research on kombucha production process from tea leaves camellia sinensis l o kuntze in thai nguyen province

i DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program: Bachelor of Food Science and Technology Thesis Title: Research on kombucha productio

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURAL AND FORESTRY

NGO NHU DUY

RESEARCH ON KOMBUCHA PRODUCTION PROCESS FROM TEA LEAVES (Camellia Sinensis (L) O kuntze) IN THAI NGUYEN PROVINCE

THESIS BECHELOR

Thai Nguyen, 2022

Study Mode : Full-time

Major : Food Technology

Faculty : Advanced EducationProgram Office Batch : 2018 - 2022

i

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Degree Program: Bachelor of Food Science and Technology

Thesis Title: Research on kombucha production process from tea

leaves (Camellia Sinensis (L) O.kuntze) in Thai

Keywords: Camellia sinensis (L) O.kuntze, fermented beverage temperature, pH, °Brix, fermentation parameter, SCOBY

Number of pages: 60

Date of submission November 11th, 2022

ii

ACKNOWLEDGMENT

At the beginning of my thesis page, This experiment was carried out at the Department of Food Technology, Faculty of Biotechnology and Food Technology, Thai Nguyen University of Agriculture and Forestry in 2022 Therefore, I would like to say thank you Sincere thanks to the teachers and lecturers of the Faculty of Biotechnology and Food Technology for creating conditions to complete this research project

In particular, I would like to acknowledge the Supervisor guidance of Mr Dinh Thi Kim Hoa, lecturer at the Faculty of Biotechnology - Food Technology, Thai Nguyen University of Agriculture and Forestry, who has enthusiastically helped, instructed, and provided me with useful knowledge so that I could complete the thesis graduation

In addition, I would like to express my sincere thanks to my friends and family, who have always been there for me, supported me when I was having difficulties, and supported me through the difficult period when completing my thesis this bachelor's project

Finally, this thesis is not immune to errors due to a lack of knowledge and practice time I look forward to receiving feedback from the committee to improve my thesis graduation

2.1 Overview about Camellia sinensis (L) O.kuntze 7

2.1.1 Characteristics of Camellia sinensis (L) O.kuntze 7

2 Overview of the chemical compositions of Camellia Sinensis (L) O kuntze 10The Composition of a Typical Tea Beverage, 10

2.4.2 Kombucha size market 17

2.5 Research about Kombucha in the world 20

2.6 Research on Kombucha in Vietnam 21

2.7 Influence of fermentation in aerobic environment 22

2.7.1 Acetic acid bacteria 22

2.7.2 Lactic acid bacteria 24

2.8 Fermentation process 25

2.8.1 Acetic fermentation in food 25

2.9 Cell morphology and structure 29

2.9.1 Yeasts 29

2.9.2 Bacterial 31

CHAPTER III 34

RESEARCH CONTENT AND METHODOLOGY 34

3.1 Material and reseach scope 34

3.1.1 The raw material 34

3.5.1 Experimental design method 37

Research content 2: Completing the process of producing Kombucha product from tea leaves (Camellia sinensis (L) O.kuntze) 42

3.6 Data statistical analysis methods 48

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CHAPTER IV RESULT AND DICUSSION 49

4.1 Result for determining the component of raw materials 49

4.2 Research results on the effect of the tea materials on the quality of products504.3 Research results to select the ratio of old tea leaves/water in the quality of product 51

4.4 Results of the study on the effect of pH on the quality of product 53

4.5 Research results on selection of added sugar ratio for Kombucha fermentation 55

4.6 Research results on the effects of yeast ratio on the quality of product 57

4.7 Research results on Kombucha fermentation time 58

CHAPTER V CONCLUSION AND RECOMMENDATION 62

5.1 Conclusion 62

5.2 Request 62

CHAPTER VI REFERENCE 63

APPENDIX 1

5.5 The results on the effects of yeast ratio on the quality of product 8

5.6 The result of effecting fermentation time on the quality of product 10

5.7 Appendix ballot for product sensory assessment score 12

1

LIST OF FIGURES

Figure 1 Flavonoid structures 11

Figure 2: Molecular structure of catechin 12

Figure 2.1 : Apple cyder product 17

Figure 2.2 : Kombucha size market 18

Figure 2.3: Global Kombucha Market 19

Figure 2.4: Main metabolic activities of yeasts, acetic acid bacteria,

and lactic acid bacteria during a kombucha fermentation.((Laureys et al., 2020) 29

Figur 3.1: The processing of produce kobucha beverages from tea leaves 42

( Camellia sinensis (L) O.kuntze) according to ( Jayabalan et al , 2014) 42

Figure 4.1: determining the component of raw materials 49

2

LIST OF TABLES

Table 2.1.: The composition of a typical tea beverage 10

Table 2.2: Some genera and species of yeast found in kombuchas 31

Table 2.3: Some genera and species of bacteria found in kombuchas 31

Table 3.1 Experiment chemicals 34

Table 3.2 Laboratory instruments 35

Table 3.3 The importance coefficient of each indicator 41

Table 3.4 Table of regulations to evaluate the level of product quality 41

Table 3.5 Experimental design to study the effect of tea material on the quality of product 43

Table 3.6 Experimental design to determine the effect of material/ distilled water ratio on the quality of product 44

Table 3.7 Experimental design to determine the effect of pH on the quality of product 45

Table 3.8 Experimental design to study the adding sugar on the quality of product 46

Table 3.9 Experimental design to study the effect of yeast ratio on the fermentation process 47

Table 3.10 Experimental design to determine the effect of fermentation time on the quality of product 48

Table 4.1: the effect of the tea materials on the quality of products 50

Table 4.2 Affecting of material/ distilled water ratio on the sensory quality of product 51

Table 4.3 Research results on the effect of the ratio old tea leaves /water in the quality of product 52

Table 4.4 Affecting of material/ distilled water ratio on the sensory quality of product 52

Table 4.5 Results of the study on the effect of pH on the quality of product fermentation 53

Table 4.6 Affecting of the effect of pH on the sensory quality of product 54

3 Table 4.7 Research results on the effect of the percentage of added sugar on the

quality of Kombucha tea products 55Table 4.8 Affecting of the percentage of added sugar on the sensory quality of

product 56Table 4.9 Efects of yeast ratio on the quality of Kombucha tea products 57Table 4.9 Affecting of yeast ratio on ratio on the sensory quality of product 57Table 4.10 The result of fermentation time on the quality of Kombucha tea

products 58Table 4.11 Affecting of fermentation time on the sensory quality of product 59Table 4.12 Results of microbiological analysis of Kombucha tea products 61Table 4.13 Quality analysis results of fermented Kombucha tea products from

extracts of old tea leaves 61Table 4.14 Preliminary calculation table of raw material prices for the

production process (For 60 liter fermentation batch) 61

4

LIST OF ABBREVIATIONS

SCOBY Symbiotic colony of bacteria and yeast

bacteria (SCOBY) “Symbiotic Colony of Bacteria and Yeast”(St-Pierre, n.d.)

When microorganisms are grown in herbal tea, provided with enough nitrogen, vitamins and minerals and other basic nutrients, they grow and convert sugars into lactic acid, gluconic acid, and produce a in small amounts of alcohol, this alcohol is then converted to acetic acid (up to about 3%), giving it a sweet and sour taste similar to that of the fermented apple cider of the tea The final tea composition contains an average of 0.5 -1% alcohol, small amounts of lactic acid, malic acid, acetic acid, malonic acid, and oxalic acid The amount of vitamin B in the finished product is quite high, there is also a small amount of vitamin C Kombucha fermented tea is a popular and popular beverage in Eastern European countries and other countries, it is known as a real food healthy product Scientific studies have shown that the consumption of Kombucha is very beneficial for health Kombucha is high in glucuronic acid (GA) (Martínez-Leal et al., 2020) GA helps detoxify the liver by combining with waste products and toxins from drugs and environmental pollution and converting them into soluble compounds that the body can easily push out In addition, Kombucha has antioxidant capabilities, so it can fight free radicals that cause digestive disorders As a digestive enzyme, kombucha helps increase the amount of beneficial bacteria in the gut, by producing lactic acid Besides, using Kombucha tea has many other uses such as boosting immunity, good for bones and joints and preventing cancer ) (Martínez-Leal et al., 2020) However, at present, the production of Kombucha tea is mainly carried out spontaneously on a small scale in a few households, there is almost no research on the production process to put it into production on a public scale And so need research on manufacturing processes to put into production on an industrial scale In addition, Thai Nguyen is a province with the second largest tea production reserves inViet Nam The

6 amount of tea leaves and old tea leaves that are discarded accounts for a very large proportion because producers only use young tea buds for dry tea production This, creating production technology, making use of old tea leaves will be an effective solution, improving the use value of tea plants in Thai Nguyen province Based on the discussion above, the research team carried out this topic

1.2 Research’s objective

1.2.1 Overall objective

Developing fermentation process to create Kombucha products with

delicious flavor from tea leaves, improving the value of tea plants (Camellia

sinensis (L) O.kuntze) in Thai Nguyen province

1.2.2 Detail objective

- Selecting the treatment mode of raw materials to produce Kombucha - Investigating the technological parameters of the fermentation process to produce Kombucha from tea leaves

- Completing the Kombucha fermentation process from Thai Nguyen tea leaves on a laboratory scale and evaluating economic efficiency

1.3 Research question

Could fresh leaves of Camellia Sinensis (L) O.Kuntze plants be used to

ferment Kombucha beverages?

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CHAPTER II

LITERATURE REVIEW

2.1 Overview about Camellia sinensis (L) O.kuntze

2.1.1 Characteristics of Camellia sinensis (L) O.kuntze

Thai Nguyen tea leaves Tea tree, scientific name Camellia sinensis (L)

O.kuntze, is a plant whose leaves and buds are used for the production of Tea, belonging to the family Theaceae Tea Leaves are simple, alternate, with a characteristic aroma; leaf blade hard and thick, oval with rounded tail and rounded or oblong base, 9-11 cm long, 4-5 cm wide, dark green on the upper surface than below, hairy on the underside; shallow serrated margins; Leaf veins are feathery, main veins are prominent, 5-6 pairs of secondary veins are connected near the leaf edge Petiole short, shallow trough, green, 0.6-0.8 cm long, no accompanying leaves.(Đỗ Ngọc Quý- NXB Nông nghiệp, 2008.)

Camellia Sinensis (L) O.kuntze is a tree capable of living in tropical and subtropical regions such as Ha Giang, Dien Bien, Lai Chau, Thai Nguyen province The living plant mainly grows in shrubs or small trees The main biological activities of Tea have been proven in the world, including antioxidant, antibacterial, anti-oxidant, anti-obesity, anti-cancer, anti-hypertensive However, currently in Vietnam there is no publications on the evaluation of the common bioactivity of this tea leaf Therefore, this study was carried out to evaluate the outstanding biological activities of old tea leaves from tea tree in order to prove the medicinal value of this folk medicinal plant Tea, commonly known by the folk name "Che" is formed mainly by large bushes or small trees, there are plants of the tea family that are climbing stems There are types of Tea that normally grow above 1,500 meters (4,900 feet) so that the tea tree grows slowly, accumulating rich flavors The main stem and roots develop below the soil layer The leaves of the tea are 4–15 cm long and about 2–5 cm wide Fresh leaves contain about 4% caffeine The young, light green leaves are harvested for

8 tea production At that stage, the underside of the leaves has short white hairs Older leaves turn dark green Depending on the age, tea leaves can be used to make different tea products because of the different chemical composition of the leaves Usually, only the budding leaves and 2 to 3 new leaves that grow near that time are harvested for processing Hand-harvesting takes place at regular intervals every 1 to 2 weeks Tea leaves are used in Oriental medicine to treat bronchial asthma (as an asthma medicine), mouth sores, angina, coronary heart disease and external vascular disease Today, green tea is popular everywhere, is a very healthy drink, contributes to preventing cancer, lowering cholesterol, killing bacteria, and losing weight Tea contains large amounts of catechins, an

antioxidant Among the activities, (-)-catechin from C.sinensis stimulates

PPARgamma, the nuclear receptor, which is the current pharmacological target for the treatment of type 2 diabetes The chemical composition of tea leaves has been investigated carefully studied The main constituents of green tea leaves belong to the polyphenol group for 25 ± 35% of the dry weight (Balentine et al., 1997; Hara, Luo, Wickremashinghe & Yamanishi, 1995c) The most important and characteristic tea polyphenols are the flavanols in which catechin (flavan-3-ols) predominates and the main ones are: epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG), (+) - catechin (C), and (+) - gallocatechin (GC) (Hara et al., 1995c) These compounds contribute to the fleshy, astringent, and sweet aftertaste of bleached tea (Hara,Luo, Wickremashinghe & Yamanishi, 1995e)

Tea also contains flavonols, mainly quercetin, kaempferol, myricetin and their glycosides In black tea, oxidation of polyphenols during proces sings leads to the formation of catechin and gallic acid complexes such as theaflavins, theavinic acids, ubigins or theasinensis, and of proanthocyanidin polymers (Balentine et al., 1997; Hara et al associates, 1995d) Methylxantine is present in 2 ± 4% as caffeine and small amounts of theophylline and theobromine (Hara et

9 al., 1995c) Tea are contains many amino acids, but theanine, specifically for the tea plant, is the most abundant, accounting for 50% of the total amino acids Amino acid degradation is involved in the biological origin of tea aroma (Balentine et al., 1997) Chlorophyll, carotenoids, lipids, and volatile compounds are not the main ingredients in a teapot but they also play an important role in the development of aroma (Hara et al., 1995c) The volatile parts of tea leaves have been studied in detail and more than 600 different molecules have been isolated (Hara et al., 1995c, e; Shi moda, Shiratsuchi & Osajima, 1995; Shimoda, Shige matsu,

Shiratsuchi & Osajima, 1995) Includes terpenoids and degradation products of amino acids, carotenoids, and linoleic acid (Hara et al., 1995c) Tea also contains carbohydrates, vitamins E, K, A, low content of vitamin B and vitamin C (only in green tea) Tea alsoprovides dietary amounts of potassium, manganese, and fluorine ions (Hara, Luo, Wick remashinghe & Yamanishi, 1995f)

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2 Overview of the chemical compositions of Camellia sinensis (L) O kuntze

The Composition of a Typical Tea Beverage

Table 2.1.: The composition of a typical tea beverage

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2.2.1 Polyphenols

In terms of human consumption, tea represents a major source of dietary polyphenols The polyphenolic fraction of tea represents 30 to 40% wt/wt of extract solids and provides astringency, the 'drying' sensation experienced in the mouth after consumption of the tea beverage A tea drinker typically consumes 180 to 240 mg of polyphenols from a strong cup of tea Recently interest in the health aspects associated with the consumption of tea beverages has grown within the scientific community and has generated much excitement about tea polyphenols The tea plant produces a diverse number of polyphenolic constituents, presumably as a means of chemical defense against insects, birds, and animals, which would consume the plant as food (Beart et aI., 1985) The evolution of salivary proline-rich proteins, which bind polyphenols effectively, has ameliorated this defense mechanism, converting it to 'astringency' (Luck et aI., 1994)

2.2.2 Caffeine, Methylxanthines, and Related Compounds

Tea has been valued historically for its caffeine content Caffeine is viewed as an important constituent of tea, bestowing mood and cognitive-enhancing properties (Bokuchava and Skobeleva, 1980)

2.2.3 Flavonoids

Figure 1 Flavonoid structures

Most phenolic compounds found in tea are polyphenols Polyphenols are compounds consisting of more than one benzene ring with each containing at least one hydroxyl group (–OH) The main polyphenols present in tea are the flavonoids There are six major classes of flavonoids in the diet including flavonols, flavones, flavanols, flavanones, anthocyanins and isoflavones The most common subclasses of flavonoids in tea are the flavanols (primarily

12 catechins) and flavanols (such as quercetin) Also present in tea, but at significantly lower concentrations, are phenolic acids such as gallic acid and cinnamic acid esters of quinic acid Research interest in tea has been primarily due to the presence of flavonoids Health benefits of tea are believed to be largely due to the consumption of these flavonoids Both green and black teas are rich in flavonoids tea (2 g of tea leaves infused in hot water for 1–3 min) will provide 150–200 mg of flavonoids A typical green tea serving contains approximately 90–100 mg of catechins (Harbowy and Ballentine, 1997) During the production of black tea most of these catechins are oxidized to condensed flavonoids, such as theaflavins and thearubigins

2.2.4 Catechin compounds

Figure 2: Molecular structure of catechin

The major catechin compounds in tea are: (-) epigallocatechin 3 - gallate (EGCG), (-) epigallocatechin (EGC), (-) epicatechin (EC), (-) epicatechin 3 - gallate (ECG), and (-) epicatechin 3 - gallate (ECG) +) cate chin (C) The main catechin in fresh tea leaves accounts for 25-30% of CK and plays an important role in performing biological functions in the life process of tea plants, it always changes in both quantity and quality during development of tea plants The most important of them is EGCG, which accounts for 50% of the total mass of tea phenols and about 8 - 12% of CK in dried tea leaves and accounts for about 10% of matcha Catechin of tea belongs to flavonoid, flavan - 3 - ol group, molecule with 15 carbons including 2 rings of 6 carbons A and B joined by 3 carbon units at positions 2, 3, 4 forming a heterocycle C contains an oxygen atom The structure of catechins contains two asymmetric carbons at positions 2 and 3

13 without a double bond at positions 2, 4, and a 4-oxo groussp.(Balentine et al., 1997; Hara, Luo, Wickremashinghe & Yamanishi, 1995c)

The group of catechin compounds is assessed to be related to the green color of the split, plays a decisive role in the formation of the color of the powder and the taste of tea, and during processing, catechins also participate in flavoring aromatic under the action of enzymes and some other compounds available in the bud

The quality of green tea depends not only on the content of catechins but also on the ratio of their constituent catechins The ratio of catechin components is not only important to evaluate tea quality, but it is also reported to inhibit tumor cell growth and the degree of impact in the order: ECG > EGCG > EGC > EC In fact, many tea varieties have low tannin content in buds, but when processing green tea, the product is not of high quality due to its acrid taste On the contrary, there are tea varieties like Shan tea, which contain quite high tannin content, but when processed, green tea gives the product a sweet, bitter taste In Japanese matcha, although the EGCG content is high, it still has a sweet taste when drinking This explains that the taste of tea always depends on the proportion of catechins If the proportion of catechin gallate component (complex catechin) is high in the molecule containing many OH groups, it will cause the tea to have a slightly bitter acrid taste, and vice versa, the high ratio of simple catechin will give the tea a mild acrid taste The content and proportion of cat echin components always depend on each tea variety, geographical location, cultivation technique, and harvest season (Higdon and Frei, 2003)

In 1966, Higginson published the first report on the epidemiology of host and cancer Many extensive studies on the cancer prevention effect of green tea on esophageal, breast, pancreatic, prostate and bowel cancers The anticancer properties of tea are mainly attributed to catechins, a group of polyphenol compounds In green tea, catechin compounds contain 4 main groups: EC, ECG, EGC and EGCG When analyzing the catechin content in young shoots and mature leaves by HPLC method, the results show that catechins are abundant in the shoots of tea leaves and higher than in mature leaves, among which EGCG has the highest content, while the concentrations of EC and C are relatively low David J Wess et al used methanol electrophoresis chromatography to analyze

14 the catechin and caffeine content in matcha and regular green tea, the results showed that the EGCG in matcha was at least 3 times higher times that of regular green tea.(Higdon and Frei, 2003)

2.2.5 Chlorophyll

Chlorophyll is the green pigment of plants in general and tea in particular There are two types of chlorophyll: chlorophyll a and chlorophyll b This substance plays an important role in the biosynthesis of plants The structural formula of chlorophyll consists of four pyrol rings connected by a methyl (-CH) bridge to form a porphyrin structure with Mg in the middle The fourth pyrol ring has a phytol group and the third pyrol ring has a cyclopental ring monospaced, double bond system in the structure makes chlorophyll a strong photochemical activity The bonding energy of the team is small, the magnetic field formed inside the pair is easy to bounce off when receiving light energy The phytol tail consists of a phytol alcohol base of 20 carbon

Scientist German chemist Richard Willstätter (1872-1942) who received the Nobel Prize in chemistry discovered that chlorophyll, consists of 2 components: a and b (green chlorophyll and yellow green chlorophyll.) Matcha contains both chlorophyll a and chlorophyll b In many European countries (Germany, UK, France ) and FDA in the US, chlorophyll and chlorophyllin are considered as a food, a safe drug, there is no limit on the dose used (FAO report - WHO No 557, 1974) Chlorophyll has the effect of preventing cancer and fighting cancer cells, which is explained by two effects: firstly, it blocks toxins that are easily combined structurally with chlorophyllin and secondly, it is a natural electron-reducing effect due to the free radicals of chlorophyllin (chlorophyllin is considered an antioxidant) Chlorophyllin has been applied in many products for people following a diet, popular in Western countries for weight loss and prevention of diseases requiring no dietary energy, degenerative diseases such as fiber atherosclerosis, hypertension, diabetes, alzhermer Chlorophyll is very similar in structure to human blood, so it is an excellent food for the cardiovascular system Research shows that it increases the production of oxygen in the blood, which in turn helps heal wounds on the body faster In

15 addition, chlorophyll is known as a detoxifier because it effectively removes

toxins and heavy metals from the body, including from the blood and liver

2.3 Overview of biological effects (pharmacology) of Kombucha

2.3.1 Detoxifying

Glucuronic acid works to detoxify the liver by combining with waste products and toxins, then converting them into soluble compounds that the body can easily push out In one study, the use of kombucha tea helped test subjects' liver cells to be protected from oxidation and maintain physiologically normal

despite exposure to a specific toxin According to the researchers, due to

“Kombucha’s antioxidant activities and may be beneficial for liver disease where oxidative stress occurs"

2.3.2 Digestion-Aid

Kombucha has antioxidant capabilities, so it can fight free radicals that cause digestive disorders As a digestive enzyme, Kombucha helps increase the amount of beneficial bacteria in the gut, by producing lactic acid This bacteria

acts like the acidophilus bacteria commonly found in yogurts In addition, two

types of lactic acid and acetic acid help accelerate fermentation in the intestines, helping to prevent constipation, intestinal infections, dysentery, digestive disorders, and curing intestinal and stomach ulcers Kombucha can also prevent

the yeast (Fungi Candida albicans) infections in the gut and help restore balance

to the digestive system.(Jakubczyk et al., 2020)

2.3.3 Extra Energy

Kombucha can supplement the human body's energy further thanks to the formation of iron during fermentation It also contains very small amounts of caffeine and B vitamins that have the ability to energize the body Iron-rich kombucha can help increase blood hemoglobin levels, improve oxygen supply to tissues, and promote energy production at the cellular level In other words, by helping the body to produce more energy (ATP), this ancient tea can also help people who drink regularly have an abundant source of energy

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2.3.4 Immune Booster

Kombucha has the ability to control free radicals through antioxidant measures, thereby helping to strengthen immunity Oxidative stress reduces the function of the immune system; however, it is suppressed by a powerful antioxidant called D-Arabino-1,4-lactone acid (DSL) that has been detected in Kombucha fermentation Scientists believe that DSL and the vitamin C present in kombucha are factors that help it fight cell damage, inflammation, tumors, and depression

2.3.5 Good for joints

Consuming Kombucha tea can aid in the healing, repair, and prevention of osteoarthritis in several ways: Kombucha combines glucosamines that increase hyaluronic acid production, aid in the maintenance of collagen, and prevent the pain due to arthritis Besides, it also supports collagen production throughout the body and reduces the appearance of wrinkles

2.3.6 Cancer Prevention

Kombucha has many uses in preventing cancer and increasing the ability to recover health in patients with this disease Regular intake of Kombucha helps prevent cancer cell growth in its early stages thanks to its glucuronic, lactic and acetic acids as well as its antibiotic compounds It may have anti-cancer effects especially on hormone-dependent tumors Many studies have shown that the rational use of Kombucha can be useful in the prevention and treatment of prostate cancer, against lung cancer, malignant bone tumors, kidney cancer

2.3.7 Weight loss

According to data from a 2005 study, scientists suggest that Kombucha improves metabolism and limits fat accumulation Although more research is needed to confirm this, according to many people, drinking Kombucha can aid weight loss because it contains acetic acid (like apple cider vinegar) and polyphenols that have been shown to help with weight loss well

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2.4 Some kind of products in the current market

2.4.1 Cyder

Figure 2.1: Apple cyder product

Cyder is a fermented alcoholic beverage prepared from any fruit juice including apple, peach, pear (Perry Perry cider) or other fruit juices The most traditional and common type of cider is cider Based on different processing conditions, cider can be classified into the following categories Hard cider - an alcoholic drink made from fermented apple cider Scrumpy - strong cider (as made in the west of England) Sweet cider - unfermented cider Cyder - single juicing of classic fruit

The average alcohol content of cider is about 1.2% to 8.5% Apples are actually mainly used to produce cider because they are richer in tannins and sharper in flavor It is widely available and popular in European countries and the UK Cider is a common ingredient for Cocktails, and it is also used to make vinegar (e.g apple cider vinegar) The anabolic substances synthesized during the fermentation of cider are responsible for most of the flavors and aromas Tannins and several other phenolic compounds are congeners found in cider.

(Blenkinsop, Philip (2012) Insight)

2.4.2 Kombucha size market

The global kombucha market size was valued at USD 2.64 billion in 2021 and is expected to expand at a compound annual growth rate (CAGR) of 15.6% from 2022 to 2030 Consumers have significantly shifted toward proactively

18 tackling their health & wellbeing, and have dedicated themselves to improving their overall longevity, which has led to higher product acceptance Kombucha is marketed as a product that eliminates toxins, boosts energy & immune system, and helps lose weight, which is driving the frequency of its consumption among consumers globally

Figure 2.2 : Kombucha size market

In September 2020, Equinox Kombucha conducted consumer research of 2000 people in the U.K on their thoughts about healthy foods and beverages, which revealed that 39% of people were eating healthier foods, 27% were eating more natural foods, and 23% were reading food labels more often Nearly 33% of people knew what kombucha was, with 12% thinking it was a food item Nearly 48% believe that probiotics are always better for improving gut health, and 14% believe that in order to cleanse the gut, detox is a must

In a similar vein, in July 2021, Remedy Drinks announced the launch of a zero-sugar shelf-stable product in the U.S The product will be offered in cans and will be available in four different flavors: peach, ginger lemon, mixed berry, and raspberry lemonade Furthermore, the United States Food and Drug Administration (FDA) stated that this product is safe for human consumption when properly prepared and has been commercialized in many countries

19 Several consumers are still unaware of the potential positive benefits of drinking authentic kombucha on their overall well-being and the state of their immune system However, in the coming years, there is a massive opportunity for kombucha brands to educate consumers in the area of gut health and fermented foods & beverages

Products like kombucha will be increasingly important to customers who are looking for food and drink items that help them stay healthy Market players are improving their understanding of specific health outcomes provided by regular drinking of kombucha, as well as engaging with customers in the process of new product development and design

Distribution Channel

Figure 2.3: Global Kombucha Market

The off-trade segment is anticipated to expand at a higher CAGR of 17.1% during the assessment period These beverages are now more readily available through various distribution channels, including supermarkets and convenience stores, health stores, and online retailers

The range of innovative flavors is also attracting an increased number of consumers, with companies offering novel flavors such as lavender, watermelon, peach, and passionfruit alongside their original beverages and marketing them through off-trade platforms

20 The on-trade segment accounted for a larger revenue share of 61.2% in the global kombucha market in 2021 and is expected to maintain its dominance during the forecast period Recently, growing inclination towards premium hand-crafted cocktails containing flavored kombuchas has led to an increase in their consumption through on-trade channels, including premium bars, pubs, cafés, restaurants, and other food service outlets

These on-trade channels are offering non-alcoholic and gut-supporting kombucha cocktails in different ingredients and novel flavors, which has increased their consumption through these channels This beverage has a very low alcohol percentage of about 0.5% to 2% which makes it a potential option for cocktails; in response, several bars and pubs are offering more affordable options to cater to the ongoing trend

2.5 Research about Kombucha in the world

Kombucha has been widely used in some countries around the world such as China, Japan, Russia, France, etc Since 1852, the research of Kombucha tea has been focused, mainly in Europe Between 1925 and 1950, several medical studies have shown some benefits of Kombucha tea These include: Bacinskaja (1914) and colleagues said that Kombucha is good for the digestive system, has the ability to regulate intestinal activities, especially against constipation By 1927, Madaus had studied and concluded that Kombucha and its metabolites had a positive effect on cell wall regeneration, an excellent remedy for atherosclerosis In 1987, research by Veronika Carstens showed: Kombucha has the effect of detoxifying the body, strengthening, and improving immune system function Studies on the identification of microbial strains in Kombucha and the application of strains isolated from Kombucha have also existed for a long time:

Since 1928, Hermann has identified two strains of Bacterium xylinum and B

xylinoides and B gluconicum

The researchers also identified Kombucha as a symbiotic association

between Bacterium xylinum and other bacteria, with yeast cells of the

Saccharomyces ketogenum strain, forming a bird’s nest-like mass In 1973, List

et al isolated Acetobacter ketogenum; Pichia fermentans; Saccharomyces

2.6 Research on Kombucha in Vietnam

In Vietnam, there have not been many research on Kombucha tea Herbalist Dinh Cong Bay, General Secretary of the Ho Chi Minh City Medicinal Association, said that he had also heard of water ginseng, because he had not studied it, he could not answer the real use of this tea In Vietnam, there have also been several research works on kombucha In 2012, Le Nguyen Thi Hong Ngoc and colleagues had a research project "Study on the microbiome of Kombucha tea and its effectiveness on some physiological parameters of rats The result of this study has depicted that the microflora in Kombucha includes: acetic bacteria, lactic acid bacteria and yeast, and at the same time investigated the effects of Kombucha on a number of physiological parameters of rats such as weight, total erythrocyte count, total white blood cell count, and health of laboratory rats through a swimming exhaustion model Kombucha products have health promoting effects, do not adversely affect the physiological parameters of rats Recently, the topic "Investigation of the effects of processing time on some properties and biological activities of Kombucha vinegar tea" by Master Ton Nu Lien Huong from Can Tho University in 2012, the topic "Research on Kombucha vinegar tea" Some strains of yeast fermenting kombucha from Ha Giang Shan Tuyet tea" by MSc Lam Thi Hong Lien and "Research on some bacterial strains fermenting Kombucha from Thai Nguyen tea" by MSc Le Thi Hue, Hanoi National University of Education February 2013

22

2.7 Influence of fermentation in aerobic environment

2.7.1 Acetic acid bacteria

Several microorganisms are common in kombucha fermentation The microorganisms found in kombucha belong to the genera Acetobacter, Gluconobacter, Gluconacetobacter and Komagataeibacter Komagataeibacter xylinus is the most characteristic microorganism of kombucha fermentation responsible for the production of cellulose coating This microorganism has been classified into many types as Acetobacter xylinus, Acetobacter xylinum, Acetobacter aceti subsp xylinus, Gluconobacter xylinus, Gluconacetobacter xylinus and some others Identification of Acetic acid microorganisms at the species level is often imprecise because phenotypic and 16S rRNA methods are often not accurate enough to identify Acetic acid bacteria Common microorganisms in kombucha are cellulose-producing acetic acid bacteria, possibly Komagataeibacter species, and possibly Komagataeibacter xylinus strains These species can include 10-20% acetic acid in the medium, while Acetobacter species can only accumulate up to 8% acetic acid Acetic acid bacteria are gram-negative (or gram-modified) bacteria belonging to the class a-Proteobacteria and the family Acetobacteraceae They are migratory between 0.5 mm and 1-4 mm long, with ellipsoid to rod-shaped and non-spore forming cells Although they are obligate aerobic, they can live and grow for quite a long time, in low oxygen conditions, such as bottled wine and cocoa fermentation These low oxygen conditions can induce a viable but non-cultured state (VBNC), which reduces their ability to recover through culturing methods Their minimum growth is 25-30 C and pH 5.0-6.5, but many species also grow at pH 3.0-4.0 and even lower AABs are not generally pathogenic to humans and they do not produce toxic compounds or biogenic amines The most important feature of AAB is the oxidation of the ectoplasm of alcohols, aldehydes, sugars and sugar alcohols in the presence of oxygen by dehydrogenases located on the surface of their cytoplasmic membranes (Greenwalt, C J.; Steinkraus 2000,) We have an example as follows, ethanol is oxidized to acetaldehyde and then oxidized to acetic acid Glucose can be oxidized to gluconic acid, glucuronic acid, 2-

23 ketogluconic acid, 5-ketogluconic acid, 2,5-diketogluconic acid and glucuronic acid Acetobacter and Gluconacetobacter species prefer the oxidation of ethanol to glucose, while the species of Gluconobacter prefer the oxidation of glucose, glycerol, gluconic acid, and sorbitol to ethanol Gluconobacter species are usually detected in high sugar environment (such as sugarcane, sweet fruit, honey, ), while Acetobacter and Gluconacetobacter species are in high alcohol environment When the ethanol runs out, the glycerol can be oxidized to dihydroxyacetone (DHA) This compound is considered by scientists to react with amino groups of amino acids and proteins to form a brown complex and is used in skin products DHA enters the Embden-Meyerhof-Parnas (EMP) pathway, and from there into the pathway that builds up gluconeogenesis, eventually building up cellulose Acetic acid bacteria no longer have a fully functional EMP pathway (with aldolase as the key enzyme) because they lack the enzyme phosphofructokinase and metabolize glucose normally via pentose phosphate (PPP; highlighted by phosphoketolase as an essential enzyme) The Acetobacter, Gluconacetobacter and Komagataeibacter species include a fully functional TCA cycle, allowing the complete (excessive) oxidation of organic acids (such as acetic or lactic acid) to carbon dioxide These do not concur with Gluconobacter which lack the enzymes a-ketoglutarate dehydrogenase and succinate dehydrogenase, and thus cannot oxidize organic acids to carbon dioxide The number of species of Gluconacetobacter and Komagataeibacter that produced cellulose polysaccharides did not change much in aqueous medium [b- (1->4) glucans] from glucose, fructose, sucrose, and other substrates such as ethanol and glycerol This is evident in kombucha fermentation as a thick film evolves on the surface of the fermented wine Many factors influence the production of cellulose by AAB, such as substrate type, substrate concentration and pH Some species of Gluconobacter, Gluconacetobacter and Komagataeibacter also produce water-soluble levans [b- (2->6) fructans] from sucrose, dextrans [a- (1->6) glucans dissolved in water with a- (1-) > 4) branches] from maltose or maltooligosaccharides, or water-soluble acetan polysaccharides (including glucose, mannose, glucuronic acid and rhamnose) (Jansson et al., 1993)

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2.7.2 Lactic acid bacteria

Lactic acid bacteria belong to the Lactobacterium family These are spore forming bacilli, cocci and are mostly non-motile, facultative respiration They are capable of fermenting a variety of simple sugars and disaccharides but are not capable of fermenting complex carbohydrates and starches Its growth requires the presence of peptones, amino acids or ammonium salts They have special nutritional requirements that are rich in vitamins, amino acids and minerals The suitable temperature for fermentation at from 15-50℃ However, each species has a different suitable temperature range, if the temperature is higher than 80℃, lactic acid bacteria will be completely destroyed

non-The presence of Lactic acid bacteria in kombucha is inconsistent Usually, they are not present or present in low abundance, but in certain industrial kombucha fermentations they have been found in higher abundance The Lactic acid bacteria recovered in the latter case were mainly Oenococcus oeni and

actobacillus nagelii, both known to be acid-tolerant Lactic acid bacteria are

gram-positive bacteria belonging to the phylum of the Firmicutes and are characterized by certain morphological, metabolic, and physiological

characteristics They are not motile, are rod-shaped (Lactobacillus) or spherical (Leuconostoc, Lactococcus, Oenococcus), and do not sporulate They are

facultatively anaerobic, but do not use oxygen for their metabolism Their optimal growth is 25-40℃ and pH 3.5-6.0 They are generally regarded as safe (GRAS), but some strains are known to produce biogenic amines, while on the other hand, many Lactic acid bacteria are regarded as probiotic microorganisms They are associated with the production of lactic acid, but they can also produce other metabolites such as ethanol, acetic acid, carbon dioxide, diacetyl, and mannitol

Lactic acid bacteria can be classified into obligately homofermenters

(Enterococcus, Lactobacillus, Lactococcus, Pediococcus, Streptococcus), obligately heterofermenters (Leuconostoc, Lactobacillus, Oenococcus, Weisella),

and facultative heterofermenters (Lactobacillus) Homofermenters metabolize glucose via the EMP pathway with lactic acid as the main end product Heterofermenters metabolize glucose via the pentose phosphate pathway with lactic acid, carbon dioxide, and ethanol as the main end products When

25 alternative electron acceptors (such as fructose) are present, acetic acid is produced instead of ethanol, whereby fructose is reduced into mannitol Facultative heterofermenters metabolize glucose via the homofermentative pathway and metabolize pentoses and gluconic acid via the heterofermentative pathway When cells have excess pyruvate available, for example due to the presence of external electron acceptors such as citrate in milk, pyruvate can be converted into a-acetolactate, which will spontaneously degrade into diacetyl Lactic acid bacteria are well known for their ability to produce exopolysaccharides (EPS) A distinction is made between homopolysaccharides, which consist of only one monosaccharide and which can be produced in large quantities (g/L); and heteropolysaccharides, which consist of a repeating oligosaccharide (often containing glucose, galactose, and rhamnose) and which are usually produced in smaller quantities (mg/ L).(Hoffman and Lowe, n.d.)

Homopolysaccharides are synthesized by an extracellular transglycosylase (such as glucan- or fructansucrases) from a glycosidic donor (such as sucrose, lactose, or maltose) and a suitable acceptor molecule (such as maltose or growing polysaccharides) Lactic acid bacteria EPS can be desirable in yogurt or sourdough bread for their stabilizing effects, but are usually undesirable in beverages, for example the production of EPS in beer by Pediococcus damnosus

2.8 Fermentation process

2.8.1 Acetic fermentation in food

Acetic fermentation is the oxidation of ethanol to acetic acid, under aerobic conditions, with the metabolic agent being acetic bacteria

The general equation for converting ethanol to acetic acid:

C H OH + 3O = 2CH COOH + 4H O + 2CO

The essence of the oxidation of ethanol to acetic acid is a series of reactions that occur under aerobic conditions Ethanol and atmospheric oxygen must be absorbed by the bacterial cells inside and then the acetic acid formed will escape In bacterial cells, ethanol is first oxidized to acetaldehyde, which is converted to hydrateacetaldehyde, and then hydrateacetaldehyde is oxidized to acetic acid

Suitable conditions for acetic fermentation are pH = 3, temperature 30oC, ethanol concentration is about 6-12% (in the nutrient medium with

28-26 glucose) Depending on the type of bacteria, when the concentration of ethanol in the medium is not enough, a loss of acetic acid will occur due to the oxidation of acetic acid by bacteria:

CH COOH + O = 2CO + 2H O

The main microbial agent in acetic fermentation is acetic bacteria, which is classified in the genus Acetobacter They are relatively large, non-motile bacilli without spores, obligate aerobic respiration, suitable temperature is 30-35℃

Acetic bacteria and the oxidation of ethanol to acetic acid They are widely used to produce vinegar, soft drinks, rye bread, vitamin C, etc However, acetic bacteria can infect many stages in food processing They cause much damage to raw materials and finished products Especially in the industry of producing wine, beer, bread, canned food, etc…

Yeasts and Acetic acid bacterial are both present during kombucha fermentation at around 106 108 CFU/mL Extensive knowledge of the microbial diversity during kombucha fermentations has already been established, but the exact dynamics of the microbial ecosystem throughout the fermentation process has not yet been examined in sufficient detail and needs further investigation Within the kombucha microbial ecosystem, several symbiotic relationships can be found The substrate for a kombucha fermentation is usually sucrose, which is hydrolyzed into glucose and fructose by an invertase enzyme in the periplasm of yeast cells, whereby the concentrations of glucose and fructose increase This

dynamic has also been described in detail for water kefir Gluconobacter species can also use sucrose, but Acetobacter and Gluconacetobacter species are

dependent on yeasts for the hydrolysis of sucrose into glucose and fructose The total quantity of Komagataeibacter xylinus is thought to depend on the particular strain, carbon source, and other variables, while the cellulose yield is thought to be proportionate to its growth rate Caffeine and other xanthines present in tea appear to boost the creation of cellulose itself Fructose, glucose, and sucrose are the main remaining substrates in kombucha, and ethanol is the main metabolite During the fermentation of kombucha, yeast, acetic acid bacteria, and lactic acid bacteria all engage in significant metabolic activity

27 acetic acid, D-saccharic acid 1,4-lactone, gluconic acid, glucuronic acid, glycerol, and occasionally lactic acid However, reports also mention citric acid, malic acid, quinic acid, and oxalic acid According to reports, ethanol content peaks 6 to 10 days after production after that, it declines due to its conversion to acetic acid and/or cellulose, but this is not always the case Final concentrations have been reported to be around 10 g/L ethanol, 3 g/L acetic acid, and 1 g/L glycerol (Liu et al., 1996)

However, conclusive data about the dynamics of the substrate consumption and metabolite production, as well as the final metabolite concentrations are not yet established, so more detailed investigations are still necessary During kombucha fermentations, not only symbiotic relationships develop, but also many selective pressures are at work that steer the microbial species diversity in kombucha, and consequently also impact the substrate consumption and metabolite production For example, the production of ethanol by yeasts inhibits the growth of certain microorganisms One of the most important selective pressures during a kombucha fermentation is the acidic stress due to the production of organic acids by yeasts (acetic acid), Acetic acid bacterial (acetic acid, gluconic acid, glucuronic acid), and sometimes LAB (lactic acid and acetic acid) The pH of kombucha tea starts at 5.0-7.0, and drops to 2.0-4.0 after 7 days of incubation, and can decrease to below 2.0 after extended incubation Kombucha fermented until high concentrations of acetic acid are present selects for Komagataeibacter species, as these microorganisms are known for their high tolerance of acetic acid The pH of industrial kombucha fermentation processes is probably more tightly controlled than household kombucha fermentations, as kombucha will become unpalatable below a certain pH (Neffe-Skocińska et al., 2017) This also explains the presence of LAB in some industrial kombucha fermentation processes and their absence in household kombucha fermentation processes, as LAB do not grow below pH 3.5 Another important selective pressure that steers the microbial community is the limited availability of nutrients such as vitamins and nitrogenous compounds The only sources of (micro) nutrients are the tea leaves used in the recipe The low nutrient concentrations explain the prevalence of Dekkera/Brettanomyces species in

28 kombucha, as these yeasts were found to be more competitive in a nutrient-poor environment Dekkera bruxellensis from kombucha did not produce glycerol and produced lower amounts of ethanol, but higher amounts of acetic acid in comparison with Saccharomyces cerevisiae from kombucha The nutrient limitation also explains the presence of nitrogen-fixing Acetic acid bacterial in kombucha The availability of oxygen will determine the growth of Acetic acid bacterial, as these microorganisms are obligate aerobes Initially, the tea will be completely oxygenated followed by a decreasing supply of oxygen, as oxygen is consumed by the ABB and its influx hampered by the growing cellulose pellicle on top of the liquid It is therefore likely that the bottom part of the fermentation vessel will become anaerobic, limiting the conversion of ethanol and glucose into acetic acid and gluconic acid, respectively Most yeasts, such as Saccharomyces and Dekkera species, will continue a fermentative metabolism as long as substrates are available, even under aerobic conditions When glucose was used as a substrate, it seemed to be preferentially converted into gluconic acid and not into ethanol, whereas fructose seemed to be preferentially converted into ethanol Lactose did not seem to yield a satisfactory fermentation, as the pH remained > 5 after 17 days of incubation The optimal concentration of sucrose was around 50 g/L The best substrate for producing kombucha was found to be sucrose and black tea

Other substrates such as glucose, fructose, and lactose did not result in a satisfactory fermentation, as well as other nutrient sources, such as green tea, peppermint tea, and lime blossom tea It is clear that the process parameters during a kombucha fermentation will exert a significant impact on the final microbial composition and therefore on the metabolite composition of kombucha For example, the dimensions of the fermentation vessel and the ratio of the surface area to the volume could influence the level of oxygenation during a fermentation This is less of a concern when the liquor is vigorously stirred or aerated during the fermentation process, as has been successfully applied by some producers Low oxygenation and thus low acid production by Acetic acid bacterial can result in a pH above 4, which might stimulate the growth of LAB and thus the production of lactic acid Fermentation time will also have a

29 substantial impact on the acidic stress to the microbial ecosystem and therefore the microbial and chemical composition of the final product Higher fermentation temperatures appear to benefit LAB, which is consistent with their higher optimal growth temperatures in comparison to yeasts and AAB Beverage composition (Oliveira et al., 2022)

Yeast is a single-celled organism, yeast cells as well as many other cell types are composed mainly of basic parts such as glucan, protein, lipid and some other small components such as chitin Protoplasmic membrane: composed of complex compounds impurities such as proteins, phospholipids, permease enzymes Protozoa: the main constituents are water, proteins, carbohydrates, lipids and mineral salts, enzymes and organs in them Cell nucleus and other suborgan components: vacuoles, mitochondria, ribosomes, etc.1995c, e; Shi moda, Shiratsuchi & Osajima, 1995; Shimoda, Shige matsu, Shiratsuchi &

30 Osajima, 1995) Includes terpenoids and degradation products of amino acids, carotenoids, and linoleic acid (Hara et al., 1995c) Tea also contains carbohydrates, vitamins E, K, A, low content of vitamin B and vitamin C (only in green tea) Tea also provides dietary amounts of potassium, manganese and fluorine ions (Hara, Luo, Wick remashinghe & Yamanishi, 1995f)

Yeasts of the genera Brettanomyces, Zygosaccharomyces and

Saccharemyces were identified in 56%, 29%, and 26% respectively The species

Saccharomycodes ludwigii and Candida kefyr were only demonstrated in isolated

cases Furthermore, the test revealed pellicle-forming yeast such as Candida

krusei or Issatchenkia orientalis/ Occidentalis as well as species of the apiculatus yeasts (Kloeckera, Hanseniaspora) This, the genus Brettanomyces may be a

typical group of yeasts that are especially adapted to the environment of the tea fungus However, to investigate further the beneficial effects of tea fungus, a spectrum of the other typical genera must be defined Only three specimens showed definite contaminations In one case, no yeasts could be isolated because

of massive contamination with Penicillium spp In the remaining two samples

(from one household), Candida albicans was demonstrated The low rate of contamination might be explained by protective mechanisms, such as formation of organic acids and antibiotic substances Thus, subjects with a healthy metabolism do not need to be advised against cultivating Kombucha However, those suffering from immunosuppression should preferably consume controlled commercial Kombucha beverages

In the case of yeast, observed biodiversity is higher than that of bacteria Adaptive mechanisms present in yeast cover various metabolic maneuvers Many yeast species present central environmental or stress responses, consisting of gene modulation and activation of stress tolerance mechanisms These mechanisms involve polyploidy induction, multiplication of large fragments in chromosomes, loci variation in families of Ty1 retrotransposons Such evolutionary maneuvers may have enabled the high diversity of yeasts to adapt to kombucha fermentation conditions (Table 1)

(Chakravorty et al., 2016

Zygosaccharomyces bailii, Schizosaccharomyces pombe, Torulospora delbreuckii, Rhodotorula mucilaginosa, Brettanomyces bruxellensis, Candida stellata

(Teoh; Heard; Cox, 2004)

Dekkera, Zygosaccharomyces, Kazachstania, Davidiella, Pichia, Wallemia, Lachancea, Leucosporidiella, Kluyveromyces, Naumovozyma, Meyerozyma, Saccharomyces, Hanseniaspor

(Marsh et al., 2014)

Candida arabinofermentans, Brettanomyces bruxellensis, Schizosaccharomyces pombe, Zygosaccharomyces bailii

(Villarrealsoto et al., 2020b

Komagataeibacter xylinus, Komagataeibacter europaeus, Komagataeibacter intermedius), Gluconacetobacter, Gluconobacter (Gluconobacter oxydans), Acetobacter (A malorum, A pasteurianus, A pomorum, A tropicalis)

(Villarrealsoto et

al., 2020b)

Acetobacter (A xylinum), Gluconacetobacter (G xylinus sin Komagataeibacter xylinus), Lactobacillus, Lactococcus, Leuconostoc, Bifidobacterium, Thermus, Allobaculum, Propionibacterium, Enterococcus

(Marsh et al.,

2014)

32

Acetobacter

Acetobacter belongs to the family Pseudomonadaceae, widely distributed

in nature and can be isolated from air, soil, water, food, vinegar, wine, beer, fruit,

etc There are about 20 species of the genus Acetobacter Acetobacter has been

isolated and described, there are many species with high economic value

The Acetobacter strain has a rod-shaped shape, depending on the culture

conditions (temperature, composition of the culture medium), the cells produced

by Acetobacter bacteria have different morphology, elongated or enlarged form

Size varies depending on species (0.3-0.6 x 1.0-8.0μm) May be mobile (with simple flagella or flagella) or non-motile (without flagellum) Obligatory aerobic,

resistant to high acidity Acetobacter bacteria have the ability to assimilate many

different carbon sources but cannot use starch Cells can be chained or stand alone Capable of forming scum on liquid medium, the ability to form scum

varies depending on the type: Acetobacter xylinum: forming thick and firm

cellulose scum Acetobacter orleanoe: forms a thin but firm scum Acetobacter

pasteurianum: forms dry and wrinkled scum Acetobacter suboxydans: forms a

thin, easily disintegrated scum Acetobacter has the ability to assimilate salts and degrade peptones Many species require certain amino acids such as pantothenic acid and minerals K, Mg, Ca, Fe, P, S in the form of inorganic salts , organic or organic compound Therefore, beer, yeast autolysis, malt water, juice are very good sources of nutrients for the growth of Acetobacter bacteria In addition to the ability to oxidize ethanol to acetic acid, some species of Acetobacter also synthesize vitamin B1, vitamin B2, oxidize sorbite to sorbose sugar (used in the vitamin C production industry) (Nguyen Thanh Dat, 2000)

The tea fungus ‘Kombucha’ is a symbiosis of Acetobacter,

including Acetobacter xylinum as a characteristic species, and various yeasts A

characteristic yeast species or genus has not yet been identified Kombucha is mainly cultivated in sugared black tea to produce a slightly acidulous effervescent beverage that is said to have several curative effects In addition to sugar, the beverage contains small amounts of alcohol and various acids, including acetic acid, gluconic acid and lactic acid, as well as some antibiotic

33 substances To characterize the yeast spectrum with special consideration for indirect pathogenic yeasts, two commercially available SCOBY samples from private households in Germany were analyzed using different methods microbiology and biochemistry

34

CHAPTER III

RESEARCH CONTENT AND METHODOLOGY

3.1 Material and reseach scope

3.1.1 The raw material

Camellia sinensis (L) O.kuntze leaves were collected in Thai Nguyen province This leaves were cleaned and removed from broken slices The

Camellia sinensis (L) O.kuntze leaves were dried after that preserved in PE plastic bags in the fridge and used for this research

SCOBY “Symbiotic Colony of Bacteria and Yeast” purchased at Foodplus

Import-Export Co., Ltd has been isolated at the Institute of Microbiology & Biotechnology - Hanoi National University

3.1.2 Research scope

- Research was carried out in the laboratory scale

3.2 Workplace and time to proceed

- Location: Laboratory of Department of Food Technology, Faculty of Biotechnology and Food Technology, at Thai Nguyen University of Agriculture and Forestry

- Implementation time: March 2021 to August 2022

3.3 Chemicals, equipment

Table 3.1 Experiment chemicals