Degree project work

Development of an energy dense, protein enriched oat-based yogurt

Author: Frida Sjöberg Supervisor: Olof Böök, Hanna Magnusson (Aventure AB), Kjell Edman (LNU) Examiner: Anna Blücher Date: May 24, 2017 Subject: Chemistry Level: First cycle Course code: 2KE01E

Abstract As of today, there is a challenge amongst the elderly to get the energy they need. Malnutrition is a fact for too many and protein is one of the most common deficiencies among macronutrients in this age group. Another group, also in the need of extra energy and protein, are those with a higher level of physical activity. Aventure AB in Lund, Sweden, has earlier developed an “in between meal beverage”, based on oats, with high energy and protein content called “Skaka & Smaka”. This beverage has with promising results been distributed to selected hospitals in Sweden as a pilot-trial. As an expansion of this product, Aventure wanted to create an oat-based, energy dense stirred yogurt. The aim of this study was to investigate the feasibility of addition of different protein sources to create an energy dense stirred yogurt with good texture and a balance of macronutrients. Four different types of protein sources were, separately or in combination, added to the original recipe of “Skaka & Smaka” (without added flavour), after which it was fermented and evaluated regarding sensory and physio-chemical properties. Three different fruit purée mixtures were added separately to the fermented oat-base in different proportions and evaluated by taste, color and flavour intensity. Salt concentration was adjusted and two final products were developed and evaluated through a sensory consumer test, at two different retirement homes in Hässleholm, Sweden. To measure the participants opinions a 9-point hedonic scale was used and attributes evaluated were first impression, color, taste, consistency, thickness, spoonability and total impression. All of the 11 individuals who participated were at the age of 75 and over, where of 2 were men and 9 were women. The proportions of added protein in the two final products were 10% casein and 90% whey. The two selected fruit purées, “skogen” and “havet”, were added in the concentration of 30%. The addition of salt was increased with 100%, resulting in a final concentration of 0.08 g per 100 grams. The sensory analysis revealed no statistical significance between any of the related attributes of the two yogurts. The attribute most important for general liking was taste, followed by texture and color. Seven individuals (64%) said they could consume this kind of product a few times a week and all participants thought that there was a need for this kind of product. The two yogurts developed in this project, “skogen” and “havet”, and the concept behind them seem to have good potential for consumer liking, though further development of taste and texture is needed.

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Keywords High-energy yogurt, casein, whey, plant-based protein, malnutrition, elderly, physically active

Thanks I would like to start off by thanking Olof Böök, my main supervisor at Aventure AB, for giving me the opportunity and experience to carry out this project and for his guided commitment trough this whole journey.

I also wish to thank Hanna Magnusson for her truly appreciated guidance and support in and outside of the lab.

I’d like to thank my internal supervisor at Linneaus University, Kjell Edman, for his kind support, shared knowledge and help during the writing process.

I also like to thank Eva Malm Körkkö (Ehrenborg residential home), Caritha Andersson (Högalid residential home), all the parcipitants and staff at these retirement homes for making the sensory analysis possible.

I would also like to thank my family for their love and encouragement.

Finally, I would like to thank all the great people at Aventure for brightening up each and every single day with good conversations and many great laughters as well as stories during our beloved Thursday “fika”.

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Swedish summary Som det ser ut idag så finns det en utmaning bland de äldre att få i sig den energi de behöver. Undernäring är ett faktum för allt för många och protein är en av de vanligaste bristerna bland makronäringsämnena inom denna åldersgrupp. En annan grupp som också är i behov av extra energi och protein är de med en hög fysisk aktivitetsnivå. Aventure AB i Lund, Sverige, har tidigare utvecklat en mellanmåls dryck, baserad på havre, med ett högt energi- och proteininnehåll, kallad ”Skaka & Smaka”. Denna dryck har i ett pilot försök och med lovande resultat distribuerats till flertalet utvalda sjukhus i Sverige. Som en vidareutveckling av denna produkt ville Aventure skapa en havrebaserad, energität, rörd yoghurt. Syftet med denna studie var att undersöka geomförbarheten i tillsättning av olika proteinkällor för att skapa en energität, rörd yoghurt med bra konsistens och en balans av makronäringsämnen. Fyra olika typer av proteinkällor tillsattes, separat eller i kombination, till originalreceptet för ”Skaka & Smaka” (utan tillsatt smak), vartefter blandningen fermenterades och utvärderades utifrån sensoriska och fysio-kemikaliska egenskaper. Tre olika fruktpurée blandningar tillsattes separat till den fermenterade havrebasen i olika proportioner och utvärderades utifrån smak, färg och smakintensitet. Saltkoncentrationen justerades och två slutliga produkter utvecklades och utvärderades genom ett sensoriskt konsumenttest, vid två olika äldreboenden i Hässleholm, Sverige. För att mäta deltagarnas åsikter användes en 9-punkt hedonisk skala och de attribut som utvärderades var första intryck, färg, smak, konsistens, tjockhet, skedbarhet och totalt intryck. Alla de 11 individer som deltog var av ålder 75 och över, varav två var män och nio var kvinnor. Proportionerna av tillsatt protein i de två slutliga produkterna var 10% kasein och 90% vassle. De två valda fruktpuréerna, ”skogen” och ”havet”, tillsattes i en koncentration på 30%. Tillsatsen av salt ökades med 100%, vilket resulterade i en slutlig koncentration på 0.08g per 100 gram. Den sensoriska analysen visade inte på någon statistisk signifikant skillnad mellan de relaterade attributen för de två olika yoghurtarna. Attributen som var av störst vikt för generellt tycke var smak, följt av konsistens och färg. Sju individer (64%) sa att de kunde konsumera en produkt som denna ett par gånger i veckan och alla deltagande tyckte att det fanns ett behov av en produkt som denna. De två yoghurtarna som utvecklades i detta projekt, ”skogen” och ”havet”, och det bakomliggande konceptet tycks ha god potential bland konsumenter, men vidare utveckling av smak och konsistens är nödvändigt.

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Contents

1 Introduction ______1 1.1 Aim ______1

2 Background ______2 2.1 Nutrition and the elderly ______2 2.1.1 Recommendations ______2 2.2 Nutrition and physical activity ______2 2.2.1 Recommendations ______3 2.3 Earlier product and further development ______4 2.4 Oat and health ______4 2.5 Protein sources ______4 2.5.1 Casein ______4 2.5.2 Whey ______5 2.5.3 Rice protein ______6 2.5.4 A mix of rice and pea protein ______6 2.6 Protein properties ______7 2.6.1 Solubility ______7 2.6.2 Emulsification ______8 2.6.3 Rheology ______8 2.6.3.1 Viscosity______8 2.6.3.2 Flowability______8 2.6.6 Texturization ______8 2.6.7 Flavour binding ______9 2.7 Fermentation ______9 2.8 Sensory analysis ______9

3 Materials and Methods ______11 3.1 Experimental trials ______11 3.1.1 Screening of starter culture and addition of whey and casein protein ____ 11 3.1.2 Effect of different volumes of transferred inoculum on fermentation time _ 11 3.1.3 Addition of vegetable proteins ______11 3.1.4 Lowered heat treatment ______11 3.1.5 Optimum ratios of whey/casein, increased LOB and addition of fruit purée 11 3.1.6 Different fruit purée concentrations and texturizer ______12 3.1.7 Lowering of casein and an increase in pH ______12 3.1.8 Adjustments to the pasteurisation step and increased content of texturizer 12 3.2 Sample preparation ______12 3.2.1 Preparation of Glucanova oat base (10 wt%) ______12 3.2.2 Preparation of “Skaka & Smaka” oat base (yogurt substrate) ______12 3.2.3 Preperation of starter culture inoculum ______13 3.4 Fermentation ______13

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3.4.1 Bacterial strains ______13 3.5 Addition of fruit purée and texturizer ______14 3.5.1 Fruit and berry purées ______14 3.6 Analytical measurements ______15 3.6.1 pH ______15 3.6.2 Dry matter ______15 3.6.3 Rheometry ______15 3.6.4 Viscosity ______15 3.6.5 Flowability ______15 3.6.6 Brix ______16 3.6.7 Theoretical analysis of nutritional composition ______16 3.7 Sensory analysis ______16 3.8 Statistical analysis ______17

4 Results ______18 4.1 Experimental trials ______18 4.1.1 Screening of starter culture and addition of whey and casein protein ____ 18 4.1.2 Effect of different volumes of transferred inoculum on fermentation time _ 18 4.1.3 Addition of vegetable proteins ______18 4.1.4 Lowered heat treatment ______18 4.1.5 Optimum ratios of whey/casein, increased LOB and addition of fruit purée 18 4.1.6 Different fruit purée concentrations and texturizer ______19 4.1.7 Lowering of casein and an increase in pH ______19 4.1.8 Adjustments to the pasteurisation step and increased content of texturizer 19 4.2 Final products ______20 4.2.1 Flowability ______20 4.2.2 Rheological measurements ______21 4.2.3 Theoretical nutritional composition ______22 4.2.4 Final recipe ______22 4.3 Sensory and statistical analysis ______24

5 Discussion ______26 5.1 Addition of casein and whey protein ______26 5.2 Addition of starter culture ______27 5.3 Addition of vegetable proteins ______27 5.4 Addition of fruit purée ______27 5.5 Final products ______27 5.6 Sensory and statistical analysis ______28 5.7 For future development ______28

6 Conclusion ______30

References ______31

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Appendices ______I Appendix A Presentation and questionnary for sensory analysis ______I

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1 Introduction A good nutritional status is important for health and to avoid sickness. As we age, the functions of our bodies and organs decrease and becomes more vulnerable to disease. A balanced intake of energy therefore becomes even more important as we grow older [1]. The need of energy also decrease with age, adding to the importance of eating a well- composed diet [2]. In the world today, 8.5 percent of people (617 million) are at the age of 65 and over. Older people are a rapidly growing population and this number is expected to rise to nearly 17 percent (1.6 billion) of the world’s population by 2050 [3]. Sweden has one of the highest life expectancy in the world, with 79.9 years for men and 83.7 for women. People in Sweden today, aged 65 and over, are 20% (1.96 million) and is projected to rise to 23% by 2040 [4].

Undernutrition is a common problem among elderly [5] and one of the main factors for the onset of frailty, which can bring many health complications [6]. Some of the reasons for this deficiency in dietary intake could be difficulties in eating due to different conditions such as loss of appetite, movement, taste and smell, long term illnesses, reduced absorbency of nutrients, medication or psychological and social aspects [7, 8]. A key nutrient, which also affect the incidence of frailty in elderly adults, is protein. Increased intake of this macronutrient could increase the quality of life among elderly through e.g. improved muscle function and prevention of the onset of chronic disease [9].

Consuming an energy balanced diet is also of big importance for individuals who have a high level of physical activity [10]. Being in a constant negative energy balance will most likely lead to a metabolic decline and reduced performance [11]. An adequate intake of protein is also crucial for muscle development, as well as for maintenance of many bodily functions [12].

Certain proteins in some foods can give rise to allergic reactions, where milk is a common source [13]. This, as well as lifestyle changes, an interest in alternative diets and an increased awareness of sustainable production, feeds the trend and need of milk substitutes on the market [14].

As for now, there is a gap in the market for a nutrient dense “in between meal snack”, having a balanced nutritional profile. Development of a product fulfilling these requests could be of important nutritional value for both elderly and individuals requiring an increased energy intake through physical activity.

This project is an expansion of the “in between-meal beverage” - Skaka & Smaka [15] and proceeds from an earlier study made on the development of an oat-based yogurt [16], with focus on nutrition density, a balance between macronutrients as well as a high protein content.

1.1 Aim In association with Aventure AB in Lund, Sweden, the aim of this project is to achieve an energy dense, oat-based stirred yogurt with a balance in macronutrient content, mainly aimed towards the elderly and to evaluate its’ physio-chemical and sensory properties after addition of different protein sources.

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2 Background The high-energy ”in between meal beverage” called ”Skaka & Smaka” was developed by Aventure in collaboration with a dietitian, as a way to offer the elderly a well- balanced nutritional snack. As a further product development, this project aims to create a yogurt on the same concept of a good nutritional composition as a whole with a high energy content. Although the original product was directed towards the older generation, people with a higher physical activity is another target group with an elevated need for high-energy snacks with a good nutritional balance. Through different marketing, layout and potentially flavor variation, the same product could reach and fit two totally different segments.

2.1 Nutrition and the elderly A combination of energy- and protein deficiency is the most common malnutrition in Sweden. The average frequency of this condition, in 25 Swedish studies, was 28% [17]. Malnutrition among elderly can lead to a decrease in general life quality through for example impaired immune and muscle function, increased morbidity, need for care and mortality [18]. The definition of malnutrition is according to the Swedish Society for Clinical Nutrition and Metabolism (SWESPEN) - “a state where deficiency in energy, protein or others nutrients have caused measurable and unfavourable changes in body composition, function or of a persons disease progress” [1].

2.1.1 Recommendations According to the Nordic Nutrition recommendations (2012) the distribution of macronutrients among individuals of age 65 years and older are as follows: fat (25- 40%), carbohydrates (45-60%) and protein (15-20%) [19]. The daily need of calories for sedimentary (limited to activities of daily living) males over 70 years of age is about 2,000. For females in the same age range and inactivity level, the calorie requirement is about 1,600. With increased physical activity, like a brisk walk for at least 4.8 km, the need of energy increases with about 400 calories for both men and women [20].

2.2 Nutrition and physical activity Regular physical activity as well as high cardiorespiratory fitness is today well known for having many health benefits. Not only can it prevent cardiovascular disease, but epidemiologic studies also show prevention of site-specific cancers, type 2 diabetes, improved bone health, reduced disability and increased longevity [21]. Making sure that the required intake of energy and necessary nutrients are met is essential for good health and performance [22], even more so during regular physical activity [23]. For building muscle, an adequate intake of both energy and protein is needed [10, 12]. Having a sufficient protein intake but eating insufficient amounts of energy will result in the proteins being used as fuel instead of building blocks [24]. An increase in muscle mass synthesis can be seen following resistance training and could last up to 48 h. This process is in turn linked to the availability of amino acids (the building blocks of proteins) in the muscle. A sufficient intake of protein after this kind of exercise, therefore assists in creating a positive muscle protein balance [25]. Some amino acids have shown indications of having regulatory effects on key metabolic pathways, necessary for maintenance, growth, reproduction and immunity. Dietary supplementation of one or several of these amino acids; arginine, cysteine,

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glutamine, leucine, proline and tryptophan, may thereby enhance muscle growth and athletic performance [26]. Branched chain amino acids (BCAA), i.e. leucine, isoleucine and valine, is taken up directly by the skeletal muscles, instead of first being metabolised by the liver, like most other amino acids. This makes them extra importan during and after strenuous physical activity [27].

2.2.1 Recommendations The recommended intake of the different macronutrients for adults and children over two years of age are, according to the Nordic Nutrition Recommendations from 2012, the same as for elderly (see 2.1.1 Recommendations above), with the only exception of protein, which has a broader interval of 10-20% (compared to 15-20% for people at the age of 65 and over). The actual intake per gram thereby becomes greater with an increased energy expenditure, like for example through physical activity. Required energy intake among men and women of different ages can be seen in table I, and are based on individuals with sedentary jobs and a restricted increase in physical activity level during spare time hours [19]. Table II show general guidelines for energy expenditure per hour for different physical activities and bodyweights [28], i.e. the extra addition of calorie intake needed if wanting to stay in an energy balanced state.

Table I. Reference values for energy intake amongst adults with a sedentary lifestyle. Based on the Nordic Nutrition Recommendations 2012 [19]. Age Average energy intake* (kcal) Women 18-30 2245 31-60 2102 61-74 1935 Men 18-30 2794 31-60 2627 61-74 2317

Table II. Energy expenditure per hour for different physical activities and bodyweight. Based on SOK [28]. These are just estimations and may vary depending on individual differences like for example body composition and gender. Activity Weight (kg) Energy expenditure (kcal/h) Strength training (free weights) 55-65 358 - 423 75-85 488 - 553 Running (12 km/h) 55-65 743 - 878 75-85 1013 - 1148

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2.3 Earlier product and further development The fermentation base of the yogurt in this project, called “Skaka & Smaka”, was originally developed by Aventure AB as an “in between meal beverage” for hospitalised elderly. This was done with the attempt of adding more energy and nutrients into their diet, thereby lowering the risk of malnutrition [29]. The product is oat-based and has a balance of fat, carbohydrate and protein content, which of the latter is a common deficiency amongst elderly [17]. It has no added sugar (sucrose) or flavourings and exists in two flavours, strawberry and raspberry/blueberry (fruit purée).

As a further product development and expansion of product portfolio, an attempt to create an oat-based yogurt came into existence. This was performed on the same “Skaka & Smaka” beverage base (without the flavours) with the addition of a fermentation process. An earlier study [16] examined the best choice of starter cultures for this process. In the current project, further trials were made on the addition of protein from different sources and in different amounts to see which gave the best physio-chemical and sensory properties. For added flavour, different fruit purées were mixed into the “Skaka & Smaka” oat-base.

2.4 Oat and health Oats are an important crop worldwide, both as a nutritious grain for human consumption but even more so as a livestock feed [30]. Production of oats are the sixth ranked cereal in the world (23,309 tonne/MT) [31, 32], where most is produced within countries of the European Union, Russia, Canada and Australia respectively [32]. There are different oat varieties, where Avena sativa L. (also referred to as the “common oat”), is the most used [33]. Oats are rich in protein, a good source of dietary fiber, lipids and many important minerals. Adding to this and its’ content of phytochemicals like flavonoids, sterols, indoles, saponins and tocols [34], oats also contain a good range of β-glucans (≈ 4.6%) [35]. This is a soluble and viscous fiber (concentrated in the bran) attributed to many of the health benefits given by consumption of oats, like lowering of bad cholesterol [36] and stabilisation of blood glucose and insulin levels [37, 38]. There’s also indications showing β-glucans having a boosting effect on the immune system and its’ defence against bacteria, viruses, fungi and parasites [39]. The dry mix of liquid oat bran (LOB10) used in these experiments contains a well- balanced macronutrient profile (20% protein, 33% carbohydrates and 8.5% fat), and a high fiber content of 19% of which 10% are β-glucans [40]. This fulfils the requirement from EFSA of 4 g of β-glucans per 30 grams of available carbohydrate per meal for reduction in post-prandial glycaemic respons [41] and at least 3 g/d for a blood cholesterol lowering effect [42].

2.5 Protein sources

2.5.1 Casein The protein in bovine milk is to 80% comprised of the four main types of caseins (α-s1, α-s2, β, κ) [43]. Most of these casein fractions exist in colloidal casein micelles (average diameter of 200 nm) together with calcium phosphate, which in definite amounts aids in casein stability against heat coagulation [44]. The interior of the micelle constitutes mainly of α-s1-, α-s2- and β-caseins, forming the hydrophobic core, whereas κ-casein (about 12-15% of total casein) exists mainly on the surface, contributing to stability and

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hindering of aggregation [45, 46]. The heat-stability of casein is relatively high and no noticeable effect has been seen within heat treatments of 70-100°C [47]. The different types of caseins are insoluble at pH 4-5, though the general pH where casein in room temperature precipitate, viz its’ isoelectrical point (Ip), is 4.6 [48]. Isoelectric precipitation of casein is the main factor of yogurt curd formation [49]. The casein used in this project came from Ewalco AB, Sweden, and was a rollerdried calcium caseinate (4574). The protein content was 87.5 g/100g. For amino acid composition, see figure1, below.

Figure 1. Amino acid composition of calcium caseinate from Ewalco AB.

2.5.2 Whey Whey constitutes the remaining 20% of the protein content in bovine milk [43], where of most are globular proteins. The two major whey proteins are β-lactoglobulin (β-lg) and α-lactalbumin (α-lac). Other proteins includes immunoglobulins, bovine serum albumin and various minor proteins and enzymes [48]. Compared to casein, whey proteins are more heat sensitive. Heat treatments at 72 and 87°C for 26 seconds has in a study [50] shown to denaturate 2.8 and 34.1% of the whey proteins respectively. During high temperature pasteurisation (90-95°C for 5 min), most of the whey proteins are denaturated [48]. This denaturation leads to an exposure of otherwise “hidden” sulphydryl groups, which in turn interacts with the κ-caseins on the exterior of the casein micelles, as well as with other serum whey proteins (aggregation). Denaturation to a certain degree may help to improve the final texture of yogurt [51] by increasing firmness and viscosity [49]. In this study a whey protein isolate (ProvonÒ 295 IP) from Glanbia Nutritionals was used, containing 88 g protein per 100 gram. For amino acid composition, see figure 2, below.

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Figure 2. Amino acid composition of Whey protein isolate (ProvonÒ 295 IP) from Glanbia Nutritionals. .

2.5.3 Rice protein Rice is a very important crop worldwide and although it ranks high in nutrional value amongst grains, its’ protein content is very modest with an average of 9.5% (though it can range from 4.3% to 18.2%) [52]. Lysine is an essential amino acid often present in very small amounts among many legume crops and cereals, including rice [64]. The rice protein used in this project was a rice protein hydrolysate (VITALPEP RICE-L NB145) from Alsiano, Sweden, which was designed for maximum compatibility in liquid matrices with an improved aminoacid digestibility. The protein content was 76 g/100g. For amino acid composition, see figure 3, below.

Figure 3. Amino acid composition of rice protein hydrolysate (VITALPEP RICE-L NB145) from Alsiano.

2.5.4 A mix of rice and pea protein Compared to rice, pea is a fairly new protein source in the industry market. Pea isolate is a good source of arginine, lysine and the branched chain amino acids (BCAA); leucine, isoleucine and valine [53]. More information on rice protein can be seen above.

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The protein sample (VegaPRO OPTIMUM) used in this study came from Alsiano, Sweden, and was a mix of rice and pea protein hydrolysates, designed to achieve a high nutritional score in protein indexes, with a PDCAAS equal to milk but free of allergens. For amino acid composition, see figure 4, below.

Figure 4. Amino acid distribution of a mixed protein powder of rice and pea (VegaPRO OPTIMUM), from Alsiano.

2.6 Protein properties

2.6.1 Solubility The solubility of a protein is dependent up on hydrophilic and hydrophobic interactions and affect many of its’ functional properties such as thickening, foaming, emulsifying and gelling [54]. The solubility increases with ionic interactions (protein-water) and depends on electrostatic repulsion at varying pH other than the isoelectric point [55]. A repulsive force between molecules in solution can also form through hydration of ionic groups. The solubility of a protein is mainly dictated by the ratio of hydrophilic and non-polar groups on its’ surface, where a decrease in the latter favours solubility [54].

Most of the proteins in food are acidic and have a minimum of solubility at pH 4-5 (e.g. casein proteins). This is due to a higher amount of aspartic- (Asp) and glutamic acid (Glu) residues compared to the residues of lysine (Lys), arginine (Arg) and histidine (His). A large ratio of hydrophilic to non-polar residues at the surface of a protein can make the electrostatic repulsion greater than those of hydrophobic interactions, making the protein soluble even at its’ pI (e.g. b-lactoglobuline and bovine serum albumin). At otherwise constant conditions, solubility generally increases with heat (0-40°C), except for some proteins like b-casein and certain cereal proteins, which exhibit a negative relationship with temperature [3, 56]. Higher temperature though, can have a negative impact on protein solubility, due to unfolding of the protein structure (denaturation), leading to an increased hydrophobicity. For example, when heating native whey protein isolate to 70°C for 1-10 min, its’ otherwise broad solubility span at pH 2-9 changes with a minimum solubility at pH 4.5 [56]. Also effecting solubility is ionic strength, which

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can bring about different outcomes depending on the ionic strength itself and the hydrophilic to non-polar surface nature of the protein [54].

Proteins are classified into four different groups depending on their solubility characteristics. Proteins soluble in water at pH 6.6 are referred to as albumins and soluble in dilute salt solutions at pH 7.0 are globulins. Soluble only in acid (pH 2) and alkaline (pH 12) solutions are glutelins and prolamines respectively, which are both highly hydrophobic proteins [54].

2.6.2 Emulsification An emulsion is a mixture of liquids that normally wouldn’t mix. Food emulsions of the oil in water type often gives a creamy texture and is created by the addition of mechanical energy and an emulsifier of some kind. This added energy breaks down the oil drops into smaller fractions and enables the emulsifier to adsorb to the droplets, driven by a lowered interfacial tension [54, 55]. This stabilizing layer around the fat droplets, forming a physical barrier to coalition, are in food emulsions [57] often made up of proteins. This interaction happens spontaneously due to the amphiphilic molecular structure of proteins [54]. Casein has a high surface activity and good adsorption characteristics which makes them potent emulsifiers [58]. Physical and chemical factors affecting emulsification are protein properties, its’ concentration, pH, ionic strength, temperature and viscosity, amongst other factors [54, 55].

2.6.3 Rheology In the development of food, measurements of physical properties such as sensory texture and processing needs, can be made through rheological methods. Although, some sensory detection of texture goes beyond rheological properties [54]. In food rheology, the key concepts are stress (force per area) and strain (relative deformation) as a response to applied force. When it comes to fluid viscosity, those referred to as Newtonian fluids show a constant viscosity, independent of time and shear rate [59]. Fluids like yogurt have a viscosity that changes in relation to shear rate. They also exhibit a so called yield stress (minimum force required to initiate flow), which automatically makes them non-Newtonian fluids [54].

2.6.3.1 Viscosity The viscosity, a solutions resistance to flow under an applied force, is of great importance for the product’s acceptability by consumers. Protein solutions exhibit a so called pseudoplastic behaviour, independent of time. An increased shear rate will decrease the viscosity of these solutions. When force no longer is applied, there is a difference in how well the solution regain its’ viscosity, depending on the types of proteins included. Solutions with globular proteins, more rapidly regain their viscosity than do fibrous and are referred to as thixotropic solutions [54].

2.6.3.2 Flowability To investigate the flow of a certain material, a consistometer can be used. It’s an easy to use benchtop instrument which provides a single parameter for a variety of flow tests. Depending on the instrument and standard use, the results in themselves are standardised [60].

2.6.6 Texturization Protein products with texture refers to expectations of chewiness, elasticity, softness and juiciness. The transformation of a proteins physical structure from a globular to a

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fibrous state is indicated to give rise to these sensory properties. Due to the lack of other desirable functional properties, often found in animal proteins, a plant based source of protein is often used to add texture [54].

2.6.7 Flavour binding Flavour and aroma compounds are often small and hydrophobic molecules [61]. The lipophilic nature of fat thereby enables interaction with the flavour and aroma compounds and are important for its’ retention over time [62]. These low mass molecules are also able to bind to hydrophobic patches at the surface of intact proteins or to the hydrophobic stretches of partially denaturated proteins [61].

2.7 Fermentation Fermentation has for a long time been used as a way to preserve different types of foods and may also enhance its’ nutritional value [63]. Depending on nutrient availability, environmental conditions and the microorganisms involved, different foods like alcoholic beverages, blue cheese, and yogurt etcetera can be produced. The most important microorganisms in biotechnology today is Lactic acid bacteria (LAB), yeasts, and molds of Aspergillus spp., Penicillium spp. and Mucorales [64].

The microorganisms used for yogurt production (fermented milk) are lactic acid bacteria. The typical starter cultures for this process include Streptococcus thermophilus and Lactobacillus bulgaricus [65]. They exert a symbiotic relationship, where L. bulgaricus produces free amino acids from the milkproteins present in milk, which are used by S. thermophiles. S. thermophiles in turn produce formic acid which stimulates the growth of L. bulgaricus. Though there are indications saying the interactions may be more complex than this. Anyhow, the production of lactic acid is greater for the mix of cultures than by the cultures alone, where the optimum ratio of the two species are 1:1 [66]. When naturally occurring or added LAB converts carbohydrates and related components to end products such as acids and carbon dioxide, it preserves the food by making it a nutrient deficient and unfavourable environment (e.g. low pH and anaerobic) for spoilage organisms [63]. The acids produced by bacteria such as S. thermophiles and L. bulgaricus, contributing to the decrease in pH and flavour, are for example lactic acid, acetone, diacetyl, aldehydes, alcohols and acetaldehyde, where of the latter is the one said to give the characteristic yogurt flavour [67].

2.8 Sensory analysis Instrumental measurements can only take you so far and to get an idea of consumer acceptance for a specific product, a sensory analysis can be of much value. It aims to identify the human response to a foods different sensory properties, such as taste, color and texture [68]. To quantify the consumers preference or acceptance, a so called affective analysis or consumer test is performed [69]. In order to retain valuable information from the consumer test, it’s essential for it to be performed on the targeted segment group to which the product is developed [68]. Due to high variability in a person’s frame of reference, depending on e.g. psychological factors, previous experience, culture and habit, the sample size should be at least 75-150 individuals [70]. To measure the consumers perception of a food, a so called 9-point hedonic scale is commonly used [71]. The participants tastes the product and are asked to choose one out of nine alternatives, which best represents their opinion in liking or disliking,

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concerning the specific attribute asked for. The design of the 9-point hedonic scale can differ in appearance, where some have verbal labels and some don’t. It can also be presented vertically or horizontally and even unbalanced, containing more likes than dislikes [70]. The results from the analysis should not, according to studies [72], be affected by the differences in design. The order off in which the samples are presented to the participants should be balanced, to minimize errors, though it is known that higher scores is often given to the sample first tried, than if tested after other products [69]. In table III below, you can see the labels and corresponding number of the 9-point hedonic scale used [68].

Table III. The 9-point hedonic scale.

9. Like extremely 8. Like very much 7. Like moderately 6. Like slightly 5. Neither like nor dislike 4. Dislike slightly 3. Dislike moderately 2. Dislike very much 1. Dislike extremely

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3 Materials and Methods

3.1 Experimental trials

3.1.1 Screening of starter culture and addition of whey and casein protein A pre-trial was initially made, based on an earlier work where the most optimal combination of bacterial starter culture for an oat-based yogurt had been examined [16]. This was done to confirm the results and also to try new combinations of proteins (whey and casein). The results from the earlier study concluded that a combination of the commercial yogurt starter cultures, Lyoflora SYAB1 and Lyofast Y350A, were best suited to achieve good taste and texture. The nine different samples of both protein and starter culture combinations is seen in table IV below.

Table IV. Combination of different protein sources and starter cultures made for the first experimental trials. Lyoflora SYAB1 / Lyofast Y350A (50/50) Lyoflora SYAB1 Lyofast Y350A Whey (100%) Whey (100%) Whey (100%) Casein (100%) Casein (100%) Casein (100%) Whey/Casein (50/50%) Whey/Casein (50/50%) Whey/Casein (50/50%)

3.1.2 Effect of different volumes of transferred inoculum on fermentation time For the next trial, the effect of transferred inoculum volume on fermentation duration was examined. A two- and four-fold (40 µL and 80 µL respectively) increase in transferred starter culture compared to the volume (20 µL) used in an earlier study [16] was tested. This was performed on samples including two different ratios of casein and whey protein; 25:75 and 50:50.

3.1.3 Addition of vegetable proteins With a desire of making the yogurt milk free and vegan friendly, the milkproteins were replaced with two different vegetable protein sources. The proteins used came from Alsiano, which of one was a pure rice protein hydrolysate (Vitalpep Rice L NB145) and the other included a mix of rice and pea protein (Vegapro optimum NB168). Half of the samples included the same weight amount (6.93 wt%) of protein as for the added whey and casein in earlier trials and the other half was adjusted (rice 7.97 wt% and vegapro 7.58 wt%) to represent the same amount of pure protein in gram, as the protein sources just mentioned (the milk proteins have a higher protein content per 100 g).

3.1.4 Lowered heat treatment With suspicion of a too extensive denaturation of vegetable proteins during high temperature pasteurisation (95°C for 5 min) due to bad off-flavour, a trial of lowering the heat exposure in this process to 72°C for 5 min was tested.

3.1.5 Optimum ratios of whey/casein, increased LOB and addition of fruit purée Further trials were made to examine the optimal ratio of casein and whey protein; 30:70 and 40:60. In an attempt to receive a thicker texture for the vegetable protein yogurts (rice and vegapro) the addition of added oat flour (LOB) was increased to 12 and 14%.

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The original recipe of added LOB is 10%. Addition of different fruit purées (20%) to all samples (after fermentation) was also made.

3.1.6 Different fruit purée concentrations and texturizer Further development of yogurts with a casein and whey ratio of 40:60 was made. Two different amounts of fruit purées were tested (25- and 30%). A re-examination of the combination of Lyoflora SYAB1 and Lyofast Y350A vs SYAB1 alone was also made during this experiment. Addition of extra salt was made to some of the samples with the aim to enhance taste. To increase viscosity, a texturizer (0.3% GENUÒ texturizer type YA-100, CPKelco) was added toghether with the added fruit purée.

3.1.7 Lowering of casein and an increase in pH The protein content in further trials constituted of samples with a casein/whey ration of 5:95 and 10:90. Samples with only whey (100%) were also tested, whereof some were increased with additional 30% whey protein. Addition of fruit purées and texturizer were made with 30% respectively 0.3%. The pH for samples including casein were slightly increased to around 5.2 in an attempt to affect the proteins’ physio-chemical properties (increase solubility and decrease protein aggregation).

3.1.8 Adjustments to the pasteurisation step and increased content of texturizer In the final experiment an adjustment to the pasteurisation process was made on samples including 10% casein and 90% whey. Changes were made through improved stirring by exchanging the handstirring of closed bottles in a pot of boiling water to having the samples in big beakers (covered with aluminium foil) with electrical stirrers in a water bath at 88°C. The samples were heated to 72°C for 5 minutes. Addition of 30% fruit and berry purée was made as above and the amount of added texturizer was increased to 1.2%. An additional increase (33%) of the salt content was also made, resulting in a total increase of 100% from the original recipe.

3.2 Sample preparation

3.2.1 Preparation of Glucanova oat base (10 wt%) To a beaker, 900 g of tap water with a temperature of 62°C was measured up on a OHAUS Pioneer™ weight scale. To this, 100 g of a commercial flour mix called LOB10 (liquid oat bran) from Glucanova AB in Lund, Sweden, was added. The mixture was then stirred with a whisk until no lumps remained. The beaker was thereafter placed in a 65°C-water bath for an hour with an electrical stirrer. After that the water and flour mix was heated in a microwave until boiling (repeated twice). Stirring of the mixture was made with a metal spoon, both in-between heating and afterwards. The beaker was then put in an ice-bath, until cooled to approximately 20°C. Finally, the mixture was homogenized with a stick blender, and measurements for pH, viscosity and dry matter was made to ensure repeatability. Target values for these measurements were as in the former in the order 6.6, 15-25 mPas and about 10% respectively.

3.2.2 Preparation of “Skaka & Smaka” oat base (yogurt substrate) All ingredients (see recipe in table V), except the protein source, was measured up and whisked together in a glass beaker until a homogeneous solution was achieved. The mixture was then poured into a sauce pan and heated to 65°C. The pan was removed from the stove and the protein source was carefully added to the hot solution under

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gentle stirring (avoiding foaming), until homogenous. The solution was then transferred to a beaker and put in an ice bath to cool down to approximately 20°C, after which pH, viscosity and dry matter was measured.

Table V. Recipe for “Skaka & Smaka”.

Removed due to confidential information

3.2.3 Preperation of starter culture inoculum 100 mL of oat milk from Oatly (1.5% fat) was weighed up in a glass bottle, the lid was fastened and the bottle was placed in a 42°C water bath until the milk reached approximately 30°C. It was then transferred to a fume cupboard for sterile working. The top of the commercially available starter culture Lyoflora SYAB1 (Kemikalia AB, Sweden) pouch, containing a dose of 5 UC, was sanitized with chlorine before opening. The whole pouch of starter culture was then poured into the lukewarm oat milk, the lid was fastened and the bottle was shaken by hand until the culture content was completely dissolved. Bottles were then stored at 4°C.

3.4 Fermentation 100 mL of “Skaka & Smaka” oat base was poured into a glass bottle (100 mL) and then heated to 95°C in a boiling water bath, where they were held for 5 min. They were then cooled down to 46°C in an ice bath, after which they were transferred to a fume cupboard for sterile transfer of the pre-prepared inoculums. Using an automatic pipette, 80 µL of the starter culture inocolum was transferred to the “Skaka & Smaka” oat base (yogurt substrate). To make sure of an even distribution of the bacteria in the oat base, the bottles were then shaken by hand.

3.4.1 Bacterial strains The commercial starter cultures used in this study were chosen based on the results from an earlier study [16] performed at Aventure and were named Lyoflora SYAB1 and Lyofast Y350A (Kemikalia, Sweden). They were both freeze dried, included 5 UC (standard units for a cultures fermentative activity) per pouch and had an optimum activity at 43°C. SYAB1 contained no animal based products and was thereby suitable for vegetarian yogurt options as well. The acidification capacity of the cultures were 3.9 and 4.1 for SYAB1 and Y350A respectively. For strains included in each starter culture, see below.

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Lyoflora SYAB1: Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, Bifidobacterium animalis ssp. lactis.

Lyofast Y350A: Streptococcus thermophiles (EPS), Lactobacillus delbrueckii ssp bulgaricus.

3.5 Addition of fruit purée and texturizer The texturizer (1.2% GENUÒ texturizer type YA-100, CPKelco) was measured up in a small plastic cup, after which a concentration of 30% fruit pureé was weighed into a glass beaker. The texturizer was carefully shifted into the fruit purée under constant stirring with a plastic/metal spoon. The mixture was then placed in a microwave and brought to boil, after which it was stirred again. The liquid was set to cool, either in an ice-bath or in the fridge. When cooled down to about 35°C, the fruit purée with texturizer was poured into the fermented oatbase and mixed with a spoon until evenly distributed.

3.5.1 Fruit and berry purées The fruits and berries used in this project, to add flavour to the yogurts, came from a family owned company Hafi, Sweden. The recipes are pre-developed in cooperation with a company called Berries by Astrid, Sweden. Although all three recipes contain both fruit and berries, it’s named as “fruit purée” throughout the text. The different content for the three flavours “skogen”, trädgård” and “havet” is seen below. The energy content differs from 76 – 100 kcal per 100 grams.

Skogen: raspberry (KRAV*), strawberry (KRAV*), blueberry (KRAV*), banana, apple juice (KRAV*).

Trädgård: redcurrants, apple purée, strawberry (KRAV*), pear juice.

Havet: mango, sea buckthorn, apple juice (KRAV*), salt.

* The KRAV label stands for organic produce with high demans on animal care, health, social responsibility and environmental impact.

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3.6 Analytical measurements

3.6.1 pH Measurents of pH were done on homogenised samples at room temperature (22°C) to make sure that the quality target values for the different oat bases were reached, as well as for controlling that a satisfied level of fermentation had been achieved, which affects the taste. The instrument used was a calibrated Mettler Toledo FE20 FiveEasyä EL20 pH-meter.

3.6.2 Dry matter Measurements of dry matter was carried out using a Mettler Toledo HB-43S moisture sensor. A filter paper was placed in the sample pan of the instrument and the lid was closed for an automatic reset. When finished, a small amount of homogenised sample (room temperature) was transferred, with a disposable plastic pipette, to the filter paper until the instrument showed a symbol that enough sample volume was reached. With the help of the disposable pipette, the sample was thereafter slightly smeared out on the filter paper to minimise the surface tension and to increase exposed sample area, in the attempt of getting a more accurate analysis result. The lid of the instrument was closed and the analysis automatically started. Results were shown on the instrument display after about 6-12 minutes.

3.6.3 Rheometry The variance in viscosity dependent on variations in temperature were measured using a Kinexus pro rheometer (Malvern). The geometry used was 4V21 SC0001 SS : PC25 C0052 AL and the settings for shear rate, test time and sampling interval were 30 s-1, 1 and 5 minutes, respectively. Starting temperature was set to 7°C and end temperature to 37°C, with a temperature interval of 5°C. The instrument was calibrated using the zero gap setting, after which the sample was loaded into the sample container and the measurement was started. Sample measurements were made in duplicates. Since the measurment at each temperature only lasted for 1 min, another measurement at only 37°C was performed during 5 min. This was to investigate if the viscosity would stabilize or not.

3.6.4 Viscosity To measure the viscosity, a Brookfield Digital Viscometer Model DV-E was used. The rotation speed was set at 30 or 60 rpm, depending on the thickness of the sample. The size of the spindle could also differ depending on the same attribute. Spindle 2 was used on thinner samples and spindle 3 and 4 was used for thicker samples. The analysis was performed on homogenised samples (room temperature) which were placed in a plastic container, filling it all the way to the top line. The container was thereafter placed under the spindle and manually moved upward until the spindle thoroughly had immersed into the sample. A plastic box was placed under the container to keep it in a good position. The motor of the instrument was turned on, correct settings were made and the viscosity value (mPas) was measured at the indication of stabilization.

3.6.5 Flowability Consistency, as a distance of flow, was measured using a standard 30 cm scaled Bostwick consistometer in accordance with ASTM F1080 - 93(2013). It was made sure that the instrument was placed on a flat surface through adjustable screws and a built in spirit level. The sample compartment (width = 5cm and height = 4 cm), which held 100

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mL of sample was filled to the top with yogurt and levelled with a dough-scraper. Flow of the fluid sample was initiated by a sharp opening of the sample compartment gate and the distance travelled over the stainless steel track, engraved with a series of graduation with 0.5 cm intervals, was measured after 30 sec. Measurements were done, both of the maximum reading at the centre of the shaped liquid and at the minimum reading at the edge of the trough, from which an average value was given. The trials were made in triplets, for samples at both refrigerator temperature (≈ 7°C) as well as room temperature (≈ 22°C).

3.6.6 Brix To measure the sugar (sucrose) content in the samples, a HI 96811 refractometer was used. The measured value is expressed in °Brix, which represents the amount of sugar content expressed in percentage, that is, 1 °Bx = 1 g sugar per 100 g of sample. Calibration of the instrument was performed by placing distilled water with a plastic pipette in the sample container and making sure there were no bubbles. To shut out any intense light, the sample container was covered (by hand), followed by pressing the “zero” button. If no error message appeared, the calibration was successful and the distilled water was removed using the same pipette and a soft tissue, after which the sample to be analysed was added in the same way as before. The sample container was covered and this time the button “read” was used and the analyse results appeared on the screen.

3.6.7 Theoretical analysis of nutritional composition The theoretical nutritional content of the final products were calculated in excel by multiplying the weight (grams) added of the specific nutrient with the percentage of which it was present in the raw material/product sample. This was done individually for the total content of protein, carbohydrates, dietary fiber, b-glucans, fat and salt. For calculations of total energy content, the results from the former calculations were used and multiplied with the corresponding value for kcal/gram. Values used were; 4.06 (protein and carbohydrates), 8.84 (fat) and 1.9 (dietary fiber). For the fat content of rapeseed oil a value of 9.00 was used in accordance with the nutrition label of the product packaging. The energy content for the fruit purées were calculated by multiplying the energy content, labeled in the specifications, with the added amount of 30%.

3.7 Sensory analysis The sensory consumer test was performed at two different retirement homes in Hässleholm, Sweden. The first one was at Ehrenborg, where only 2 individuals were able to participate (both women). At the second retirement home, called Högalid, 9 individuals participated (seven women and two men). A total of 11 individuals participated. All were at the age of 75 or above. The ambition was to have a sensory analysis at a gym as well, but due to time limitations, it just wasn’t possible.

The participants were introduced to the concept of the products, as well as the project behind it, and were asked to fill out a related questionnaire, one sample at a time. The design of the hedonic scale used in the questionnaire was presented in a horizontal way, with some questions only having three alternatives with a verbal label and some being all verbally labeled. This was to reduce the information needed to be processed by the participants, but still understanding the proportion of the scale. The order of in which the two yogurts selected for testing, “skogen” and “havet”, were served was balanced

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among the participants. Without trying the product, the participants were first asked to give an opinion of their first impression. Following consumption, they then proceeded to answering different questions about specific attributes related to the yogurt tested. All these questions were answered, based on a 9-point hedonic scale, by putting an “X” in the box which best correlated with the participants opinion. The label represented by box number 1 = Dislike extremely, 5 = Neither like nor dislike and 9 = Like extremely (for full version, see table III). After trying both products, the end of the questionnaire contained some general questions regarding sample preference, ranking of attributes, consumption frequency, the need of this kind of product and general appetite. For questionnaire and presentation sheet, see Appendices A.

3.8 Statistical analysis To see if there were any significant statistical differences in the average values (no separation between men and women) of attribute score, between the two final yogurts, a two-tailed and paired Student t-test was performed (excel). Due to the low sample size of individuals, there was no reasons to preform a more advanced statistical analysis. Evaluation of the results originated from the null hypothesis that there was no significant difference between the samples, with a confidence interval of 95%.

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4 Results

4.1 Experimental trials

4.1.1 Screening of starter culture and addition of whey and casein protein Trying different combinations of the two starter cultures, Lyflora SYAB1 and Lyofast Y350A, gave no detectable difference in taste or texture. It was obvious though that Lyoflora SYAB1 results in a faster decrease in pH with as much as 0,85 - 1,1 (depending on included protein sources) during 8,5 hours of fermentation. The combination of different ratios of casein and whey protein gave a too sturdy texture for samples including 50:50 of casein and whey as well for the ones which included 100% casein. All nine samples displayed a more or less grainy texture.

4.1.2 Effect of different volumes of transferred inoculum on fermentation time Comparing inoculum volumes of 40 versus 80 µL (2- and 4-fold increase) didn’t have a huge difference on the rate of which the pH decreased, that is, no significant difference in fermentation time. After 8 hours of fermentation the difference in pH between 40 and 80 µL of added inoculum to samples including 25:75 casein to whey were only 0.07. For samples including a ratio of 50:50 of the above mentioned protein sources, the dissimilarity in pH lowering were 0.36. The lower pH values were in both cases attributed to the added inoculum volume of 80 µL. The decreased (25%) addition of casein in favour of the same increase in whey, giving a ratio of 25:75, gave a less sturdier texture than samples with a 50:50 ratio. All samples still displayed a grainy texture.

4.1.3 Addition of vegetable proteins In an attempt to make the yogurt vegan, the milkproteins (casein and whey) were replaced with two different vegetable proteins; rice and a mix of rice and pea (vegapro). All samples had a very undesirable after-taste and it didn’t seem to make any difference between the lower and higher amounts (100% vs. 115% for rice and 109.4% for vegapro) of added protein. A relative normal lowering in pH was observed with a mean value, after 8 hours of fermentation, of 4.63 (rice 100%), 4.65 (rice 115%), 4.87 (vegapro 100%), and 4.86 (vegapro 109.4%). The sensory viscosity (by eye and mouthfeel) were very low for all samples and had a somewhat shiny/elastic appearence. Vegapro had a slightly thicker texture but the difference was minor. Compared to the samples with milkproteins the color of the samples including only vegetable proteins were darker/browner.

4.1.4 Lowered heat treatment To see if the unfavourable after-taste for the vegetable proteins were due to too much denaturation of the proteins during pasteurisation, the temperature in this process was lowered from 95 - 72°C. No effect on either taste nor viscosity was observed.

4.1.5 Optimum ratios of whey/casein, increased LOB and addition of fruit purée In an attempt to increase the viscosity of the yogurts with added vegetable proteins, an increase of oat flour (LOB10, Glucanova) to the oat-base was made with 2- and 4%. The viscosity of all samples increased when the percentage of added oat flour increased. A slightly higher sensory viscosity (by eye and mouthfeel) was seen with the vegapro protein compared to the ones only having added rice protein. The undesirable after-taste was still very noticeable, although it seemed to be less in samples containing vegapro

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protein. Vegapro with 14% LOB exhibited the least off-taste, although it was still very bad. In this experiment two different ratios of casein and whey protein; 30:70 and 40:60 was also examinated. They were both similar in taste and viscosity. The 40:60 ratio were slightly more acidic and had a smoother texture. Both ratios had a grainy texture, although this feature was less noticeable within the samples with 40% casein and 60% whey. Addition (20%) of three different and separately added fruit purées (“havet”, “trädgård” and “skogen”) were not able to disguise the unwanted after-taste of the rice and rice/pea proteins. Neither did it anything on minimizing the grainy texture of the yogurts with added casein and whey. It gave the yogurts a nice color (yellow, pink- peach and purple-pink) and taste, though the latter would favour from a bit more intensity. The flavours “skogen” and “trädgård” were preferred over “havet”. A slight decrease in pH and dry matter was observed after addition of the purées.

4.1.6 Different fruit purée concentrations and texturizer Further trials were done on yogurts with the addition of 40% casein and 60% whey. An introduction of the starter culture Lyofast Y350A was also made to make sure that no hasty decisions had been made by excluding it in the early beginning. Addition of either 25- or 30% of fruit-purée were added to the yogurts as well as 0.3% texturizer. An increase by 67% of added salt was made to a few samples to investigate if it made any difference to the intensity of the flavour. Once again, no noticeable difference was observed with the addition of Lyofast Y350A. The viscosity by taste and visual inspection was quite high, similar to that of a mildly set yogurt, though the texture was still grainy. Addition of 30% fruit purée was preferred, by the conductor, over the addition of 25% because of a better intensity in flavour. The increase in added purée favoured the taste of the flavour “havet”, which led to a change of preference, by the conductor and supervisor, for “skogen” and “havet” instead of the earlier choice of “skogen” and “trädgård”. The experienced acidity of all yogurts were quite high. The mean pH value after fermentation and the addition of fruit purée was 4.22. No increase in flavour enhancement was observed by the addition of extra salt.

4.1.7 Lowering of casein and an increase in pH The casein/whey ratios were altered to 5:95 and 10:90 in an attempt to minimize the grainy effect of precipitated casein. Samples including either 100% or an extra addition by 30% (more than the original recipe) whey, were also included. Samples including casein gave a viscosity similar of that of a “pourable yogurt” and had a fairly smooth and soft texture but the grainy consistency was still present, though less than in previous samples. The casein/whey ratio of 10:90 gave a somewhat thicker yogurt, a less sandy consistency and appeared slightly smoother to the eye than the 5:95 ratio. Samples including 100% whey had similar properties to those above but exhibited a more grainy texture. Samples including 130% whey were even more grainy. The acidity of samples having added casein were good and had a mean pH value of 5.14 after four hours of fermentation, which decreased to 4.37 after having added the fruit purées and having the samples stored in the fridge overnight. The mean pH value for those containing only whey after six hours of fermentation were 4.73 and decreased to 4.26 after the addition of the purées and stored under the same conditions just mentioned.

4.1.8 Adjustments to the pasteurisation step and increased content of texturizer Changes in the pasteurisation method resulted in a smooth yogurt with no grainy texture (without added purée or texturizer). This at the expense of a huge loss in viscosity, generating a “half-thick” liquid consistency. Having added the fruit purée and texturizer (0.12%) as well as letting the yogurt sit in the fridge for about a day, the texture was a

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bit granular. This was mostly seen for the flavour “skogen” and could be compensated for by mixing the cold (6°C) yogurt with a stick mixer. It was hard to evaluate the specific affect on flavour enhancement by the extra addition of salt, but the taste of salt itself couldn’t be identified and the taste was fairly good.

4.2 Final products The two final products developed had an addition of 10% casein and 90% whey, as the primary protein source. The fruit purées selected were “skogen” and “havet”, which were added in the amount of 30%. The average pH value for “skogen” and “havet” was 4.24 and 4.13, respectively. Dry matter and sugar content (sucrose) for “skogen” was 27.7% and 20% respectively. For “havet”, the dry matter was 29.2% and the sugar content 20.2%. The viscosity (measured with a Brookfield viscometer) for “skogen” was slightly higher than that of “havet”, with a value of 118,7 versus 67,5 mPas (60 rpm, spindle 4). The change in pH during fermentation was also measured and is seen in figure 5.

Figure 5. The change in pH during the fermentation process of the final product.

4.2.1 Flowability The average values when measuring the flow properties of the two yogurts “skogen” and “havet” at 7°C and 22°C can be seen in table VI below.

Table VI. Average values for flowability of “skogen” and “havet” in cm. 7 °C 22 °C Skogen 10.93 12.15 Havet 15.29 16.05

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4.2.2 Rheological measurements To see the change in viscosity from a refrigerator (≈ 7°C) temperature until it reaches the mouth (≈ 37°C), rheological measurements were performed under this temperature interval at 30 rpm. The results clearly show a decrease in viscosity at increased temperature. For each temperature measurement, which lasted for 1 min under constant shear rate, a decrease in viscosity was seen. As the instrument was stabilizing from one temperature interval (5°C) to the next, there was no applied force to the sample. When stabilized at the right interval temperature, force was once again applied and during this “hold”, a slight increase in viscosity (see arrow in figure 6 for example) was observed. This increase in viscosity became less obvious with an increase in temperature. As also can be seen in figure 6, the viscosity (during all temperatures) of “skogen” was quite higher than that of “havet”.

Figure 6. Results for viscosity from rheometrical measurements during a temperature interval of 7 – 37°C at 30 rpm. Product “havet” is seen furthest down (yellow) and “skogen” is on top (pink). The grey arrow represent the slight increase in viscosity when share rate is null and the instrument is stabilizing to the next temperature.

After performing measurements at only 37°C for five minutes, from what can be seen in figure 7, it seemed like the first 10-20 seconds were most crusial to the decrease in viscosity, after which it begun to stabilize. Figure 7 only show the data for “havet” because of problems in transfer of data between computers for “skogen”, but both yogurts exhibited a similar behavior.

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Figure 7. Results for viscosity from rheometrical measurements over time at 37°C for the product “havet”.

4.2.3 Theoretical nutritional composition The theoretical protein content were the same for both “skogen” and “havet, as were the content of salt, dietary fiber and b-glucans. A minor difference in fat, carbohydrate and energy content was seen between the two yogurts, where “havet” had the higher values for all parameters just mentioned. The energy percentage for the macronutrients were all within the interval of the Nordic Nutrition Recommendations (2012), except the fat content for “skogen” which were 1% above the higher limit of the recommended interval. For compilation of data, see table VII below.

Table VII. Theoretical nutritional value and energy percentage of the macronutrients for the two final products, “skogen” and “havet”. Nutritional component Skogen E% Havet E% Energy (kcal) 135 142 Protein (g) 5.6 17 5.6 16 Carbohydrate (g) 14.2 43 15.1 44 Dietary fiber (g) 2.2 2.2 - β-glucans (g) 0.53 0.53 Fat (g) 6.3 41 6.5 40 Salt (g) 0.23 0.23

4.2.4 Final recipe The specified ingredients for the final products can be seen in the ingredient list just below. In table VIII, a more general recipe in g per 100 grams, is shown. In figure 8, the appearance of the actual products, used for sensory analysis, can be seen.

Ingredients: Removed due to confidential information

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Table VIII. General recipe for the final yogurt products.

Removed due to

confidential information

Figure 8. The two final yogurt products, used for sensory analysis. Shown to the left is “havet” and to the right is “skogen”.

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4.3 Sensory and statistical analysis The sensory analysis consisted of 11 induviduals at the age of 75 and over. Among these participants there were 9 woman (82%) and 2 men (18%). When comparing the answers from the seven related attributes for each yogurt sample, no significant statistical difference was observed. The p-values ranged from 0.28 – 0.85, where the lower value was related to opinions about taste, in favour of “skogen”, and the upper value concerned first impression. The average values for all attributes of the different yogurts tested is seen in table VIIII.

Table VIIII. Average values of different attributes from the sensory analysis of the two yogurts “skogen” and “havet”, where 9 is the highest score (like extremely) and 1 the lowest (dislike extremely).

First Total impression Color Spoonability Taste Consistency Thickness impression Skogen 5.91 6.27 5.91 5.73 5.82 6.00 5.80 Havet 5.82 6.09 5.55 4.82 5.45 5.55 5.90

When the participants were asked to give suggestions for further improvements of the yogurts, most answers concerned taste and texture. For all the participants comments, see table X below.

Table X. Participant’s comments about suggestions for improvements of the yogurts “skogen” and “havet” (not everyone gave an answer). Suggestions for improvements

Skogen Havet Could be a little bit thicker Could be a little bit thinner Remove the bitter taste Could be a little bit thicker "Floury" taste "Floury" taste Needs more taste Needs better taste Gruff feeling on the tongue Gruff feeling on the tounge (2) A little strong (2)

When asked which of the yogurts they rather preferred, 6 individuals (55%) answered “skogen”, and the remaining 5 people (45%) answered “havet” as their preference of choice. They were also asked to give a reason to their preference, see table XI. When asked to rank which of the attributes; color, taste and consistency, that were most important for general liking, taste was in 73% of the case marked as the most important attribute. Second most important in 45% of the case was consistency, and color was in 55% of the case marked as the least important attribute for liking. Seven of the participants (64%) said that they could consume a product of this kind a few times a

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week, 2 individuals (18%) said more seldom, 1 participant (9%) said daily, and 1 (9%) wouldn’t be able to consume it at all. All individuals participating thought that this kind of product was needed and when asked about their general appetite, 9 claimed they had a good appetite (I feel hunger and I enjoy eating) and 2 induviduals said their appetite were neither good nor bad (I sometimes feel hunger and enjoy eating from time to time).

Table XI. Reasons given by participants from the sensory analysis for their choice of preference (not everyone gave an answer). Reasons for preference

Skogen Havet Like the color Viable Has a better taste (2) Nicer color when served It was a little bit weaker in taste Best taste Good thickness and nice taste The other one was too thick More flavour

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5 Discussion Having tried addition of different protein sources to create a balance of macronutrients to an oat-based yogurt, it’s clear that there are many different factors and properties of the proteins, affecting the final results. The struggle in an earlier study [16] of developing an oat-base yogurt, conducted at Aventure, was to reach a good viscosity and flavour.

5.1 Addition of casein and whey protein Most of the trials in this project gave a higher viscosity compared to the earlier study mentioned above (although they only added whey), but it also came with a grainy texture. It was thought to be an effect of too much denaturation of the proteins, but there was also a suspicion of precipitation of the casein proteins. Whey protein is less heat- stable than casein [47, 50], and having a pasteurisation step that was too stagnant (no stirring), most possibly led to an uneven heat distribution, making the whey proteins denaturate (unfold), hence, creating a grainy structure. Since the samples with only casein exhibited a much sturdier and more grainy texture than the samples containing only whey, and with the fact in mind that casein should be able to withstand denaturation under the temperatures exerted [47], it’s indicated that there is a precipitation of the casein proteins. Having the yogurts ferment to a pH around 4.4 – 4.7, this is very reasonable since the isoelectric point (minimum solubility) for casein is 4.6 [48]. In an attempt to gain some of the benefits of caseins ability to create a viscous network within the solution and to lower the effect of precipitation, the amount of added casein was lowered and so was the time of fermentation, to give the yogurt a higher pH (around 5.2). These changes, as well as an optimization of the stirring during pasteurisation and a lowering of temperature (72°C for 5 min), to reduce denaturation of the whey proteins, resulted in a very smooth texture of the yogurt. The viscosity, on the other hand, was a bit more runny than aimed for. In an attempt to compensate for this loss in viscosity, the amount of added texturizer was increased by 400% from 0.3 to 1.2 g per 100 grams of flavoured yogurt. Unfortunately this wasn’t enough to receive a desirable viscosity.

After storing the samples with a 10:90 casein to whey ration in the fridge for a couple of days, which were produced using the more stagnant version of pasteurisation (possibly causing denaturation of whey proteins to a certain extent), the grainy texture seemed to diminish, while still having a good viscosity (this discovery though was made after making the final yogurts for the sensory analysis). This may be an indication of that a certain amount of whey denaturation may favour the viscosity and that this could be done while still getting a smooth texture. The adjustment to the lower temperature of 72°C during pasteurisation as well as increased stirring (a more even heat-distribution), might have decreased the denaturation of whey proteins too much, resulting in a too loose yogurt. In an earlier study [50], temperatures of 72°C only denaturated 2.8% of the whey proteins. Time is also of importance when it comes to heat treatments, and though the duration of the heat treatment in that study only lasted for 26 seconds, compared to 5 min used in this project, it still may be a clue to that 72°C generates an insufficient denaturation of whey proteins when it comes to creating a yogurt with a relatively high viscosity.

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5.2 Addition of starter culture In every trial performed to compare the two different starter cultures; Lyoflora SYAB1 and Lyofast Y350A, the conclusion was made that the latter made no noticeable difference in either taste or texture. Lyoflora SYAB1, had in comparison, a much better ability to decrease the pH over time, which made the fermentation process more effective and therefore the starter culture of choice.

5.3 Addition of vegetable proteins Two different protein samples of vegetable origin were tested, where one included only rice protein and the other one was a mixture of both rice and pea protein. Although these protein sources had been used before (different form or supplier) at Aventure in similar projects, these specific kinds were developed to have a good solubility, which had been a challenge in the past. Both the rice and rice/pea proteins had a good solubility but didn’t contribute to an increase in viscosity other than that created by an increase of dry matter. A further increase of dry matter was used to improve the viscosity by adding a higher amount of oat flour (12 and 14%). An increase in viscosity was seen (by eye and mouthfeel) in correlation with the amount of added oat flour, but it was still far from the viscosity desired. The most distinctive attribute for the samples with added vegetable proteins were its’ unfavourable and bitter after taste. An addition of 30% fruit purée were not able to disguise it, although an increase of oat flour seemed to lower this off-taste to some degree. The vegetable protein source containing both rice and pea, had less of this undesirable attribute than rice alone. The lowering in temperature during the pasteurisation process, did nothing on minimizing this undesired after taste. When tasting the protein powder itself, the same bitter taste was observed, indicating that the unfavourable taste of these proteins could be due to earlier extraction and/or processing steps made by the supplier. Because of time limitations, further investigations to improve these properties were not able to be carried out.

5.4 Addition of fruit purée From the three different fruit purées added; “trädgård”, “skogen” and “havet”, the two last mentioned were selected for having the best taste as well as color. From the three different proportions of 20, 25 and 30% of added purée, the latter was chosen for giving the best taste intensity. The taste might benefit from being even more intense, i.e. adding more fruit purée, but then one must also take into consideration its’ effect on viscosity. Although both flavours gave a fairly good taste, it still had a fairly distinct taste of oat. This was mostly seen for the taste “skogen”, where as “havet” better disguised the taste of oat.

5.5 Final products The two final products of this project, “skogen” and “havet”, were both good in color and acidity. The taste was fairly good, but needs further development. As for the viscosity, it needs to be increased to receive the desired thickness of a more spoonable yogurt.

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The average values received for the two yogurts from the measurments of flowability using a consistometer are standardized within the ASTM F1080 - 93(2013) standard and clearly shows a lower viscosity for “havet” compared to “skogen” since it travels a further distance.

From the data received through rheological measurements, the relationship of a decreased viscosity with an increase in temperature is obvious. Also observed is a decrease in viscosity during the measurments by the rheometer instrument at a set temperature with constant applied force. When the instrument stabilized from one temperature to another, there shear rate was null. During this time, a slight increase in viscosity was seen. These to phenomenon indicate on a pseudoplastic and thixotropic kind of behaviour, which is to be expected for yogurt solutions. Figure 6, clearly also show a lower viscosity for “havet” than that of “skogen”, which is due to the different fruit and berry compositions of the fruit purées. The slight decrease in viscosity during the first 10-20 seconds at a constant applied force at 37°C, adds to the importance of physical analyses in combination with sensory testing, though the physio-chemical properties of a product may change during consumption.

The theoretical nutritional composition exhibit a good balance between protein, carbohydrates and fat, of which all are in good correlation with the energy percentage recommended by NNR (2012). A yogurt portion of 250 g represents 17.5% (“skogen”) and 18.3% (“havet”) of the daily energy need (in accordance with NNR 2012) and 14.7- 19.6% of the recommended daily intake of protein for women in the age of 61-74. The same yogurt portion covers 14.6% (“skogen”) and 15.3% (“havet”) of the energy need and 12.3-16.4% of the protein content needed on a daily basis for men in the same age group. The average calori content for 250 g of the two yogurts covers, according to SOK [28], the energy expenditures of 43-49 minutes of strength training or 21-39 minutes of running (12 km/h) for people at the weight of 75-65 kg.

5.6 Sensory and statistical analysis The sample size of individuals participating in the sensory analysis was very low and far from the 75-150 people needed for a representable outcome. The consumer test performed was still of important value, though it gave an indication of that the product is needed and of in what direction further product development needs to proceed. The results regarding thickness of the products were somewhat contradictory and might have been due to the viscosity being a bit “in between” that of a yogurt beverage and a stirred one, which could have confused the frame of reference for some individuals. More obvious was that a further development in taste is needed and important, though it was the most favoured attribute for general liking. Overall, the average value of the seven different attributed for both yogurts were quite good, ranging from 4.82 – 6.27 on a scale of 9 representing “extremely like”. The sensory analysis and statistics show potential for this kind of product in the target group of elderly people.

5.7 For future development If further developments were to be made on these products, an attempt to disguise some of the intense oat flavour should be made. There might also be an interest to look for alternative flavours/purées which works in a better harmony with the oat base, though

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the fruit purées used in this project somewhat enhanced the taste of oat. A multiple choice of flavour might also be of value to fit the different segments of the elderly and those who are very physically active. Due to time limitation, no analytical measurements were made on total energy- (bomb calorimeter) nor protein content, which would be of interest if further development were to be continued. Also of interest, would be to analyse the sustainability of the product over time, as well as bacterial survival.

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6 Conclusion Although the individuals of the sensory analysis were few, it gave a clear indication of that a product, as the one developed in this project, is needed in the care of elderly. It’s an energy dense yogurt with a balanced macronutrient composition. Meant as a healthy “in between snack”, to help individuals in this age group to get the sufficient energy and nutrients they need, on a daily basis. This kind of product is also considered to fit a segment of individuals with a higher level of physical activity, but a sensory analysis still remains to be performed on this target group.

The products developed, “havet” and “skogen”, is thought to have good potential, although further development in taste and texture is necessary. Further development should focus on finding a flavour, better able to disguise the after taste of oat and an optimization of the pasteurisation process (time and temperature), for a desirable denaturation of the milk proteins, which seemed to have the most impact on thickness and texture.

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References

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Appendices Appendix A Presentation and questionnary for sensory analysis

Hello! My name is Frida Sjöberg and I study the Nutrition and Food Science bachelor program at Linnaeus University in Kalmar.

At the moment, I am doing my final degree project at Aventure AB in Lund, a company that scientifically develops products with a good health impact.

UTVECKLAD PRODUKT The product you will taste today is an energy-dense yogurt with a balanced nutritional value of protein, fat and carbohydrates. The yogurt is oat-based and flavored with real fruit- and berry purées, without any additives. The source of protein is whey and casein (milk proteins). Note that there may be traces of soy and lactose.

The product is primarily designed as a healthy and balanced snack for individuals in need of increased daily intake of energy, such as older and physically active.

FACT THAT SAYS BECAUSE!

150 140 ENERGY (KCAL/100 G)

PROTEIN (G/100 G)

FAT (G/100 G)

CARBOHYDRATES (G/100 G)

FIBER (G/100 G)

45 34

15 11 5 ,6 6 2 4 0,4

OAT PROVIVA FRESUBIN BASED (ROSE HIP) JUICY YOGURT DRINK

I

Consumer test

Welcome to a yogurt taste test!

Gender: Woman Man

Age: 65–75 75 +

Number of the first yogurt you try: ______

1. Start with one sample at a time 2. Place an ”X” in the box that best fit your opinion (1 = Dislike extremely and 9 = Like extremely) 3. Please drink some water in between the samples 4. It may be wise to save some of the product in order to make a final comparison in the end. 5. In case of questions, please contact the test leader.

Participation in the taste test is voluntary and the answers are anonymous!

Welcome to taste your first yogurt!

Please proceed to the next page

II

What is your first impression?

Neither Dislike like nor Like extremely dislike extremely

1 9

What do you think about the color?

Neither Dislike like nor Like extremely dislike extremely

1 9

What do you think about the spoonability?

Neither

Dislike like nor Like extremely dislike extremely

1 9

Please proceed to the next page

III

What do you think about…

Dislike Dislike Dislike Dislike Neither Like Like Like Like extremely very moderately slightly like slightly moderately very extremely much nor much dislike Taste

Consistency (mouthfeel)

Thickness

Total impression

1 9

Do you think the product could be improved in any way?

That was all the questions for your first yogurt, you’re welcome to taste the second one and answer the questions on the upcoming pages!

Please proceed to the next page

IV

Number on the yogurt that you’re testing: ______

What is your first impression?

Neither Dislike like nor Like extremely dislike extremely

1 9

What do you think about the color?

Neither

Dislike like nor Like extremely dislike extremely

1 9

What do you think about the spoonability?

Neither like nor Dislike Like extremely dislike extremely

1 9

Please proceed to the next page

V

What do you think about…

Dislike Dislike Dislike Dislike Neither Like Like Like Like extremely very moderately slightly like slightly moderately very extremely much nor much dislike Taste

Consistency (mouthfeel)

Thickness

Total impression

1 9

Do you think the product could be improved in any way?

Final questions:

Which yogurt did you like the best?

Nr. 14 Nr. 20

Why do you prefer this one?

Please proceed to the next page

VI

Rank which of the attributes color, taste och consistency (mouthfeel) that is of most importance for you to like the product. Rank from 1 - 3, where 1 = most importance och 3 = least importance.

Color ______Taste ______Consistency (mouthfeel) ______

How often could you consume a product like this…

Daily

A few times a week

More seldom

Not at all

Do you think a product like this is needed?

Yes

No

How is your general appetite?

Good appetite, I feel hunger and I enjoy eating

Neither good nor bad appetite, I feel hunger sometimes and enjoy eating occasionally

Bad appetite, I rarely feel hunger and do not like to eat

Thank you for your participation!

VII