(Đồ án HCMUTE) developing low alcoholic kefir beverage with passion fruit, gac fruit and ginger

Experiment 1: Effect of different sugar types on biomass growth and alcoholic content of water kefir .... Experiment 2: Effect of different ratios of two sugar types on biomass growth an

MINISTRY OF EDUCATION AND TRAINING

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

SKL 0 0 8 9 4 1

DEVELOPING LOW ALCOHOLIC KEFIR

BEVERAGE WITH PASSION FRUIT, GAC FRUIT

AND GINGER

GRADUATION THESIS FOOD TECHNOLOGY

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

GRADUATION PROJECT

2022-18116021

Ho Chi Minh city, August 2022

DEVELOPING LOW ALCOHOLIC KEFIR BEVERAGE WITH PASSION FRUIT, GAC FRUIT AND GINGER

PHAN NGUYEN THANH TRUC Student ID: 17116040

BUI MAI UYEN LINH Student ID: 18116021 Major: FOOD TECHNOLOGY Supervisor: CAO THI THANH LOAN, Ph.D

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

GRADUATION PROJECT

2022-18116021 DEVELOPING LOW ALCOHOLIC KEFIR BEVERAGE WITH PASSION FRUIT, GAC FRUIT AND GINGER

PHAN NGUYEN THANH TRUC Student ID: 17116040

BUI MAI UYEN LINH Student ID: 18116021 Major: FOOD TECHNOLOGY Supervisor: CAO THI THANH LOAN, Ph.D

Ho Chi Minh city, August 2022

i

THE SOCIALIST REPUBLIC OF VIETNAM

Independence – Freedom– Happiness

-

GRADUATION THESIS ASSIGNMENT

Student name: Phan Nguyen Thanh Truc Student ID: 17116040 Class: 17116CLA1 Student name: Bui Mai Uyen Linh Student ID: 18116021 Class: 18116CLA2

Supervisor: Cao Thi Thanh Loan, Ph.D Email: thanhloanafu@gmail.com

Date of assignment: 20/01/2022 Date of submission: 08/08/2022

1 Thesis title: Developing low alcoholic kefir beverage with passion fruit, gac fruit and ginger

- Determine shelflife of the passion gac ginger kefir beverage

The content and requirements of the graduation thesis have been approved by the Chair of the

Food Technology program

CHAIR OF THE PROGRAM

(Sign with full name)

Ho Chi Minh City, August 08, 2020

SUPERVISOR

(Sign with full name)

We declare that the entire content of this thesis is our original work We declare that all references in the graduation thesis were properly cited in accordance with regulations

iii

ACKNOWLEDGEMENTS

The successful completion of this thesis has everything to do with the support and guidance given by many people Among them, first and foremost we extend our heartfelt gratitude to Ph.D Cao Thi Thanh Loan, our supervisor for the enthusiastic support and conscientious guidance We would like to acknowledge our supervisor for giving us precious suggestions If not for the motivation provided by our supervisor, this thesis would not have been completed

We must be thankful to Assoc Prof Nguyen Tan Dung, Head of the Faculty of Chemical and Food Technology All the theories we applied to complete this thesis successfully were taught to

us by the excellent panel of lecturers in our department Therefore, we are forever in debt to them for providing us the wisdom to accomplish this task successfully

We would also like to express our deep thanks to the School Board of Ho Chi Minh University

of Technology and Education, the Dean Board, lecturers, and staff from the Faculty of Chemical and Food Technology, who allowed and created favorable conditions for us to do the research at the laboratory

We would like to send the most respectful thanks to Nurturing Foods Co Ltd (Vietnam), and all staff members for providing us with all the necessary resources for the implementation of the thesis Thanks to everyone for helping us with their suggestions to make the project better, especially Ms Hoang Thi Thu and Mr Tran Trung Tin

Last but not least, we would like to thank our parents and friends for always being with us and supporting us overcome difficulties in the process of implementing the thesis

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TABLE OF CONTENTS

GRADUATION THESIS ASSIGNMENT i

DISCLAIMER ii

ACKNOWLEDGEMENTS iii

TABLE OF CONTENTS xvi

LIST OF FIGURES xix

LIST OF TABLES xx

LIST OF ABBREVIATIONS xxii

ABSTRACT xxiii

Chapter 1 INTRODUCTION 1

1.1 Problem statement 1

1.2 Research objectives 1

1.3 Research contents 1

Chapter 2 LITERATURE REVIEW 3

2.1 Water kefir 3

2.1.1 Introduction 3

2.1.2 Historical development of water kefir 4

2.1.3 Health benefits of water kefir beverage 4

2.1.4 Microorganisms and yeasts in water kefir 5

2.1.5 The kefir market 8

2.2 Substrates and solvents for F1 water kefir fermentation 10

2.2.1 Sugars 10

2.2.2 Water 12

2.2.3 Natri Bicarbonat 13

2.3 Passion fruit 13

2.4 Gac fruit 15

2.5 Ginger 16

2.6 Pectinase 17

Chapter 3 MATERIALS AND METHODS 19

xvii

3.1.1 Production of F1 water kefir beverage 19

3.1.2 Preparation of passion gac ginger juice 21

3.1.3 Production of passion gac ginger kefir beverage 24

3.2 Research contents 25

3.2.1 Experiment 1: Effect of different sugar types on biomass growth and alcoholic content of water kefir 25

3.2.2 Experiment 2: Effect of different ratios of two sugar types on biomass growth and alcoholic content of water kefir 26

3.2.3 Experiment 3: Effect of sugar concentration and kefir grains rate on biomass growth and alcohol content of water kefir 26

3.2.4 Experiment 4: Investigation of the appropriate fruit juice mixing ratio for kefir beverages and evaluate sensory of kefir beverages 27

3.2.5 Experiment 5: Determination the physiochemical and microbiological properties of the passion gac ginger kefir beverage 30

3.2.6 Experiment 6: Determination the shelf-life of passion gac ginger kefir beverage 31

3.3 Analysis methods 31

3.3.1 Determination of the alcohol content of kefir water after each fermentation 31

3.3.2 Determination of the biomass of water kefir grains 31

3.3.3 Microbiological analysis 32

3.3.4 Determination of pH 32

3.3.5.Determination of total soluble solids (TSS) 32

3.3.6 Sensory analysis 32

Chapter 4 RESULTS AND DISCUSSION 33

4.1 Effect of different sugar types on biomass growth and alcoholic content of water kefir 33

4.2 Effect of different ratios of two sugar types on biomass growth and alcoholic content of water kefir 35

4.3 Effect of sugar concentration and kefir grain rate on biomass growth and alcoholic content of kefir water 35

4.4 Investigation of the appropriate fruit juice mixing ratio for kefir beverages and evaluate sensory of kefir beverages 38

4.4.1 Investigation of gac fruit powder portions 38

4.4.2 Investigation of ginger portions to minimize the off-odor 39

4.4.3 Investigation the application of the mixed juice portions on the alcohol production

and sensory quality of the kefir drink 40

4.5 Determination the physiochemical and microbiological properties of the passion gac ginger kefir beverage 42

4.5.1 Physicochemical criteria of the kefir beverage 42

4.5.2 Microbiological characteristics of the kefir beverage 43

4.6 Determination the shelflife of passion gac ginger kefir beverage 45

Chapter 5 CONCLUSION 47

5.1 Conclusion of main findings 47

5.2 Recommendations 47

REFERENCE 48

APPENDIX A 58

APPENDIX B 59

APPENDIX C 60

APPENDIX D 61

APPENDIX E 62

xix

LIST OF FIGURES

Figure 2 1 Some overseas kefir fermented water products 8 Figure 2 2 Vinamilk unsweetened kefir yogurt 9 Figure 2 3 Vinamilk drinkable yogurt kefir without sugar 9 Figure 2 4 Kefir home's kefir soda products 9 Figure 2 5 The chemical formula of sodium bicarbonate 13 Figure 2 6 Passion fruit (Passiflora edulis Sims) 13 Figure 2 7 Ginger (Zingiber officinale) 16 Figure 2 8 Chemical structure of active ingredients of Ginger [96] 17 Figure 2 9 The main properties of ginger [97] 17

Figure 3 1 Activated water kefir grains (Nurturing Foods Co., Ltd, Vietnam) 19 Figure 3 2 Types of sugars used in water kefir fermentation 20 Figure 3 3 Production of F1 water kefir beverage [8], [27] 21 Figure 3 4 Production of passion fruit juice 22 Figure 3 5 Production of gac fruit solution 23 Figure 3 6 Production of ginger juice 23 Figure 3 7 Production of passion gac ginger juice 24 Figure 3 8 Production of passion gac ginger kefir beverage 25 Figure 3 9 Kefir beverages made from passion fruit juice and gac fruit powder 0.5%; 1.0%;

1.5% from left to right 28

Figure 3 10 Mixing ratio of fruit juice 10%; 20%; 30% with F1 kefir water 29

Figure 4 1 Total aerobic plate count and Lactobacillus spp of the F1 kefir water and the passion

gac ginger kefir beverage after 24h storing at 3-6oC 43

Figure 4 2 Total yeast count of the F1 kefir water and the passion gac ginger kefir beverage

after 24h storing at 3-6oC 44

Figure 4 3 Total soluble solids (TSS), pH and ethanol content changes of kefir drinks during the

storage time (10days) 45

Figure 4 4 Color parameters changes of the passion gac ginger kefir beverage during storage

time (10days) 45

LIST OF TABLES

Table 1 1 Overview of genera and species of yeasts and bacteria found in water kefir via

culture-dependent and -independent methods 5

Table 2 1 Composition of sugars [61] 10 Table 2 2 Chemical composition of honey [66] 11 Table 2 3 The of composition of Lavie natural mineral water ( Lavie product’s label) 12 Table 2 4 Composition of passion fruit [76] Nutritional value per 100 g (3.5 oz) 14 Table 2 5 Composition of 100g gac fruit aril [86] 15

Table 3 1 Formula of gac fruit powder ratios in the fruit juice 27 Table 3 2 Formula for examination juice added to enhance the aroma 28 Table 3 3 Formula of ingredients used in juice mixture 29 Table 3 4 Ratios of fruit juice examined for mixing with F1 water kefir solution 29 Table 3 5 Methods for chemical components analysis 30 Table 3 6 Methods for microbiology analysis 30

Table 4 1 Results effect of sugar types on kefir water growth 33 Table 4 2 Composition of sugars [61] 33 Table 4 3 Results effect of sugar ratio on kefir water growth 35 Table 4 4 The initial total soluble solids and pH of sugary medium 35 Table 4 5 Results effect of sugar concentration on kefir water growth 36 Table 4 6 Results effect of kefir grain rate on kefir water growth 36 Table 4 7 Microbiological analysis of the kefir fermented with 5% (12g) kefir grains 37 Table 4 8 Microbiological analysis of the kefir fermented with 10% (24g kefir grains) 37 Table 4 9 Sensory evaluation of kefir drinks from passion fruit juice and three different ratios of

gac fruit powder ratios (0.5%; 1.0%; 1.5%) 38

Table 4 10 Sensory evaluation of kefir drinks from passion fruit juice, gac fruit powder and

three different ratios of ginger juice ratios (5%; 10%; 15%) 39

Table 4 11 Total soluble solids (TSS), pH and ethanol content of three kefir samples at 0h 40

xxi

Table 4 12 Total soluble solids (TSS), pH, ethanol content of kefir samples after storage at 24h.

40

Table 4 13 Sensory evaluation of kefir drinks made from the final fruit juice mixture formula

with three different ratios (10%; 20%; 30%) 41

Table 4 14 Chemical components of the kefir beverage 42

LIST OF ABBREVIATIONS

AOAC : Association of Official Agricultural Chemists

xxiii

ABSTRACT

This study aimed to solve the dilemmatic situation of high-alcohol kefir soda in reaching the entire market, particularly children and women The fermentation of different sugar types, ratios and concentrations, using kefir grains was proposed to produce a low-alcoholic kefir beverage

The microbiological analysis (total aerobic plate count and lactobacillus spp and yeasts), chemical

properties (pH, total solids, ethanol content) were investigated at the end of fermentation and after 24h of the final product The results showed that among 4 sugar types of grape molasses, honey, brown cane sugar and refined sugar, the latter two produced the lowest alcohol content, with the optimal concentration of sugar is 5% (12g) with 75% and 25% of brown cane sugar and refine sugar, respectively Regarding sensory analysis, kefir drink containing 20% juice mixtures of passion fruit, gac fruit, and ginger received high marks for flavor and overall acceptability

Furthermore, with a density of Lactobacillus spp in the final product of 2.2 x 106cfu/ml, this kefir drink can be considered a promising functional food During storage, low decreases of pH, total soluble solids, and color were observed, while the alcohol content slightly increased at day 10 Therefore, the shelf life of passion gac ginger kefir drinks made from natural kefir grains is suggested to be 8 days based on the alcohol content and sensory results

Keywords: low alcoholic kefir beverage, passion fruit, gac fruit, ginger

Chapter 1 INTRODUCTION

1.1 Problem statement

The current COVID-19 virus epidemic has necessitated the usage of non-traditional antiviral agents to lower the risk of infection and enhance patient recovery The market demand for nutritious and functional foods has also increased due to the development of economy and the health-concious improvement in awareness of customers Probiotic drinks were highlighted factors among many choices because of theirs healthy benefits for digestion and immune system Probiotic beverages are good sources of beneficial microorganisms which can contribute to the disease recovery and enhance the immune system However, the probiotic drinks were commonly produced from milk which might cause some problems with the ingestion of lactose-intolerant consumers Therefore, water kefir drinks made from sugary solution was a potential alternative approach Scientific evidence supported the claim that regular sugary kefir ingestion was associated with a wide range of health-promoting effects, including anti-carcinogenic [1], anti-oxidant [2], anti-inflammatory [3], antifibrotic [4], anti-microbial [5] Several scientific studies backed up the health benefits of kefir drinking, and its global use has been steadily increasing [1], [6]

Kefir drinks have numerous health benefits [1], [2], [3], [4], [5], [6] However, current water kefir products had a relative high alcoholic content, which would be a limitation if this healthy beverage wanted to reach a large market of customers from all ages and regions Therefore, the study was conducted with the aim of resolving this dilemmatic problem in order to broaden the market of water kefir drinks

Passion fruit is a nutritious tropical fruit that is rich in antioxidants, vitamins, and fiber that could benefit human health [74], [75], [77] Gac fruit has a high concentration of antioxidants and carotenoids such as lycopene and beta-carotene help benefit many different parts of the human body [83], [84], [87], [88], [121], [122], [123] Ginger might have anti-inflammatory, antibacterial, and antiviral properties [92], [93], [94], [95] With these enormous advantages, the application of these fruits and herbs in water kefir production created a health-promoting beverage that meets the custom’s demands about healthy fluid intake

1.2 Research objectives

The overall goal of this thesis was to assess the effect of sugar type and ratio on the alcohol content of fermented kefir drink The results obtained were then used to propose a suitable production process for developing a low alcoholic kefir product with passion fruit, gac fruit, and ginger that is suitable for a wider range of customers while maintaining the desired quality and nutritional value

1.3 Research contents

The topic “Developing low alcoholic kefir beverage with passion fruit, gac fruit and ginger” included the following specific contents:

2

Experiment 1: Effect of different sugar types on biomass growth and alcoholic content of water

kefir

Experiment 2: Effect of different ratios of two sugar types on biomass growth and alcoholic

content of water kefir

Experiment 3: Effect of sugar concentration and kefir grains rate on biomass growth and

alcohol content of water kefir

Experiment 4: Investigation of the appropriate fruit juice mixing ratio for kefir beverages and

evaluate sensory of kefir beverages

Experiment 5: Determination the physiochemical and microbiological properties of the

passion gac ginger kefir beverage

Experiment 6: Determination the shelflife of the passion gac ginger kefir beverage

Chapter 2 LITERATURE REVIEW

2.1 Water kefir

2.1.1 Introduction

While milk kefir contains a lot of protein as well as probiotics and prebiotics, water kefir can

be a good source of probiotics and prebiotics for vegans and those who are allergic to dairy [135] Water kefir is a probiotic or Lacto-fermented beverage made from sugar water and water kefir grains [7], [8], [9] These do not seem to be grains such as rye, wheat, or rice Water kefir is composed of a water-soluble polysaccharide gel known as 'kefiran’ [10], which has a mild cord-like texture in the mouth and ranges from pale white to yellowish grains, resembling cauliflowers and increasing to the size of walnuts [11], [12]

Water kefir grains contain a specific biofilm, which is a colony of various yeast and healthy bacterial strains or species [13], [14] living in a stable, growing culture of collaboration, where their cells attach to a surface embedded within a dextran, or a polysaccharide matrix of sugars, lipids, and proteins [15] produced by the microorganisms through sucrose fermentation [16] This communal harmony of microorganisms, formally known as the Symbiotic Culture of Bacteria and Yeasts (SCOBY) [17], maintains by carbohydrate feeding, which allows them to produce enzymes, carbon dioxide, alcohol (ethanol), and, most importantly, the beneficial lactic acid that propagates probiotics Although some interconnected colony of lactic acid bacteria

predominates within each grain, the helpful probiotic Lactobacillus species are ubiquitous and

always reign [17], [24] The microbial ecosystem which constitutes the grains, and the fermented consumed beverage can vary according to the fermentation conditions (time and temperature) and especially with the use of different substrates [136],[137]

Water kefir has typically been produced on a modest scale [8], [18], and the use of specified starter cultures is uncommon However, as water kefir grows in popularity as a beverage, due in part to consumer lifestyle trends and in part to water kefir being viewed as a healthy drink with purported health benefits [138], the need for a thorough understanding of the biology and dynamics

of water kefir, as well as defined and controlled production processes, will dramatically increase F1 water kefir is the solution obtained after being fermented for 48h at 25oC [27] This solution has diverse microflora [13], [14] and a slightly sweet, sour taste [7], [8], [9] This F1 water kefir solution could be used for the second fermentation to increase the intensity of flavor and alcoholic content or mixed with fruit and vegetable juice for constant consumption

Kefir beverages contain an abundance of health advantages [1], [2], [3], [4], [5], [6] Current water kefir products, on the other hand, had a rather high alcoholic content, which would be a constraint if this healthy beverage intended to reach a huge market of customers of different ages and areas As a result, developing low-alcohol kefir drinks was necessitated to address this issue and expand the market for water kefir beverages

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2.1.2 Historical development of water kefir

Early documentation [19] from the second half of the 19th century indicated the use of water kefir grains in a fermented drink prepared from the sweetened juice of tibicos or water kefir growing on the pads of the nopal, opuntia, in Mexico [8] When reconstituted in a solution of sugar and water, these newly found forms formed hard granules that spread tibicos [20], [22]

These speculations have also targeted the southern peninsula of Ukraine, the Caucasus mountains, and the northeastern Himalayas in Tibet, China [21], [22] In many cases, it is difficult

to pinpoint the place of origin of water kefir just by considering the shared symbiotic nature of aggressively active yeast and bacteria, which tend to provide different variations of the right culture for making this bubbly beverage In short, manifestations of water kefir cultures were prevalent around the world [8], [18] and it seemed that these kefir grains had existed and been used for thousands of years [20], [24]

Nevertheless, there is another reliable scientific article [22] that relates to the origins of the Tibicos The literature refers to a specific bacteria cultured from known strains or species that resemble properties identical to those of Tibicos The bacterium mentioned above was responsible for the formation of dextran, similar to the polysaccharide matrix produced in particular by the

fermentation of sucrose by Lactobacillus hilgardii and strains of the Leuconostoc genus, both

predominant bacterial species in water kefirs

Around the world, water kefir grains have a variety of names alongside sugar grains or tibicos

in Mexico [22], [23], which are made from piloncillo or brown sugar, pineapple skins, and cinnamon to make their popular fermented drink tepache The French named these grains Graines Vivantes; The Italians referred to them as Kefir di Frutta; the Germans called them Piltz [22], [25], [26]

Moreover, to name a few of the most common references for water kefir grains, are tibi or Japanese water crystals, African Bees, Aqua Gems, Australian Bees, California Bees, Ginger Beer Plant or Ginger Bees, and Sea Rice [27], [28] However, regardless of its name or the precise composition of its culture, the main technique of using water kefir grains remains essentially the same around the world [23], [27]

In recent years, there has been more interest in different probiotic products such as yogurt, kefir, koumiss, and Yakult With the market’s trend of consumption, the popularity of kefir has also expanded worldwide and it is considered a healthy product with high nutritional value in Europe, Asia, and South and North America [27], [29], [30]

2.1.3 Health benefits of water kefir beverage

Consumers nowadays prefer products with functional characteristics that can improve their health The definition of "functional foods" [31], [32] has influenced consumer views of nutritious foods Studies have been developed with the goal of reinforcing fundamental scientific knowledge

on kefir and kefir grains [20], [26], [27] as well as further exploring the potential for the manufacture of new consumer-friendly nutritional foods Additionally, researches have revealed

that frequent drinking of kefir may have enormous health benefits Improved digestion [32], antioxidant activity [2], antibacterial effects [3], [4], [35], gut microbiome modulation [32], [33], control of plasma glucose [8], alleviation of obesity [33], improvement of cholesterol levels [33], anti-inflammatory effects [4], [36], [37], reduced cardiac hypertrophy [1], and reduced kidney hypertrophy [1], as well as anti-mutagenic [1], [4], and anti-allergenic activity [35], are among the many reported health benefits [1], [6], [8], [18], [34] Furthermore, kefir has considerable antibacterial properties against numerous foodborne pathogens [3], [4], and it has recently been shown to serve as a defensive mechanism, closing the immune system against viral viruses [4], [34]

One of the most successful ways for preventing infections caused by a variety of pathogens, including viruses, is to boost and stimulate the immune system [38] It is widely known that probiotic microflora in fermented milk drinks can exert their positive benefits via two mechanisms:

as a direct action of the live probiotic culture or through their metabolites [39] Kefir is one of the most essential functional products accessible because of its multiple health-promoting and disease-prevention properties Kefir's antimicrobial properties are due to its capacity to modify the gut flora by increasing the amount of probiotic bacterial strains [38], [40] Kefir consumption can give health-promoting benefits caused by probiotics such as lactic acid, acetic acid, and antimicrobial proteins with bacteriostatic actions against many diseases [3], [4], [40] Furthermore, the high concentration of bio-functional metabolites in kefir highlights the growing need to investigate these immune-boosting and antiviral elements [40] Given these advantages, there is a growing desire for rigorous clinical trials to better understand the impact of frequent use of fermented functional drinks as part of the diet or as a dietary supplement [34], [40]

2.1.4 Microorganisms and yeasts in water kefir

2.1.4.1 Representative microorganisms in water kefir

The variety of microbial species in water kefir has been studied multiple times using available

techniques Lactobacillus species were isolated and/or detected in all, but no single unifying or

characteristic species were identified [22], [26], [27], [28], [41], [42] Table 1.1 summarizes the microorganisms discovered in water kefir using culture-dependent or culture-independent species

diversity investigations Lactobacillus hilgardii and Lactobacillus nagelii have been identified as important Lactobacillus species in water kefir grain communities, owing to their production of exopolysaccharides (EPS) [8], [18] Acetobacter sicerae, a unique AAB species, was recently

discovered in water kefir [43]

Table 1 1 Overview of genera and species of yeasts and bacteria found in water kefir via

culture-dependent and -inculture-dependent methods

Reference Microbial species found

Ward (1892) [44] Acetobacteraceae, Lactobacillus hilgardii

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Horisberger (1969) [45] Lactobacillus brevis, Lactococcus lactis

Pidoux et al (1988) [23] Lactobacillus casei, Lactobacillus hilgardii, Lactobacillus

plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides

Magalhães et al (2011)

[48]

Acetobacter lovaniensis, Bacillus cereus, Gluconobacter liquefaciens, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus kefiri, Lactobacillus paracasei, Lactobacillus satsumensis, Lactobacillus sunkii

Gulitz et al (2011) [49] Acetobacter fabarum, Acetobacter orientalis, Lactobacillus

casei, Lactobacillus hilgardii, Lactobacillus hordei, Lactobacillus nagelii, Leuconostoc citreum, Leuconostoc mesenteroides

Hsieh et al (2012) [50] Lactobacillus hordei, Lactobacillus mali, Leuconostoc

Mesenteroides, Pseudomonas, Sporolactobacillus, Zymomonas mobilis

Gulitz et al (2013) [28] Acetobacter, Bifidobacterium, Clostridium,

Gluconacetobacter, Gluconobacter, Lactobacillus, Leuconostoc

Marsh et al (2013) [51] Acetobacter, Bifidobacterium, Gluconacetobacter,

Gluconobacter, Lactobacillus, Leuconostoc, Zymomonas

Diosma et al (2014) [52] Pichia occidentalis, Saccharomyces cerevisiae

2.1.4.2 Representative yeasts in water kefir

The diversity of yeast species in water kefir grains appear to be smaller than that of bacteria

Numerous studies have evaluated the composition of water kefir yeast, Saccharomyces cerevisiae

was discovered in all, indicating that this species is an important part of the grain community [26],

[27], [28], [41], [42], [47], [49], [51], [53], [54] Other Saccharomyces species, such as S florentinus and S pretoriensis, have been identified on rare occasions; however, the former is now classified as Zygotorulaspora florentina [42] S bayanus has also been found, according to [7] According to the number of studies identifying the genus, Dekkera (anamorph, Brettanomyces)

appears to be the second most commonly found yeast in water kefir grains; however, except for one study [51], this yeast has been found almost exclusively in Belgian water kefir grains [26], [27], [51], [54], [55] As previously discussed, given the potential lack of diversity within kefir grain samples due to the possibility that research groups will continue to use the same grain lineage

for further study, while Dekkera occurs as a common yeast in water kefir grains, the finding may

be skewed due to geographically restricted distribution In addition, D bruxellensis was the only species found in Belgian grains, but Marsh et al [51] discovered both D bruxellensis and D anomala in grains from the United States, Canada, and the United Kingdom, showing that the

genus is more widely distributed than simply Belgium [51] As these authors indicate, the limited

detection of Dekkera in most investigations may be due to the yeasts' slow doubling period when

cultivated on

Species of Zygotorulaspora and Hanseniaspora are frequently assigned to the water kefir grain

community, with a greater distribution among grains from various regions; however, neither genus

has been identified in grains from South America Zygotorulaspora is represented by a single species Z florentina, which was formerly recognized as Saccharomyces florentinus and/or Zygosaccharomyces florentinus [22], [27], [42], [49] In addition, Marsh et al [51] did not

discover this yeast in water kefir grains from the USA, Canada or the UK [51] Species of

Hanseniaspora have not been detected in Belgian or South American investigations While yeast variety is abundant in Brazilian grains [47], [53], S cerevisiae and D bruxellensis are often

dominant in Belgian water kefir grains [26], [54], [55] Again, this might be the result of a limited

grain lineage Hanseniaspora valbyensis and Hanseniaspora vinae have been discovered [42],

[49], [51]

Species of Lachancea, primarily Lac fermentati, have also been identified in grains from

different locations, including Brazil, Germany and the UK [47], [49], [51], [53]

Candida species have been found primarily in water kefir grains from South America, but also

in studies from France and Thailand; species detected include Candida ethanolica [56], C californica [57], C lambica, C valida [22] and C valdiviana [53]

Other less commonly found yeast includes Torulaspora, Kazachstania, Pichia, Kluyveromyces and Yarrowia Torulaspora pretoriensis is the sole species of this genus identified in water kefir grains, being found in grains from France, Italy, UK and USA [22], [42], [51] Kazachstania (Ka

8

Kluyveromyces (Kl lactis) and Yarrowia (Y lipolytica) have been detected primarily in Brazilian

water kefir grains, these grains having the most diverse yeast microbiota compared to grains from

other geographical areas The discovery of yeast such as Candida and Kluyveromyces, which are

more commonly associated with milk kefir, may indicate that they originated as milk kefir grains and were later adapted for the production of sugary kefir drinks [8]

2.1.5 The kefir market

The kefir market of the world

In 2020, the worldwide kefir market was estimated at $1,627.8 million [58] Globalization trends have a significant influence on the expansion of kefir Modern customers, particularly Millennials and Generation Z, are more concerned about health and nutrition in their everyday meals and seek out items that are helpful to human health This demographic accounts for a sizable proportion of kefir product purchasers Dairy consumption may be avoided for health (lactose intolerance, allergies) or ethical (animal welfare) reasons [59] As a result, water kefir is a great non-dairy alternative to milk kefir, and it's not just for vegetarians and vegans

Figure 2 1 Some overseas kefir fermented water products

The Vietnamese kefir food market

Although it has been around for a long time, kefir products are still quite new to consumers

The main products at present are kefir yogurt, drinkable yogurt kefir and kefir soda

Figure 2 2 Vinamilk unsweetened kefir yogurt

Figure 2 3 Vinamilk drinkable yogurt kefir without sugar

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2.2 Substrates and solvents for F1 water kefir fermentation

2.2.1 Sugars

The term sugar describes the chemical class of carbohydrates of the general formula (CH2O)n

or Cn(H2O)n-1 for monosaccharides Sugar is the colloquial word for sucrose, a solid crystalline sweetener used in foods and beverages Sucrose, a disaccharide, is present in most plants, but only

in sugarcane and sugar beet plants in adequate amounts for commercial recovery Sucrose is the sweetness standard, and other sweeteners are compared to sucrose as 100% [60]

Color, taste, sweetness, antioxidant capabilities, and aqueous solution reactions are all chemical features of cane sugar that impact daily use [60] The color of cane sugar is used to determine its purity; the lowest color sugars are the purest sucrose with the least amount of color and flavor molecules, as well as other organic and inorganic components Brown sugar is often manufactured from cane sugar for flavor reasons

Table 2 1 Composition of sugars [61]

Components White refined cane sugar Unrefined brown cane sugar

Refined white cane sugar

Cane sugar refining converts raw cane sugar into extremely pure white sugars Water, ash, and lowering sugar content are all managed Products are of constant quality and are safe for use at home and in the food and beverage industries Refined sugars may be stored for lengthy periods; white refined sugars held at room temperature for more than 60 years show just a modest rise in color These refineries can save money by returning low-grade products to a manufacturer rather than processing it It is believed that the procedures used in the production phase of white refined sugar, particularly the refining process, and the additional chemical compounds such as sulfur dioxide, blanket, and hydrogen peroxide for whitening, affect the development of microorganisms [62], [63]

Brown cane sugar

Impurities are removed from all cane sugars throughout the manufacturing process (refining process) The unrefined sweeteners are the least refined since they contain the majority of the natural cane molasses It comprises both conventional brown sugars (muscovado, panela, jaggery, and piloncillo) and those made using a more complex drying procedure, such as Sucanat

Honey

Sugars are the primary ingredients of honey, accounting for approximately 95% of the dry mass content The monosaccharides hexose, fructose, and glucose are the main sugars produced by the hydrolysis of the disaccharide sucrose [64], [65] The main oligosaccharides found in blossom honey include disaccharides such as sucrose, maltose, turanose, and erlose Honeydew honey also includes the trisaccharides melezitose and raffinose

Table 2 2 Chemical composition of honey [66]

12

Bacillus species Bacillus bacteria in honey cause serious bee pests, although they are not hazardous to humans As a result, honey should not be disposed of in open areas where bees may readily get it in order to prevent bee problems Honey, on the other hand, has naturally occurring osmotolerant yeast that might produce unwanted fermentation Osmotolerant yeasts may thrive in honeys with a high moisture content

Grapes are one of the greatest sources of nutrients and phenolic chemicals in fruits, and the presence of phenolic substances in these fruits contributes to their antioxidant activity [71] The macroelement composition of grape molasses samples generated from various grape varieties ranges from 48.70mg/kg to 5109.56 mg/kg [72]

2.2.3 Natri Bicarbonat

Figure 2 5 The chemical formula of sodium bicarbonate

Sodium bicarbonate, often known as baking soda or bicarbonate of soda, is a crystalline white solid with the formula NaHCO3 that often appears as a fine powder The pH, water kefir grain development, microbial species diversity, and metabolite synthesis were all affected by the buffer capacity (-HCO3) concentration of the water used for water kefir fermentation [73]

Sodium bicarbonate, often known as baking soda or bicarbonate of soda, is a white crystalline solid with the formula NaHCO3 that is commonly found as a fine powder The buffer capacity (-HCO3) concentration of the water used for water kefir fermentation affects the pH, water kefir grain formation, microbial species diversity, and metabolite production The high water buffer capacity (-HCO3) was beneficial for the development and metabolism of the lactic acid bacteria (LAB) compared to the yeasts, resulting in high LAB to yeast ratios on the grains, which were mirrored in high lactic acid to ethanol concentration ratios [134] The water kefir grain growth significantly decreased due to the poor water buffer capacity

2.3 Passion fruit

Chemical composition of passion fruit

Figure 2 6 Passion fruit (Passiflora edulis Sims)

Passion fruit (Passiflora edulis Sims) is a tropical and subtropical fruit recognized for its

enticing flavor, nutritional properties, and therapeutic characteristics [74] It is said to have been widely used in folk medicine to treat anxiety, asthma, urinary tract infections, diabetes, and other diseases [75]