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
Research rationale
Kombucha is also known as water ginseng tea, mushroom tea or Manchurian mushroom tea It is a drink fermented by sugar tea by the symbiosis of yeast and 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.
Research’s 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
- 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.
Research question
Could fresh leaves of Camellia Sinensis (L) O.Kuntze plants be used to ferment Kombucha beverages?
Limitation
- The quality of yeast strains decreased due to long-term storage during the research period
- Raw materials are harvested off-season, so the quality of raw materials is not as good as expected
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)
Overview of the chemical compositions of Camellia Sinensis (L) O kuntze 10
The Composition of a Typical Tea Beverage
Table 2.1.: The composition of a typical tea beverage
Chemical composition Green tea Black tea
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)
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
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)
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 Willstọtter (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
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"
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)
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
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
Chemicals, equipment
No Chemicals Origin of products
No Experimental equipment Origin of products
10 PH meter HANNA HI 2210-02 China
18 Test tube cleaning brush Vietnam
Research content
Research content 1 Investigation of tea leaves (Camellia Sinensis (L) O.kuntze) components
Research content 1.1 To study some chemical compositions of the tea leaves (Camellia Sinensis (L) O.kuntze)
Research content 1.1.1 Determination method of moisture content followed by TCVN 10788:2015
Research content 1.1.2 Determination method of Ash content followed by TCVN 8124: 2009
Research content 1.1.3 Determiniation of total tanin content followed the
Research content 1.1.4 Method for determination of total acid by titration with 0.1N NaOH with 0.1% phenolphthalein as indicator
Research content 1.1.5 Sensory method followed by TCVN 3215-79
Research content 2 Completing the process of producing Kombucha product from tea leaves (Camellia sinensis (L) O.kuntze)
Research content 3 Investigation of some factors affecting the quality of fermented product
Research content 3.1.Research on the effect of the tea matrials on the quality of products
Research content 3.2 Research on the effect of material/distilled water ratio on the quality of product
Research content 3.3 Research on the effect of pH on the quality of product
Research content 3.4 Research on the adding sugar on the quality of product
Research content 3.5 Research on the effects of yeast ratio on the quality of product
Research content 3.6 Research on the effect of fermentation time on the quality of product
Research content 4 Evaluation the quality and preliminary cost estimation of final product
Research methods
Research content 1 To study some chemical compositions of the tea leaves (Camellia Sinensis (L) O kuntze )
3.5.1.1 Determination method of moisture content follwed by TCVN
Use the heat to separate the moisture in the material, while retaining all the substances in the material Therefore, the drying temperature should not be too high or too low If the temperature is too high, the moisture will evaporate quickly, but it is easy to change the chemical properties of the substances, especially volatile substances, which will affect the analysis results If the temperature is too low, the steam is released slowly, causing time and substances to change Moisture was determined by drying to constant mass
To conduct moisture determination must use - Analytical balance with permissible deviation not exceeding 0.001g - The oven can be controlled to adjust the temperature (105˚C) or (120˚C)
- The weighing bowl is made of glass, porcelain or aluminum with a diameter of 50mm and a lid
Take samples and crush them, use an analytical to weigh 5 grams of
Angelica sinensis placing it in the crucible, knowing the mass before, putting the sample in the drying oven at 105 C, and removing it after a specified period of time (usually 4-5 hours) Weigh and record the results after placing it in a desiccator at room temperature Continue until the weight is constant
Moisture in % (W) calculated by the formula:
38 G1: Weight of crucible and sample before drying (g)
G2: Weight of crucible and sample after drying (g)
The test result is the mean of two parallel determinations, the difference between them not exceeding 0,2%
3.5.1.2 Determination method of Ash content followed by TCVN 8124: 2009
Organic materials are incinerated at elevated temperatures (550 °C) in a muffle furnace, and inorganic matter (ash) remains The weight of inorganic materials left is used to calculate ash content Instruments and equipment used to conduct the test should be used:
- Analytical balance with error not exceeding ± 0.01g
- The crucible is made by porcelain or metal
- The furnace can be adjusted to 500 - 600ºC
- Desiccator, below to desiccant matter
- Electric stove or alcohol lamp
Fire a porcelain crucible or metal bowl washed, in the oven with from 500 to 600ºC to constant weight Cool in a desiccator and weigh on the analytical balance to the nearest 0.0005g Place in the crucible about 5 gram of sample (Angelica sinensis (Oliv.) Diels) weigh them all on the analytical balance with the above accuracy Put everything in the muffle furnace and increase the temperature slowly from 500 to 600ºC Ignite until the ash is white, which means that common organic matter has been removed for 6-7 hours Turn off the muffle furnace and allow it to cool (might take a few hours), remove crucibles from the muffle furnace and place into a desiccator to cool
Note: If in the case inorganic matter (ash) still has a dark color, make it cool, add a few drops of concentrated H2O2 or HNO3, and fire it to white ash
Make it cool in the desiccator and weigh on the analytical balance Continue firing at the above temperature for 30 minutes, and then make it cool in the desiccator After that, weigh and repeating until the weight remains constant The process is continued until the mass is maintained at the same level
39 The results between the two firing times and the next weighing should not be more than 0.0005g apart
Ash content in % (X) calculated by the formula:
G: Weight of (g) crucible G1: Weight of crucible and sample before burning(g)
G2: Weight of crucible and sample after burning (g)
3.5.1.3.Determiniation of total tanin content followed the Loventan followed by TCVN 9745-1:2013
Using boiling water to extract the solute of tea to obtain a solution with a concentration of 8-10g tea/liter
Step 1: Weigh accurately 10gram tea leaves by analytical balance Step 2: Put the chopped tea leaves and transfer them all into a 250ml conical flask, add 100ml of boiling distilled water, soak for 15 minutes, shake gently every 5 minutes and then filter through a filter funnel into a 1-liter volumetric flask After that adding 100ml of boiling water to the conical flask, soak for 15 minutes, filter again and so on after 5-7 times to drain all the solutes out of the tea, all the filtered water is transferred to the original level, then cooled down., add distilled water to the bottle, shake well Use this solution (solution A) to determine the chemical composition of tea
Determination of tannin content in tea leaves
Step 1: Using a pipette to accurately 10ml of solution A into a white porcelain bowl with a capacity of 1 liter, add 750ml of distilled water and 10ml of Indigocacmin indicator solution 0.1% and 10ml , H2SO4 1/4
Step 2: Adding 0.1N KMnO4 to the burette to the mark Step 3: Titrate the solution with 0.1N KMnO4 During titration, stir vigorously with a glass rod Titrate at 1 drop/s
40 Step 4: When the solution appears yellow green (the mixture color in the porcelain bowl gradually changes from green to gray-blue, light blue, yellow green) stop titration and result in 0.1N KMnO4 concentration was consumed
Note: - It is necessary to titrate 2-3 samples under the same conditions - Titrate a blank in parallel (replacing 10ml of solution A with 10ml of distilled water)
Percentage tannin content on dry matter of tea is calculated according to the formula:
+ a: The volume of 0.1N KMnO4 used to titrate the tea water sample, ml + b: The volume of 0.1N KMnO4 used to titrate the blank, ml
+ V1: Volume of solution A: V2 = 1000ml + W: Amount of dry matter of tea in the original sample
3.5.1.4 Method for determination of total acid by titration with 0.1N NaOH with 0.1% phenolphthalein as indicator
- In a glass beaker (100ml or 200ml type) fermentation solution, add 1-2 drops of phenolphthalein 0.1%, titrate with 0.1N NaOH solution
Note: Titration requires gentle shaking until the solution turns pale pink
- Calculate the number of grams of total acid in 100ml of fermentation solution according to the formula:
V: Number of ml of 0.1 N NaOH used to titrate 10 ml of fermentation solution
K: Amount of total acid corresponding to 1ml of 0.1N NaOH (K=0.006) 10: Number of ml of titrated fermentation solution
3.5.1.5 Sensory method followed by TCVN 3215-79
In this thesis, the method of frointingmark and assigning grades was utilized, followed by TCVN standards, and a sensory assessment group encompass with 10 peoples was created to work on the project (5 men and 5 women) Using a score
41 factor of 20 that was built on a unified scale of 6 levels 5 points (scores from 0-5)
This method evaluates a sensory indicator or all sensory criteria 0 points indicate defective and unsatisfactory products and a scale of 1-5 corresponds to a product that enhance in perfection On a scale of 5, the product has almost no defects, indicating the characteristics of the final product
Table 3.3 The importance coefficient of each indicator
Table 3.4 Table of regulations to evaluate the level of product quality
Minimum requirement for the sensory panel's unweighted grade point average
6 broken type 0 – 3,9 Each indicator is less than 1.0
Research content 2: Completing the process of producing Kombucha product from tea leaves (Camellia sinensis (L) O.kuntze)
The research of producing Kombucha from Tea leaves will be followed by the process
Figur3.1: The processing of produce kobucha beverages from tea leaves ( Camellia sinensis (L) O.kuntze) according to ( Jayabalan et al , 2014) Research content 3 Investigation of some factors affecting the quality of fermented product
Experiment 3.1: Research on the effect of the tea matrials on the quality of products
43 The experiment was designed according to a single-factor, in which tea raw materials were changed according to two types of tea leaves (fresh old tea leaves, dried old tea leaves) and fixed tparameters such as: material/ water ratio( g/L) ;initital sugar ; pH; fermentation time, fermentation temperature
Table 3.5 Experimental design to study the effect of tea material on the quality of product
Formula Tea material Fixed factor Monitored factor
-Material/water: 200 (g/L) - Additional sugar: 200 grams pH = 6.5 -Yeast ratio :2.5% (w/v) -Fermentation time: 7 days, - Fermentation temperature: room temperature
- Sensory quality of final product
- Total acidity (g/L) - Residue sugar content (g/L) F2
Based on, brix concentration, sensory quality, total acidity and residue sugar content of final product, the most suitable types of tea leaves will be chosen
Experiment 3.2: To study the effect of material/distilled water ratio on the quality of product
The experiment was carried out various tests in a totally randomized design
Each experiment was repeated three times and the parameters are described in the table 3.6
Table 3.6 Experimental design to determine the effect of material/ distilled water ratio on the quality of product
Material/distilled water (Kg/mL) ratio
- Selection tea leaves ratio will be achieved from experiment 2 - Additional sugar:
- Sensory quality of final product
- Total acidity (g/L) - Residue sugar content (g/L)
- Based on, brix concentration total acidity, residue sugar content (g/L) sensory quality of final product, the most suitable material /distilled water (w/v) ratio will be chosen
Experiment 3.3: Research on the effect of pH on the quality of product
The experiment was carried out various tests in a totally randomized design
Each experiment was repeated three times and the parameters are described in the table 3.7
Table 3.7 Experimental design to determine the effect of pH on the quality of product
- Types of material was obtained in experiment 1
- Material /distilled water ratio will be achieved from experiment 2
- Yeast ratio: 2.5 %(w/v) - Fermentation temperature: room temperature - Fermentation period:7 days
- Brix concentration ( o Bx) - Sensory quality of final product
Based on brix concentration ( o Bx), sensory quality of product, total acidity and residue sugar content , the most suitable pH will be selected
Experiment 3.4 Research on the adding sugar on the quality of product
The experiment was carried out various tests in a totally randomized design
Each experiment was repeated three times and the parameters are described in the table 3.8
Table 3.8 Experimental design to study the adding sugar on the quality of product
- Types of material was obtained in experiment 1 - Material / distilled water (w/v) ratio was obtained in experiment 2 - pH was chosen in experiment 3 - Yeast ratio: 2.5%(w/v) - Fermentation temperature: room temperature - Fermentation period: 7 days
-Sensory quality of final product -Total acidity (g/L) -Residue sugar - content (g/L)
Based on alcohol content, brix concentration, sensory quality of product, total acidity (g/L) , residue suage content (g/L) the most suitable sugar adding will be selected
Experiment 3.5: Research on the effects of yeast ratio on the quality of product
The experiment was carried out various tests in a totally randomized design Each experiment was repeated three times and the parameters are described in the table 3.9
Table 3.9 Experimental design to study the effect of yeast ratio on the fermentation process
- Types of material was obtained in experiment 1 - Material / distilled water (w/v) ratio was obtained in experiment 2
- pH was chosen in experiment 3 - Sugar adding ( o Brix) was achieved in experiment 4 - Fermentation temperature: room temperature - Fermentation period: 7 days
- Sensory quality of final product
- Total acidity (g/L) - Residue sugar content (g/L)
Based on the brix concentration, sensory quality of final product, total acidity, residue sugar content, the most suitable yeast ratio will be selected
Experiment 3.6: Research on the effects of fermentation time on the quality of product
The experiment was carried out various tests in a totally randomized design Each experiment was repeated three times and the parameters are described in the table 3.10
Table 3.10 Experimental design to determine the effect of fermentation time on the quality of product
F19 1 - Types of material was obtained in experiment 1 - Material/distilled water (w/v) ratio was obtained in experiment 2
- pH was chosen in experiment 3 - Sugar adding ( o Brix) was achieved in experiment 4 - Yeast ratio was obtained in experiment 5
- Sensory quality of final product
- Total acidity (g/L) - Residue sugar content (g/L)
Based on brix concentration, sensory quality of finalproduct, total acidity and residue sugar content, the most suitable fermentation time will be selected.
Data statistical analysis methods
The SPSS 20.0 software is used for Data statistical analyses
RESULT AND DICUSSION
Result for determining the component of raw materials
The results of moisture, ash content, tanin of Camellia Sinensis (L) O.kuntze are shown in the figure 4.1 below
Figure 4.1: determining the component of raw materials
Effect of raw material moisture on fermentation performance
The moisture content of raw materials is one of the factors affecting the fermentation performance of plant organic compounds Its effect is often expressed through the binding of water to proteins and other hydrophilic components of the material, thereby affecting the movement of the solvent into the material, slowing the diffusion process and affect the fermentation performance (Giang trung khoa & CTV, 2017)
Giang Trung Khoa found that in the studied humidity range, the higher the humidity, the higher the solvent penetration into the material, which reduces and affects the polarity of the water-solvent system in the material, thereby affecting the progress of the process
50 At the same time, it was also found that, when extracting in the production of soluble products, the soluble content of tea and lotus both decreased by about 3%, the tannin content decreased by 1/2 compared to extraction by Voronsov method This proves that the regimes of temperature, time, material/solvent ratio applied in this study to extract tea samples are also relatively suitable for kombucha fermentation (Hoàng thị Minh Nguyệt, 2019)
The ash content of Camellia Sinensis (L) O.kuntze Leaves in this study was relatively high It accounted 4.27% of the initial weight (including moisture)
This means the raw root has much microelements as well as other inorganic matters.
Research results on the effect of the tea materials on the quality of products50 4.3 Research results to select the ratio of old tea leaves/water in the quality of
The two type of tea leaves were collected, removed impurities, cleaned, then studied according to two experimental formulas: fresh tea leaves and dried tea leaves that were dried to safe moisture (10-13%) Raw materials of tea leaves are extracted hot with purified water at 100℃ for 15 minutes and the extract after filtering is used to ferment to produce Kombucha products Results of sensory quality assessment, total acid content The number and residual sugar content of the two samples are described in Table 4.1
Table 4.1: the effect of the tea materials on the quality of products
Residue sugar (g/L) Alcohol (%V) Total score
Note: Values in the same column with different exponentials have a significant difference at the α= 0.05 level
Table 4.2 Affecting of material/ distilled water ratio on the sensory quality of product
Quality assessment Color Smell Taste
From the results of Table 4.1, it shows that when Kombucha tea is fermented from fresh tea leaf extract for better organoleptic value, the organoleptic quality is classified as good, while Kombucha tea is made from tea extract From the dried tea leaves, the sensory scores are only good With both experimental formulas, the product has an alcohol content within the threshold of alcohol content to create a harmonious taste for Kombucha products (under 1%V), In general, bacterial yeast strain NL1 ferments more favorably from juice
Extracted from old tea leaves when freshly extracted showed a higher total acid content and lower residual sugar content when compared with the fermentation formula from the extract of dried old tea leaves
This, choosing fresh old tea leaves to extract then fermenting it to produce Kombucha tea
4.3 Research results to select the ratio of old tea leaves/water in the quality of product
The ratio of water/tea leaf material affects the extract, thereby affecting the sensory quality of the finished Kombucha tea The results of the study on the influence of the ratio of water/materials of old tea leaves on the quality of Kombucha beverages are shown in Table 4.3
Table 4.3 Research results on the effect of the ratio old tea leaves /water in the quality of product
Alcohol (%V) Total acid content (g/L) Total score
Table 4.4 Affecting of material/ distilled water ratio on the sensory quality of product
Quality assessment Color Smell Taste
From the results of Table 4.3, it is shown that the amount of old tea leaves used has a great influence on the sensory quality of Kombucha tea products
Sensory quality was the most preferred and had the highest total score (19.5) and was ranked as good when the ratio of fresh leaves/water used was 200grams/1000ml water (F5) With a lower ingredient ratio (100grams/1000ml of ingredients) or higher (500 grams/1000ml of water) both give a good overall sensory score Besides, when extracted, it is fresh tea with a ratio of water
53 /Ingredients are 100 grams/1000ml of water (F6), the resulting product has a lower total acid content and a higher residual sugar content when compared to the F5 result, giving the product an over-taste sweet, not harmonious This can be explained by the high content of extracted polyphenol compounds, which will hinder the growth of the symbiotic microflora, so the fermentation efficiency is not high With the experimental formula F3 (500 grams/1000ml of water), the residual sugar content is much less than the two formulas F4, F5, but the total acid content of the product is not high, so the product still has harmonious taste, giving a higher sensory score than F3 With all 4 research formulas on the ratio of water / ingredients are equal for alcohol content is not high, lower than 1% and there is almost no difference in alcohol with significance level α = 0.5%
From the comments on (ratio water of fresh old tea leaves) was used to study for the next experiments.
Results of the study on the effect of pH on the quality of product
The results of the study on the percentage of pH on the quality of product for Kombucha fermentation are shown in Table 4.5
Table 4.5 Results of the study on the effect of pH on the quality of product fermentation
Note: Values in the same column with different exponents are significantly different at the α= 0.05 level
Table 4.6 Affecting of the effect of pH on the sensory quality of product
Quality assessment Color Smell Taste
Table 4.5 shows the influence of pH on the quality of the product
The lowest alcohol levels were found at a pH value of 2.5 This environment has a high concentration of H + ions, which causes the charge state of the yeast cell wall to be changed As a result, yeast is inhibited and alcoholic fermentation is weak, resulting in low alcohol content Moreover, the product has a strong bitter taste which makes a bad impression on customers and has poor sensory value
As the pH rises to 3.5 or higher, the alcohol is likely to drop Because this is a great environment for bacteria to multiply, it will outnumber the activity, growth, and development of yeast, and lower the alcohol content pH has a significant effect on fermentation, as well as yeast growth, activity, and development Although yeast can grow in the pH range of 2 to 8, the most consistent and greatest growth occurs in the pH range of 3.5 to 4.5 If the pH is too high or too low, this can inhibit yeast activity and growth, disrupt fermentation, and create a host of unwanted by-products that reduce the quality of the product Therefore, a pH value of 3.5 can be considered as the operating limit for quality control of the product
From the above discussion, a pH value of 3.5(F8) was optimal for fermentation and was applied to the subsequent experiments
Research results on selection of added sugar ratio for Kombucha
The fermentation process is affected by many factors such as temperature, pH, amount of oxygen, dissolved CO2, operating system, precursor supply, cutting speed in the fermenter, as well as nature and composition of the environment (Marsh et al,, 2014) Any variation in these factors can affect the fermentation rate, organoleptic properties, nutritional quality and other physicochemical properties of the Kombucha tea product The concentration of added sugar has a different effect on the quality of the finished product because the biological activities of the microflora will also be affected (Wolfe & Dutton, 2015) The percentage of added sugar on the quality of Kombucha tea products is shown in Table 4.7
Table 4.7 Research results on the effect of the percentage of added sugar on the quality of Kombucha tea products
Note: Values in the same column with different exponents are significantly different at the α= 0.05 level
Table 4.8 Affecting of the percentage of added sugar on the sensory quality of product
Quality assessment Color Smell Taste
The results of table 4.7 show that if the added sugar content is low, for Kombucha tea product has not high organoleptic quality, only average grade The resulting product has low acid, residual sugar and alcohol content, The product has no obvious taste, does not exhibit the characteristic mild sour taste of Kombucha tea, When the added sugar content reaches for The product has outstanding sensory quality, showing a total sensory score of 18.3 good rating points Products with alcohol content 0.63%, total acid content of 5.41 g/L is similar to the composition of some Kombucha tea products that have been announced and commercialized on the market If continuing to increase the initial sugar ratio up to F12, the residual sugar content is high, the total acid content does not increase much compared to F11, making the product taste too sweet, not typical for Kombucha product, so the sensory score of the product is only good
From the above comments, choose F12 with the initial sucrose ratio of to conduct further studies F13: initial sugar ratio Extract
Research results on the effects of yeast ratio on the quality of product
Table 4.9 Efects of yeast ratio on the quality of Kombucha tea products
Note: Values in the same column with different exponents are significantly different at the α= 0.05 level
Table 4.10 Affecting of yeast ratio on ratio on the sensory quality of product
Quality assessment Color Smell Taste
From the results of Table 4.9, it shows that, when increasing the percentage of microorganisms added from 1.5% to 2.5%, the sensory quality of
58 the product increases markedly, with the ratio of strains of 1.5 Percentage of sensory quality of the product is only average, but when this ratio is 2.5% the product quality is classified as good with a total organoleptic score of 18.6 With F15 due to the low percentage of micro-organisms added, the total organic acid content is low, the residual sugar content is high, making the product taste too sweet, not showing the characteristic mild sour taste of the product Kombucha tea products But continuing to increase the percentage of fermenting microorganisms, the sensory quality of the product decreased and only reached a good level This can be explained by the high total acid content, reduced residual sugar, much higher alcohol content than the other two formulas, making the product taste unbalanced, the product has a sour taste harsh when compared to F15 and F16 From the above comments, the percentage of additional microbial strains of 2.5% (v/v) was selected for further studies.
Research results on Kombucha fermentation time
Fermentation time is also a factor affecting the composition and quality of Kombucha tea products The research topic fermented for a period of respectively The effect of fermentation time on the quality of Kombucha tea products is shown in Table 4.10
Table 4.10 The result of fermentation time on the quality of Kombucha tea products
Note: Values in the same column with different exponents are significantly different at the α= 0.05 level
Table 4.11 Affecting of fermentation time on the sensory quality of product
Quality assessment Color Smell Taste
The experimental results showed that, with the NL1 symbiotic strain used, the fermentation time of 7 days will give Kombucha tea products with the best organoleptic quality, which is classified as good, with a fermentation time of approx After 6 days, the product has only good sensory quality, the high residual sugar content makes the product have a sweet, unbalanced taste In contrast, the fermentation time is 8 days, the total acid content is large, the residual sugar content is low, the alcohol content is the highest (0.72%), making the product slightly sour, the sensory score is low average rating only
According to Chu & Chen, 2006, Kombucha tea fermentation usually ranges from 6 to 60 days and biological activities can be increased during this process; however, the best results were achieved in an average of 15 days
Although most of the obtained antioxidant activities increased with incubation time, prolonged fermentation is not recommended due to the accumulation of organic acids, which can reach harmful levels if used drink directly Choosing the fermentation time also depends on the expected organoleptic properties of the product, Reiss (1994) reported that within 6 to 10 days of fermentation, the resulting Kombucha tea product had characteristics resembling a According to Coton et al, (2017), the fermentation process to create Kombucha tea should not last more than 10 days when used as drinking water for humans and it is recommended to study growth of industrial-scale Kombucha tea microbial populations during 0, 2, 4 and 8 days
60 From the above comments, the most suitable fermentation time to produce Kombucha tea from old tea leaves is 7 days, which was selected to study for the process of optimizing the production process on an industrial scale
Perfecting kombucha beverage fermentation research process
Table 4.12 Results of microbiological analysis of Kombucha tea products
Criteria name Test method Result method
Coliform, CFU/mL ISO 4832: 2006 No colonies
E coli, CFU/mL ISO 16649 – 2: 2001 No colonies
Enterococci, Streptococci faecal, CFU/mL ISO 7899 – 2: 2000 No colonies Pseudomonas aeruginosa, CFU/Ml ISO 16266: 2006 No colonies Staphylococcus aureus, CFU/mL AOAC 2016 (975.55) No colonies Clostridium perfringens, CFU/Ml ISO 7937: 2004 No colonies
Table 4.13 Quality analysis results of fermented Kombucha tea products from extracts of old tea leaves
Table 4.14 Preliminary calculation table of raw material prices for the production process (For 60 liter fermentation batch)
Material Weight Cost (Vnd) Price (Vnd)
So the average cost is: 17,700 (VND/500 ml bottle) This production cost is completely suitable for the commercialization of the product
CONCLUSION AND RECOMMENDATION
Conclusion
From the experimental research results within the framework of the topic, we come to some conclusions as follows:
Using fresh old tea leaves in Thai Nguyen to extract fermented juice to produce Kombucha tea will give products with better sensory quality than dried old tea leaves
Using a single-factor study to determine the technological parameters for the process of extracting tea leaves by water solvent, using the extract to ferment Kombucha tea production with specific technological parameters such as: following: Ratio of water/tea leaves: 5/1; extraction temperature is 100℃ for 15 minutes; the ratio of added sugar to the extract before fermentation is:
200g/1000mL; rate of microorganism strain NL1 used: 2.5% (v/v); pH of fermentation solution: 6.5 fermentation time: 7 days at room temperature;
Kombucha tea products are obtained from the optimization process for good sensory quality, natural sweet and sour taste, characteristic harmony, organic acid composition, total sugar content, equivalent alcohol content with Kombucha tea products being commercialized in the market.
Request
In order to improve the quality of the product and commercialize the product, we make the following recommendations:
Continue to test the product when mixing more fruit extracts, to diversify products and create a unique flavor for fruit Kombucha products, especially using seasonal fruits with high yield of Kombucha Vietnam, but the output is still precarious such as red dragon fruit, litchi, longan, frozen guava