Developing low alcoholic kefir beverage with passion fruit, gac fruit and ginger

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 grai

MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY

UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH

QUALITY TRAINING

GRADUATION THESIS FOOD TECHNOLOGY

DEVELOPING LOW ALCOHOLIC KEFIR

BEVERAGE WITH PASSION FRUIT, GAC FRUIT

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

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

THE SOCIALIST REPUBLIC OF VIETNAM

Independence – Freedom– Happiness

-GRADUATION THESIS ASSIGNMENT

Student name: Phan Nguyen Thanh Truc

Student name: Bui Mai Uyen Linh

Major: Food Technology

Supervisor: Cao Thi Thanh Loan, Ph.D

- 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

Ho Chi Minh City, August 08, 2020

(Sign with full name) (Sign with full name)

i

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

ii

The successful completion of this thesis has everything to do with the support and guidancegiven by many people Among them, first and foremost we extend our heartfelt gratitude toPh.D Cao Thi Thanh Loan, our supervisor for the enthusiastic support and conscientiousguidance We would like to acknowledge our supervisor for giving us precious suggestions Ifnot 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 andFood 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 themfor 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 MinhUniversity of Technology and Education, the Dean Board, lecturers, and staff from the Faculty ofChemical and Food Technology, who allowed and created favorable conditions for us to do theresearch at the laboratory

We would like to send the most respectful thanks to Nurturing Foods Co Ltd (Vietnam), andall staff members for providing us with all the necessary resources for the implementation of thethesis 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 andsupporting us overcome difficulties in the process of implementing the thesis

iii

iv

v

vi

vii

viii

ix

x

xi

xii

xiii

xiv

xv

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

3.1 Materials 19

xvi

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

xvii

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

xviii

LIST OF FIGURES

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 passiongac ginger kefir beverage after 24h storing at 3-6 C 43o

Figure 4 2 Total yeast count of the F1 kefir water and the passion gac ginger kefir beverageafter 24h storing at 3-6o C 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

xix

LIST OF TABLES

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

xx

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

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

xxi

: Colony Forming per Unit: Exopolysaccharides: Lactic Acid Bacteria: Total Soluble Solids: Brown can sugar Water Kefir: Refined white sugar Water Kefir: Garape molasses Water Kefir: Honey Water Kefir

: Tiêu chuẩn Việt nam: Passion gac ginger: De Man, Rogosa and Sharpe: Potato Dextrose Agar

: 3,5-dinitrosalicylic acid

: Species

xxii

This study aimed to solve the dilemmatic situation of high-alcohol kefir soda in reaching theentire market, particularly children and women The fermentation of different sugar types, ratios andconcentrations, 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 after24h 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 theoptimal 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 beconsidered 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 shelflife 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.

xxiii

Chapter 1 INTRODUCTION

The current COVID-19 virus epidemic has necessitated the usage of non-traditional antiviralagents to lower the risk of infection and enhance patient recovery The market demand fornutritious and functional foods has also increased due to the development of economy and thehealth-concious improvement in awareness of customers Probiotic drinks were highlightedfactors 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 thedisease recovery and enhance the immune system However, the probiotic drinks werecommonly 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 potentialalternative approach Scientific evidence supported the claim that regular sugary kefir ingestionwas 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 scientificstudies backed up the health benefits of kefir drinking, and its global use has been steadilyincreasing [1], [6]

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

Passion fruit is a nutritious tropical fruit that is rich in antioxidants, vitamins, and fiber thatcould benefit human health [74], [75], [77] Gac fruit has a high concentration of antioxidantsand carotenoids such as lycopene and beta-carotene help benefit many different parts of thehuman 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, theapplication of these fruits and herbs in water kefir production created a health-promotingbeverage 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 alcoholcontent of fermented kefir drink The results obtained were then used to propose a suitableproduction process for developing a low alcoholic kefir product with passion fruit, gac fruit, andginger that is suitable for a wider range of customers while maintaining the desired quality andnutritional value

1.3 Research contents

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

1

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.

2

Chapter 2 LITERATURE REVIEW

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 waterkefir grains [7], [8], [9] These do not seem to be grains such as rye, wheat, or rice Water kefir iscomposed 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 cauliflowersand increasing to the size of walnuts [11], [12]

Water kefir grains contain a specific biofilm, which is a colony of various yeast and healthybacterial strains or species [13], [14] living in a stable, growing culture of collaboration, wheretheir 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 ofBacteria and Yeasts (SCOBY) [17], maintains by carbohydrate feeding, which allows them toproduce enzymes, carbon dioxide, alcohol (ethanol), and, most importantly, the beneficial lacticacid 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 fermentedconsumed 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 specifiedstarter 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 purportedhealth benefits [138], the need for a thorough understanding of the biology and dynamics of waterkefir, 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] Thissolution has diverse microflora [13], [14] and a slightly sweet, sour taste [7], [8], [9] This F1water kefir solution could be used for the second fermentation to increase the intensity of flavorand 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] Currentwater kefir products, on the other hand, had a rather high alcoholic content, which would be aconstraint if this healthy beverage intended to reach a huge market of customers of different agesand areas As a result, developing low-alcohol kefir drinks was necessitated to address this issueand expand the market for water kefir beverages

3

2.1.2 Historical development of water kefir

Early documentation [19] from the second half of the 19th century indicated the use of waterkefir grains in a fermented drink prepared from the sweetened juice of tibicos or water kefirgrowing on the pads of the nopal, opuntia, in Mexico [8] When reconstituted in a solution ofsugar 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 Caucasusmountains, and the northeastern Himalayas in Tibet, China [21], [22] In many cases, it isdifficult to pinpoint the place of origin of water kefir just by considering the shared symbioticnature of aggressively active yeast and bacteria, which tend to provide different variations of theright culture for making this bubbly beverage In short, manifestations of water kefir cultureswere prevalent around the world [8], [18] and it seemed that these kefir grains had existed andbeen used for thousands of years [20], [24]

Nevertheless, there is another reliable scientific article [22] that relates to the origins of theTibicos The literature refers to a specific bacteria cultured from known strains or species thatresemble properties identical to those of Tibicos The bacterium mentioned above wasresponsible 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, andcinnamon to make their popular fermented drink tepache The French named these grainsGraines 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 orJapanese water crystals, African Bees, Aqua Gems, Australian Bees, California Bees, GingerBeer Plant or Ginger Bees, and Sea Rice [27], [28] However, regardless of its name or theprecise composition of its culture, the main technique of using water kefir grains remainsessentially 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 hasalso expanded worldwide and it is considered a healthy product with high nutritional value inEurope, 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 kefirand kefir grains [20], [26], [27] as well as further exploring the potential for the manufacture of newconsumer-friendly nutritional foods Additionally, researches have revealed

4

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 kidneyhypertrophy [1], as well as anti-mutagenic [1], [4], and anti-allergenic activity [35], are amongthe many reported health benefits [1], [6], [8], [18], [34] Furthermore, kefir has considerableantibacterial properties against numerous foodborne pathogens [3], [4], and it has recently beenshown 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 probioticmicroflora in fermented milk drinks can exert their positive benefits via two mechanisms: as a directaction of the live probiotic culture or through their metabolites [39] Kefir is one of the most essentialfunctional products accessible because of its multiple health-promoting and disease-preventionproperties Kefir's antimicrobial properties are due to its capacity to modify the gut flora byincreasing 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 proteinswith 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 boosting and antiviral elements [40] Given these advantages, there is a growing desire for rigorousclinical trials to better understand the impact of frequent use of fermented functional drinks as part ofthe diet or as a dietary supplement [34], [40]

immune-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 themicroorganisms 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 -independent methods

Reference Microbial species found

Ward (1892) [44] Acetobacteraceae, Lactobacillus hilgardii

5Horisberger (1969) [45]

Pidoux et al (1988) [23]

Franzetti et al (1998)[46]

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 Belgianwater kefir grains [26], [27], [51], [54], [55] As previously discussed, given the potential lack ofdiversity 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 aerobia), Pichia (P cecembensis, P membranifaciens, P caribbica, P fermentans),

7

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 milkkefir 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] Globalizationtrends have a significant influence on the expansion of kefir Modern customers, particularlyMillennials and Generation Z, are more concerned about health and nutrition in their everydaymeals and seek out items that are helpful to human health This demographic accounts for asizable 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

8

Figure 2 2 Vinamilk unsweetened kefir yogurt

Figure 2 3 Vinamilk drinkable yogurt kefir without sugar

Figure 2 4 Kefir home's kefir soda products

9

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 crystallinesweetener used in foods and beverages Sucrose, a disaccharide, is present in most plants, butonly in sugarcane and sugar beet plants in adequate amounts for commercial recovery Sucrose isthe sweetness standard, and other sweeteners are compared to sucrose as 100% [60]

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

Table 2 1 Composition of sugars [61]

Refined white cane sugar

Cane sugar refining converts raw cane sugar into extremely pure white sugars Water, ash, andlowering sugar content are all managed Products are of constant quality and are safe for use athome 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 incolor These refineries can save money by returning low-grade products to a manufacturer ratherthan processing it It is believed that the procedures used in the production phase of white refinedsugar, particularly the refining process, and the additional chemical compounds such as sulfurdioxide, blanket, and hydrogen peroxide for whitening, affect the development ofmicroorganisms [62], [63]

10

Brown cane sugar

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

Table 2 2 Chemical composition of honey [66]

Components

WaterFructoseGlucoseSucroseOther sugarsMineralsProteinAcids (as gluconic acid)Phenolic compounds

Mineral compounds range in concentration from 0.02 to 1.03g/100g in honey [67] Honeycontains numerous different elements, the most important of which being potassium Thepredominant mineral element is potassium, which accounts for almost one-third of the totalelements Several studies have revealed that the trace element concentration of honey isdetermined by its botanical origin, with light flower honeys having a lower content than darkhoneys, such as honeydew, chestnut, and heather [67]

Honey is a highly concentrated sugar solution with a high osmotic pressure, makingmicroorganism development difficult It includes fewer germs than other natural foods, and

11

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

in honeys with a high moisture content

Grape molasses

Grape molasses contain sugar, such as saccharose, glucose, fructose, a small amount ofmaltose, and raffinose [68] The amount of fructose changed from 22.34% to 34.69% and wasdetermined to be 28.42% on average On the other hand, the glucose ratios ranged from 27.57%

to 41.11%, with an average value of 31.67% [69] Simşek & Artık (2002) [70] reported that thefructose and glucose contents of 25 commercially produced grape molasses samples changedfrom 30.14 to 34.42% and from 30.73 to 34.99%, respectively

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

12

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 whitesolid with the formula NaHCO3 that often appears as a fine powder The pH, water kefir graindevelopment, microbial species diversity, and metabolite synthesis were all affected by the buffercapacity (-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 crystallinesolid 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 kefirgrain formation, microbial species diversity, and metabolite production The high water buffercapacity (-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 weremirrored in high lactic acid to ethanol concentration ratios [134] The water kefir grain growthsignificantly 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 beenwidely used in folk medicine to treat anxiety, asthma, urinary tract infections, diabetes, and otherdiseases [75]

13

Table 2 4 Composition of passion fruit [76] Nutritional value per 100 g (3.5 oz)

EnergyCarbohydratesSugars

Dietary fiberFat

Protein

Vitamins

Vitamin A equiv

beta-CaroteneRiboflavin (B2)Niacin (B3)Vitamin B6Folate (B9)CholineVitamin CVitamin K

Minerals

CalciumIronMagnesiumPhosphorusPotassium