hugenholtz 1993).

Figure 3- Pathways for the alternative fates of pyruvate (Lahtinen et al. 2012).

*1. diacetyl synthase, 2. acetolactate synthase, 3. pyruvate–formate lyase, 4. pyruvate dehydrogenase, 5. pyruvate oxidase, 6. acetate kinase; dashed arrow represents a non-enzymatic reaction

2.2.2.2. Impact of media characteristics Beer is usually recognized as a beverage with a good microbiological stability, being that mainly caused by the conditions settled in terms of oxygen level, pH, temperature or substrate composition that avoid presence of spoiled LAB (Suzuki et al. 2006). Oxygen is an important factor during LAB fermentation because it can affect the metabolism of pyruvate and lactate, being necessary its presence for an effective fermentation process. As mentioned before, bacteria from the genus Leuconostoc are heterofermentative; some studies proved that they produce more acetate and less ethanol during aerobic growth (presence of oxygen), without affecting

8 production of lactate, which is a quite important point for the production of low-to-no alcoholic beverages using this bacteria genus (Condon 1987) . It has been described that for the fermentation of some LAB strains, when pH is adjusted within the range 5.0-6.5 and temperature is settled between 30 ºC and 37 ºC, there is an increase on the ratio of lactic acid produced, being possible to observe a tendency to just follow the homolactic pathway (Hofvendahl, Van Niel, and Hahn-Hägerdal 1999). In terms of substrate composition, the four main ingredients of wort are water, hops, malted barley and adjuncts. The malted barley, often called just “malt” is main source of starch, providing also enzymes to break down the starch into fermentable sugars (Barth 2013). In general, the sugar concentration in the fermentation media also affects LAB fermentation because, for example, when the glucose concentration is limited, some strains as Lc. lactis (that usually is homofermentative) turn heterofermentative, thus resulting in a so-called mixed-acid fermentation. Some other similar cases are reported in the literature, being also stated that once the glucose level turns back to non-limiting values, bacteria return to the homofermentative pathway (Liu 2003). The presence of hops in the wort composition is another factor that limits the growth and fermentation of LAB, due to their inability to survive in the presence of this bitter flavour inductor. Nowadays, there are already some LAB strains showing hop resistance due to appearance of the genes horA and horC, but a few number of (spoilage) strains have these genes; hence, for a successful fermentation of the LAB, the wort used should be unhoped (Suzuki et al. 2006).

2.2.3. Flavour In terms of nose and mouth sense, the terms aroma, taste and flavour are often used, sometimes even as synonymous. However, according to the experts on this topic, aroma is related with the odours perceived by the nose, taste corresponds to the sense during mouth passage of the product and flavour describes the overall sensation of aroma and taste together. In the case of food, the consistency (tactile sensation) is also included in the flavour description, but in beverages it does not make sense to be considered (Rothe 1988). In food fermentation processes, flavours use to be caused by the accumulation of volatile and non- volatile aroma compounds, as well as other compounds related to taste (bitterness, umami, sweetness, sourness, and saltiness). In terms of aroma compounds, the volatile compounds are divided in different chemical classes such as alcohols, aldehydes, ketones, esters, phenolic compounds, organic acids and terpenes, being their precursors provided by the three main food elements: proteins, carbohydrates and lipids. Generally, the conversion of the precursors in aroma components is not carried out by a single enzyme, but through a functional metabolic pathway (Figure 4). Regarding taste compounds, these can also be divided in different chemical classes, such as aminoacids (responsible for sweetness and umami), oligopeptides (related with bitterness) or simple organic acids (sourness taste) (Smid and Kleerebezem 2014).

Figure 4- Functional metabolic pathways for the generation of aroma compounds from existing precursors, during lactic acid fermentation of malt wort (Nsogning Dongmo et al. 2016).

*PKP, phosphoketolase pathways; EMPP, Embden-Meyerhof-Parnas pathways; TCA, tricarboxylic acid cycle; PC, pyruvate carboxylase; CS, citrate synthase; SCS, succinyl-CoA synthase; SDH, succinate dehydrogenase; FA, fumarase; MDH, malate dehydrogenase; OAD, oxaloacetate decarboxylase; PO, pyruvate oxidase; PFL, pyruvate formate lyase; ALS, acetolactate synthase; LDH, lactate dehydrogenase; PTA, phosphate acetyltransferase; ALDH, acetaldehyde dehydrogenase; ALDC, acetolactate decarboxylase; PDC, pyruvate decarboxylase; AK, acetate kinase; AAT, alcohol acyltransferase; DR, diacetyl reductase; DS, diacetyl synthase; BDH, 2,3-butanediol dehydrogenase; MLF, malolactic fermentation; AT, aminotransferases; DC, decarboxylase; DH; dehydrogenase; H, hydrogenase; TA, threonine aldolase; OX, oxidation; KMBA, 2-oxo-4-methylthiobutyric acid; CR, chemical reaction; LP, lipase; FAD, fatty acid desaturase; BOX, b-oxidation; HPL, hydroperoxide lyase; KADC, b-ketoacyldecarboxylase; R, reductase; ES, esterase; GS, glycosylation; DO, dioxygenase; BGs, b-glucosidase; ACR, acidic chemical reaction; PADs, phenolic acid decarboxylases; PARs, phenolic acid reductases: in bold are major aroma compounds.

In Table 2 are presented some of the main aroma active compounds and their typical odours detected in cereal based beverages (Nsogning Dongmo et al. 2016) and in other cereal related products (Pico, Bernal, and Gómez 2015), fermented by LAB. Until a few years ago, there were no literature data about the key aroma compounds of cereal beverages fermented by LAB. However, some recent studies identified β-damascenone, furaneol, phenylacetic acid, 2-phenylethanol, 4-vinylguaiacol, sotolon, methional, vanillin, acetic acid, nor-furaneol, guaiacol and ethyl 2-methylbutanoate as the twelve key aroma compounds that present a major contribution to the flavour profile of malt wort beverages fermented with LAB; it was also stated that acetaldehyde has a great importance on the aroma profile. Among all these compounds, the ones considered more appealing and with high acceptance by the consumers are β-damascenone, acetaldehyde, furaneol, phenylacetic acid and ethyl 2-methylbutanoate; however, further studies about exclusion and recombination of aromas can help realize the real contribution of each key compound to the final flavour, probably being also interesting to develop some studies about their synergistic effect (Nsogning Dongmo et al. 2017).

Table 2- Common aroma active compounds produced by LAB fermentation

Compounds class Compound Aroma descriptor Ethanol Alcohol (1) Isobutanol Malty, wine (1) Alcohols 2-Ethoxyethanol Sweet, ether (1) Propanol Alcohol, pungent (1) Isoamyl alcohol Malty, balsamic, alcoholic (2) Aldehydes Acetaldehyde Fresh, green (1) Acetone Ether, grape (1) β-Damascenone Baked apple (1) Ketones 2-Pentanone Pungent, fish like (1) Diacetyl Butter (1) Ethyl 2-methylbutanoate Fruity (1) Isobutyl acetate Fruity, floral (2) Isoamyl acetate Banana (2) Esters Ethyl caproate Fruity (2) Ethyl caprylate Sweet, fresh, fruity (2) Ethyl acetate Fruity (1) 2-Phenylethyl acetate Rose (2) 2-Phenylethanol Rose (1) 4-Vinylguaiacol Clove (1) Phenolic compounds Phenylacetic acid Honey (2) Guaiacol Smoky (1) Vanillin Vanilla, sweet (1) Furaneol Caramel (1) Heterocyclic compounds Sotolon Seasoning-like (1) Nor-furaneol Caramel (1) Formic acid Pungent (1) Acetic acid Sour (1) Lactic acid Sour (1) Organic acids Hexanoic acid (caproic acid) Sweaty (1) Octanoic acid (caprylic acid) Sweaty (1) Decanoic acid (capric acid) Rancid, fatty (2) Sulfurous compounds Methional Boiled/cooked potatoes (2) Linalool Flowery (1) Terpenes Limonene Citrus (2) (1) (Nsogning Dongmo et al. 2016), (2) (Pico, Bernal, and Gómez 2015)

2.3. Non-conventional yeasts Non-conventional yeasts are usually undomesticated strains, performing spontaneous fermentation, but there is a growing interest in isolating and characterising them for development of starter cultures that increase flavour diversity in different type of beverages, including beer. They usually exhibit low fermentation yields, and are more sensitive to ethanol stress than Saccharomyces cerevisiae strains. However, non-conventional yeast have a good control of microbial spoilage and can provide a characteristic aroma and taste, thus resulting in new variations and styles of beer (Varela 2016; Steensels and Verstrepen 2014).

2.3.1. Overview Some of the most popular examples of non-conventional yeasts that show brewing and non-alcoholic beer production potential are Brettanomyces anomalus, Brettanomyces bruxellensis, Candida tropicalis, Saccharomycodes ludwigii, Torulaspora delbrueckii, Pichia kluyveri or Zygosaccharomyces rouxii (Michel et al. 2016). However, since this project is focused on non-to-low alcoholic beverages with a unique flavour profile, the selection of the non-conventional yeasts was based on previous in-house experimental tests that showed some interesting results. In this way, the group of yeasts selected include some strains isolated from kefir grains (Saccharomyces kefir, Saccharomyces unisporus and Kluyveromyces marxianus), a small group of confidential yeasts and strains of the species Galactomyces geotrichum, Kazachstania gamospora and Hanseniaspora guilliermondii. The kefir grains consist on a complex microbial symbiotic mixture of lactic acid bacteria (Lactobacillus, Lactococcus, Leuconostoc, and Streptococcus spp.), acetic acid bacteria (Acetobacter) and yeasts (Kluyveromyces, Saccharomyces and Torula) involved in a polysaccharide-protein matrix (Figure 5), being the species Saccharomyces kefir, Saccharomyces unisporus and Kluyveromyces marxianus frequently found in this mixture (Piermaria, de la Canal, and Abraham 2008).

Figure 5- Kefir grains (Kniesel 2017)

The species Galactomyces geotrichum does not have a defined isolation source, but Kazachstania gamospora and Hanseniaspora guilliermondii can be find on soil and in fruits, respectively (Gamero et al. 2016). Galactomyces geotrichum is used in food fermentation, but in the last years, it has been

12 extensively described in the literature as being very efficient in the removal/cleaning of azo dyes (major class of synthetic colorants) in the textile industry. This species is described as an excellent alternative to the several physicochemical methods developed in the last few decades, since they produce a lot of toxic sludge (aromatic amines) and secondary waste products that are not environmental recommended (Govindwar et al. 2014; Waghmode et al. 2012; Waghmode, Kurade, and Govindwar 2011). In the field of the cereal based products, the species Kazachstania gamospora was quite recently described as a good contributor in the bakery field, showing better overall results that typical baker’s yeast (Saccharomyces cerevisiae) (Zhou et al. 2017). The species Hanseniaspora guilliermondii is commonly used in wine fermentation due to many pleasant characteristics that it provides to the final product (Moreira et al. 2011; Zironi et al. 1993).

2.3.2. Growth and metabolism In general, yeast can obtain energy from sugars through respiration or (alcoholic) fermentation (Figure 6). Respiration is usually performed in the presence of abundant amounts of oxygen and results in a high yield of ATP, being possible for S. cerevisiae to obtain 38 ATPs per glucose molecule. However, when the oxygen conditions are limited, fermentation is performed and usually only results in 2 ATPs per glucose molecule, having the advantage of not requiring oxygen (Pfeiffer and Morley 2014).

Figure 6- Respiration (left) and fermentation (right) pathways of yeast (Walker 1998)

In the presence of oxygen, a few types of yeast as S. cerevisiae tend to perform fermentation instead of respiration when glucose concentrations are sufficiently high, even with a so discrepant difference in terms of energy yield. This is called the Crabtree effect, and the yeasts that display it are named Crabtree- positive. However, when respiration is exclusively used it is called the Pasteur Effect, and it happens due to an end product of the aerobic glucose utilization that inhibits one of the main enzymes involved in the

13 fermentation process. These yeasts that exclusively use respiration can be also called Crabtree-negative (De Deken 1966). The Crabtree effect is considered successful and a good advantage because, besides the low production of cell-biomass, there is production of ethanol that is considered an excellent tool to compete with other microorganisms - thus avoiding contaminations and allowing an exponential yeast growth (Hagman et al. 2013).

2.3.3. Flavour The demand for niche products with distinctive aroma profiles has led to a strong interest on non- conventional yeasts, due to the capacity of many of them to produce unique aroma compounds that might be perceived as desirable in particular fermented products. In Table 3 are presented some of the main aroma active compounds produced by yeast and their typical odours detected in cereal-based products fermented by non-conventional yeast (Aslankoohi et al. 2016; Pozo-Bayón, Guichard, and Cayot 2006).

Table 3- Common aroma active compounds produced by non-conventional yeasts fermentation

Compounds class Compound Aroma descriptor Dihydromyrcenol Citrus (1) Isobutanol Glue, alcohol (1) Alcohols 1-Vinylhexanol Mushroom (1) Propanol Alcohol strong (2) Isoamyl alcohol Whiskey, malt, alcohol (1) Acetaldehyde Fresh, grass (2) Decanal Soap, orange peel (1) Aldehydes Safranal Herb, sweet (1) Phenylacetaldehyde Honey, sweet (1) Benzaldehyde Almond (1) Isophorone Peppermint (1) 2-Nonanone Fruit, flower (1) Ketones Sulcatone Green, citrus (1) 2-Heptanone Soap, fruit, cinnamon (1) Isobutyl acetate Fruity (2) Ethyl decanoate Grape, fruit (1) Isoamyl acetate Banana (1) Ester Ethyl acetate Fruity (2) Ethyl caprylate Fruit, sweet, soap, fat (1) Ethyl caproate Fruity, sweet (2) 2-Phenylethyl acetate Rose, honey, flower (1) 2-Phenylethanol Honey, rose, flower (1) Phenolic compounds 4-Vinylguaiacol Clove, spices (1)

2-Pentyl furan Fruit, flower (1) Heterocyclic compounds 2-Furanmethanol Burnt, warm oil (1) Furfural Bread, almond, sweet (1) Hexanoic acid (caproic acid) Sweaty (2) Octanoic acid (caprylic acid) Sweat, fruit-acid, soap (1) Organic acids Nonanoic acid Green, fat Decanoic acid (capric acid) Soapy (2) Caryophyllene-E Wood, spice (1) Terpenes Linalool Flowers, fresh (2) Limonene Citrus, lemon, mint (1) (1) (Aslankoohi et al. 2016), (2) (Pozo-Bayón, Guichard, and Cayot 2006)

2.4. Aims of the project Besides what is described in the literature and presented previously, there is still a scarce knowledge about the use of LAB and some non-conventional yeast for the production of non-to-low alcoholic beverages. Therefore, the main objective of this study was to evaluate the biodiversity of bacteria and yeast strains in terms of VOCs production during fermentation of cereal-based liquids, and how they can contribute to unique flavour profiles. More specifically, this study aimed at:

- Select bacterial strains of GRAS/QPS status (Generally Recognized as Safe/ Qualified Presumption of Safety, respectively) and non-conventional yeast strains that present high production of VOCs associated with a fresh and sour profile (without off-flavours), as well as low ethanol production when fermenting unhopped high fan glucose wort and maltose wort.

- Apply multivariate statistical analysis (CA, PCA and PLS regression) to explain possible correlations between bacteria/yeast strains growth and VOCs profiles, with regard to the sensory evaluation.

3. Material and Methods

3.1. Media During this project, bacteria were inoculated in de Man, Rogosa and Sharpe (MRS) medium (de Man, Rogosa, and Sharpe 1960) or Glucose M17 (GM17) medium (Terzaghi and Sandine 1975), depending on the specifications of the strain; the non-conventional yeasts tested were inoculated in yeast extract peptone dextrose (YEPD or YPD) medium. For the fermentations of all LAB and non-conventional yeasts, the media used was high fan glucose wort (HFGW). After running these fermentations, the best candidates in terms of aroma profile were also inoculated in maltose wort (MW).

3.1.1. MRS medium The MRS media used was supplied by Oxoid (Oxoid Ltd. 2017) and the solution was prepared following the proportions of 52 g of MRS broth in 1 L of deionised (DI) water at approximately 60 ºC. After mixing the solution until it was completely dissolved, it was autoclaved at 121 ºC during 15 min to make it sterile.

3.1.2. GM17 medium The GM17 media used was also supplied by Oxoid (Oxoid Ltd. 2017), and the solution was prepared by mixing 48.25 g of GM17 broth in 950 mL of DI water that was then slightly boiled. To make it sterile, it was autoclaved at 121 ºC during 15 min. Once it cooled down and reached 50 ºC, 50 mL of sterile glucose solution (0.5% w/v) was added.

3.1.3. YPD medium The YPD media used was obtained by mixing bacto yeast extract (1% w/v, 10 g/L) and bacto peptone (2% w/v, 20 g/L), both from Becton, Dickinson and Company (Becton, Dickinson and Company 2017), with agar (2% w/v, 20 g/L) from PanReac AppliChem (PanReac AppliChem 2017) and glucose- monohydrate (2% w/v, 0,22 g/mL) from Merck Millipore (Merck Millipore 2017). After mixing all these ingredients with DI water, the final product was autoclaved at 121 ºC during 15 minutes.

3.1.4. High fan glucose wort The initial wort was HFGW 14.5 ºP, produced at the Brewing Pilot Plant at Carlsberg Research Laboratory and stored in barrels at 10 ºC. This wort was unhoped, enriched with some aminoacids and glucose was the major sugar. To ensure a consistent quality of the wort, some enzymatic solutions as Ultraflo Max (0,1 g/kg) , Attenuzyme Pro (1 g/kg), AMG 300 L BrewQ (6 g/kg) and Neutrase 0.8 L BrewQ (2 g/kg) were added, all supplied by Novozymes (Novozymes 2017). In order to avoid possible contaminations, after extraction from the barrel (Annex A: Tapping wort out from a barrel), all the wort used was centrifuged in 1L bottle assemblies during 40 min, at 5000 rpm on a Avanti J-26S XPI centrifuge from Beckman Coulter (Beckman Coulter 2017), and filtered under sterile conditions (Annex B: Sterile filtration of wort) using a Pellicon3 Cassette Holders (XX42PMINI) filter from Merck

Millipore (Merck Millipore 2017), supported by a MCP-Z Process pump from Ismatec (Ismatec 2017). Its final composition is presented on Table 4.

Table 4- Chemical composition of the high fan glucose wort

Concentration Concentration Concentration Aminoacids Sugars Minerals (mg/L) (g/L) (mg/L) Leucine 206.00 Glucose 104.20 Phosphate 977.60 Phenylalanine 170.00 Maltose 4.90 Potassium 648.00 Valine 161.00 Sucrose 3.10 Phosphorus 437.10 Arginine 159.00 Fructose 2.20 Chloride 314.40 Glutamic acid 145.00 Maltotriose 2.00 Magnesium 91.00 Alanine 134.00 TOTAL 116.40 Sulphate 47.70 Tyrosine 133.00 Calcium 38.22 Lysine 102.00 Free oxalic acid 34.80 Serine 92.00 Silica 32.58 Isoleucine 88.00 Sodium 30.80 Aspartic acid 88.00 Nitrate 2.40 Threonine 85.00 Zinc 0.38 Histidine 64.00 Iron 0.29 Methionine 52.00 Manganese 0.07 Glycine 48.00 Copper 0.06 TOTAL 1727.00 Aluminium 0.004 TOTAL 2655.404

Due to previous optimization studies, all the wort used in the performed fermentations was adjusted to pH 6 and all strains were tested at 7 ºP, except a few of them that were also tested on 5 ºP. To adjust the Plato (ºP), the wort was mixed with autoclaved DI water in the appropriate proportion and it was measured using a Density Meter DMA 35N from Anton Paar (Anton Paar 2017). Usually, the pH of the HFGW 7 ºP was between 5.2 and 5.7 so some drops of potassium hydroxide (46%) were added to increase this value, being controlled by a Lab 850 pH meter from SI Analytics (SI Analytics 2017). To guarantee that the wort was sterile, after pH and Plato adjustments, it was all passed through a 0.2 µm filter to sterile glass flasks, always close to the flame.

3.1.5. Maltose wort The initial MW was at 15 ºP and it was obtained directly from the brewing kettle before addition of the hops (unhopped), at the Brewing Pilot Plant at Carlsberg Research Laboratory. It was composed of 70% pilsner malt and 30% barley, being also added 0.5 g/kg of calcium chloride. As mentioned before, to ensure a consistent quality of the wort, some enzymatic solutions as Ultraflo Max (0,15 g/kg) and Attenuzyme Pro (0.15 g/kg) were added, all supplied by Novozymes (Novozymes 2017). Since the MW

17 wort was quite dense - and even after centrifugation the pellet was not stable at the bottom of the bottle, it was not possible to filtrate it. In this way, to avoid contaminations, it was pasteurized in a 20 L water bath from GFL (GFL 2017), during 1h at 100 ºC. As well as for the HFGW, the pH and the Plato of the MW used for the fermentations were adjusted to 6 and 7 ºP, respectively. However, since it was not possible to filter this wort, the adjustments in terms of pH and Plato were done inside of the flow bench to keep it sterile.

3.2. Microorganisms In this project, both LAB and non-conventional yeasts were taken for wort fermentation during the experimental trials.

3.2.1. Lactic acid bacteria The LAB strains used in this project were mainly obtained from Carlsberg Research Laboratory collection. The strains tested are all considered food-grade and the majority of them are from genera Lactococcus (Lc.), Lactobacillus (Lb.), Leuconostoc (Leuc.) or Pediococcus (P.); the genus Fructobacillus (Fr.) is a reclassification of the genus Leuconostoc (Endo and Okada 2008). Apart from these, one species from the Streptococcus (Strep.) genus was also tested. The number, the genus code and the corresponding taxonomic name of each strain are presented in Table 5.

Table 5- Code and taxonomic name of the LAB tested

Code Strain Code Strain Code Strain Lc.1 Lc. lactis ssp. cremoris Lb.17 Lb. farraginis Lb.62 Lb. rhamnosus Lc.2 Lc. lactis ssp. lactis Lb.18 Lb. uvarum Lb.63 Lb. rhamnosus Lc.3 Lc. lactis ssp. lactis Lb.19 Lb. silagei Lb.64 Lb. casei ssp. casei Lc.4 Lc. lactis ssp. lactis Lb.20 Lb. oeni Lb.65 Lb. zeae Lc.5 Lc. lactis ssp. lactis Lb.21 Lb. zeae Lb.66 Lb. casei Lc.6 Lc. lactis ssp. lactis Lb.22 Lb. paracasei ssp. paracasei Lb.67 Lb. plantarum Lc.7 Lc. lactis ssp. lactis Lb.23 Lb. parakefiri Lb.68 Lb. rhamnosus Lc.8 Lc. lactis ssp. lactis Lb.24 Lb. gasseri Lb.69 Lb. plantarum Lc.9 Lc. lactis ssp. hordniae Lb.25 Lb. pentosus Lb.70 Lb. plantarum Lc.10 Lc. lactis ssp. lactis Lb.26 Lb. alimentarius Lb.71 Lb. harbinensis Lc.11 Lc. lactis ssp. lactis Lb.27 Lb. farciminis Lb.72 Lb. paracasei Lc.12 Lc. lactis ssp. lactis Lb.28 Lb. gallinarum Lb.73 Lb. rhamnosus Lc.13 Lc. lactis ssp. lactis Lb.29 Lb. hilgardii Lb.74 Lb. casei Lc.14 Lc. lactis ssp. lactis Lb.30 Lb. johnsonii Lb.75 Lb. paracasei Lc.15 Lc. lactis ssp. lactis Lb.31 Lb. pasteurii Lb.76 Lb. plantarum ssp. plantarum Lc.16 Lc. lactis ssp. lactis Lb.32 Lb. plantarum ssp. plantarum Lb.77 Lb. plantarum Lc.17 Lc. raffinolactis Lb.33 Lb. casei Lb.78 Lb. rhamnosus Lc.18 Lc. plantarum Lb.34 Lb. curvatus Lb.79 Lb. plantarum Lc.19 Lc. lactis ssp. tructae Lb.35 Lb. brevis Lb.80 Lb. plantarum ssp. plantarum Lc.20 Lc. fujiensis Lb.36 Lb. buchneri Lb.81 Lb. plantarum Lb. coryniformis ssp. Lc.21 Lc. lactis ssp. lactis Lb.37 Lb.82 Lb. rossiae coryniformis Lc.22 Lc. lactis ssp. lactis Lb.38 Lb. malefermentans Lb.83 Lb. paracasei

Lc.23 Lc. lactis Lb.39 Lb. delbrueckii ssp. sunkii Lb.84 Lb. plantarum Lc.24 Lc. lactis Lb.40 Lb. pentosus Lb.85 Lb. rhamnosus Lc.25 Lc. lactis ssp. lactis Lb.41 Lb. pentosus Lb.86 Lb. zeae Lc.26 Lc. lactis Lb.42 Lb. paraplantarum Lb.87 Lb. plantarum Lc.27 Lc. lactis Lb.43 Lb. leichmannii Leuc.1 Leuc. mesenteroides ssp. cremoris Lc.28 Lc. lactis ssp. lactis Lb.44 Lb. paracasei Leuc.2 Leuc. citreum Lc.29 Lc. lactis Lb.45 Lb. acidophilus Leuc.3 Leuc. fallax Lb. delbrueckii ssp. Lb.1 Lb.46 Lb. casei Leuc.4 Leuc. citreum bulgaricus Lb. delbrueckii ssp. Lb.2 Lb.47 Lb. fermentum Leuc.5 Leuc. citreum delbrueckii Leuc. mesenteroides ssp. Lb.3 Lb. delbrueckii ssp. lactis Lb.48 Lb. gasseri Leuc.6 dextranicum Lb.4 Lb. helveticus Lb.49 Lb. plantarum Leuc.7 Leuc. lactis Lb.5 Lb. dextrinicus Lb.50 Lb. rhamnosus Leuc.8 Leuc. palmae Lb.6 Lb. mali Lb.51 Lb. rhamnosus Leuc.9 Leuc. miyukkimchii Lb.7 Lb. acidophilus Lb.52 Lb. rhamnosus Leuc.10 Leuc. holzapfelii Lb.8 Lb. nagelii Lb.53 Lb. rhamnosus Leuc.11 Leuc. carnosum Lb.9 Lb. salivarius Lb.54 Lb. acidophilus Fr.12 Fr. durionis Lb.10 Lb. fermentum Lb.55 Lb. casei ssp. casei Fr.13 Fr. ficulneus Lb.11 Lb. sakei ssp. sakei Lb.56 Lb. salivarius Fr.14 Fr. fructosus Lb.12 Lb. sakei ssp. carnosus Lb.57 Lb. acidophilus Fr.15 Fr. pseudoficulneus Lb.13 Lb. sanfranciscensis Lb.58 Lb. rhamnosus Pe.1 Pe. acidilactici Pe. pentosaceus Lb.14 Lb. amylolyticus Lb.59 Lb. rhamnosus Pe.2

Pe. claussenii Lb.15 Lb. amylovorus Lb.60 Lb. rhamnosus Pe.3

Strep. salivarius ssp. Lb. delbrueckii ssp. Lb.16 Lb.61 Lb. salivarius Strep.1 thermophilus jakobsenii

The strains were kept at -80 ºC in glycerol and from there they were inoculated (500 µL) in glass tubes with 9 mL of MRS or GM17 - being then set to grow during 24 h at 30 ºC or 37 ºC, depending on the optimal incubation temperature of each strain. After 24 h, the tubes were vortexed and the strains were sub-cultured using an inoculation loop of 10 µL; for the strains that were not growing sufficiently, 2/3 mL of the media was removed, in order to have it more concentrated after vortexing, thus increasing the chances of growth. The subculture on the growth media was repeated for two or three days, depending on how fast the strains were growing, being then ready for inoculation on the fermentation media.

3.2.2. Non-conventional yeast The yeast strains tested in this project are also part of the Carlsberg Research Laboratory collection; they were specifically selected because previous projects showed their potential as non-to-low ethanol producers with an interesting flavour profile. The strains tested include species from the genus Saccharomyces and Kluyveromyces, which were isolated from Kefir grains, but also Galactomyces, Kazachstania and Hanseniaspora strains. The remaining yeasts are confidential, so their taxonomic name will be kept in secret. The full list of yeasts tested, as well as the corresponding code of each strain, is presented in Table 6.

Table 6- Code and taxonomic name of the non-conventional yeasts tested

Code Strain S.1 Saccharomyces kefyr 1 S.2 Saccharomyces kefyr 2 S.3 Saccharomyces unisporus Kl. Kluyveromyces marxianus Y1 Yeast 1 Y2 Yeast 2 Y3 Yeast 3 Y4 Yeast 4 Gal.1 Galactomyces geotrichum 1 Gal.2 Galactomyces geotrichum 2 Gal.3 Galactomyces geotrichum 3 Kaz. Kazachstania gamospora Hans. Hanseniaspora guilliermondii

These yeast strains were kept in the fridge at 5 ºC, and inoculation was done through transfer of 500 µL of each to a plastic tube with 6 mL of YPD media, being then set to growth for 24 h at 25 ºC. Like bacteria strains, the inoculation on growth media of the yeast strains was repeated during two or three days, depending on how fast they were growing, being then ready for inoculation on the fermentation media.

3.3. Fermentation and samples processing Fermentations were performed in glass cylinders of 250 mL filled with HFGW at pH 6 and, apart from LAB strains Lc.2, Lc.23, Lb.52, Leuc.2, Leuc.5, Pe.1 and Pe.2 that were run both on 5 ºP and 7 ºP, all the others were just tested in HFGW 7 ºP. All cylinders were inoculated with 2.5 mL (1% v/v) of the corresponding bacteria or yeast strain (T0). Once the glass cylinders were inoculated, they were placed in Cimarec i Poly 15 multipoint stirrers from ThermoFisher Scientific (Thermo Fisher Scientific 2017), at 25 ºC for yeast and at 30 ºC or 37 ºC for

LAB, during 24 h. After this incubation period (T24), the content of each cylinder was split in five 50 mL conical centrifuge tubes, which were then centrifuged during 20 min at 4700 rpm and 20 ºC on a Heraeus Multifuge X3R centrifuge, from Thermo Fisher Scientific (Thermo Fisher Scientific 2017). Once the tubes were all centrifuged, the supernatant was transferred to new 50 mL conical centrifuge tubes. Hence, five tubes per sample were produced, being one of them used for the sensory analysis, two for VOCs analysis, another for ABV analysis and the fifth one kept frozen in-house. After all these fermentations, the best candidates in terms of aroma profile were picked. All the bacteria strains selected were inoculated again in HFGW at pH 6 and 7 ºP, but also in MW at pH 6 and 7 ºP. The inoculation and fermentation conditions were exactly the same as described before, but two cylinders per

20 strain were used instead of only one (more volume for sensory analysis). After 24 h of the inoculation, two cylinders’ content per strain was poured in 1L bottle assemblies and centrifuged during 20 min at 5000 rpm and 20 ºC, on a Avanti J-26S XPI centrifuge from Beckman Coulter (Beckman Coulter 2017). When centrifuged, the HFGW samples were all passed through a 0.2 µm filter to sterile glass flasks, always close to the flame, and kept in the cold room. However, for the MW, after centrifugation, the volume was split equally in two glass bottles (250 mL), being one of them pasteurized for 40 min at 65 ºC. In both cases, 25 mL was taken to conical centrifuged tubes for ABV analysis (the remaining 25 mL of the tube where filled with DI water, in order to save more volume for the sensory analysis). In terms of non-conventional yeasts, they were also inoculated in HFGW and MW at pH 6 and 7 ºP, but instead of 24 h-fermentation, one week-fermentation was tested. After this 7-day period, sample processing for both types of wort was exactly the same as explained for the LAB candidates, except that samples for ABV analysis were also taken from the fermentations in HFGW due to the different fermentation time of this trial. In all laboratorial trials, there was one cylinder just filled with wort, for each incubation temperature tested, that was used as a control.

3.4. Analysis In order to obtain as much information as possible to well characterize all the strains tested in this project, several different analyses were performed.

3.4.1. Bacterial growth monitoring - optical density The optical density (OD) of each sample was measured just right after inoculation of the strain in the glass cylinders with HFGW (pH 6, 5 ºP or 7 ºP) (T0), and after 24 h incubation at the optimal growth temperature (25 ºC, 30 ºC or 37 ºP) of each strain (T24), in order to follow growth of all strains. The measurements were performed at 600 nm (OD600), using 1,5 mL cuvettes in a Spectronic BioMate 3 UV- Vis spectrophotometer from Thermo Electron Corporation, part of Thermo Fisher Scientific (Thermo Fisher Scientific 2017).

Since at T0 there was no fermentation, the samples were not turbid, so there was no dilution for OD measurements. However, the samples collected at T24 were considerably turbid, so they were diluted 10 times (100 µL of sample and 900 µL of autoclaved DI water). In both cases, the blank of each sample was obtained by centrifugation of 1 mL at 13 000 rpm for 3 min, using a 260D brushless microcentrifuge from Denville Scientific Inc. (Denville Scientific Inc. 2017).

For the LAB candidates, the OD was measured at T0 and T24 on the MW (because the data on HFGW were already obtained before); for the non-conventional yeast, the OD was measured in both fermentation media, at T0 and at T7days. The procedure and conditions were exactly the same as described previously, being the dilution at T7days for the non-conventional yeast also 10 times.

3.4.2. Acidifying activity - pH In order to control how much acidic the media became after fermentation with each strain, the final pH value (T24) was checked. As mentioned before, the equipment used was a Lab 850 pH meter from SI

Analytics (SI Analytics 2017). For the candidates, the same approach was performed at T24 for LAB and

T7days for the non-conventional yeast.

3.4.3. Ethanol content - alcohol by volume The ethanol content (%v/v) of all samples was analysed in aliquots of 50 mL for each strain, on an Alcolyzer Beer Analysing System from Anton Paar (Anton Paar 2017) (Annex C: ABV measurement). In the case of both bacteria and yeast candidates, the ABV value obtained by the equipment was duplicated due to the 1:1 dilution of the samples.

3.4.4. Sensory evaluation The sensory evaluation included both aroma and taste analysis. The aroma evaluation of all strains tested was performed by five experienced members of the Yeast and Fermentation group, which sniffed all samples at T24 directly from the conical centrifuged tube. The data resulting from this evaluation was recorded, using binary code (0 and 1), in an aroma evaluation table (Annex D.1: Aroma evaluation), which was adapted from the literature (Delgerzaya et al. 2009). In a second round of sensory analysis, taste evaluation (Delgerzaya et al. 2009) was also performed further to aroma evaluation (Annex D.2: Taste evaluation) to the LAB and non-conventional yeast candidates fermented in HFGW and MW. For this flavour analysis of the candidates, besides the the members of the Yeast and Fermentation team, also a group of experienced people in sensory analysis from the Carlsberg Research Laboratory evaluated the samples.

3.4.5. Aroma profiles - volatile organic compounds analysis The volatile organic compounds (VOCs), produced by the bacteria and yeast strains tested in this project, were analysed at the Central Laboratory of the Carlsberg Group Development Center in Obernai (France). The compounds analysed are tabulated in Table 7; the two techniques used to analyse these compounds were Gas Chromatography Headspace (GC Headspace) and GC Carbon Disulfide (GC-CS2) extraction. All VOCs analysed by GC-Headspace were also analysed using GC-CS2 extraction.

Table 7- Volatile compounds screened in all samples

VOCs Abbreviation Acetaldehyde Acet_Ald Ethylacetate Et_Ac Propanol Prop_ol GC-Headspace Isobutanol Isob_ol Isoamyl acetate Isoam_Ac

Isoamyl alcohol Isoam_ol Ethyl caproate Et_Cap Acetaldehyde Acet_Ald Ethylacetate Et_Ac Isobutylacetate Isobut_Ac 2-Phenylethyl acetate 2_Phe_Ac Isoamylacetate Isoam_Ac Propanol Prop_ol Isobutanol Isob_ol Isoamyl alcohol Isoam_ol

2-Phenylethanol 2-Phe_ol GC-CS2 extraction Ethyl caproate Et_Cap Ethyl caprylate Et_Capr Caproic acid C6 Caprylic acid C8 Capric acid C10 4-Vinylguaiacol 4-VG Limonene Lim Linalool Linal

3.4.6. Fermentation rate - carbon dioxide release Since the non-conventional yeast candidates carried out fermentation for 7 days, a carbon dioxide release test was performed to assess the behaviour of these strains. The evaluation was done by weighting all the cylinders during some of the fermentation days, and then plot the weight lost to see the tendency over the week.

3.5. Statistical analysis To analyse the data generated during this project, the software Unscrambler X 10.4 from CAMO was used and it consists on a program for multivariate analysis (MVA) and design of experiments (DoE) (CAMO 2017c). Among the wide range of statistical methods offered by the software, the ones selected were Cluster Analysis (CA), Principal Component Analysis (PCA) and Partial Least Squares (PLS) regression.

3.5.1. Cluster analysis The CA includes many techniques to discover patterns within complex data sets. Hence, the goal of this statistical method is grouping the data in clusters, having the elements within each cluster a high level of association, but the clusters by themselves at the end are clearly distinctive from one another. The concept

23 of association or distinctiveness among a data set can vary a lot depending on the algorithm applied to it (Anderberg 1973). The clustering method selected was Hierarchical Average-linkage and, in terms of distance measure, the option selected was Squared Euclidean, after testing all the options presented by the software and according with the suggestions of the instructions manual (Esbensen et al. 2002). The number of clusters selected was adapted according with the data and software suggestions.

3.5.2. Principal component analysis The PCA is widely used to reduce the dimensionality of a data set containing a large number of interrelated variables, retaining at the same time as much variation as possible. Basically, a new set of variables is created, the so-called principal components (PCs), which are uncorrelated and order in a decreasing way in terms of variation (the first PC retains most of the variation present in all of the original variables, then the second retains less and so on) (Jolliffe 2002). PCA helps in finding out what is most different from one sample to another, which variables are more related to these differences, and if the variables are correlated or not, being also possible to detect putative patterns. It is a quite useful statistical method because it quantifies the amount of useful information, thus removing meaningless variation (CAMO 2017b). The validation option selected for the PCAs performed in this project was the cross validation in a systematic method (112233); in terms of algorithm, the model input selected was NIPAL, after testing all the options presented by the software and according with the suggestions of the instructions manual (Esbensen et al. 2002).

3.5.3. Partial least squares regression The PLS regression is a recent statistical method that generalizes and combines features from PCA and Multiple Regression (MR). The objective of this method is to analyse and predict a set of dependent variables that arise from a set of independent variables or predictors. It can be considered a powerful method of analysis because of the low number of requirements in terms of measurement scales, sample size and residual distributions; it can be used for theory confirmation, but also to guide later testing based on how it suggests where relationships might exist or not. More recently, some authors suggested the acronym PLS as Projection to Latent Structures (CAMO 2017a; Abdi 2010). In terms of validation, the option selected for the PLS regression performed in this project was the cross validation in a systematic method (112233), the algorithm selected was Kernel PLS and, to obtain a better view of the results, an Hoteling’s T2 ellipse was also performed, after testing all the options presented by the software and according with the suggestions of the instructions manual (Esbensen et al. 2002).

4. Results and Discussion

The data obtained in this project (Annex E: Lactic acid bacteria and non-conventional yeast data) were grouped in three categories, VOCs, sensory and growth parameters (OD, pH and ABV), and were then introduced in the UnscramblerX to run CA, PCA and PLS.

4.1. Lactic acid bacteria From all VOCs analysed, in any LAB strain, the presence of isobutyl acetate and limonene were recorded, so these two compounds do not appear in any of the plots presented below.

4.1.1. Cluster analysis In order to show an overview on the association level of the bacteria tested, a CA was performed to explore how the strains are grouped in terms of VOCs concentration. The final dendrogram obtained for the LAB in terms of VOCs is presented in Figure 7. For this dendrogram, the strains Lc.9 and Lb.19 were excluded because of their very high level of isoamyl alcohol (5.11 mg/L) and propanol (6.01 mg/L), respectively. These two values were so far away from the average (0.42 mg/L for isoamyl alcohol and 0.05 mg/L for propanol) that each of them was forming its own cluster.

Figure 7- Cluster analysis plot for LAB, based on VOCs concentration

*Lc.2 I, Lc.23 I, Lb.52 I, Leuc.2 I, Leuc.5 I, Pe.1 I, Pe.2 I correspond to the strains fermented in HFGW 5 ºP

After analysing the data (Annex E.1: Volatile organic compounds) and comparing it with the clusters obtained in Figure 7, it was possible to grasp the pattern in each cluster. The first cluster (purple) includes Lb.32, Lb.49, Lb.73, Lb.76 and Lb.87, being all characterized by a high concentration of acetaldehyde (between 8.90 and 12.60 mg/L for a general average of 2.48 mg/L). About the second cluster (brown), it is composed by Lc.25, Lb.67, Lb.68, Lb. 70, Lb.71, Lb.77, Lb.79, Lb.80 and Lb.84 and it presents an even higher concentration of acetaldehyde (between 13.50 and 16.70 mg/L), as well as a slightly higher concentration of isoamyl alcohol (between 0.65 and 0.85 mg/L) when comparing with the general average (0.42 mg/L). Still looking at acetaldehyde concentrations, the third cluster (green), which is constituted by Lc.24, Lc.26, Lb.25, Lb.27, Lb.40, Lb.41, Lb.45 and Lb.81, represents the strains that have a concentration of this volatile between 4.25 and 7.85 mg/L. Almost all Lb. plantarum strains tested in this project are included in one of these three clusters (11 out of 14), forming the biggest species group; this makes sense according with other studies that describe Lb. plantarum as one of the LAB strains that produce higher amounts of acetaldehyde in fermented malt beverages (Salmerón, Thomas, and Pandiella 2014). The forth cluster (red) includes the strains Lc.1 Lc.2, Lc.2 I, Lc.4, Lc.5, Lc.7, Lc.8, Lc.10-16, Lc.19 Lc.21, Lc.22, Lc.28, Lc.29, Lb.12 and Pe.3. In this case, these strains are clustered due to the above- average concentration of 4-vinylguaiacol in all them, with values between 2.70 and 6.70 mg/L for a general average of 0.94 mg/L. Almost all strains included in this cluster are from the species Lc. lactis (18 out of 20); in the literature, in terms of dairy products, they are characterized as producers of high concentrations of alcohols and aldehydes (Morales et al. 2003; Ayad et al. 1999), but no studies have been found about the volatiles produced by this species when inoculated in cereal-based media. The fifth cluster (light blue) comprises the remaining strains that, in general, present average volatile concentrations. In terms of relative distance, one observes that the three first clusters are quite related, due to the high concentration of acetaldehyde that differentiates these strains from the remainder. The largest relative distance is found between the clusters that include the species with higher concentrations of acetaldehyde (purple and brown) and the cluster with LAB that exhibits high concentration of 4-vinylguaiacol (red), because there is a considerable difference in volatile pattern. Since the concentration values of acetaldehyde in the third cluster (green) and of 4-vinylguaiacol in the fourth cluster (red) are not so far from the average value - like the acetaldehyde concentrations in the first and second clusters, their relative distant to the “average-cluster” (light blue) is considered intermediate. Therefore, after analysing the CA obtained for the LAB tested in this project, it is possible to conclude that acetaldehyde and 4-vinylguaiacol are the most prominently volatile compounds. This is consistent with what is described in the literature because these two compounds are considered key aroma compounds that bring about a major contribution to the flavour profile of malt wort beverages fermented with LAB (Nsogning Dongmo et al. 2017).

4.1.2. Principal component analysis Since a large number of microorganisms and different parameters were analysed, a PCA was performed in order to understand which factors have more influence upon the variance observed in the data. Considering VOCs concentration and growth parameters, the PCA bi-plot obtained is presented in Figure 8. In Figure 9 is presented the PCA plot with the strains represented by their metabolic pathway.

Figure 8- Principal component analysis bi-plot obtained for LAB, based on VOCs and growth parameters (PC-1 (83%) vs PC-2 (10%))

Figure 9- Principal component analysis plot obtained for LAB metabolism (PC-1 (83%) vs. PC-2 (10%))

In Figure 8 is possible to see that acetaldehyde (Acet_Ald) is the main principal compound (PC-1), being responsible for most variance observed in the data (83%), and 4-vinylguaiacol (4-VG) is the second parameter (PC-2) most responsible for variance (10%); these two PCs can explain 93% of data variance. This is easy to identify in the plot because both components are close to the corresponding axis, and isolated from the rest of the volatiles tested. In terms of data similarities, three very distinct groups can be identified in Figure 8. On a PCA, the points close to the intersection of the axis usually present average results (without a considerable variance); upon analysis of the VOCs (Annex E.1: Volatile organic compounds) and growth parameters (Annex E.3: Growth parameters) data, it is possible to confirm that in fact the group of points in the centre of the plot correspond to strains with average results. On the right, the strains with higher acetaldehyde concentration lie all together (close to the x-axis) and, on the top, the strains with higher concentration of 4-vinylguaicol are grouped (close to the y-axis). Comparing with the CA presented in Figure 7 and its discussion, it is possible to recognize the agreement in terms of strains grouping and main responsible for data differentiation (acetaldehyde and 4-vinylguaicol) – which, as mentioned before, belong to the list of the key aroma compounds in fermented cereal based beverages described in the literature (Nsogning Dongmo et al. 2017). When comparing Figure 8 and Figure 9, it is possible to observe that there is a tendency for the strains with higher production of acetaldehyde being mainly heterofermentative or facultative heterofermentative; this agrees with the literature (Figure 2), because acetaldehyde is an intermediate compound in ethanol production, which does not take part in the homofermentative pathway. The strains with higher production of 4-vinylguaiacol tend to be mainly homofermentative and the species next to the intersection of the axis (average VOCs and growth parameters values) can be both homofermentative and heterofermentative because, even in small concentrations, there is a wide variety of volatile profiles. Besides PC-1 and PC-2, the PCA obtained for LAB in terms of VOCs concentration and growth parameters also showed a PC-3 and PC-4 (Annex F.1: Lactic acid bacteria). The main responsible for variance in PC-3 is pH and in PC-4 is propanol (Prop_ol), being 3% and 1% the levels of variance covered by them, respectively (Figure F.1. 1). When visualizing the 3D-plot of PC-1, PC-3 and PC-4 (Figure F.1. 2), the predominance of the PC-1 can be observed, since the majority of the strains tend to agglomerate close to the PC-1 edges instead of next to the PC-3 and/or PC-4 edges. This plot, once more, shows the low level of variance covered by PC-3 and PC-4 - being clear that the results obtained for pH and for propanol concentration are not considered significant to justify the majority of the variance existent in the data.

4.1.3. Partial least squares regression In order to check whether the sensory evaluation (dependent variable) performed in this project is consistent with the aroma described in the literature (Table 2) for the VOCs analysed in each LAB strain (independent variable), a PLS regression was performed. In Figure 10, it is possible to observe the resulting plot, as well as a closer view of the data agglomerate, being the sensory data presented in annexes (Annex E.2: Sensory analysis). Acetaldehyde (Acet_Ald) and 4-vinylguaiacol (4-VG), as

28 mentioned before, are the main effectors upon data variance and this can be also confirmed in the PLS plot because they are the compounds closer to the borders of the ellipse, showing extreme values of Factor-1 and Factor-2. For Factor-1 (x-axis), acetaldehyde is responsible for 84% of the variance while for Factor-2 (y-axis) it is responsible for 10% of the variance. In terms of 4-vinylguaiacol, in both Factor- 1 and Factor-2, this compound is responsible for 1% variance in each axis. Since the sensory analysis was performed using binary code (0 and 1) and not a more accurate quantification method, the results are not so clear as in the CA and in the PCA.

Figure 10- Partial least squares regression plot obtained for LAB, based on VOCs and sensory analysis (Factor-1 (85%,1%) vs Factor-2 (10%,1%))

Starting with the alcohols, and according to literature, the aroma of isoamyl alcohol (Isoam_ol) and isobutanol (Isob_ol) is referred as malty. Looking at the plot, isoamyl alcohol is relatively close to the malt and roasted barley (Roasted ba) characterization, but the isobutanol is far from this classification, being closer to fruity and floral descriptors. Upon inspection of the data, it is possible to observe that the strain with higher concentrations of isobutanol shows an even higher concentration of acetaldehyde. Since the acetaldehyde aroma is referred to as fresh and green, which can be associated with fruity and floral, it looks like the acetaldehyde aroma overtook the isobutanol aroma. In the sensory analysis performed, the vinegar descriptor was related to the strong and not so pleasant aroma, fitting very well the aroma profile of propanol (Prop_ol) - since its aroma is described in the literature as strong/pungent. In terms of fruity and floral aroma, this characterization has often been associated with esters; observing the results plot, ethyl acetate (Et_Ac), ethyl caprylate (Et_Capr), 2-phenylacetate (2-Phe_Ac), ehtyl caproate (Et_Cap) and isoamyl acetate (Isoam_Ac) follow this association, because they are nearby the fruity and floral aroma classification on the plot. The aroma of the hexanoic (C6) and octanoic (C8) acids are classified as sweety in the literature. Brown sugar (Brown suga) and honey aroma are correlated with a sweet profile, but in Figure 10 is possibly to observe that these two organic acids are not nearby the expected classification, being closer to fruity and floral. After analyzing the data, it is possible to notice a situation similar to that of isobutanol: the concentrations recorded for C6 and C8 are quite low compared with the other volatiles, and the strains with higher concentration of these two acids also present a much higher concentration of acetaldehyde, so it looks like that there is again a case of aroma overtake. However, the decanoic acid (C10) is not classified as swetty in the literature, but as something stronger and not so pleasant. This apparent in the results plot, because C10 is not so close to C6 and C8, showing a tendency to be closer to the vinegar classification - which makes sense, because the vinegar classification was used for stronger and not so pleasant aroma. The aroma of 2-phenylethanol (2_Phe_ol) is classified as floral (rose) in the literature, and this information can be confirmed by the plot generated, because the compound occupies a position close to the floral classification. Linalool (Linal) aroma is often described as floraly, and in the plot it is not so far from the floral classification; yet, the roasted barley, honey, brown sugar and malt classifications are closer. The reasons for this not so clear association between the presence of linalool and the floral aroma can be related with the not so high concentration of this VOC in the different strains, entertaining again a case of aroma overtake by other volatiles. About acetaldehyde and 4-vinylguaiacol: since they are settled quite far form the rest of the data, it is more difficult to associate them with a specific classification.

However, as mentioned before, acetaldehyde aroma is classified as fresh and green - and in Figure 10 it is located on the right side of the plot, so it is possible to see that fruity and floral are the classifications closer to it. Also as mentioned before, 4-vinylguaiacol aroma is classified in the literature as clove-like, being often considered as a beer off-flavour (Vanbeneden et al. 2008), so it was expected to be next to the vinegar classification in the plot (strong and not so pleasant aroma); in fact, it is closer to the malt classification. This compound is often found in wheat malt beers in high concentrations (Coghe et al.

2004), being a characteristic aroma of this beer - meaning that eventually it was associated with a malt aroma due to similiarities with wheat beer.

4.2. Non-conventional yeast From all VOCs analysed, the presence of limonene and linalool was not detected in any non- conventional yeast strain, so these two compounds are not presented in the plots below.

4.2.1. Cluster analysis The final dendrogram for the non-conventional yeasts used in this project is presented in Figure 11. The strains Y4 and Kl. were not excluded of the plot, even occupying a full cluster each. The reason for this decision is related with the few strains of yeast tested, and the attempt to understand the differences between all them.

Figure 11- Cluster analysis plot for non-conventional yeast, based on VOCs concentration

Comparing the VOCs data for yeast (Annex E.1: Volatile organic compounds) with the dendrogram obtained, the strain Y4 is occupying a full cluster (red) because it presents a very high concentration of isoamyl alcohol (101.34 mg/L, given for a grand average of 26.71 mg/L). For this strain, is also observed a high concentration of isobutanol (30.99 mg/L for a grand average of 9.04 mg/L) and 2-phenylethanol (25.86 mg/L for a grand average of 9.48 mg/L), resulting in the highest ABV registered among all bacteria and yeast tested in this project (2.92% v/v). According to the literature, experimental trials with Kl. showed that acetaldehyde, ethyl acetate and ethanol (alcohol smell) were the major compounds produced (Medeiros et al. 2001). However, more

31 recently was demonstrated that ethyl acetate is one of the main volatile compounds, but isoamyl acetate is the major ester identified, being isoamyl alcohol and 2-phenylethylethanol also aroma compounds with high concentration after fermentation (Padilla et al. 2014). In this project, Kl. (purple) presents high concentrations of isoamyl alcohol (58.84 mg/L), isobutanol (21.54 mg/L) and 2-phenylethanol (34.88 mg/L) like Y4, but the concentrations of isoamyl acetate (0.00 mg/L) and ethyl acetate (0.55 mg/L) are quite low comparing to the general average of the different non-conventional yeast (0.40 mg/L for isoamyl acetate and 5.14 mg/L for ethyl acetate). The cluster occupied by this strain is closely-related with the previous one, showing a relative distance between 3 and 3.5, due to the high concentration in the alcohols tested. The third cluster (green), which is composed by the strains Kaz., S.1 and S.3, is characterized by a high level of acetaldehyde (concentration on these three strains is between 43.25 and 64.50 mg/L, for an overall average of 21.01 mg/L); this differentiates them from the other strains. In the baking industry, Kaz. is described as high producer of 3-methyl-1-butanol (isoamyl acetate precursor), 1-hexyl-acetate (pear aroma) and phenetyl-2-methylbutyrate (floral aroma) (Zhou et al. 2017), yet other studies more related with industrial beer fermentation showed that the acetaldehyde concentration produced by this strain was quite high, especially comparing with the results obtained for the other yeasts tested (Gamero et al. 2016). In terms of kefir yeasts (S.1 and S.3), acetaldehyde, diacetyl and acetoin (the latter two being responsible for a buttery aroma) are described as the main volatiles produced during fermentation (Seydim et al. 2000). The fourth and last cluster (light blue) includes the remaining yeast strains that display average values in terms of all VOCs. In terms of relative distance, the first two clusters have the maximum relative distance (10) to the third and fourth clusters due to very high level of alcohols produced during wort fermentation. The third cluster (green) presents an intermediate relative distance to the fourth cluster (light blue), because the high levels of acetaldehyde are not as distant from the corresponding average as the concentrations of alcohols in the first and second clusters. In the fourth cluster, it is also possible to observe an intermediate relative distant (~1.5) between the first three strains and the remaining ones, thus justifying a slightly higher concentration of alcohols and esters in Y3, Hans. and S.2. In the literature, Hans. is described as high producer of 2-phenylethyl acetate, propanol and 3-(methylthio)propionic acid (rancid, pungent aroma) during fermentation (Moreira et al. 2011); however, in the results obtained for this project, the concentration of propanol obtained for this strain (3.46 mg/L) is lower than the general average (4.99 mg/L), with the concentration 2-phenylethyl acetate (6.12 mg/L) being even higher than the overall average (1.19 mg/L). As expected, the strains of the genus Galactomyces are close to each other, as well as Y1 and Y2. Since the taxonomic name of the Y strains were not revealed, it is not possible to justify why Y1 and Y2 are intermediately distant to Y3 and why they show a so different behaviour compared with Y4. Furthermore, the Saccharomyces species S.1 and S.3 are not so close to S.2 as they are from each other, due to the high level of acetaldehyde produced by S.1 and S.3 that is not observed in S.2; given the results of this three

32 strains, was also possible to observe that S.2 exhibits a final sum of esters (7.10 mg/L) higher than in S.1 and S.3 (0.43 and 0.38 m/L, respectively).

4.2.2. Principal component analysis In Figure 12 includes the PCA plot obtained for the non-conventional yeast tested, in terms of VOCs concentration and growth parameters. The total percentage of variance covered by the PC-1 is 63% and for the PC-2 is 31%, which means that they can explain 94% of the variance of the data. Inspecting the plot more carefully, it is possible to understand that PC-1 corresponds to isoamyl alcohol (Isoam_ol) and PC-2 corresponds to acetaldehyde (Acet_Ald). This makes sense when comparing with the CA on Figure 11, because the compounds that mainly differentiate the clusters are also isoamyl alcohol and acetaldehyde.

Figure 12- Principal component analysis plot obtained for non-conventional yeast, based on VOCs and growth parameters (PC-1 (63%) vs PC-2 (31%))

Comparing the data obtained for VOCs (Annex E.1: Volatile organic compounds) and growth parameters (Annex E.3: Growth parameters) in yeast with the plot in Figure 12, it is possible to devise four distinctive groups. On the left side, Y4 and Kl. are settled around isoamyl alcohol because, as mentioned before for the CA, they show a much higher concentration of this compound than the average for all yeast strains tested (especially Y4). On the top, S.1, S.3 and Kaz. are grouped next to acetaldehyde because they also show a much higher concentration of this compound when compared with the remaining ones. In the centre, just near the intersection of the plot axis, the strains Y3, S.2 and Hans. appear and they are together because their volatile concentrations and growth parameters are around the general average. According with the CA displayed before for the non-conventional yeast, the strains Gal.1-3, Y1 and Y2 also belong to the cluster of strains that present average values for the different

VOCs. However, since the growth parameters are also included in the PCA performed, when observing the data, it is possible to perceive that they are differentiated due to their slow-growing profile: low OD values (between 0.14 and 1.26 for a general average of 1.88) and high pH (between 4.90 and 5.54 for a general average of 4.71). As mentioned before, the growth of fermentative microorganisms can be affected by the initial oxygen level, pH, temperature or composition of the substrate, being a low growth when the microorganism exhibits a lower excretion of organic acids (e.g. succinate) - which then results in a lower decrease of the medium pH (Coote and Kirsop 1976). On the PCA obtained for LAB, only four PC were identified, and 97% of the variance could be explained. However, for non-conventional yeast, the variance is fully explained, and five PC are presented (Annex F.2: Non-conventional yeast). Since the first two PCs already explain 94% of the data variance, PC-3, PC-4 and PC-5 present a very low percentage of data variance explanation: 3%, 2% and 1%, respectively. Observing the plots PC-3 vs PC-4 (Figure F.2. 1) and PC-4 vs PC-5 (Figure F.2. 2), it is clear that PC-3 corresponds to ethyl acetate (Et_Ac), PC-4 corresponds to 2-phenylethanol (2-Phe_ol) and PC-5 corresponds to isobutanol (Isob_ol).

4.2.3. Partial least squares regression In order to check whether the sensory evaluation performed (dependent variable) is in agreement with the aroma described in the literature (Table 3) for the VOCs analysed in each non-conventional strain (independent variable), a PLS regression was performed. However, in this case the data are even more difficult to interpret because the number of strains tested was quite low, besides the difficulties faced due to the binary scale used for sensory analysis. The final plot obtained for this statistical method is labelled as Figure 13. Isoamyl alcohol and acetaldehyde, as mentioned before, are the main contributors to data variance, and this can be also confirmed in the present plot because they are the two VOCs at the border of the ellipse. For Factor-1 (x-axis), isoamyl alcohol is responsible for 40% of the variance, and for Factor-2 (y-axis) it is responsible for 54% of the variance. In terms of acetaldehyde, it is responsible for 11% of the variance in the x-axis for 5% of the variance in the y-axis.

Figure 13- Partial least squares regression plot obtained for non-conventional yeast, based on VOCs and sensory analysis (Factor-1 (40%,11%) vs Factor-2 (54%,5%))

In terms of alcohols, the aroma of isoamyl alcohol is characterized in the literature as malty, but in the plot obtained it is close to the honey characterization. Checking the data obtained, it is possible to observe that the compounds showing higher concentration of this VOC also exhibit high concentrations of other volatiles like acetaldehyde, isobutanol, 2-phenylethanol or propanol. Since these other compounds are associated with fruity, floral and vinegar classifications, the mixed of odours in the samples is apparent and it can be the reason for a not so clear association of isoamyl alcohol with the malt aroma. In terms of isobutanol and propanol, their aromas are described in the literature as something strong/non-pleasant so they should be next to the vinegar classification. However, in the plot they are also associated with the honey characterization, being the explanation possibly similar to the one presented for isoamyl alcohol (mixed of high concentration of different compounds). The esters aroma, as well as the aroma of 2-phenylethanol (phenolic compound), are characterized in the literature as fruity, flower and sweet/honey. In the plot obtained, it is possible to observe some proximity between these compounds and the honey characterization, not that far from the floral and fruity classifications. The aroma of the hexanoic and octanoic acids are also described in the literature as fruity and sweet; it is possible to detect this association in Figure 13, because they are represented just next to some of the esters (isoamyl acetate, isobutyl acetate and ethyl caproate), so not far from the fruity and honey classfications. The brown sugar (Brown suga) profile is also associated to a sweet aroma, but in this case any of the VOCs referred to is next to this classification in the plot. In contrast, the decanoic acid is not classified as fruity and/or swetty in the literature, but as something stronger and non-pleasant (soapy). The same happens with 4-vinylguaicol, mentioned in the literature as a compound that presents a non-pleasant aroma. Analyzing the plot presented in Figure 13, C10 and 4-VG

35 are just next to the vinegar classification, which is logical because this is the characterization attributed to the non-pleasant samples. Also for yeast, acetaldehyde aroma is characterized in the literature as fresh and grass/green, being expected the association with a fruity and floral classification. However, this compound is clearly far from the fruity and floral classification in the plot; this can be explained, once again, by the mix of high concentration compounds that can be found in the strains with a considerable level of acetaldehyde, resulting in a mix of aromas that is difficult to classify in a proper way.

4.3. Candidates From the sensory analysis performed for all LAB and non-conventional yeast tested, a short list (Table 8) was done with the strains showing the most interesting and agreeable aroma.

4.3.1. Lactic acid bacteria From the 136 LAB strains screened in this project, the eight best candidates were selected. As mentioned before, this selection was done by the team during the aroma evaluation of all strains tested, since in many cases the aroma was clearly non-pleasant. As mentioned previously, the candidates were fermented again in HFGW, but also in MW because it is the most widely used fermentation medium in beer manufacture (Ernandes et al. 1993); furthermore, it could come up with an interesting flavour profile. Pasteurization was performed for the MW samples because it is an alternative of filtration to extend the shelf life of the product (Dymond 1997); some previous in-house tests confirmed that at 65 ºC during 40 min, did not affect to a large extent the sensory profile of other cereal-based beverages. After all fermentations in HFGW and MW, the team and a group of experienced staff in sensory analysis from the Carlsberg Research Laboratory evaluated the flavour of all samples; the summary of the aroma and taste analyses is presented in Table 8. In the literature, it is claimed that pasteurization can affect beverages sensory profile (Ortega-Rivas and Salmerón-Ochoa 2014), but it is also stated that its intensity/level is quite important for the extent of how the product is affected (Cao et al. 2011). In this study, no sensory differences were noticed between the pasteurized and non-pasteurized MW samples, meaning that the conditions of 65 ºC and 40 min are also not affecting the flavour characteristics of this type of cereal-based beverages.

Table 8- LAB candidates selected from the sensory analysis

Code Strain Medium Aroma evaluation Taste evaluation HFGW Fruity, malt (fresh/citrus) Sour Lb.25 Lb. pentosus MW Malt, honey Sweet, malt Lb. plantarum ssp. HFGW Fruity, malt (fresh/citrus) Sour (strong) Lb.32 plantarum MW Fruity, malt (fresh/citrus) Sour (strong) HFGW Honey, fruity Sour (strong) Lb.49 Lb. plantarum MW Fruity, honey Sour, vinegar

HFGW Malt, honey (stable/neutral) Sour, vinegar, slightly fruity Lb.56 Lb. salivarius MW Brown sugar, roasted barley Sweet, sour, honey HFGW Fruity, malt Sour, malt Lb.67 Lb. plantarum MW Fruity, honey Sour HFGW Fruity, honey, vinegar Sour (quite acidic) Lb.68 Lb. rhamnosus MW Malt, honey, roasted barley Sour (soft) HFGW Malt, roasted barley Sweet, malt, slightly sour Lb.73 Lb. rhamnosus MW Malt, roasted barley Sweet, malt, slightly sour HFGW Vinegar, fruity Sour Lb. 84 Lb. plantarum MW Malt, fruity Sour

As it can be observed, there is a pattern on the best candidates selected, because the aroma tends to be mainly related with malt and/or fruity and the general taste is sour. When looking for the PCA plot presented for LAB in HFGW (Figure 8), it is possible to observe that all candidates belong to the group of strains with high concentration of acetaldehyde, except the strain Lb.56 that belongs to the VOCs average concentrations group. However, Lb.56 is close to the borderline between these two groups, which means that its acetaldehyde concentration is one of the highest among the average strains. As mentioned before, the aroma associated with acetaldehyde is described as fresh and green for LAB (Table 2), which validates the aroma evaluation of the candidates, because malt/cereals and fruits are in general associated with fresh and green aromas. Analysing the data obtained for the growth parameters of the samples fermented in MW (Annex G: Growth parameters data for candidates), one realizes that the data obtained for the pasteurized and non- pasteurized samples is quite similar (pH average for pasteurized samples is 4.48, and for non-pasteurized samples is 3.98; ABV average is 0.02 %v/v for pasteurized samples, and 0.03 %v/v for non-pasteurized samples), which also confirms that the conditions used do not destabilize the product.

4.3.2. Non-conventional yeasts In this project, 13 non-conventional yeast strains were tested. Among them, eight candidates were chosen and their selection, as well as for the LAB strains, was done based on the aroma evaluation performed by the team. Since both LAB and non-conventional yeast should be fermented under similar conditions, the non-conventional yeast candidates were also inoculated in HFGW and MW (with subsequent results for pasteurized and non-pasteurized conditions) - but instead of 24 h, the fermentations were performed for 7 d. Usually in standard beer production, the yeast fermentation use to take 6-7 d (Kunze 2014), so it was decided to try a full week fermentation and check if more interesting flavour profiles were coming up. After running all fermentations in HFGW and MW, the team and some members of the sensory panel evaluated the samples; the summary of the aroma and taste evaluation is presented in Table 9. As mentioned for LAB, the aroma and flavour profiles of the pasteurized and non-pasteurized MW samples were quite similar, so it was proven that the pasteurization conditions tested do not affect that much the flavour of the MW fermented with the selected non-conventional yeast strains.

Table 9- Non-conventional yeasts candidates selected from the sensory analysis

Code Strain Medium Aroma evaluation Taste evaluation HFGW Malt, vinegar Sour, vinegar S.1 S. kefyr 1 MW Malt, fruity Sour HFGW Malt, fruity Sour, vinegar S.2 S. kefyr 2 MW Malt, fruity Sour, slightly sweet HFGW Malt, honey Sour, slightly sweet Y1 Yeast 1 MW Vinegar (quite acidic) Sour, pungent HFGW Malt, fruity Sour Y2 Yeast 2 MW Fruity, slightly malt Sweet HFGW Malt, vinegar Sour Y4 Yeast 4 MW Malt, vinegar, slightly fruity Sour, vinegar HFGW Malt, honey Sweet, honey Gal.1 G. geotrichum 1 MW Malt, honey Sweet, honey HFGW Malt, honey Sweet Gal.2 G. geotrichum 2 MW Malt, honey Sweet, malt, honey HFGW Malt, slightly vinegar Sour, vinegar (strong) Kaz. K. gamospora MW Malt, slightly vinegar Sour, vinegar (strong)

As can be observed, there is a pattern on the best candidates selected because the aroma tends to be mainly related to malt, honey and/or vinegar and the general taste is sour. Going back to the PCA plot of the non-conventional yeasts in HFGW (Figure 12), it is possible to observe that the strains S.1 and Kaz. are next to acetaldehyde, which has is aroma classified as fresh and grass, and fits the malt and vinegar evaluation. Y4 is next to isoamylalcohol, being its aroma classified as malt, alcohol and whisky (strong); it also fits the classification of malt and vinegar mainly attributed to the respective samples. The remaining strains display average values for the tested VOCs and four of them also exhibit a low growth, which means that they should tend to present a wort aroma profile. The general classification attributed to the aroma of these strains is malt, honey and fruity, with some remarks of wort tasting, which means that the data and the evaluations obtained are positively correlated. Comparing the sensory evaluation performed previously for all strains in HFGW (Annex E.2: Sensory analysis) with the evaluation presented in Table 9 for the samples run in HFGW, some changes are apparent. After the 7 d fermentation, the aroma evaluation tends to be more malt, honey and fruity (sweet pattern), while before it was more related with fruity and floral (fresh/green). Comparing also the ABV values, the HFGW fermented during 7 d (Annex G: Growth parameters data for candidates) present much higher results than the HFGW fermented during 24 h (Annex E.3: Growth parameters), which means that for a non-to-low alcoholic beverage, a full week fermentation of these strains in HFGW is not recommended. After analysing the data obtained for the growth parameters of the samples fermented in MW (Annex G: Growth parameters data for candidates), it is possible to observe that for the pasteurized and non- pasteurized samples there are some similarities (pH average for pasteurized samples is 5.22 and for non- pasteurized samples is 5.20; ABV average is 0.73 %v/v for pasteurized samples and 0.70 %v/v for non- pasteurized samples); this also confirms that the conditions used do not destabilize the product. However,

38 when comparing these results with the ones obtained for HFGW, one finds that the OD and ABV averages for the samples fermented in HFGW are higher than for MW, and the pH average is lower for HFGW than for MW. According to the literature, this is justified by the yeast tendency to ferment glucose much faster than maltose (D’Amore, Russell, and Stewart 1989), which means that they grow faster in glucose wort (higher OD) and, consequently, they produce more ethanol (higher ABV) and succinate (lower pH).

4.3.2.1. Yeast fermentation rate

As showed before (Figure 6), during fermentation yeasts tend to release CO2 to the atmosphere and ethanol to the medium, in the stoichiometric 1:1 molar relation. To check whether this happens with the strains tested, the CO2 release of the yeasts that were fermenting in HFGW and MW during 7-days was controlled. The results obtained are presented in Figure 14 for HFGW and in Figure 15 for MW. Comparing the plots obtained and the growth parameters data of the non-conventional yeast candidates (Annex G: Growth parameters data for candidates), it is possible to realize that strains with high values of

CO2 released also present high ABV results, both for HFGW and MW. In this way, it is proven that the results obtained agree with the information available in the literature.

Figure 14- CO2 release plot for non-conventional yeast candidates in HFGW

Figure 15- CO2 release plot for non-conventional yeast candidates in MW

After reviewing both plots, one finds that, both in HFGW and MW, the species of the genus

Galactomyces (Gl.1 and Gl.2) tend to have a lower CO2 release than all the others (less ethanol production), the Saccharomyces kefir strains (S.1 and S.2) and the Kazachstania strain are always in the top group and the confidential yeasts (Y1, Y2 and Y4) vary between the top and the middle positions.

One also perceives that, in general, the CO2 release in the first 5 days is more significant and visible, while the curve tends to stabilize in the last days of the week. This behaviour was also reported in other studies (Bizaj et al. 2009; Landaud and Latrille 2001).

5. Conclusions

In terms of LAB, it can be concluded that acetaldehyde and 4-vinylguaiacol are the volatile compounds that present the broadest range of concentration values, being able to explain 93% of the data variation in terms of volatile organic compounds. The majority of the strains associated with high levels of acetaldehyde belong to the species Lb, plantarum, while 4-vinylguaiacol was mainly produced by Lc. lactis strains. In the literature, acetaldehyde is described as giving fresh and green aroma; in this project, it was accordingly associated with fruity and floral notes. In contrast, 4-vinylguaiacol is defined in the literature as clove-like, but in this study it was perceived as malty. For non-conventional yeast, isoamyl alcohol and acetaldehyde were the volatiles with a broader range of concentration values, being able to explain 94% of the variation in terms of volatile organic compounds produced. Since few strains were tested, it is not possible to draw conclusions in terms of synthetized VOCs patterns at species level. In the literature, isoamyl alcohol is described as accounting for a malty aroma; however, in this project it was more associated with other notes. Similarly, acetaldehyde aroma was not consistent with literature data. The LAB candidates selected were characterized by a high concentration of acetaldehyde in HFGW. Sensory evaluation performed by the panellists of HFGW and MW, showed that they are characterized by a fresh and sour profile. Hence, it might be concluded that acetaldehyde is the volatile compound related to a fresh and sour profile. In terms of non-conventional yeasts candidates: except the strains Gal.1 and Gal.2 that were recognized as sweet when fermenting HFGW and MW, all others showed a fresh and sour profile. However, in HFGW, the remaining yeast candidates presented high concentrations of isoamyl alcohol (Y4), acetaldehyde (Kaz. and S.1) and average results for all volatiles (S.2, Y1 and Y2). Therefore, it is hard to clearly conclude which compound(s) can be specifically associated with a fresh and sour profile, in wort fermented by the selected non-conventional yeast. In terms of wort medium, there were almost no aroma and taste differences between the candidates fermented in HFGW and MW, except for Lb.25, Lb.56 and Y2 that showed a sweeter (i.e. less fresh and sour) profile in MW, with noticeable banana flavour in the case of Y2. After comparison of the pasteurized and non-pasteurized maltose wort samples, no sensory differences were noticed. This means that pasteurization at 65 ºC for 40 min did not affect the organoleptic properties of the product. About the different fermentation times tested in the non-conventional yeast candidates, it is possible to conclude that in HFGW, on average, there was an increase of 1% in the ABV of the samples fermented for 7 d when compared with those fermented during 24 h, and the aroma was also different. Among the non- conventional yeast candidates, it was possible to conclude that the confidential yeast strains (Y1, Y2 and Y4) did not present the same metabolic pathway in HFGW and MW.

6. Future Work

In this project, selected food grade LAB from Carlsberg Research Laboratory collection were tested, but only a few non-conventional yeasts were included. Hence, it would be interesting to search for more yeast strains with unique characteristics (i.e. pleasant flavour profile and low production of ethanol). In order to achieve this goal, new screening assays can be conducted with relation to volatiles synthesis, souring activities in wort with different cereals composition, etc. Furthermore, more precise or comprehensive volatiles analysis can be performed, including gas chromatography (GC) coupled with other powerful detectors like mass spectrometry (MS), Fourier transform infrared spectroscopy (FTIR) or nuclear magnetic resonance (NMR). More insightful sensory evaluation using other olfactory descriptors and/or a quantification system can also be applied. Once obtained more detailed data, it should be possible to have a clearer idea of the correlation between the volatiles profile and the flavour analysis, and to check whether this correlation agrees with the data obtained in this project for HFGW and/or if there are other volatiles that justify the fresh and sour profile in HFGW and MW. About the results obtained in this project, the candidates that match the fresh and sour profile will be evaluated and potentially applied to develop new non-to-low alcoholic beverages.

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Annexes

Annex A: Tapping wort out from a barrel

Tapping procedure: 1. Clean the inlet of the keg with EtOH and cover the inlet with paper tissue to soak the EtOH. 2. Clean the keg beer tap with water and EtOH. 3. Clean the tube with water and EtOH. 4. Connect the tube to the TOP valve of the keg beer tap. 5. Close the valves on the keg beer tap before attaching it on the keg. When valves are closed they are perpendicular, when they are opened the valves are parallel. 6. Attach the keg beer tap to the keg by screwing. When firm, press down handle. 7. Connecting CO2. a. Adjust pressure of the CO2 tank by opening the valve on the top of the tank and the small valve on the tube to let out CO2. b. Close the small valve and adjust pressure to 0.5 bar. c. Connect the CO2 to the SIDE valve of the keg beer tap d. Open the small valve on the CO2 tube and the side valve of the keg beer tap and wait for the keg to be filled with CO2. 8. Tap the wort by opening the TOP valve of the keg beer tap.

Disconnecting procedure: 1. Close all valves (top and side valve on keg beer tap, small and large valve on CO2 tank). 2. Disconnect CO2 tank. 3. Release CO2 from CO2 tube by opening small valve. 4. Release CO2 from keg by opening the SIDE valve. 5. Remove the keg beer tap. 6. Repeat step 1-3 from the tapping procedure

Keg cleaning: 1. If keg is emptied, carry the keg to the pilot hall for cleaning.’ 2. Connect the keg to the cleaner by screwing – check if it is firm by lifting. 3. Wash with hot water for 2-3 minutes (black handle on the right side of the keg) 4. Sterilize by steaming for 4-5 minutes (grey handle behind the keg) 5. Unscrew keg and place bottom up to the left of the cleaning device

Annex B: Sterile filtration of wort

Preparation of the wort: 1. Centrifuge the glucose wort in 1 litre food grade jars (REED lab), at 5000 rpm for 40 min. 2. Balance jars to maximally 30 grams difference. 3. Transfer supernatant to green cap bottle of appropriate size. 4. Wash jars with water and ethanol and leave to dry – do not send for machine wash.

Principles of the filtration unit: The filtration unit consists of a feed tube, a sterile filter and two output tubes: retentate and permeate. Permeate is the clean liquid that has passed through the filter, retentate is the liquid that was passed around the filter for recirculation. The distribution of liquid to permeate/retentate is controlled by the valve on top of the permeate. This distribution combined with the speed of the pump determines the pressure on the filter membrane. This pressure should not exceed 1 bar. Pump speed is set between around 1050 rpm.

Preparation of the filtration unit: 1 Connect the feed tube (thickest) to the pump and leave the permeate and retentate open. 2 Flush system with 1 litre of water – washes away alkaline storage solution – pressure on the membrane will drop. 3 Clean with diluted phosphoric acid (1-0.5 ml concentrate acid/litre water). Flush system with 500ml by inserting the feed tube. Recirculate the remaining by placing the retentate and permeate tubes in the acid vial. Recirculate for approximately 5 minutes. 4 Flush with water as in step 2). 5 Clean with dilute peracetic acid (5ml of 40 % peracetic acid in 1 L) as described in step 3). 6 Flush with water as described in step 2).

Filtration 1 Transfer the feed tube to the wort bottle and clean the tubes of water by pumping wort through until the outflow is clearly wort coloured. 2 Start filtration by moving the retentate tube to the wort bottle and the permeate tube to the clean container. If this is a Biostat tank, connect the permeate tube directly to the sampling tube on the fermentation tank. 3 After filtering disconnect the tube and cover the tube on the fermenter with blue filter, blue side to the tube.

Cleaning filtration unit 1 Flush the filtering machine with water as described under preparation step 2). 2 Clean with dilute alkaline solution (1 ml 46% KOH in 1 litre H20) as described under preparation step 3. During the wash the output liquid can turn reddish – this is normal as impurities are washed off the membrane. 3 Flush the filtering machine with water as described under preparation step 2). 4 Fill all tubes with dilute KOH and seal by connecting them together. Leave the feed tube in water. 5 Place the filtration unit at 5 degrees in the cold room in the pilot plant.

Annex C: ABV measurement

Preparation of the testing:

1. Filter twice one beer can (Tuborg) with a paper filter to use as a control during the measurement of the samples. 2. Connect the pH electrode to the chamber that is between the machine and the samples’ carousel. 3. Disconnect the 2 white cables from each other and connect each of them to the respective entrance on the chamber. 4. Start Excel file and click on AP-Soft Print. 5. Click on Start Data Collection, change the file name and click on start. 6. Click on File à Page Setup à Sheet à Ok.

Calibration: 1. Place 1 cup of deionized water on position 1. 2. Press start on the alcolyzer and check if the result showed on excel for density is between 0,998153 and 0,998253. 3. Refill the cup of deionized water on position 1 and place one cup of buffer pH 4 on position 4, one cup of buffer pH 7 on position 5 and one cup of alcohol 96% on position 6. 4. On the alcolyzer screen, press Favourites à Daglig SOP à Ok.

Measurement of the samples: 1. Fill one of the tubes close to the alcolyzer with filtrated beer and place it on position 1 of the carousel (control). 2. Fill tubes with the samples to analyse (at least 50 mL each(!); there is a slightly trace in the majority of the tubes that represent the 50 mL); its optional cover the tubes with the lids. 3. Place all the samples on the carousel. 4. Click Start on the screen of the alcolyzer. 5. Usually each sample takes 5/6 minutes to be analysed; once all the samples placed in the carousel are analysed, the alcolyzer will stop and it is possible analyse more samples without calibrate again the equipment.

After all the measurements 1. Remove all the samples’ tubes from the carousel. 2. Disconnect the pH electrode from the chamber and place it in the tube on the top of the alcolyzer; 3. Disconnect the white cables from the chamber and connect them to which other; 4. Place two cups of 3% Mugasol on position 1 and 2, one cup of 2% RBS 50 on position 3, one cup of 1% RBS 50 on position 4 and then between four and six cups of deionized water starting on position 5.

III

Annex D: Sensory analysis Annex D.1: Aroma evaluation

Table D. 1- Aroma evaluation table

Stain number Malt Honey Vinegar Brown sugar Roasted barley Fruity Floral Comments

NOTES: ______

DATE:

Annex D.2: Taste evaluation

Table D. 2- Taste evaluation table

Strain number Sweet Salty Sour Bitter Malt Honey Vinegar Roasted barley Burnt Pungent Comments

NOTES: ______

DATE:

Annex E: Lactic acid bacteria and non-conventional yeast data Annex E.1: Volatile organic compounds

Table E.1. 1- Volatile organic compounds data for LAB (part I)

Strains Volatile Organic Compunds (VOC's) Genus Species Code Number Fermentation Acet_Ald (mg/L) Et_Ac (mg/L) Isobut_Ac (mg/L) Prop_ol (mg/L) Isob_ol (mg/L) Isoam_Ac (mg/L) Isoam_ol (mg/L) Et_Cap (mg/L) Et_Capr (mg/L) 2-Phe_Ac (mg/L) C6 (mg/L) 2-Phe_ol (mg/L) C8 (mg/L) 4-VG (mg/L) C10 (mg/L) Sum_Acids Sum_Alc Sum_Esters Lim (mg/L) Linal (ug/L) Lactococcus lactis subsp. cremoris Lc. 1 9 Ho 3.53 0.08 0.00 0.00 0.00 0.00 0.25 0.00 0.00 0.00 0.02 0.12 0.02 5.30 0.00 0.04 0.12 0.08 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 2 10 Ho 2.04 0.00 0.00 0.00 0.00 0.00 0.77 0.00 0.00 0.00 0.04 0.20 0.00 6.67 0.00 0.04 0.20 0.00 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 2 I 10_5P Ho 1.82 0.05 0.00 0.00 0.00 0.00 0.62 0.00 0.00 0.00 0.03 0.14 0.00 4.40 0.00 0.03 0.14 0.05 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 3 14 Ho 1.56 0.06 0.00 0.00 0.00 0.00 0.18 0.00 0.00 0.00 0.03 0.16 0.05 2.40 0.01 0.09 0.16 0.06 0.00 0.00 Lactococcus lactis subsp. Lactis Lc. 4 16 Ho 2.18 0.12 0.00 0.00 0.00 0.00 0.31 0.00 0.00 0.00 0.04 0.17 0.04 3.57 0.01 0.09 0.17 0.12 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 5 17 Ho 2.32 0.09 0.00 0.00 0.00 0.00 0.27 0.00 0.00 0.00 0.03 0.15 0.03 2.70 0.01 0.07 0.15 0.09 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 6 18 Ho 1.86 0.09 0.00 0.00 0.00 0.00 0.25 0.00 0.00 0.00 0.02 0.08 0.02 1.51 0.01 0.05 0.08 0.09 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 7 19 Ho 2.04 0.08 0.00 0.00 0.00 0.00 0.29 0.00 0.00 0.00 0.02 0.12 0.03 3.22 0.01 0.06 0.12 0.08 0.00 0.00 Lactococcus lactis subsp. Lactis Lc. 8 20 Ho 1.68 0.07 0.00 0.00 0.00 0.00 0.23 0.00 0.00 0.00 0.03 0.14 0.03 3.14 0.01 0.07 0.14 0.07 0.00 0.00 Lactococcus lactis subsp. hordniae Lc. 9 96 Ho 2.86 0.17 0.00 0.00 0.40 0.01 5.11 0.00 0.00 0.00 0.03 0.49 0.01 0.18 0.00 0.04 0.89 0.18 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 10 97 Ho 1.17 0.16 0.00 0.00 0.00 0.00 0.46 0.00 0.00 0.00 0.03 0.16 0.02 5.15 0.00 0.05 0.16 0.16 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 11 98 Ho 2.16 0.11 0.00 0.00 0.00 0.00 0.25 0.00 0.00 0.00 0.04 0.12 0.03 2.66 0.00 0.07 0.12 0.11 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 12 99 Ho 2.99 0.13 0.00 0.00 0.00 0.00 0.32 0.00 0.00 0.00 0.04 0.14 0.03 3.46 0.00 0.07 0.14 0.13 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 13 100 Ho 3.30 0.18 0.00 0.00 0.00 0.05 0.50 0.00 0.00 0.00 0.04 0.13 0.10 2.66 0.00 0.14 0.13 0.23 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 14 101 Ho 1.79 0.15 0.00 0.00 0.00 0.00 0.40 0.00 0.00 0.00 0.03 0.14 0.03 3.55 0.00 0.06 0.14 0.15 0.00 0.00 Lactococcus Lactococcus lactis subsp. lactis Lc. 15 102 Ho 1.58 0.15 0.00 0.00 0.00 0.00 0.56 0.00 0.00 0.00 0.04 0.15 0.03 4.76 0.00 0.07 0.15 0.15 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 16 103 Ho 3.33 0.16 0.00 0.00 0.00 0.00 0.41 0.00 0.00 0.00 0.04 0.13 0.03 3.13 0.00 0.07 0.13 0.16 0.00 0.00 Lactococcus raffinolactis Lc. 17 104 Ho 2.29 0.15 0.00 0.00 0.00 0.00 0.38 0.00 0.00 0.00 0.03 0.14 0.02 0.14 0.00 0.05 0.14 0.15 0.00 0.00 Lactococcus plantarum Lc. 18 105 Ho 1.68 0.14 0.00 0.00 0.00 0.00 0.38 0.00 0.00 0.00 0.03 0.15 0.01 0.14 0.00 0.04 0.15 0.14 0.00 0.00 Lactococcus lactis subsp. tructae Lc. 19 106 Ho 3.17 0.10 0.00 0.00 0.00 0.00 0.63 0.00 0.00 0.00 0.04 0.18 0.02 4.94 0.00 0.06 0.18 0.10 0.00 0.00 Lactococcus fujiensis Lc. 20 107 Ho 0.26 0.12 0.00 0.00 0.00 0.00 0.37 0.00 0.00 0.00 0.04 0.16 0.03 2.67 0.00 0.07 0.16 0.12 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 21 118 Ho 2.14 0.14 0.00 0.00 0.00 0.00 0.31 0.00 0.00 0.00 0.04 0.14 0.03 4.49 0.01 0.08 0.14 0.14 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 22 119 Ho 2.51 0.17 0.00 0.00 0.00 0.00 0.81 0.00 0.00 0.00 0.04 0.19 0.02 6.13 0.00 0.06 0.19 0.17 0.00 0.00 Lactococcus lactis Lc. 23 122 Ho 1.60 0.08 0.00 0.00 0.00 0.00 0.22 0.00 0.00 0.00 0.05 0.13 0.00 2.06 0.00 0.05 0.13 0.08 0.00 0.00 Lactococcus lactis Lc. 23 I 122_5P Ho 1.73 0.06 0.00 0.00 0.00 0.00 0.16 0.00 0.00 0.00 0.03 0.09 0.00 1.03 0.00 0.03 0.09 0.06 0.00 0.00 Lactococcus lactis Lc. 24 154 Ho 7.15 0.40 0.00 0.00 0.23 0.01 0.65 0.00 0.00 0.00 0.06 0.21 0.04 0.45 0.01 0.11 0.44 0.41 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 25 156 Ho 16.41 0.55 0.00 0.00 0.35 0.01 0.85 0.00 0.00 0.00 0.07 0.25 0.04 0.46 0.02 0.13 0.60 0.56 0.00 0.00 Lactococcus lactis Lc. 26 160 Ho 7.83 0.46 0.00 0.00 0.27 0.01 0.72 0.00 0.00 0.00 0.07 0.21 0.05 0.46 0.02 0.14 0.48 0.47 0.00 0.00 Lactococcus lactis Lc. 27 170 Ho 2.71 0.35 0.00 0.00 0.20 0.00 0.33 0.00 0.00 0.00 0.04 0.14 0.02 0.37 0.00 0.06 0.34 0.35 0.00 0.00 Lactococcus lactis subsp. lactis Lc. 28 174 Ho 3.93 0.49 0.00 0.00 0.19 0.00 0.75 0.00 0.00 0.00 0.06 0.18 0.03 3.34 0.00 0.09 0.37 0.49 0.00 0.00 Lactococcus lactis Lc. 29 178 Ho 3.14 0.47 0.00 0.00 0.19 0.00 0.57 0.00 0.00 0.00 0.05 0.17 0.02 3.11 0.00 0.07 0.36 0.47 0.00 0.00 Lactobacillus delbrueckii subsp. bulgaricus Lb. 1 1 Ho 3.01 0.08 0.00 0.00 0.00 0.00 0.20 0.00 0.00 0.00 0.03 0.12 0.02 0.26 0.00 0.05 0.12 0.08 0.00 0.00 Lactobacillus delbrueckii subsp. delbrueckii Lb. 2 2 Ho 0.82 0.07 0.00 0.00 0.00 0.00 0.17 0.00 0.00 0.00 0.03 0.12 0.02 0.12 0.01 0.06 0.12 0.07 0.00 0.00 Lactobacillus delbrueckii subsp. lactis Lb. 3 3 Ho 0.08 0.07 0.00 0.00 0.00 0.00 0.17 0.00 0.00 0.00 0.03 0.11 0.01 0.38 0.00 0.04 0.11 0.07 0.00 0.00 Lactobacillus helveticus Lb. 4 4 Ho 2.36 0.07 0.00 0.00 0.00 0.00 0.15 0.00 0.00 0.00 0.04 0.11 0.03 0.14 0.00 0.07 0.11 0.07 0.00 0.00 Lactobacillus dextrinicus Lb. 5 6 Ho 1.15 0.08 0.00 0.00 0.00 0.00 0.28 0.00 0.00 0.00 0.03 0.16 0.01 0.11 0.00 0.04 0.16 0.08 0.00 0.00 Lactobacillus mali Lb. 6 7 Ho 0.07 0.08 0.00 0.00 0.00 0.00 0.28 0.00 0.00 0.00 0.04 0.19 0.02 0.32 0.01 0.07 0.19 0.08 0.00 0.00 Lactobacillus acidophilus Lb. 7 8 Ho 1.15 0.06 0.00 0.00 0.00 0.00 0.17 0.00 0.00 0.00 0.02 0.11 0.01 0.26 0.00 0.03 0.11 0.06 0.00 0.00 Lactobacillus nagelii Lb. 8 21 Ho 0.05 0.06 0.00 0.00 0.00 0.00 0.27 0.00 0.00 0.00 0.04 0.22 0.02 0.32 0.00 0.06 0.22 0.06 0.00 0.00 Lactobacillus salivarius Lb. 9 22 Ho 2.55 0.08 0.00 0.00 0.00 0.00 0.18 0.00 0.00 0.00 0.03 0.16 0.02 0.31 0.00 0.05 0.16 0.08 0.00 0.00 Lactobacillus fermentum Lb. 10 23 He 0.16 0.16 0.00 0.00 0.00 0.00 0.31 0.00 0.00 0.00 0.04 0.18 0.03 0.49 0.00 0.07 0.18 0.16 0.00 0.00 Lactobacillus sakei subsp. sakei Lb. 11 25 Fa He 1.07 0.10 0.00 0.00 0.00 0.00 0.34 0.00 0.00 0.00 0.06 0.20 0.12 1.47 0.40 0.58 0.20 0.10 0.00 0.00 Lactobacillus sakei subsp. carnosus Lb. 12 26 Fa He 0.76 0.07 0.00 0.00 0.00 0.00 0.28 0.00 0.00 0.00 0.03 0.18 0.02 4.01 0.00 0.05 0.18 0.07 0.00 0.00 Lactobacillus sanfranciscensis Lb. 13 28 He 0.89 0.10 0.00 0.00 0.00 0.00 0.26 0.00 0.00 0.00 0.07 0.18 0.14 1.36 0.03 0.24 0.18 0.10 0.00 0.00 Lactobacillus amylolyticus Lb. 14 30 Ho 1.01 0.08 0.00 0.00 0.00 0.00 0.18 0.00 0.00 0.01 0.03 0.13 0.03 0.29 0.01 0.07 0.13 0.08 0.00 0.00 Lactobacillus amylovorus Lb. 15 31 Ho 1.05 0.09 0.00 0.00 0.00 0.00 0.24 0.00 0.00 0.00 0.02 0.15 0.02 0.33 0.00 0.04 0.15 0.09 0.00 0.00 Lactobacillus delbrueckii subsp. jakobsenii Lb. 16 32 Ho 0.63 0.09 0.00 0.00 0.00 0.00 0.38 0.00 0.00 0.00 0.04 0.18 0.07 0.11 0.00 0.11 0.18 0.09 0.00 0.00 Lactobacillus farraginis Lb. 17 34 Fa He 0.11 0.16 0.00 0.00 0.00 0.00 0.47 0.00 0.00 0.00 0.05 0.20 0.08 0.29 0.02 0.15 0.20 0.16 0.00 0.00 Lactobacillus uvarum Lb. 18 38 Ho 0.10 0.08 0.00 0.00 0.00 0.00 0.17 0.00 0.00 0.00 0.04 0.17 0.02 0.28 0.01 0.07 0.17 0.08 0.00 1.11 Lactobacillus Lactobacillus silagei Lb. 19 39 He 0.08 0.19 0.00 6.01 0.00 0.00 0.24 0.00 0.00 0.00 0.03 0.16 0.02 0.15 0.01 0.06 6.17 0.19 0.00 0.00 Lactobacillus oeni Lb. 20 40 Ho 0.00 0.09 0.00 0.00 0.00 0.00 0.20 0.00 0.00 0.00 0.04 0.16 0.02 0.29 0.01 0.07 0.16 0.09 0.00 0.00 Lactobacillus zeae Lb. 21 41 Fa He 0.06 0.08 0.00 0.00 0.00 0.00 0.25 0.00 0.00 0.00 0.03 0.15 0.01 0.25 0.00 0.04 0.15 0.08 0.00 0.00 Lactobacillus paracasei subsp. paracasei Lb. 22 42 Fa He 0.14 0.08 0.00 0.00 0.00 0.00 0.20 0.00 0.00 0.00 0.03 0.12 0.01 0.24 0.00 0.04 0.12 0.08 0.00 0.00 Lactobacillus parakefiri Lb. 23 43 He 0.00 0.24 0.00 0.00 0.00 0.00 0.22 0.00 0.00 0.00 0.01 0.13 0.01 0.16 0.00 0.02 0.13 0.24 0.00 0.00 Lactobacillus gasseri Lb. 24 45 Ho 0.14 0.08 0.00 0.00 0.00 0.00 0.20 0.00 0.00 0.00 0.03 0.12 0.01 0.24 0.00 0.04 0.12 0.08 0.00 0.00 Lactobacillus pentosus Lb. 25 46 Fa He 4.29 0.09 0.00 0.00 0.08 0.00 0.38 0.00 0.00 0.00 0.04 0.20 0.03 0.35 0.01 0.08 0.28 0.09 0.00 0.00 Lactobacillus alimentarius Lb. 26 48 Fa He 0.65 0.00 0.00 0.00 0.00 0.00 0.19 0.00 0.00 0.00 0.02 0.17 0.01 0.37 0.01 0.04 0.17 0.00 0.00 0.00 Lactobacillus farciminis Lb. 27 49 Ho 5.06 0.05 0.00 0.00 0.00 0.00 0.33 0.04 0.00 0.00 0.03 0.16 0.01 0.31 0.00 0.04 0.16 0.09 0.00 0.00 Lactobacillus gallinarum Lb. 28 50 Ho 0.47 0.07 0.00 0.00 0.00 0.00 0.13 0.00 0.00 0.00 0.03 0.14 0.01 0.20 0.00 0.04 0.14 0.07 0.00 0.00 Lactobacillus hilgardii Lb. 29 51 He 0.08 0.09 0.00 0.00 0.00 0.00 0.54 0.00 0.00 0.00 0.03 0.16 0.06 0.26 0.01 0.10 0.16 0.09 0.00 0.00 Lactobacillus johnsonii Lb.30 52 Ho 0.71 0.08 0.00 0.00 0.00 0.00 0.13 0.00 0.00 0.00 0.02 0.11 0.01 0.20 0.00 0.03 0.11 0.08 0.00 0.00 Lactobacillus pasteurii Lb. 31 55 Ho 0.24 0.07 0.00 0.00 0.00 0.00 0.24 0.04 0.00 0.00 0.02 0.20 0.01 0.12 0.00 0.03 0.20 0.11 0.00 0.00 Lactobacillus plantarum subsp. plantarum Lb. 32 56 Ho 11.09 0.06 0.00 0.00 0.00 0.00 0.33 0.00 0.00 0.00 0.03 0.19 0.03 0.21 0.02 0.08 0.19 0.06 0.00 0.00 Lactobacillus casei Lb. 33 57 Fa He 0.12 0.07 0.00 0.00 0.00 0.05 0.18 0.00 0.00 0.00 0.03 0.13 0.01 0.22 0.00 0.04 0.13 0.12 0.00 0.00 Lactobacillus curvatus Lb. 34 58 Fa He 0.64 0.08 0.00 0.00 0.00 0.00 0.26 0.00 0.00 0.00 0.02 0.16 0.01 0.31 0.00 0.03 0.16 0.08 0.00 0.00 Lactobacillus brevis Lb. 35 59 He 0.11 0.10 0.00 0.00 0.00 0.00 0.52 0.00 0.00 0.00 0.05 0.15 0.09 0.56 0.01 0.15 0.15 0.10 0.00 0.00 Lactobacillus buchneri Lb. 36 61 He 0.09 0.00 0.00 0.00 0.00 0.00 0.21 0.00 0.00 0.00 0.03 0.11 0.05 0.24 0.01 0.09 0.11 0.00 0.00 0.00

Table E.1. 2- Volatile organic compounds data for LAB (part II)

Strains Volatile Organic Compunds (VOC's) Genus Species Code Number Fermentation Acet_Ald (mg/L) Et_Ac (mg/L) Isobut_Ac (mg/L) Prop_ol (mg/L) Isob_ol (mg/L) Isoam_Ac (mg/L) Isoam_ol (mg/L) Et_Cap (mg/L) Et_Capr (mg/L) 2-Phe_Ac (mg/L) C6 (mg/L) 2-Phe_ol (mg/L) C8 (mg/L) 4-VG (mg/L) C10 (mg/L) Sum_Acids Sum_Alc Sum_Esters Lim (mg/L) Linal (ug/L) Lactobacillus coryniformis subsp. coryniformis Lb. 37 62 Fa He 0.20 0.00 0.00 0.00 0.00 0.00 0.20 0.00 0.00 0.00 0.03 0.16 0.01 0.26 0.00 0.04 0.16 0.00 0.00 0.00 Lactobacillus malefermentans Lb. 38 63 He 0.06 0.09 0.00 0.00 0.00 0.00 0.34 0.00 0.00 0.00 0.03 0.15 0.04 0.61 0.00 0.07 0.15 0.09 0.00 0.00 Lactobacillus delbrueckii subsp. sunkii Lb. 39 108 Ho 1.92 0.10 0.00 0.00 0.00 0.00 0.27 0.00 0.00 0.00 0.02 0.12 0.02 0.10 0.00 0.04 0.12 0.10 0.00 0.00 Lactobacillus pentosus Lb. 40 109 Fa He 6.18 0.11 0.00 0.00 0.00 0.00 0.47 0.00 0.00 0.00 0.04 0.17 0.03 0.14 0.01 0.08 0.17 0.11 0.00 0.00 Lactobacillus pentosus Lb. 41 110 Fa He 4.96 0.11 0.00 0.00 0.00 0.00 0.50 0.00 0.00 0.00 0.05 0.20 0.03 0.17 0.01 0.09 0.20 0.11 0.00 0.00 Lactobacillus paraplantarum Lb. 42 111 Fa He 2.53 0.11 0.00 0.00 0.00 0.00 0.46 0.00 0.00 0.00 0.04 0.20 0.03 0.20 0.01 0.08 0.20 0.11 0.00 0.00 Lactobacillus leichmannii Lb. 43 120 He 1.74 0.06 0.00 0.00 0.00 0.03 0.12 0.00 0.00 0.00 0.01 0.10 0.01 0.19 0.00 0.02 0.10 0.09 0.00 0.00 Lactobacillus paracasei Lb. 44 121 Fa He 0.06 0.17 0.00 0.00 0.00 0.00 0.31 0.00 0.00 0.00 0.04 0.14 0.01 0.19 0.00 0.05 0.14 0.17 0.00 0.00 Lactobacillus acidophilus Lb. 45 123 He 4.43 0.08 0.00 0.00 0.00 0.00 0.18 0.00 0.00 0.01 0.02 0.19 0.01 0.27 0.01 0.04 0.19 0.08 0.00 0.00 Lactobacillus casei Lb. 46 124 He 0.07 0.21 0.00 0.00 0.00 0.00 0.35 0.00 0.00 0.00 0.04 0.16 0.02 0.20 0.00 0.06 0.16 0.21 0.00 0.00 Lactobacillus fermentum Lb. 47 125 He 0.16 0.14 0.00 0.00 0.00 0.00 0.25 0.00 0.00 0.00 0.04 0.15 0.03 0.26 0.01 0.08 0.15 0.14 0.00 0.00 Lactobacillus gasseri Lb. 48 126 Ho 0.86 0.07 0.00 0.00 0.00 0.00 0.18 0.00 0.00 0.00 0.01 0.15 0.01 0.20 0.00 0.02 0.15 0.07 0.00 0.00 Lactobacillus plantarum Lb. 49 127 Fa He 12.56 0.19 0.00 0.00 0.00 0.00 0.50 0.00 0.00 0.00 0.06 0.19 0.04 0.17 0.02 0.12 0.19 0.19 0.00 0.00 Lactobacillus rhamnosus Lb. 50 128 Fa He 0.06 0.09 0.00 0.00 0.00 0.00 0.37 0.00 0.00 0.00 0.03 0.20 0.02 0.27 0.00 0.05 0.20 0.09 0.00 2.47 Lactobacillus rhamnosus Lb. 51 129 Fa He 0.18 0.07 0.00 0.00 0.00 0.01 0.29 0.00 0.00 0.00 0.04 0.18 0.02 0.29 0.00 0.06 0.18 0.08 0.00 1.50 Lactobacillus rhamnosus Lb. 52 130 Fa He 0.07 0.07 0.00 0.00 0.00 0.00 0.33 0.00 0.00 0.00 0.05 0.19 0.03 0.40 0.00 0.08 0.19 0.07 0.00 1.93 Lactobacillus rhamnosus Lb. 52 I 130_5P Fa He 0.05 0.07 0.00 0.00 0.00 0.04 0.25 0.00 0.00 0.00 0.04 0.14 0.03 0.30 0.00 0.07 0.14 0.11 0.00 1.92 Lactobacillus rhamnosus Lb. 53 131 He 0.05 0.16 0.00 0.00 0.00 0.00 0.24 0.00 0.00 0.00 0.01 0.21 0.01 0.17 0.00 0.02 0.21 0.16 0.00 1.44 Lactobacillus acidophilus Lb. 54 132 Ho 0.68 0.17 0.00 0.00 0.00 0.00 0.35 0.00 0.00 0.00 0.03 0.14 0.01 0.14 0.00 0.04 0.14 0.17 0.00 0.00 Lactobacillus casei ssp. casei Lb. 55 133 He 0.17 0.19 0.00 0.00 0.00 0.00 0.30 0.00 0.00 0.00 0.01 0.19 0.01 0.19 0.00 0.02 0.19 0.19 0.00 1.49 Lactobacillus salivarius Lb. 56 134 Ho 2.62 0.12 0.00 0.00 0.00 0.00 0.25 0.00 0.00 0.00 0.04 0.15 0.02 0.13 0.00 0.06 0.15 0.12 0.00 0.00 Lactobacillus acidophilus Lb. 57 135 Ho 1.16 0.13 0.00 0.00 0.00 0.00 0.12 0.00 0.00 0.00 0.01 0.14 0.01 0.17 0.00 0.02 0.14 0.13 0.00 0.00 Lactobacillus rhamnosus Lb. 58 136 He 0.08 0.00 0.00 0.00 0.00 0.00 0.24 0.00 0.00 0.00 0.02 0.19 0.01 0.20 0.00 0.03 0.19 0.00 0.00 1.55 Lactobacillus rhamnosus Lb. 59 137 He 0.36 0.13 0.00 0.00 0.00 0.00 0.93 0.00 0.00 0.00 0.02 0.18 0.01 0.19 0.00 0.03 0.18 0.13 0.00 1.98 Lactobacillus rhamnosus Lb. 60 138 He 0.13 0.09 0.00 0.00 0.00 0.00 0.23 0.04 0.00 0.00 0.02 0.15 0.01 0.16 0.00 0.03 0.15 0.13 0.00 1.74 Lactobacillus salivarius Lb. 61 139 Ho 2.73 0.11 0.00 0.00 0.00 0.00 0.27 0.00 0.01 0.00 0.04 0.14 0.01 0.11 0.02 0.07 0.14 0.12 0.00 0.00 Lactobacillus Lactobacillus rhamnosus Lb.62 140 He 0.09 0.12 0.00 0.00 0.00 0.00 0.32 0.00 0.00 0.00 0.02 0.16 0.01 0.17 0.00 0.03 0.16 0.12 0.00 1.93 Lactobacillus rhamnosus Lb. 63 141 He 0.06 0.10 0.00 0.00 0.00 0.01 0.26 0.00 0.00 0.00 0.02 0.16 0.01 0.18 0.00 0.03 0.16 0.11 0.00 1.72 Lactobacillus casei ssp. casei Lb. 64 142 He 0.08 0.12 0.00 0.00 0.00 0.00 0.31 0.00 0.00 0.00 0.02 0.20 0.01 0.20 0.00 0.03 0.20 0.12 0.00 1.74 Lactobacillus zeae Lb. 65 143 Ho 0.07 0.15 0.00 0.00 0.00 0.00 0.26 0.00 0.00 0.00 0.02 0.18 0.01 0.21 0.00 0.03 0.18 0.15 0.00 1.34 Lactobacillus casei Lb. 66 144 Ho 0.22 0.36 0.00 0.00 0.17 0.00 0.54 0.00 0.00 0.00 0.06 0.21 0.02 0.46 0.01 0.09 0.38 0.36 0.00 0.00 Lactobacillus plantarum Lb. 67 145 He 14.34 0.38 0.00 0.00 0.23 0.00 0.68 0.00 0.00 0.00 0.06 0.22 0.04 0.45 0.02 0.12 0.45 0.38 0.00 0.00 Lactobacillus rhamnosus Lb. 68 147 He 14.86 0.13 0.00 0.00 0.00 0.00 0.38 0.00 0.00 0.00 0.04 0.19 0.02 0.18 0.02 0.08 0.19 0.13 0.00 1.20 Lactobacillus plantarum Lb. 69 148 He 0.46 0.43 0.00 0.00 0.17 0.00 0.55 0.00 0.00 0.00 0.05 0.20 0.02 0.45 0.01 0.08 0.37 0.43 0.00 0.00 Lactobacillus plantarum Lb. 70 149 He 15.77 0.40 0.00 0.00 0.18 0.00 0.65 0.00 0.00 0.00 0.06 0.23 0.04 0.39 0.02 0.12 0.41 0.40 0.00 0.00 Lactobacillus harbinensis Lb. 71 150 n.d. 14.34 0.47 0.00 0.00 0.20 0.00 0.23 0.00 0.00 0.00 0.06 0.24 0.04 0.49 0.02 0.12 0.44 0.47 0.00 0.00 Lactobacillus paracasei Lb. 72 155 Ho 0.43 0.50 0.00 0.74 0.28 0.00 0.70 0.00 0.00 0.00 0.07 0.22 0.09 0.73 0.02 0.18 1.24 0.50 0.00 0.00 Lactobacillus rhamnosus Lb. 73 158 He 9.83 0.10 0.00 0.00 0.00 0.00 0.35 0.03 0.00 0.01 0.02 0.22 0.02 0.41 0.00 0.04 0.22 0.13 0.00 1.74 Lactobacillus casei Lb. 74 159 Ho 0.57 0.50 0.00 0.00 0.22 0.00 0.78 0.00 0.00 0.00 0.05 0.22 0.02 0.47 0.00 0.07 0.44 0.50 0.00 0.00 Lactobacillus paracasei Lb. 75 161 Ho 0.49 0.43 0.00 0.00 0.19 0.00 0.63 0.00 0.00 0.00 0.05 0.19 0.02 0.42 0.00 0.07 0.38 0.43 0.00 0.00 Lactobacillus plantarum subsp. pantarum Lb. 76 162 He 11.43 0.28 0.00 0.00 0.22 0.00 0.61 0.00 0.00 0.00 0.06 0.20 0.04 0.39 0.02 0.12 0.42 0.28 0.00 0.00 Lactobacillus plantarum Lb. 77 163 He 16.66 0.46 0.00 0.00 0.26 0.01 0.82 0.00 0.00 0.00 0.08 0.26 0.00 0.43 0.02 0.15 0.52 0.47 0.00 0.00 Lactobacillus rhamnosus Lb. 78 164 He 0.05 0.17 0.00 0.00 0.00 0.00 0.49 0.00 0.00 0.00 0.04 0.18 0.02 0.13 0.00 0.06 0.18 0.17 0.00 1.55 Lactobacillus plantarum Lb. 79 165 He 15.43 0.47 0.00 0.00 0.28 0.01 0.69 0.00 0.00 0.00 0.08 0.26 0.05 0.44 0.02 0.15 0.54 0.48 0.00 1.10 Lactobacillus plantarum subsp. pantarum Lb. 80 166 He 13.55 0.47 0.00 0.00 0.34 0.00 0.83 0.00 0.00 0.00 0.07 0.23 0.04 0.44 0.02 0.13 0.57 0.47 0.00 0.00 Lactobacillus plantarum Lb. 81 167 He 6.63 0.28 0.00 0.00 0.10 0.00 0.33 0.00 0.00 0.00 0.06 0.16 0.04 0.41 0.01 0.11 0.26 0.28 0.00 0.00 Lactobacillus rossiae Lb. 82 168 n.d. 0.43 0.47 0.00 0.00 0.05 0.24 0.75 0.00 0.00 0.00 0.04 0.20 0.02 0.31 0.00 0.06 0.25 0.71 0.00 0.00 Lactobacillus paracasei Lb. 83 171 Ho 0.39 0.30 0.00 0.00 0.00 0.00 0.71 0.00 0.00 0.00 0.06 0.21 0.03 0.48 0.00 0.09 0.21 0.30 0.00 0.00 Lactobacillus plantarum Lb. 84 172 He 16.62 0.29 0.00 0.00 0.18 0.02 0.98 0.00 0.00 0.00 0.06 0.25 0.04 0.35 0.01 0.11 0.43 0.31 0.00 1.04 Lactobacillus rhamnosus Lb. 85 173 He 0.09 0.18 0.00 0.00 0.00 0.00 0.49 0.00 0.00 0.00 0.02 0.21 0.01 0.20 0.00 0.03 0.21 0.18 0.00 1.86 Lactobacillus zeae Lb. 86 176 Ho 0.22 0.30 0.00 0.00 0.23 0.00 1.03 0.00 0.00 0.00 0.06 0.27 0.02 0.46 0.00 0.08 0.50 0.30 0.00 0.00 Lactobacillus plantarum Lb. 87 177 He 8.93 0.29 0.00 0.00 0.21 0.00 0.81 0.00 0.00 0.00 0.07 0.23 0.05 0.47 0.02 0.14 0.44 0.29 0.00 0.02 Leuconostoc mesenteroides subsp. cremoris Leuc. 1 35 He 2.14 0.08 0.00 0.00 0.00 0.00 0.16 0.00 0.00 0.00 0.03 0.11 0.02 0.22 0.01 0.06 0.11 0.08 0.00 0.00 Leuconostoc citreum Leuc. 2 36 He 0.17 0.10 0.00 0.00 0.00 0.00 0.36 0.00 0.00 0.00 0.03 0.14 0.00 0.35 0.00 0.03 0.14 0.10 0.00 0.00 Leuconostoc citreum Leuc. 2 I 36_5P He 0.11 0.07 0.00 0.00 0.00 0.00 0.22 0.00 0.00 0.00 0.02 0.10 0.00 0.27 0.00 0.02 0.10 0.07 0.00 0.00 Leuconostoc fallax Leuc. 3 37 He 0.00 0.10 0.00 0.00 0.00 0.00 0.32 0.00 0.00 0.00 0.03 0.14 0.01 0.26 0.01 0.05 0.14 0.10 0.00 1.12 Leuconostoc citreum Leuc. 4 77 He 0.09 0.17 0.00 0.00 0.00 0.00 0.24 0.00 0.00 0.00 0.02 0.12 0.01 0.13 0.00 0.03 0.12 0.17 0.00 0.00 Leuconostoc citreum Leuc. 5 78 He 0.13 0.09 0.00 0.00 0.00 0.00 0.35 0.00 0.00 0.00 0.04 0.16 0.00 0.42 0.00 0.04 0.16 0.09 0.00 0.00 Leuconostoc citreum Leuc. 5 I 78_5P He 0.09 0.06 0.00 0.00 0.00 0.00 0.15 0.00 0.00 0.00 0.03 0.11 0.00 0.29 0.00 0.03 0.11 0.06 0.00 0.00 Leuconostoc mesenteroides subsp. dextranicum Leuc. 6 79 He 0.10 0.19 0.00 0.00 0.00 0.00 0.24 0.00 0.00 0.00 0.02 0.12 0.01 0.11 0.00 0.03 0.12 0.19 0.00 0.00 Leuconostoc Leuconostoc lactis Leuc. 7 80 He 0.18 0.19 0.00 0.00 0.00 0.00 0.25 0.00 0.00 0.00 0.00 0.10 0.00 0.07 0.00 0.00 0.10 0.19 0.00 0.00 Leuconostoc palmae Leuc. 8 81 He 0.10 0.20 0.00 0.00 0.00 0.00 0.67 0.00 0.00 0.00 0.04 0.17 0.02 0.11 0.01 0.07 0.17 0.20 0.00 0.00 Leuconostoc miyukkimchii Leuc. 9 82 He 0.07 0.17 0.00 0.00 0.00 0.00 0.57 0.00 0.00 0.00 0.04 0.23 0.02 0.11 0.01 0.07 0.23 0.17 0.00 0.00 Leuconostoc holzapfelii Leuc. 10 83 He 0.03 0.19 0.00 0.00 0.00 0.00 0.57 0.00 0.00 0.00 0.04 0.18 0.02 0.11 0.00 0.06 0.18 0.19 0.00 0.00 Leuconostoc carnosum Leuc. 11 86 He 0.36 0.19 0.00 0.00 0.00 0.00 0.57 0.00 0.00 0.00 0.04 0.19 0.02 0.14 0.10 0.16 0.19 0.19 0.00 0.00 Fructobacillus durionis Fruc. 12 87 He 0.15 0.15 0.00 0.00 0.00 0.00 0.41 0.00 0.00 0.00 0.03 0.17 0.02 0.12 0.00 0.05 0.17 0.15 0.00 0.00 Fructobacillus ficulneus Fruc. 13 88 He 0.17 0.13 0.00 0.00 0.00 0.00 0.36 0.00 0.00 0.00 0.02 0.13 0.02 0.09 0.00 0.04 0.13 0.13 0.00 0.00 Fructobacillus fructosus Fruc. 14 89 He 0.12 0.08 0.00 0.00 0.00 0.00 0.21 0.00 0.00 0.00 0.02 0.13 0.02 0.10 0.00 0.04 0.13 0.08 0.00 0.00 Fructobacillus pseudoficulneus Fruc. 15 90 He 0.13 0.16 0.00 0.00 0.00 0.00 0.46 0.00 0.00 0.00 0.02 0.10 0.01 0.09 0.00 0.03 0.10 0.16 0.00 0.00 Pediococcus acidilactici Pe. 1 11 Ho 0.11 0.07 0.00 0.00 0.00 0.00 0.34 0.00 0.00 0.00 0.04 0.19 0.00 0.48 0.00 0.04 0.19 0.07 0.00 0.00 Pediococcus acidilactici Pe. 1 I 11_5P Ho 0.09 0.05 0.00 0.00 0.00 0.00 0.24 0.00 0.00 0.00 0.03 0.14 0.00 0.37 0.00 0.03 0.14 0.05 0.00 0.00 Pediococcus Pediococcus pentosaceus Pe. 2 12 Ho 0.57 0.07 0.00 0.00 0.00 0.00 0.29 0.00 0.00 0.00 0.04 0.19 0.00 0.56 0.00 0.04 0.19 0.07 0.00 0.00 Pediococcus pentosaceus Pe. 2 I 12_5P Ho 0.39 0.04 0.00 0.00 0.00 0.00 0.15 0.00 0.00 0.00 0.03 0.14 0.00 0.44 0.00 0.03 0.14 0.04 0.00 0.00 Pediococcus claussenii Pe. 3 112 Ho 3.23 0.13 0.00 0.00 0.00 0.00 1.02 0.00 0.00 0.00 0.05 0.20 0.03 5.21 0.00 0.08 0.20 0.13 0.00 0.00 Extras Streptococcus salivarius subsp. thermophilus Strep. 1 13 Ho 0.97 0.08 0.00 0.00 0.00 0.00 0.24 0.00 0.00 0.01 0.04 0.43 0.07 0.29 0.03 0.14 0.43 0.08 0.00 0.00 Average values 2.87 0.18 0.00 0.01 0.05 0.00 0.42 0.00 0.00 0.00 0.04 0.18 0.02 0.34 0.01 0.06 0.24 0.19 0.00 0.43

VII

Table E.1. 3- Volatile organic compounds data for non-conventional yeast

Strains Volatile Organic Compunds (VOC's) Genus Species Code Acet_Ald (mg/L) Et_Ac (mg/L) Isobut_Ac (mg/L) Prop_ol (mg/L) Isob_ol (mg/L) Isoam_Ac (mg/L) Isoam_ol (mg/L) Et_Cap (mg/L) Et_Capr (mg/L) 2-Phe_Ac (mg/L) C6 (mg/L) 2-Phe_ol (mg/L) C8 (mg/L) 4-VG (mg/L) C10 (mg/L) Sum_Acids Sum_Alc Sum_Esters Lim (mg/L) Linal (ug/L) Saccharomyces kefyr 1 S. 1 64.50 0.43 0.00 6.22 4.80 0.00 17.56 0.00 0.00 0.01 0.04 3.19 0.17 0.35 0.13 0.34 14.21 0.43 0.00 0.00 Saccharomyces kefyr2 S. 2 5.73 7.03 0.00 2.86 8.61 0.07 27.75 0.00 0.00 1.20 0.03 6.85 0.11 0.31 0.11 0.25 18.32 7.10 0.00 0.00 Saccharomyces unisporus S. 3 60.27 0.38 0.00 5.56 4.21 0.00 15.94 0.00 0.00 0.74 0.04 6.32 0.15 0.36 0.11 0.30 16.09 0.38 0.00 0.00 Kluyveromyces marxianus Kl. 22.07 0.55 0.00 3.27 21.54 0.00 58.84 0.00 0.01 0.48 0.03 34.88 0.10 0.41 0.01 0.14 59.69 0.56 0.00 0.00 Yeast 1 Y1 9.46 0.69 0.00 2.61 1.16 0.07 9.09 0.00 0.01 0.02 0.05 1.35 0.46 0.45 0.58 1.09 5.12 0.77 0.00 0.00 Yeast 2 Y2 10.34 1.33 0.00 3.34 1.30 0.13 10.86 0.03 0.04 0.01 0.09 0.86 0.69 0.39 0.51 1.29 5.50 1.53 0.00 0.00 Yeast Yeast 3 Y3 22.97 7.62 0.00 6.71 15.03 0.05 33.81 0.00 0.00 0.01 0.08 12.57 0.34 0.39 0.27 0.69 34.31 7.67 0.00 0.00 Yeast 4 Y4 4.49 11.32 0.15 13.77 30.99 3.44 101.34 0.37 0.09 0.53 1.66 25.86 5.77 0.50 0.72 8.15 70.62 15.37 0.00 0.00 Galactomyces geotrichum 1 Gal. 1 4.76 1.26 0.00 2.90 3.83 0.00 2.95 0.00 0.00 0.00 0.02 0.46 0.07 0.44 0.02 0.11 7.19 1.26 0.00 0.00 Galactomyces geotrichum 2 Gal. 2 10.23 1.28 0.00 3.65 4.57 0.00 2.87 0.00 0.00 0.05 0.02 0.38 0.02 0.51 0.01 0.05 8.60 1.28 0.00 0.00 Galactomyces geotrichum 3 Gal. 3 5.36 0.24 0.00 0.00 6.76 0.00 11.81 0.00 0.00 0.03 0.01 0.90 0.03 0.33 0.06 0.10 7.66 0.24 0.00 0.00 Kazachstania gamospora Kaz. 43.26 13.82 0.01 10.51 2.82 1.02 30.43 0.03 0.02 6.27 0.46 20.58 1.23 0.41 0.10 1.79 33.91 14.90 0.00 0.00 Hanseniaspora guilliermondii Hans. 9.75 20.83 0.00 3.46 11.92 0.36 24.00 0.03 0.02 6.12 0.07 9.06 0.13 0.42 0.21 0.41 24.44 21.24 0.00 0.00 Average value 21.01 5.14 0.01 4.99 9.04 0.40 26.71 0.04 0.01 1.19 0.20 9.48 0.71 0.41 0.22 1.13 23.51 5.59 0.00 0.00

VIII

Annex E.2: Sensory analysis Table E.2. 1- Sensory analysis data for LAB (part I)

Strains Sensory Analysis Genus Species Code Number Fermentation Malt Honey Vinegar Brown sugar Roasted barley Fruity Floral Lactococcus lactis subsp. cremoris Lc. 1 9 Ho 1 0 0 0 0 0 0 Lactococcus lactis subsp. lactis Lc. 2 10 Ho 1 0 0 0 0 0 0 Lactococcus lactis subsp. lactis Lc. 2 I 10_5P Ho 1 0 0 0 0 0 0 Lactococcus lactis subsp. lactis Lc. 3 14 Ho 0 1 0 0 0 0 0 Lactococcus lactis subsp. lactis (Isolate1) Lc. 4 16 Ho 1 0 0 0 0 0 0 Lactococcus lactis subsp. lactis (Isolate2) Lc. 5 17 Ho 0 0 0 0 1 0 0 Lactococcus lactis subsp. lactis (Isolate3) Lc. 6 18 Ho 0 1 0 0 0 0 0 Lactococcus lactis subsp. lactis (Isolate4) Lc. 7 19 Ho 0 1 0 0 0 0 0 Lactococcus lactis subsp. lactis (Isolate5) Lc. 8 20 Ho 0 0 1 0 0 0 0 Lactococcus lactis subsp. hordniae Lc. 9 96 Ho 0 1 0 0 0 0 0 Lactococcus lactis subsp. lactis Lc. 10 97 Ho 1 0 0 0 0 0 0 Lactococcus lactis subsp. lactis Lc. 11 98 Ho 0 0 1 0 0 0 0 Lactococcus lactis subsp. lactis Lc. 12 99 Ho 1 0 0 0 0 0 0 Lactococcus lactis subsp. lactis Lc. 13 100 Ho 1 0 0 0 0 0 0 Lactococcus lactis subsp. lactis Lc. 14 101 Ho 0 1 0 0 0 0 0 Lactococcus Lactococcus lactis subsp. lactis Lc. 15 102 Ho 1 0 0 0 0 0 0 Lactococcus lactis subsp. lactis Lc. 16 103 Ho 0 1 0 0 0 0 0 Lactococcus raffinolactis Lc. 17 104 Ho 1 0 0 0 0 0 0 Lactococcus plantarum Lc. 18 105 Ho 0 0 1 0 0 0 0 Lactococcus lactis subsp. tructae Lc. 19 106 Ho 0 0 0 1 0 0 0 Lactococcus fujiensis Lc. 20 107 Ho 1 0 0 0 0 0 0 Lactococcus lactis subsp. lactis Lc. 21 118 Ho 1 0 0 0 0 0 0 Lactococcus lactis subsp. lactis Lc. 22 119 Ho 1 0 0 0 0 0 0 Lactococcus lactis Lc. 23 122 Ho 0 0 0 1 0 0 0 Lactococcus lactis Lc. 23 I 122_5P Ho 0 0 0 1 0 0 0 Lactococcus lactis Lc. 24 154 Ho 1 0 0 0 0 0 0 Lactococcus lactis subsp. lactis Lc. 25 156 Ho 1 0 0 0 0 0 0 Lactococcus lactis Lc. 26 160 Ho 1 0 0 0 0 0 0 Lactococcus lactis Lc. 27 170 Ho 0 0 0 0 0 0 1 Lactococcus lactis subsp. lactis Lc. 28 174 Ho 0 0 0 0 0 0 1 Lactococcus lactis Lc. 29 178 Ho 0 0 0 0 0 1 0

Table E.2. 2- Sensory analysis data for LAB (part II)

Strains Sensory Analysis Genus Species Code Number Fermentation Malt Honey Vinegar Brown sugar Roasted barley Fruity Floral Lactobacillus delbrueckii subsp. bulgaricus Lb. 1 1 Ho 1 0 0 0 0 0 0 Lactobacillus delbrueckii subsp. delbrueckii Lb. 2 2 Ho 0 1 0 0 0 0 0 Lactobacillus delbrueckii subsp. lactis Lb. 3 3 Ho 0 0 0 0 1 0 0 Lactobacillus helveticus Lb. 4 4 Ho 0 1 0 0 0 0 0 Lactobacillus dextrinicus Lb. 5 6 Ho 0 1 0 0 0 0 0 Lactobacillus mali Lb. 6 7 Ho 0 1 0 0 0 0 0 Lactobacillus acidophilus Lb. 7 8 Ho 1 0 0 0 0 0 0 Lactobacillus nagelii Lb. 8 21 Ho 0 0 0 0 0 0 1 Lactobacillus salivarius Lb. 9 22 Ho 0 0 0 1 0 0 0 Lactobacillus fermentum Lb. 10 23 He 0 0 0 0 1 0 0 Lactobacillus sakei subsp. sakei Lb. 11 25 Fa He 0 0 1 0 0 0 0 Lactobacillus sakei subsp. carnosus Lb. 12 26 Fa He 1 0 0 0 0 0 0 Lactobacillus sanfranciscensis Lb. 13 28 He 1 0 0 0 0 0 0 Lactobacillus amylolyticus Lb. 14 30 Ho 0 1 0 0 0 0 0 Lactobacillus amylovorus Lb. 15 31 Ho 0 1 0 0 0 0 0 Lactobacillus delbrueckii subsp. jakobsenii Lb. 16 32 Ho 0 0 0 1 0 0 0 Lactobacillus Lactobacillus farraginis Lb. 17 34 Fa He 1 0 0 0 0 0 0 Lactobacillus uvarum Lb. 18 38 Ho 0 0 0 1 0 0 0 Lactobacillus silagei Lb. 19 39 He 0 0 1 0 0 0 0 Lactobacillus oeni Lb. 20 40 Ho 1 0 0 0 0 0 0 Lactobacillus zeae Lb. 21 41 Fa He 0 0 1 0 0 0 0 Lactobacillus paracasei subsp. paracasei Lb. 22 42 Fa He 0 0 0 1 0 0 0 Lactobacillus parakefiri Lb. 23 43 He 0 0 0 1 0 0 0 Lactobacillus gasseri Lb. 24 45 Ho 1 0 0 0 0 0 0 Lactobacillus pentosus Lb. 25 46 Fa He 0 0 0 0 0 0 1 Lactobacillus alimentarius Lb. 26 48 Fa He 1 0 0 0 0 0 0 Lactobacillus farciminis Lb. 27 49 Ho 0 1 0 0 0 0 0 Lactobacillus gallinarum Lb. 28 50 Ho 1 0 0 0 0 0 0 Lactobacillus hilgardii Lb. 29 51 He 1 0 0 0 0 0 0 Lactobacillus johnsonii Lb.30 52 Ho 1 0 0 0 0 0 0 Lactobacillus pasteurii Lb. 31 55 Ho 1 0 0 0 0 0 0 Lactobacillus plantarum subsp. plantarum Lb. 32 56 Ho 0 0 0 0 0 0 1 Lactobacillus casei Lb. 33 57 Fa He 0 0 0 1 0 0 0

Table E.2. 3- Sensory analysis data for LAB (part III)

Strains Sensory Analysis Genus Species Code Number Fermentation Malt Honey Vinegar Brown sugar Roasted barley Fruity Floral Lactobacillus curvatus Lb. 34 58 Fa He 0 1 0 0 0 0 0 Lactobacillus brevis Lb. 35 59 He 1 0 0 0 0 0 0 Lactobacillus buchneri Lb. 36 61 He 0 0 1 0 0 0 0 Lactobacillus coryniformis subsp. coryniformis Lb. 37 62 Fa He 1 0 0 0 0 0 0 Lactobacillus malefermentans Lb. 38 63 He 0 0 1 0 0 0 0 Lactobacillus delbrueckii subsp. sunkii Lb. 39 108 Ho 0 0 0 1 0 0 0 Lactobacillus pentosus Lb. 40 109 Fa He 0 0 1 0 0 0 0 Lactobacillus pentosus Lb. 41 110 Fa He 0 0 1 0 0 0 0 Lactobacillus paraplantarum Lb. 42 111 Fa He 1 0 0 0 0 0 0 Lactobacillus leichmannii Lb. 43 120 He 1 0 0 0 0 0 0 Lactobacillus paracasei Lb. 44 121 Fa He 0 0 0 0 0 1 0 Lactobacillus acidophilus Lb. 45 123 He 1 0 0 0 0 0 0 Lactobacillus casei Lb. 46 124 He 1 0 0 0 0 0 0 Lactobacillus fermentum Lb. 47 125 He 0 0 1 0 0 0 0 Lactobacillus gasseri Lb. 48 126 Ho 1 0 0 0 0 0 0 Lactobacillus plantarum Lb. 49 127 Fa He 0 0 0 0 0 1 0 Lactobacillus Lactobacillus rhamnosus Lb. 50 128 Fa He 0 0 0 1 0 0 0 Lactobacillus rhamnosus Lb. 51 129 Fa He 1 0 0 0 0 0 0 Lactobacillus rhamnosus Lb. 52 130 Fa He 1 0 0 0 0 0 0 Lactobacillus rhamnosus Lb. 52 I 130_5P Fa He 1 0 0 0 0 0 0 Lactobacillus rhamnosus Lb. 53 131 He 1 0 0 0 0 0 0 Lactobacillus acidophilus Lb. 54 132 Ho 0 0 0 1 0 0 0 Lactobacillus casei ssp. casei Lb. 55 133 He 1 0 0 0 0 0 0 Lactobacillus salivarius Lb. 56 134 Ho 1 0 0 0 0 0 0 Lactobacillus acidophilus Lb. 57 135 Ho 0 0 0 1 0 0 0 Lactobacillus rhamnosus Lb. 58 136 He 0 1 0 0 0 0 0 Lactobacillus rhamnosus Lb. 59 137 He 1 0 0 0 0 0 0 Lactobacillus rhamnosus Lb. 60 138 He 1 0 0 0 0 0 0 Lactobacillus salivarius Lb. 61 139 Ho 1 0 0 0 0 0 0 Lactobacillus rhamnosus Lb.62 140 He 1 0 0 0 0 0 0 Lactobacillus rhamnosus Lb. 63 141 He 1 0 0 0 0 0 0 Lactobacillus casei ssp. casei Lb. 64 142 He 1 0 0 0 0 0 0 Lactobacillus zeae Lb. 65 143 Ho 1 0 0 0 0 0 0

Table E.2. 4- Sensory analysis data for LAB (part IV)

Strains Sensory Analysis Genus Species Code Number Fermentation Malt Honey Vinegar Brown sugar Roasted barley Fruity Floral Lactobacillus casei Lb. 66 144 Ho 1 0 0 0 0 0 0 Lactobacillus plantarum Lb. 67 145 He 1 0 0 0 0 0 0 Lactobacillus rhamnosus Lb. 68 147 He 0 0 1 0 0 0 0 Lactobacillus plantarum Lb. 69 148 He 1 0 0 0 0 0 0 Lactobacillus plantarum Lb. 70 149 He 1 0 0 0 0 0 0 Lactobacillus harbinensis Lb. 71 150 n.d. 1 0 0 0 0 0 0 Lactobacillus paracasei Lb. 72 155 Ho 1 0 0 0 0 0 0 Lactobacillus rhamnosus Lb. 73 158 He 0 0 1 0 0 0 0 Lactobacillus casei Lb. 74 159 Ho 1 0 0 0 0 0 0 Lactobacillus paracasei Lb. 75 161 Ho 1 0 0 0 0 0 0 Lactobacillus plantarum subsp. pantarum Lb. 76 162 He 1 0 0 0 0 0 0 Lactobacillus Lactobacillus plantarum Lb. 77 163 He 0 0 1 0 0 0 0 Lactobacillus rhamnosus Lb. 78 164 He 1 0 0 0 0 0 0 Lactobacillus plantarum Lb. 79 165 He 0 0 1 0 0 0 0 Lactobacillus plantarum subsp. pantarum Lb. 80 166 He 1 0 0 0 0 0 0 Lactobacillus plantarum Lb. 81 167 He 1 0 0 0 0 0 0 Lactobacillus rossiae Lb. 82 168 n.d. 0 1 0 0 0 0 0 Lactobacillus paracasei Lb. 83 171 Ho 0 1 0 0 0 0 0 Lactobacillus plantarum Lb. 84 172 He 0 0 0 0 0 1 0 Lactobacillus rhamnosus Lb. 85 173 He 1 0 0 0 0 0 0 Lactobacillus zeae Lb. 86 176 Ho 0 0 0 1 0 0 0 Lactobacillus plantarum Lb. 87 177 He 0 0 0 0 0 0 1 Leuconostoc mesenteroides subsp. cremoris Leuc. 1 35 He 1 0 0 0 0 0 0 Leuconostoc citreum Leuc. 2 36 He 0 0 1 0 0 0 0 Leuconostoc citreum Leuc. 2 I 36_5P He 0 0 1 0 0 0 0 Leuconostoc fallax Leuc. 3 37 He 0 0 1 0 0 0 0 Leuconostoc Leuconostoc citreum Leuc. 4 77 He 0 0 1 0 0 0 0 Leuconostoc citreum Leuc. 5 78 He 1 0 0 0 0 0 0 Leuconostoc citreum Leuc. 5 I 78_5P He 1 0 0 0 0 0 0 Leuconostoc mesenteroides subsp. dextranicum Leuc. 6 79 He 1 0 0 0 0 0 0 Leuconostoc lactis Leuc. 7 80 He 1 0 0 0 0 0 0

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Table E.2. 5- Sensory analysis data for LAB (part V)

Strains Sensory Analysis Genus Species Code Number Fermentation Malt Honey Vinegar Brown sugar Roasted barley Fruity Floral Leuconostoc palmae Leuc. 8 81 He 0 1 0 0 0 0 0 Leuconostoc miyukkimchii Leuc. 9 82 He 1 0 0 0 0 0 0 Leuconostoc holzapfelii Leuc. 10 83 He 1 0 0 0 0 0 0 Leuconostoc carnosum Leuc. 11 86 He 1 0 0 0 0 0 0 Leuconostoc Fructobacillus durionis Fruc. 12 87 He 1 0 0 0 0 0 0 Fructobacillus ficulneus Fruc. 13 88 He 0 1 0 0 0 0 0 Fructobacillus fructosus Fruc. 14 89 He 0 0 0 1 0 0 0 Fructobacillus pseudoficulneus Fruc. 15 90 He 1 0 0 0 0 0 0 Pediococcus acidilactici Pe. 1 11 Ho 0 0 1 0 0 0 0 Pediococcus acidilactici Pe. 1 I 11_5P Ho 0 0 1 0 0 0 0 Pediococcus Pediococcus pentosaceus Pe. 2 12 Ho 0 0 1 0 0 0 0 Pediococcus pentosaceus Pe. 2 I 12_5P Ho 0 0 1 0 0 0 0 Pediococcus claussenii Pe. 3 112 Ho 1 0 0 0 0 0 0 Extras Streptococcus salivarius subsp. thermophilus Strep. 1 13 Ho 0 0 0 0 0 1 0

XIII

Table E.2. 6- Sensory analysis data for non-conventional yeasts

Strains Sensory Analysis Genus Species Code Malt Honey Vinegar Brown sugar Roasted barley Fruity Floral Saccharomyces kefyr 1 S. 1 1 0 0 0 0 0 0 Saccharomyces kefyr2 S. 2 0 0 0 0 0 1 0 Saccharomyces unisporus S. 3 0 0 0 1 0 0 0 Kluyveromyces marxianus Kl. 0 1 0 0 0 0 0 Yeast 1 Y1 0 0 0 0 0 0 1 Yeast 2 Y2 0 0 0 0 0 0 1 Yeast Yeast 3 Y3 0 1 0 0 0 0 0 Yeast 4 Y4 0 0 0 0 0 0 1 Galactomyces geotrichum 1 Gal. 1 0 0 0 0 0 0 1 Galactomyces geotrichum 2 Gal. 2 0 0 0 0 0 1 0 Galactomyces geotrichum 3 Gal. 3 0 0 0 0 1 0 0 Kazachstania gamospora Kaz. 0 0 0 0 0 1 0 Hanseniaspora guilliermondii Hans. 0 0 1 0 0 0 0

XIV

Annex E.3: Growth parameters Table E.3. 1- Growth parameters data for LAB (part I)

Strains Genus Species Code Number Fermentation ΔOD pH ABV (%v/v) Lactococcus lactis subsp. cremoris Lc. 1 9 Ho 0.502 3.78 -0.02 Lactococcus lactis subsp. lactis Lc. 2 10 Ho 0.441 3.67 0.00 Lactococcus lactis subsp. lactis Lc. 2 I 10_5P Ho 0.335 3.60 0.00 Lactococcus lactis subsp. lactis Lc. 3 14 Ho 0.772 3.66 -0.02 Lactococcus lactis subsp. lactis (Isolate1) Lc. 4 16 Ho 1.014 3.67 0.00 Lactococcus lactis subsp. lactis (Isolate2) Lc. 5 17 Ho 0.756 3.64 -0.02 Lactococcus lactis subsp. lactis (Isolate3) Lc. 6 18 Ho 0.788 3.68 -0.02 Lactococcus lactis subsp. lactis (Isolate4) Lc. 7 19 Ho 0.665 3.65 -0.02 Lactococcus lactis subsp. lactis (Isolate5) Lc. 8 20 Ho 0.751 3.60 -0.02 Lactococcus lactis subsp. hordniae Lc. 9 96 Ho 0.289 4.05 -0.01 Lactococcus lactis subsp. lactis Lc. 10 97 Ho 0.358 3.62 0.00 Lactococcus lactis subsp. lactis Lc. 11 98 Ho 0.465 3.68 0.02 Lactococcus lactis subsp. lactis Lc. 12 99 Ho 0.417 3.66 0.01 Lactococcus Lactococcus lactis subsp. lactis Lc. 13 100 Ho 0.539 3.69 0.02 Lactococcus lactis subsp. lactis Lc. 14 101 Ho 0.295 3.73 0.02 Lactococcus lactis subsp. lactis Lc. 15 102 Ho 0.573 3.67 0.01 Lactococcus lactis subsp. lactis Lc. 16 103 Ho 0.547 3.69 0.01 Lactococcus raffinolactis Lc. 17 104 Ho 0.312 3.85 0.00 Lactococcus plantarum Lc. 18 105 Ho 0.147 3.63 0.01 Lactococcus lactis subsp. tructae Lc. 19 106 Ho 0.564 3.70 0.01 Lactococcus fujiensis Lc. 20 107 Ho 0.312 3.85 0.01 Lactococcus lactis subsp. lactis Lc. 21 118 Ho 0.761 3.71 0.01 Lactococcus lactis subsp. lactis Lc. 22 119 Ho 0.384 3.64 0.01 Lactococcus lactis Lc. 23 122 Ho 0.288 3.60 0.00 Lactococcus lactis Lc. 23 I 122_5P Ho 0.466 3.47 -0.02 Lactococcus lactis Lc. 24 154 Ho 1.525 3.28 0.03

Lactococcus lactis subsp. lactis Lc. 25 156 Ho 1.532 3.29 0.04

Table E.3. 2- Growth parameters data for LAB (part II)

Strains Genus Species Code Number Fermentation ΔOD pH ABV (%v/v) Lactococcus lactis Lc. 26 160 Ho 1.610 3.27 0.03 Lactococcus lactis Lc. 27 170 Ho 0.495 3.77 0 Lactococcus Lactococcus lactis subsp. lactis Lc. 28 174 Ho 0.527 3.63 0.01 Lactococcus lactis Lc. 29 178 Ho 0.500 3.62 0.01 Lactobacillus delbrueckii subsp. bulgaricus Lb. 1 1 Ho 0.608 4.01 -0.02 Lactobacillus delbrueckii subsp. delbrueckii Lb. 2 2 Ho 0.234 4.37 -0.04 Lactobacillus delbrueckii subsp. lactis Lb. 3 3 Ho 0.511 4.28 -0.02 Lactobacillus helveticus Lb. 4 4 Ho 0.843 3.44 0.00 Lactobacillus dextrinicus Lb. 5 6 Ho 0.409 4.15 -0.02 Lactobacillus mali Lb. 6 7 Ho 1.196 3.48 0.00 Lactobacillus acidophilus Lb. 7 8 Ho 0.184 4.19 -0.02 Lactobacillus nagelii Lb. 8 21 Ho 2.075 3.33 0.00 Lactobacillus salivarius Lb. 9 22 Ho 1.647 3.28 0.02 Lactobacillus fermentum Lb. 10 23 He 1.379 3.55 0.14 Lactobacillus sakei subsp. sakei Lb. 11 25 Fa He 0.665 3.85 -0.02 Lactobacillus Lactobacillus sakei subsp. carnosus Lb. 12 26 Fa He 0.551 3.78 -0.02 Lactobacillus sanfranciscensis Lb. 13 28 He 0.627 3.83 -0.02 Lactobacillus amylolyticus Lb. 14 30 Ho 0.258 4.19 -0.02 Lactobacillus amylovorus Lb. 15 31 Ho 0.208 4.46 -0.04 Lactobacillus delbrueckii subsp. jakobsenii Lb. 16 32 Ho 1.107 3.36 0.00 Lactobacillus farraginis Lb. 17 34 Fa He 1.154 3.40 0.18 Lactobacillus uvarum Lb. 18 38 Ho 0.977 3.47 0.00 Lactobacillus silagei Lb. 19 39 He 0.588 3.80 0.08 Lactobacillus oeni Lb. 20 40 Ho 1.205 3.47 0.00 Lactobacillus zeae Lb. 21 41 Fa He 1.331 3.25 0.02 Lactobacillus paracasei subsp. paracasei Lb. 22 42 Fa He 1.377 3.33 0.00

Lactobacillus parakefiri Lb. 23 43 He 0.098 5.48 -0.02

XVI

Table E.3. 3- Growth parameters data for LAB (part III)

Strains Genus Species Code Number Fermentation ΔOD pH ABV (%v/v) Lactobacillus gasseri Lb. 24 45 Ho 0.116 4.66 -0.04 Lactobacillus pentosus Lb. 25 46 Fa He 1.849 3.40 0.00 Lactobacillus alimentarius Lb. 26 48 Fa He 0.254 4.00 -0.02 Lactobacillus farciminis Lb. 27 49 Ho 0.466 3.86 -0.02 Lactobacillus gallinarum Lb. 28 50 Ho 0.216 3.94 -0.02 Lactobacillus hilgardii Lb. 29 51 He 0.907 3.73 0.08 Lactobacillus johnsonii Lb.30 52 Ho 0.173 4.30 -0.02 Lactobacillus pasteurii Lb. 31 55 Ho 0.075 4.01 -0.02 Lactobacillus plantarum subsp. plantarum Lb. 32 56 Ho 1.479 3.28 0.00 Lactobacillus casei Lb. 33 57 Fa He 0.865 3.55 0.00 Lactobacillus curvatus Lb. 34 58 Fa He 0.215 3.97 -0.02 Lactobacillus brevis Lb. 35 59 He 1.113 3.90 0.04 Lactobacillus buchneri Lb. 36 61 He 0.977 3.70 0.08 Lactobacillus Lactobacillus coryniformis subsp. coryniformis Lb. 37 62 Fa He 1.041 3.46 0.00 Lactobacillus malefermentans Lb. 38 63 He 0.373 4.24 0.02 Lactobacillus delbrueckii subsp. sunkii Lb. 39 108 Ho 0.481 3.93 -0.01 Lactobacillus pentosus Lb. 40 109 Fa He 0.828 3.43 0.02 Lactobacillus pentosus Lb. 41 110 Fa He 1.172 3.37 0.03 Lactobacillus paraplantarum Lb. 42 111 Fa He 0.971 3.40 0.02 Lactobacillus leichmannii Lb. 43 120 He 0.270 4.46 -0.04 Lactobacillus paracasei Lb. 44 121 Fa He 2.071 3.40 0.02 Lactobacillus acidophilus Lb. 45 123 He 0.321 4.23 -0.04 Lactobacillus casei Lb. 46 124 He 2.171 3.57 0.02 Lactobacillus fermentum Lb. 47 125 He 1.403 3.41 0.12 Lactobacillus gasseri Lb. 48 126 Ho 0.178 4.43 -0.02 Lactobacillus plantarum Lb. 49 127 Fa He 1.373 3.30 0.03 Lactobacillus rhamnosus Lb. 50 128 Fa He 1.956 3.13 -0.02

XVII

Table E.3. 4- Growth parameters data for LAB (part IV)

Strains Genus Species Code Number Fermentation ΔOD pH ABV (%v/v) Lactobacillus rhamnosus Lb. 51 129 Fa He 1.800 3.11 0.02 Lactobacillus rhamnosus Lb. 52 130 Fa He 2.453 3.18 0.02 Lactobacillus rhamnosus Lb. 52 I 130_5P Fa He 2.339 3.12 0.02 Lactobacillus rhamnosus Lb. 53 131 He 1.092 3.18 0.04 Lactobacillus acidophilus Lb. 54 132 Ho 0.322 4.19 -0.01 Lactobacillus casei ssp. casei Lb. 55 133 He 0.843 3.17 0.04 Lactobacillus salivarius Lb. 56 134 Ho 4.464 3.31 0.04 Lactobacillus acidophilus Lb. 57 135 Ho 0.181 3.98 0.00 Lactobacillus rhamnosus Lb. 58 136 He 1.184 3.20 0.04 Lactobacillus rhamnosus Lb. 59 137 He 1.229 3.21 0.04 Lactobacillus rhamnosus Lb. 60 138 He 1.108 3.22 0.02 Lactobacillus salivarius Lb. 61 139 Ho 2.971 3.47 0.03 Lactobacillus rhamnosus Lb.62 140 He 1.091 3.22 0.04 Lactobacillus Lactobacillus rhamnosus Lb. 63 141 He 1.165 3.17 0.02 Lactobacillus casei ssp. casei Lb. 64 142 He 0.783 3.22 0.04 Lactobacillus zeae Lb. 65 143 Ho 0.963 3.19 0.04 Lactobacillus casei Lb. 66 144 Ho 1.414 3.33 0.03 Lactobacillus plantarum Lb. 67 145 He 1.626 3.29 0.17 Lactobacillus rhamnosus Lb. 68 147 He 4.165 3.35 0.03 Lactobacillus plantarum Lb. 69 148 He 1.675 3.30 0.02 Lactobacillus plantarum Lb. 70 149 He 1.523 3.28 0.03 Lactobacillus harbinensis Lb. 71 150 n.d. 1.502 3.28 0.03 Lactobacillus paracasei Lb. 72 155 Ho 1.696 3.72 0.14 Lactobacillus rhamnosus Lb. 73 158 He 0.809 3.30 0.02 Lactobacillus casei Lb. 74 159 Ho 1.307 3.34 0.03 Lactobacillus paracasei Lb. 75 161 Ho 1.371 3.33 0.03

Lactobacillus plantarum subsp. pantarum Lb. 76 162 He 1.532 3.28 0.04

XVIII

Table E.3. 5- Growth parameters data for LAB (part V)

Strains Genus Species Code Number Fermentation ΔOD pH ABV (%v/v) Lactobacillus plantarum Lb. 77 163 He 1.524 3.29 0.03 Lactobacillus rhamnosus Lb. 78 164 He 4.308 3.22 0.04 Lactobacillus plantarum Lb. 79 165 He 1.658 3.30 0.04 Lactobacillus plantarum subsp. pantarum Lb. 80 166 He 1.555 3.30 0.03 Lactobacillus plantarum Lb. 81 167 He 1.642 3.25 0.02 Lactobacillus Lactobacillus rossiae Lb. 82 168 n.d. 0.192 4.23 0.16 Lactobacillus paracasei Lb. 83 171 Ho 1.337 3.29 0.02 Lactobacillus plantarum Lb. 84 172 He 1.589 3.27 0.03 Lactobacillus rhamnosus Lb. 85 173 He 1.199 3.20 0.04 Lactobacillus zeae Lb. 86 176 Ho 1.291 3.33 -0.01 Lactobacillus plantarum Lb. 87 177 He 1.730 3.24 0.03 Leuconostoc mesenteroides subsp. cremoris Leuc. 1 35 He 0.414 3.85 0.04 Leuconostoc citreum Leuc. 2 36 He 0.805 3.55 0.14 Leuconostoc citreum Leuc. 2 I 36_5P He 0.559 3.42 0.14 Leuconostoc fallax Leuc. 3 37 He 1.074 3.53 0.10 Leuconostoc citreum Leuc. 4 77 He 0.374 3.76 0.10 Leuconostoc citreum Leuc. 5 78 He 0.717 3.63 0.10 Leuconostoc citreum Leuc. 5 I 78_5P He 0.678 3.50 0.12 Leuconostoc mesenteroides subsp. dextranicum Leuc. 6 79 He 0.314 3.75 0.08 Leuconostoc Leuconostoc lactis Leuc. 7 80 He 0.295 3.79 0.06 Leuconostoc palmae Leuc. 8 81 He 0.069 3.60 0.10 Leuconostoc miyukkimchii Leuc. 9 82 He 0.902 3.65 0.07 Leuconostoc holzapfelii Leuc. 10 83 He 0.573 3.68 0.09 Leuconostoc carnosum Leuc. 11 86 He 0.206 4.08 0.04 Fructobacillus durionis Fruc. 12 87 He 0.072 4.34 -0.02 Fructobacillus ficulneus Fruc. 13 88 He 0.130 4.32 0.02 Fructobacillus fructosus Fruc. 14 89 He 0.293 4.37 -0.01

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Table E.3. 6- Growth parameters data for LAB (part VI)

Strains Genus Species Code Number Fermentation ΔOD pH ABV (%v/v) Leuconostoc Fructobacillus pseudoficulneus Fruc. 15 90 He 0.094 4.33 -0.01 Pediococcus acidilactici Pe. 1 11 Ho 0.672 3.87 -0.02 Pediococcus acidilactici Pe. 1 I 11_5P Ho 0.531 3.83 -0.02 Pediococcus Pediococcus pentosaceus Pe. 2 12 Ho 0.736 3.68 -0.04 Pediococcus pentosaceus Pe. 2 I 12_5P Ho 0.556 3.73 -0.02 Pediococcus claussenii Pe. 3 112 Ho 1.665 3.55 0.02 Extras Streptococcus salivarius subsp. thermophilus Strep. 1 13 Ho 0.436 3.92 -0.02 Average values 0.94 3.84 -0.02

Table E.3. 7- Growth parameters data for non-conventional yeast

Strains Genus Species Code ΔOD pH ABV (%v/v) Saccharomyces kefyr 1 S. 1 2.434 4.62 0.88 Saccharomyces kefyr2 S. 2 2.636 4.62 0.82 Saccharomyces unisporus S. 3 1.889 4.62 1.05 Kluyveromyces marxianus Kl. 1.394 4.78 0.57 Yeast 1 Y1 1.259 5.00 0.41 Yeast 2 Y2 1.097 4.90 0.59 Yeast Yeast 3 Y3 2.035 4.32 1.12 Yeast 4 Y4 3.813 4.04 2.92 Galactomyces geotrichum 1 Gal. 1 0.222 5.54 0.01 Galactomyces geotrichum 2 Gal. 2 0.143 5.36 0.02 Galactomyces geotrichum 3 Gal. 3 0.748 5.14 0.15 Kazachstania gamospora Kaz. 5.342 3.97 2.03 Hanseniaspora guilliermondii Hans. 1.456 4.37 0.88 Average values 1.88 4.71 0.88

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Annex F: PCA analysis Annex F.1: Lactic acid bacteria

Figure F.1. 1- Principal component analysis plot obtained for LAB, based on VOCs and growth parameters (PC-3 (3%) vs PC-4 (1%)).

Figure F.1. 2- Principal component analysis 3D-plot obtained for LAB, based on VOCs and growth parameters (PC-1 (83%) vs PC-3 (3%) vs PC-4(1%))

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Annex F.2: Non-conventional yeast

Figure F.2. 1- Principal component analysis plot obtained for non-conventional yeast, based on VOCs and growth parameters (PC-3 (3%) vs PC-4 (2%)).

Figure F.2. 2- Principal component analysis plot obtained for non-conventional yeast, based on VOCs and growth parameters (PC-4 (2%) vs PC-5 (1%)).

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Annex G: Growth parameters data for candidates

Table G. 1- Growth parameters data for LAB candidates

Strains Candidates (MW) Genus Species Code Number Fermentation ΔOD pH (Past.) ABV (%v/v) (Past.) pH (Non-past.P) ABV (%v/v) (Non-past.) Lactobacillus pentosus Lb. 25 46 Fa He n.d. 6.121 -0.02 4.849 0 Lactobacillus plantarum subsp. plantarum Lb. 32 56 Ho n.d. 3.74 0.02 3.809 0.02 Lactobacillus plantarum Lb. 49 127 Fa He n.d. 3.708 0.04 3.672 0.02 Lactobacillus salivarius Lb. 56 134 Ho n.d. 3.816 0.02 3.782 0.04 Lactobacilus Lactobacillus plantarum Lb. 67 145 He n.d. 3.706 0.04 3.659 0.04 Lactobacillus rhamnosus Lb. 68 147 He n.d. 3.841 0.02 3.818 0.02 Lactobacillus rhamnosus Lb. 73 158 He n.d. 7.051 -0.02 4.407 n.d. Lactobacillus plantarum Lb. 84 172 He n.d. 3.84 0.04 3.805 0.04 Average values 4.48 0.02 3.98 0.03

Table G. 2- Growth parameters data for non-conventional yeast candidates

Strains Candidates (MW) Candidates (HFGW) Genus Species Code ΔOD pH (Past.) ABV (%v/v) (Past.) pH (Non-past.P) ABV (%v/v) (Non-past.) ΔOD pH ABV (%v/v) Saccharomyces kefyr 1 S. 1 2.380 4.02 0.84 3.99 0.84 3.374 3.57 2.72 Saccharomyces kefyr2 S. 2 1.945 4.53 0.68 4.43 0.70 2.785 3.62 2.88 Yeast 1 Y1 1.763 3.73 1.04 3.75 1.04 1.069 3.56 1.18 Yeast 2 Y2 1.139 6.45 1.36 6.66 1.40 1.396 3.91 2.30 Yeast Yeast 4 Y4 2.345 3.80 0.96 3.83 0.98 3.933 3.63 3.06 Galactomyces geotrichum 1 Gal. 1 -0.016 7.98 -0.02 7.81 -0.02 0.147 4.74 0.30 Galactomyces geotrichum 2 Gal. 2 1.397 7.43 0.04 7.26 0.04 0.107 4.72 0.24 Kazachstania gamospora Kaz. 3.576 3.82 0.96 3.82 0.96 5.071 3.69 3.02 Average values 1.82 5.22 0.73 5.20 0.74 2.24 3.93 1.96

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