KR101918643B1 - Manufacturing Method of Kombucha Containing High Amounts of Glucuronic Acid and D-saccharicacid-1,4-lactone (DSL) - Google Patents

Abstract

The present invention relates to a method for producing kombucha containing high concentration of glucuronic acid and D-saccharic acid-1,4-lactone (DSL). More specifically, provided is kombucha with high content of organic acid by producing the same via a production method which comprises the following steps: a primary extraction step of mixing black tea, raw sugar, and water at a weight ratio of 1 : 1 : 8, extracting the mixture at 75-100°C for 2 hours, and carrying out a reaction by adding 1 wt% of viscozyme with respect to the weight of the black tea added therein; a secondary fermentation step of adding 5-25% kombucha starter yeast with respect to the total weight of an extract obtained in the primary extraction step and adding 10-50 ml of Maxinvert L10000 per 1 kg of the raw sugar so as to ferment the same at 20-32°C for 17 days; a tertiary fermentation step of adding 1.0-2.0 wt% of galacto-oligosaccharide (GOS) with respect the total weight of a ferment obtained in the secondary fermentation step and then fermenting the same at 10-15°C for 2 days; and a quaternary fermentation step of adding an acidity adjustment agent to the ferment obtained in the tertiary fermentation step to adjust pH of the ferment to pH 5.5-6 and then fermenting and aging the same at 5-10°C for two days.

Description

{Manufacturing Method of Kombucha Containing High Amounts of Glucuronic Acid and D-Saccharic Acid-1,4-Lactone (DSL)} with Increased Glucuronic Acid and D-

The present invention relates to a method for producing combat carrageenan, which contains a physiologically active substance glucuronic acid and a D-saccharic acid-1,4-lactone (DSL) component.

Recently, systematic researches have been conducted to search for new functional new materials having physiological activity and to scientifically verify their functionality, and interest in the development of new materials for preventing diseases from plant resources and food materials is increasing.

The physiological activity of tea has been scientifically proved, and it has become a favorite food to be consumed because of refreshing fragrance and unique fragrance produced during the manufacturing process, and consumer interest is also increasing. In addition, modern cars are widely used as new functional materials such as daily necessities such as tea, food, food, etc., besides their use as drinking beverages.

Tea is largely produced according to the manufacturing method, such as non-fermented tea (green tea), semi-fermented tea (10% - 65% fermentation, white tea, Boi Cha, etc.). Processes that use the word 'fermentation' in relation to tea today include oxidative fermentation (enzyme fermentation), microbial fermentation, and fermentation (Han et al., 2010). That is, the meaning of 'fermentation' is widely used. Of these, microbial fermentation is the perfect match for conventional fermentation.

Kombucha is known as black tea mushroom in Korea. It is a fermented drink obtained by fermentation of bacteria and yeast. It mainly contains black tea and sugar. Com Bucha originated in ancient China's Qin Dynasty, and was said to have been called "sacred tea" because of its detoxification effect and tonic effect. After that, it passed through Korea in 414 AD to Japan, and now mainly "Tea Kvass" (Dufresne and Farnworth, 2000).

Kombucha is a slightly sweet and sour fermented beverage that can be obtained by fermentation for about 14 days (Greenwalt et al., 2000), inoculated with a sugar-added black tea extract and synbiomes of bacteria and yeast. Acetobacter xylinum is known to act as a cellulose film called "tea fungus" in the upper part of the comb. In addition, bacterial fermenting a comb secondary is the major species in Acetobader xylinum and symbiote variety of yeast, such as Brettanomyces, Zygosaccharomyces and Saccharomyces.

In recent years, kombucha has attracted a lot of scientific interest because it is an abundant source of glucuronic acid (GlcUA, glucuronic acid) synthesized by acetic acid bacteria during fermentation. GlcUA is a carboxylic acid and acts as an antidote to remove exogenous chemicals from the body. Therefore, many studies have focused on improving kombucha production of many food and medicinal materials by modifying substrates and culturing them into Lactobacillus species to promote simultaneous fermentation or optimize fermentation conditions.

(Research Papers 0001) Han SK, Song YS, Lee JS, Bang JK, Suh SJ, Choi JY, Moon JH, and Park KH. 2010. Changes of the chemical constituents and antioxidant activity during microbial-fermented tea (Camellia sinensis L.) processing. Korean J Food Sci Technol 42: 21-26. (Research papers 0002) Dufresne C, Farnworth E. 2000. Tea, Kombucha, and health: a review. Food Res Int 33: 409-421. (Research paper 0003) Greenwalt CJ, Steinkraus KH, Ledford RA. 2000. Kombucha, the fermented tea: microbiology, composition, and claimed health effects. J Food Prot 63: 976-981. (Research paper 0004) Vina I, Linde R, Patetko A, Semjonovs P. 2013. Glucuronic acid from fermented beverages: Biochemical functions in humans and its role in health protection. Int J Res Rev Appl Sci 14: 217-230. (Research paper 0005) Yavari N, Assadi MM, Moghadam MB, Larijani K. 2011. Optimizing glucuronic acid production using tea fungus on grape juice by response surface methodology. Aus J Basic Appl Sci 5 (11): 1788-1794.

The present invention relates to a method for producing comb-carcass having a high content of organic acid, and a method for producing a comb-carcass having a high content of glucuronic acid and DSL through a fourth fermentation process and a comb- do.

In order to achieve the above object, the present invention relates to a method for manufacturing a combat carcass having an increased content of organic acids, comprising mixing tea, raw sugar and water at a weight ratio of 1: 1: 8 and extracting the mixture at 75 ° C to 100 ° C for 2 hours, A primary extraction step of adding 1% by weight of the added black tea to react; A secondary fermentation step in which 5% to 25% of combus-terminal seed yeast and 10 ml to 50 ml of Maxinvert L10000 per 1 kg of the raw material are added to the total weight of the extract of the primary extraction step and fermented at 20 ° C to 32 ° C for 17 days ; A third fermentation step in which galacto-oligosaccharide (GOS) is added at a weight ratio of 1.0% to 2.0% based on the total weight of the fermentation broth of the second fermentation step and fermented at 10 ° C to 15 ° C for 2 days; And a fourth fermentation step in which the pH of the fermentation broth is adjusted to 5.5 to 6 by adding an acidity regulator to the fermentation broth of the third fermentation stage and then aged fermentation at 5 to 10 ° C for 2 days.

In an embodiment of the present invention, an additional raw material is further added in the fourth fermentation step.

In one embodiment of the present invention, the sub ingredient includes at least one of ginseng, hibiscus, pomegranate, lemon and herb.

In one embodiment of the present invention, the organic acid is acetic acid, glucuronic acid, D-saccharic acid-1,4-lactone (DSL), and lactic acid.

The present invention relates to a method for producing a combat carcass having a high content of organic acids, including a first extraction step, a second fermentation step, a third fermentation step and a fourth fermentation step, The purpose is to provide secondary.

Figure 1 shows total sugar content and polyphenol content before and after viscozyme treatment after primary fermentation.
FIG. 2 shows the results of analysis of total sugar content, reducing sugar content, and alcohol content with or without Maxinvert treatment during the second fermentation.
FIG. 3 shows changes in the microflora of yeast and acetic acid bacteria with and without Maxinvert treatment during the secondary fermentation.
Fig. 4 shows changes in the number of microflora of yeast, acetic acid bacteria and lactic acid bacteria with or without GOS treatment during the third fermentation.
FIG. 5 is a result of comparing the content of organic acid with the presence or absence of GOS treatment in the third fermentation.
FIG. 6 shows the changes of the microflora of yeast, acetic acid bacterium and lactic acid bacteria depending on the presence or absence of the treatment with the acidity controlling agent during the fourth fermentation.
FIG. 7 shows the results of comparative analysis of the contents of organic acids with and without the treatment of the acidity regulator during the fourth fermentation.

Hereinafter, the present invention will be described in detail based on examples. It is to be understood that the terminology, examples and the like used in the present invention are merely illustrative of the present invention in order to more clearly explain the present invention and to facilitate understanding of the ordinary artisan, and should not be construed as being limited thereto.

Technical terms and scientific terms used in the present invention mean what the person skilled in the art would normally understand unless otherwise defined.

Fermentation proceeds by the complex strain of the comb. When the yeast grows with the sugar added as the raw material in the first extraction step, alcohol is produced, and the fermentation bacteria contained in the fermentation strain of the combus act on the bacteria to produce the fermented products of organic acid, glucuronic acid and DSL. Each fermentation step plays an important role in the quality of the combat quality by maintaining predominant strains capable of imparting the combat fermentation characteristics.

The main components of the comb-fermentation broth are known as glucuronic acid, fructose, and acetic acid.

Fermented kombucha contains organic acids, sugars, vitamins and tea polyphenols, and its main components are glucuronic acid, fructose and acetic acid.

The monosaccharides formed during the combing step fermentation process are the main substrate for the production of various organic compounds including acetic acid, gluconic acid, some micronutrients and glucuronic acid.

Glucuronic acid, one of the major active ingredients in combat, has become a subject of interest in recent years due to its detoxifying properties. Urinary system has been reported to remove several types of toxic substances such as pollutants, exogenous chemicals, excess steroid hormones and bilirubin (Vina et al. 2013). In addition, glucuronic acid can be converted to glucosamine, a beneficial substance related to cartilage, collagen and fluids associated with osteoarthritis treatment, and acts as a precursor for the biosynthesis of vitamin C.

D-saccharic acid-1, 4-lactone (DSL), one of the active substances in combat, is a competitive inhibitor of β-glucuronidase and can inhibit the production of toxic and carcinogenic substances by β-glucuronidase.

Therefore, glucuronic acid, which is a physiologically active substance of Combauca, and a microbial symbiosis model were formed by changing the fermentation conditions to enhance DSL.

The present invention relates to a process for preparing a tea, comprising: a first extraction step of mixing black tea, raw sugar, and water at a weight ratio of 1: 1: 8, extracting the mixture at 75 ° C to 100 ° C for 2 hours, adding viscozyme at a weight ratio of 1% A secondary fermentation step in which 5% to 25% of combus-terminal seed yeast and 10 ml to 50 ml of Maxinvert L10000 per 1 kg of the raw material are added to the total weight of the extract of the primary extraction step and fermented at 20 ° C to 32 ° C for 17 days ; A third fermentation step in which galacto-oligosaccharide (GOS) is added at a weight ratio of 1.0% to 2.0% based on the total weight of the fermentation broth of the second fermentation step and fermented at 10 ° C to 15 ° C for 2 days; And a fourth fermentation step of adding an acidity regulator to the fermentation broth of the third fermentation step to adjust the pH of the fermentation broth to 5.5 to 6 and fermenting the fermentation broth for aging at 5 to 10 ° C for 2 days. It is an object of the present invention to provide a method for manufacturing the comb tooth portion.

In the primary extraction step, black tea, raw sugar and water are mixed at a weight ratio of 0.5: 1: 9.5 to 1: 1: 8 and extracted at 75 to 100 ° C for 2 hours.

After the first extraction, viscozyme can be added to increase the extraction amount of polyphenol component of black tea such as catechin and gallic acid. The dosage of viscozyme may be added in a weight ratio of 0.1% to 10% of the amount of black tea.

In the present invention, the raw sugar may be white sugar, brown sugar, sulfur sugar, fructose, maltose, coconut sugar, and the like, but organic sugars rich in minerals and vitamins are preferred.

The secondary fermentation step is aimed at producing alcohol which is a raw material of acetic acid bacteria which plays an important role in post-fermentation by facilitating propagation of yeast strains of yeast strains belonging to the comb-tertiary fermenting bacteria. Therefore, in the second fermentation, Maxinvert L10000 supplied by DSM participates to increase the growth of yeast and increase the reducing sugar content, which is a raw material of alcohol production.

The secondary fermentation step is a step of adding fermentation yeast and Maxinvert L10000 to the primary extract to ferment the fermentation yeast, and the fermentation yeast can be used as a commercially available supernatant or isolated strain. The input amount of the yeast strain is 5% to 30% based on the total weight of the primary extract and the fermentation temperature is 20 to 40 캜 for 15 to 20 days. Maxinvert L10000 is added at 10 ml to 50 ml per kg of raw sugar.

The third fermentation step aims to produce organic acids and glucuronic acid from the alcohol produced in the second fermentation as a raw material. For this purpose, in the third fermentation step, galacto-oligosaccharide (GOS) is added to the second fermentation broth to prepare the fourth fermentation by promoting the growth of acetic acid bacteria and lactic acid bacteria in the broth fermentation broth. In the tertiary fermentation stage, when acetic acid is produced from the alcohol produced in the secondary fermentation, glucuronic acid production is significantly increased by the acetic acid bacteria, which is the main strain in the tertiary fermentation stage, and increased by the addition of galacto-oligosaccharide (GOS) The content of D-saccharic acid-1,4-lactone (DSL), which is a metabolite of glucuronic acid, is also significantly increased by lactic acid bacteria.

In the third fermentation step, galacto-oligosaccharide (GOS) is added to the second fermentation broth and fermented at 5 to 20 ° C for 1 to 2 days, and GOS is added at a weight ratio of 1 to 5% based on the total weight.

The fourth fermentation step is aimed at promoting the growth of lactic acid bacteria for D-saccharic acid-1,4-lactone (DSL) enhancement. For this purpose, the pH of the third lactic acid bacteria was adjusted to 5.5-6.0 after the third fermentation, thereby proliferating the lactic acid bacteria. An acidity regulator was added to adjust the pH. As the acidity controlling agent, any of those which can be used as food additives such as potassium carbonate, sodium hydrogen carbonate, sodium polyphosphate, sodium pyrophosphate, formic acid-CS, potassium phosphate dibasic, sodium phosphate and calcium hydroxide can be used.

In the fourth fermentation step, an acidity controlling agent is added, followed by aging fermentation at 5 ° C to 10 ° C for 2 days to increase the content of organic acid.

In the fourth fermentation step, in order to enhance the taste and flavor of the ginseng root, ginseng, hibiscus, pomegranate, lemon and herb may be further added.

The present invention may further include a step of filtering and packaging after the fourth fermentation step.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples are intended to illustrate the practice of the present invention and are not intended to limit the scope of the present invention.

<Test Example>

1. Analysis of total sugar content

Measurement of compositional change during fermentation of Kombucha fermentation: To determine the changes in the components during the fermentation of Kombucha and Kombucha fermentation, the total sugar content was measured using the phenol-sulfuric acid method (Dubois et al., 1956) D-glucose was diluted to an appropriate concentration and a calibration curve was prepared. The total sugar content of the standard substance was determined.

2. Reducing sugar content analysis

The content of reducing sugar was modified by Miller (1959), and 3 ml of DNS (3,5-dinitrosalicylic acid) reagent was added to 1 ml of the sample. The mixture was heated in boiling water for 5 minutes, cooled, and then absorbance was measured at 540 nm Respectively. As a standard substance, glucose was used.

The total polyphenol content was modified by the method of Singleton (1999), adding 10 μl of sample to 800 μl of distilled water, mixing 50 μl of 0.9 N Folin-Ciocalteu reagent and 150 μl of 20% sodium carbonate solution, then left in the dark for 2 hours . The absorbance of the Folin-Ciocalteu reagent was measured at 750 nm using Molybdenum blue color as a result of the reduction of the polyphenolic compound in the supernatant. The total polyphenolic compound content after the calibration curve was expressed as mg gallic acid equivalent (GAE) in 1 g of the sample.

3. Analysis of organic acid content

The organic acids were analyzed by HPLC (Agilent HPLC series 1100, Agilent, Waldbronn, Germany) by passing through a Sep-pak C18 cartridge and filtering through 0.45 ㎛ membrane filter. At this time, YMC Triart C18 (250 × 4.6 ㎜, 5 ㎛) column was used for the analysis of organic acids and detected at 210 nm using a UV detector. The mobile phase was a mixture of KH ₂ PO ₄ (pH 2.4) and methanol (97: 3) and the flow rate was 1.0 ㎖ / min. Acetic acid, malic acid, succinic acid, and lactic acid (Sigma Chemical Co., St. Louis, Mo., USA) were used as the organic acid standards. Glucuronic acid and DSL were also analyzed under organic acid analysis.

4. Statistical Analysis

The results were statistically processed using SPSS 12.0 (SPSS Inc., IL, USA) and the mean and standard deviation (SD) were calculated for the items not indicated separately. The significance of the ANOVA test was tested by Duncan's multiple range test at p <0.05.

&Lt; Example: Fermentation of combux tea >

Primary extraction stage

The extraction tank was charged with the raw materials of black tea, organic sugar cane sugar and UV sterilized water at a weight ratio of 1: 1: 8 and fermented at 75 to 100 ° C for 2 hours. After cooling, viscozyme was added at 1% by weight of black tea at 70 ℃.

As a result of measuring and comparing the total sugar content and the content of polyphenol before and after the treatment with viscozyme enzyme in the extraction process, it was confirmed that the total sugar content and polyphenol content after viscozyme treatment were significantly increased as compared with before treatment as shown in FIG.

Second fermentation step

The main purpose of the secondary fermentation is to produce the alcohol which is the raw material of the acetic acid bacteria which plays an important role in the post fermentation by facilitating the growth of the yeast bacteria. Maxinvert L10000 supplied by DSM was added to increase the yeast growth and alcohol production in the secondary fermentation, and the increase of the yeast and the reducing sugar content, which is the raw material for the alcohol production, were attempted.

After the first fermentation, 25% of the total weight of the primary extract was added, and Maxinvert L10000 was added in an amount of 10 to 50 ml per 1 kg of the organic sugar cane, followed by fermentation at 20 to 32 ° C for 17 days. The comb-bacteriocin-derived yeast strain was used as a commercial solution for comb-aquaculture.

As a result of measuring the total sugar content and the reducing sugar content after the second fermentation, the total sugar content was slightly decreased after the Maxinvert treatment as shown in FIG. 2, but there was no significant difference between them before the treatment. The content of reducing sugar was increased to 10.9 ㎎ / ㎖ after Maxinvert treatment, compared with 6.7 ㎎ / ㎖ before treatment, and the alcohol content was also significantly increased compared to before treatment.

As shown in FIG. 3, the yeast after the Maxinvert treatment tended to increase, whereas the bacterial strain tended to decrease slightly.

Third fermentation step

The purpose of the tertiary fermentation is to promote the growth of lactic acid bacteria by adding organic acid and glucuronic acid as a raw material of alcohol produced in the secondary fermentation and adding galacto-oligosaccharide (GOS).

The tertiary fermentation is the main fermentation process which gives the characteristics of combat, which is the main strain, and the amount of organic acid and glucuronic acid is increased by acetic acid bacteria.

After the secondary fermentation, galacto-oligosaccharide (GOS) was added to the fermentation broth at a weight ratio of 1.0-2.0% of the total fermentation broth and fermented at 10 ° C to 15 ° C for 2 days.

Fig. 4 shows the results of measurement of changes in the main microflora involved in the third fermentation. Acetic acid bacteria, which plays an important role in the production of organic acids, was increased compared to the secondary fermentation, and yeast was slightly decreased compared to the secondary fermentation. Lactic acid bacteria were significantly increased in tertiary fermentation, and all strains were increased with the addition of GOS.

As a result of measuring the content of organic acids after the third fermentation, the addition of acetic acid, lactic acid, glucuronic acid and its metabolite D-saccharic acid-1,4-lactone (DSL) was increased.

Fourth fermentation step

The 4th fermentation is a step of high production of DSL using lactic acid bacteria as a main strain.

To accelerate the growth of the lactic acid bacteria producing DSL, the pH of the fermentation broth was adjusted to 5.5 to 6.0 and then aged at 5 to 10 ° C for 2 days by adding an acidity regulator to the fermentation broth after the third fermentation.

FIG. 6 shows that the change in the microflora of the yeast of the fermentation broth after the fourth fermentation showed no significant difference before and after the pH adjustment, and the number of yeast bacteria decreased as compared with the third fermentation. In addition, the changes of acetic acid and lactic acid bacteria showed a tendency to increase significantly after pH adjustment.

As a result of measuring the content of organic acid after the fourth fermentation, acetic acid, DSL and lactic acid contents were increased compared to the third fermentation as shown in FIG. 7, while glucuronic acid was significantly decreased after the fourth fermentation Respectively. Acetic acid, glucuronic acid, DSL and lactic acid showed the tendency to increase with the addition of acid modifier.

Claims (4)

A first extraction step of mixing black tea, raw sugar and water at a weight ratio of 1: 1: 8, extracting the mixture at 75 ° C to 100 ° C for 2 hours, adding viscozyme at a weight ratio of 1%
A secondary fermentation step in which 5% to 25% of combus-terminal seed yeast and 10 ml to 50 ml of Maxinvert L10000 per 1 kg of the raw material are added to the total weight of the extract of the primary extraction step and fermented at 20 ° C to 32 ° C for 17 days ;
A third fermentation step in which galacto-oligosaccharide (GOS) is added at a weight ratio of 1.0% to 2.0% based on the total weight of the fermentation broth of the second fermentation step and fermented at 10 ° C to 15 ° C for 2 days; And
And a fourth fermentation step of adding an acidity regulator to the fermentation broth of the third fermentation step to adjust the pH of the fermentation broth to 5.5 to 6, followed by aging fermentation at 5 to 10 ° C for 2 days. Increased comb secondary manufacturing method.
The method according to claim 1,
Characterized in that an additional raw material is further added in the fourth fermentation step,
Wherein the additive material comprises at least one of ginseng, hibiscus, pomegranate, lemon and herb.
delete 3. The method according to claim 1 or 2,
Wherein the organic acid is acetic acid, glucuronic acid, D-saccharic acid-1,4-lactone (DSL) and lactic acid.

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