KR20130057806A - Method for increasing content of slime-polysaccharides using xylitol - Google Patents

Abstract

PURPOSE: A method for enhancing the content of slime polysaccharides by fermenting Weissella sp. using xylitol as a carbon source is provided to ensure stable fermentation rate of a fermented food containing xylitol and physiological functionality and new physical properties of polysaccharides which produce lactic acid bacteria. CONSTITUTION: A method for enhancing the content of slime polysaccharides comprises a step of fermenting Weissella sp. using xylitol as a carbon source. Weissella sp. is selected from a group consisting of Weissella confusa, Weissella cibaria, Weissella koreensis, and Weissella paramesenteroides. A fermentation composition with enhanced slime polysaccharides is prepared by fermenting with Weissella sp. on xylitol. A method for a Kimchi composition with enhanced slime polysaccharides comprises a step of adding xylitol to Kimchi as a carbon source and fermenting Weissella sp.

Description

Method of increasing the content of viscous polysaccharides using xylitol {METHOD FOR INCREASING CONTENT OF SLIME-POLYSACCHARIDES USING XYLITOL}

The present invention relates to a method of increasing the content of viscous polysaccharides using xylitol.

Xylitol (Xylitol) is xylan in the plant - the hemicellulose made by digesting (Xylan-hemicellulose) the enzyme xylose teeth Wu inoculated (Streptococcus species) as the alcohol (pentiol) per pentose obtained by a hydrogenation reaction in the (xylose) There is a big connection with growth inhibition. Streptococcus mutans , a major causative agent of dental caries, synthesizes glucan, a cohesive polysaccharide from sugar, and glucan is a basic substance of dental plaque, helping bacteria adhere to the surface of teeth. attached to the tooth surface St. Mutans produce large amounts of organic acids during metabolism, which can corrode tooth surfaces and cause caries. However, sugar alcohols including xylitol, due to the structural features described earlier oral St. and mutans are not able to degrade the viscous glucan does not generate widely used as a sugar substitute in preventing dental caries, St. In addition to mutans , some other streptococci have been reported to inhibit growth (Vadeboncoeur C. Trahan L. Mouton C. Effect of xylitol on the growth and glycolysis of acidogenic oral bacteria. J Dent Res. 62: 882- 884. 1983).

On the other hand, lactic acid bacteria are mainly fermentable sugars such as glucose (glucose), fructose (fructose), mannose (mannose), sucrose (sucrose) and maltose (proliferation) as a substrate, such as organic acids such as lactic acid Create However, non-fermentable sugars such as xylitol are used as a substrate for lactic acid bacteria to proliferate, and thus have low availability and are involved in regulating fermentation and aging of foods, thus affecting storage, sensory and quality.

Among the products produced by the growth of lactic acid bacteria, exopolysaccharides produced in the form of a viscous form on the outer wall of the cells are divided into homopolysaccharides and heteropolysaccharides according to the types of constituent sugars. May be involved in the physical properties of The substrate used by lactic acid bacteria to best produce polysaccharides is known as sugar, and these polysaccharides are emulsifiers, tackifiers, physical property regulators, plasma extenders and syrups and candies to enhance moisturization and to prevent sugar crystals, to chew gum and jelly, and to freeze ice cream. It can be used in various food industries such as prevention and control of physical properties of fluid foods.

As the effects of xylitol suppressing the growth of dental caries and controlling the fermentation rate of food have been discovered, research on the development of fermented foods containing xylitol has been reported. So far, “the effect of xylose and xylitol addition on the fermentation of organic acids of kimchi. Kim Dong-kyung, Kim Sang-yong, Lee Jung-gull. Korean Journal of Food Science 32: 889-895. 2000 ”,“ The Effect of Xylitol Addition on the Taste and Fermentation of Dongchimi. Moon Sung-won, Jang Myung-sook. Korean Journal of Culinary Science. 20: 42-48. 2004 ”and“ Effects of Xylitol and Grapefruit Seed Extracts on Sensory and Fermentation of Chinese Cabbage Kimchi. Sung-Won Moon, Hyun-Kyung Shin, Geun-Eok Ji. Korean Society of Food Science and Technology. 35: 246-253. The fermentation rate control Kimchi, such as "2003" and "Method for extending the storage period of kimchi using xylose and xylitol (see Korean Patent Application No. 10-2000-0019982).

Like this, in Korea, there are various fermented foods, and research on lactic acid bacteria or lactic acid fermentation foods related to various sensory, quality and health physiological activities is active. There is no research on this. Until now, the application of xylitol to foods was limited to the control of fermentation rate and prevention of tooth decay. Fermented foods, such as kimchi, were made from lactic acid bacteria that produce polysaccharides from xylitol, which inhibits oral flora, which is a disadvantage due to the ability to produce viscous polysaccharides such as glucan. It is not used as a seed of.

Accordingly, the inventor of the present invention has come to the present invention by noting that lactic acid bacteria isolated from kimchi have excellent characteristics of producing viscous substances by using xylitol.

It is an object of the present invention to identify a lactic acid bacterium having excellent polysaccharide production from xylitol by checking the ability of lactic acid bacteria to produce xylitol, and to provide a method of using the same as a food seed.

In order to achieve the above object, in one embodiment of the present invention, xylitol is used as a carbon source, Bisella ( Weissella ) It provides a method of increasing the content of viscous polysaccharides by fermenting the genus microorganisms. In addition, the Weissella ( Weissella ) The genus microbe is Weissella confusa ) , Weissella cibaria ) , Weissella koreensis ) and Weissella paramesenteroides, characterized in that any one selected from the group consisting of, the Weissella genus microorganisms from the group consisting of KACC 91669, KACC 91670, KACC 91671 and KACC 91672 Characterized in that it is any one selected.

In one embodiment Weissella on xylitol Fermentation with a genus microorganism provides a fermentation composition with increased viscous polysaccharide. In addition, the fermentation composition is characterized in that the kimchi, the fermentation composition is characterized in that the water kimchi.

In one embodiment, xylitol is added to kimchi as a carbon source, and Weissella It provides a method for producing a kimchi composition with increased viscosity of the polysaccharide by fermenting the microorganisms.

'Viscous polysaccharide' of the present invention refers to a viscous polysaccharide material produced by the growth of lactic acid bacteria using xylitol as a substrate.

In the present invention, lactic acid bacteria are collected by diluting a certain amount of kimchi and diluting a certain amount, and then colonized colonies formed by smearing and culturing on selective medium according to the morphological characteristics such as size, shape, color, API kit and 16s-rRNA Lactobacillus was isolated and finally isolated according to the sequence.

In the present invention, the polysaccharide was recovered and quantified using the ethanol precipitation method and the phenol sulfate method, and the polysaccharide content generated after the growth of the cells was confirmed for each strain, and the strains having high polysaccharide production ability were selected by comparing the production difference with the control. Strains used in food fermentation microorganisms is the double as high 1.5 or more in xylitol utilization compared to the control of lactic acid bacteria Weissella KFT94 Weissella confusa KACC 91672, KFT96 Weissella cibaria KACC 91671 KFT98 Weissella koreensis KACC 91670, KFT99 Weissella paramesenteroide KACC 91669 is provided.

It provides lactic acid bacteria produced in the form of Kimchi fermented seedlings in the form of Kimchi fermented seedlings in order to control the physical properties of kimchi that can be retracted during fermentation, improve viscosity, and enhance the sensory taste.

Weissella in the present invention The genus lactic acid bacterium is a simple bacillus type heterofermenting fermentation strain that produces not only lactic acid but also other substances such as carbon dioxide, acetic acid or alcohol. In addition, it is detected in various places such as fermented food, animal digestive tract, sausage, etc., and it is different from other lactic acid bacteria of Lactobacillus and Leuconostoc genus depending on fermentation temperature and sugar type. Lactobacillus of the genus Weissella may form a dominant species of fermented foods at low temperatures and exhibit low irritant flavor due to low acid production (Lee Kang-wook et al., The importance of the Weissella genus in kimchi fermentation and future research. Kor J Microbiol Biotechnol 38: 341-348 (2010)). To date, the research on Weissella has been developed to improve the flavor of Kimchi. However, the research on Weissella 's functionality and development of food materials for the viscous polysaccharides is insignificant.

The present invention can increase the content of viscous polysaccharides by fermenting microorganisms using xylitol as a carbon source. In addition, it is possible to achieve a stable fermentation rate of xylitol-containing fermented foods and to add health physiological function and new physical properties to the polysaccharides produced by lactic acid bacteria. ( St. mutans ) can exhibit inhibitory functionality.

A in FIG. 1 is a polysaccharide generated look when proliferation using the lactic acid bacteria of the genus Weissella xylitol b and c is St. a polysaccharide produced form the addition of xylitol mutans.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  One. Xylitol  Preparation of culture solution and inoculation of lactic acid bacteria

1-1. Xylitol  Preparation of Wheat Microbes

Xylitol (xylitol, Dae-jung, Korea), sucrose (sucrose, CJ, Korea), tryptic soy agar (Tryktic soy agar, Merck, Germany) were used for the experiment. The viscous polysaccharide-producing microorganism was lactic acid bacteria isolated from kimchi. Streptococcus as a Control mutans received five species (KCTC3065, KCTC3289, KCTC3298, KCTC3300, KCTC3302) from the Korea Collection for Type Culture (KCTC), Brain Heart infusion agar (Difco, USA). It was cultured in and used. Lactic acid bacteria and five Streptococcus mutans were passaged twice in heart broth (MRS broth, Merck, Germany) and heart brain infusion broth (Merck, Germany), respectively.

1-2. Inoculation of Lactic Acid Bacteria

Xylitol cultures were prepared by using TSB (Tryptic soy broth), a total bacterial growth medium, by sterilizing xylitol solution aseptically 2% of the total amount of the medium, and inoculating 1% of each microorganism thereto at 30 ° C, 24 Time incubation.

In the case of solid medium, 2% xylitol was added to TSA (Tryptic soy agar), followed by immersing the strain culture in a sterilized wooden rod and dropping the medium into a medium of 3mm size and confirming the formation of colonies and the formation of viscous substances. It was.

Example  2. Analysis of Polysaccharide Production

2-1. Cell culture and Viscous  Polysaccharide recovery

Lactic acid bacteria and five Streptococcus The polysaccharide produced in the mutans culture was recovered and the amount thereof was measured. Polysaccharide precipitation was used for ethanol precipitation. To remove the cells in the strain culture, centrifugation (J-251, Beckman, Germany) for 15 minutes at 12,000 xg, remove the precipitate, and take the supernatant obtained from the separation to remove twice the volume of ethanol (95%, Daejung, Korea) After addition, it precipitated at 4 degreeC for 24 hours. The precipitated polysaccharide was recovered after removing the supernatant by centrifugation at 12,000 xg for 15 minutes, and diluted with DW of the same volume as the initial culture and used as an experimental solution.

2-2. Analysis of Sugar Production in Lactic Acid Bacteria

Production of the recovered polysaccharides was measured by the phenol-sulfuric acid method (Dubois et al. 1956). First, dilute the experimental solution twice, then take 150 uL, add 5% phenolic solution in the same amount, add 750 uL concentrated sulfuric acid (conc. 96%), stir for 30 minutes at room temperature, and measure absorbance at 470 nm. It was. The measured value was confirmed by calculating g / L according to the standard curve. The results are shown in Table 1 below.

Strain name Polysaccharide Production
(g / L) Polysaccharide Creation Cost Xylitol Control KFT94 Weissella confusa (KACC 91672) 25.93 17.16 1.51 KFT96 Weissella cibaria (KACC 91671) 16.48 12.53 1.31 KFT98 Weissella koreensis (KACC 91670) 19.42 11.81 1.65 KFT99 Weissella paramesenteroides (KACC 91669) 25.80 15.32 1.68

* Polysaccharide production ratio = polysaccharide amount produced in xylitol medium / polysaccharide amount produced in control medium

As a result of Table 1, four strains of the genus Bisella ( Weissella ) was confirmed to produce more than 1.3-1.7 times the polysaccharide compared to the control, respectively. TSB (tryptic soy broth) used as a control was 0.25% glucose, 0.3% papic digest of soybean, 1.7% pancreatic digest of casein, sodium chloride chloride) and 0.5% dipotassium phosphate. The polysaccharides produced here are produced by the interaction of complex sugars such as soybean and casein, as well as simple glucose, glucose. Importantly, the addition of 2% xylitol based on TSB increased the polysaccharide production of 4 strains of Weissella spp. Isolated from Kimchi.

2-3. Streptococcus mutans Sugar production of

St experiments under the same conditions. The polysaccharide content of mutans was tested in the same manner as the lactic acid bacteria polysaccharide obtained and quantitative analysis and the results are shown in Table 2 below.

Strain name (St. Mutans) Polysaccharide Production (g / L) Xylitol Control group Comparative example glucose Sugar KCTC 3065 -0.010 -0.010 1.70 35.77 KCTC 3289 -0.010 -0.008 3.52 20.73 KCTC 3298 -0.011 -0.008 9.24 25.61 KCTC 3300 -0.010 -0.008 8.89 61.83 KCTC 3302 -0.009 -0.010 11.02 17.27

St. In the case of mutans , polysaccharide production in the control (TSB medium) was insignificant, and the xylitol added groups showed little sugar production. Glucose added for comparison showed a polysaccharide production of 1.70 ~ 19.24 g / L. In the Sucrose added group, the KCTC3300 strain showed high sugar production of 61.83 g / L, and the other strains produced more than 17 g / L polysaccharide.

St. mutans, which is to create a cohesive materials plaque with sugar and polysaccharide substance for different oral and jeomjilmul that lactic acid is produced, St. Unlike mutans does not use the functions Cathedral of xylitol, lactic acid is basically a high increase in sugar availability Sugar resolution, and some of which take actively creating a viscous polysaccharide as xylitol and at the same time expressing the functional characteristics of a novel food St . Inhibition of the growth of mutans may reduce oral risk.

2-4. Xylitol  Community Type of Strains Used

2% xylitol containing TSA (Tryptic soy agar) in each of the lactic acid bacteria and St. After dropping and incubating mutans , the results of confirming whether viscous substances were formed are shown in Table 3 and FIG. 1.

Strain name Characteristic Remarks KFT94 W. confusa (KACC 91672)
KFT96 W. cibaria (KACC 91671)
KFT98 W. koreensis (KACC 91670)
KFT99 W. paramesenteroides (KACC 91669) Size: 5 ~ 6mm
Color: black, edge opaque
Viscous at the edge of the colony Figure 1 (a) KCTC 3065 St. mutans
KCTC 3289 St. mutans
KCTC 3300 St. mutans Size: 3mm or less
Color: black
No viscous product
Colony small Figure 1 (b) KCTC 3298 St. mutans
KCTC 3302 St. mutans Size: 3mm or less
Shape: Transparent
No viscous product
Colony small Figure 1 (c)

As shown in Table 3, the strain cultured using xylitol was confirmed to produce a transparent viscous material at the edge of the colony. St. In the case of mutans , when xylitol was added, the growth was low, which was clearly different from the colony form of lactic acid bacteria.

Combining the above results, Lactobacillus of four species of Weissella, KFT94 Weissella confusa kacc 91672, KFT96 Weissella cibaria KACC 91671, KFT98 Weissella koreensis KACC 91670, KFT99 Weissella paramesenteroides It was confirmed that KACC 91669 was an excellent polysaccharide-producing strain when grown using xylitol. When xylitol, which was used for the purpose of preventing tooth decay and inhibiting lactic acid fermentation rate, was used with this strain, it increased the resolution of xylitol and actively proliferated it. to produce a large amount of jeomjilmul as well as the effect of the physical property control can be expected for healthy physiological functionality, the remaining xylitol is used in lactic acid as a carbon source for growth is St. It can be used as a new functional food material and spawn that can be applied to various fields because it can show caries effect on mutans .

Xylitol-using lactic acid bacteria can be used as a seed for food fermentation. Depending on the type of food, lactic acid bacteria fermented products can be lyophilized and supplied in a live state, or used in liquid or powder form. In addition, because the genus Bisella (Weissella) strain is not only a viscous functional but also a heterogeneous fermentation strain, it produces a variety of acids such as lactic acid, succinic acid and acetic acid, so it can be used as a xylitol-using lactic acid spawn with excellent flavor and change in physical properties. .

Example  3. Xylitol  Use Slime  produce Lactic acid strain  Kimchi Preparation and Analysis

3-1. Manufacture of Kimchi

Kimchi was prepared using a strain of lactic acid bacteria of the genus Weiisella, which produces a viscous substance using xylitol as a fermentation seed. The kimchi prepared was measured by physicochemical analysis to investigate the storage and physical properties of kimchi. Kimchi was prepared in the content of the thermal radish water kimchi that can confirm the kimchi broth characteristics.

material Content (g) Yeolmu 100 chili powder 5.0 garlic 3.0 ginger 0.8 Red pepper 5.2 Sugar 0.6 Glutinous rice paste 10.0 Brine 3% 100

3-2. Kimchi Analysis

Kimchi spawn inoculation was prepared by dividing as shown in Table 5, and then analyzed every 10 days while storing at 5 ℃. Basically, the sugar and xylitol added to kimchi are considered to confirm the difference in the production of viscous substances.In order to compare the control of physical properties, the fermentation rate and The change in physical properties was confirmed.

Kinds Characteristics (Yellow Water Kimchi) Sample 1 Glutinous rice paste + added radish water kimchi with sugar Sample 2 Glutinous rice paste + xylitol added radish water kimchi Sample 3 Weissella spawn + hot radish water kimchi with sugar Sample 4 Weissella spawn + heated radish water kimchi with xylitol

3-2-1. Saving Kimchi pH  change

Kimchi pH was measured by using a pH meter (ThermoOrion, 720, USA) 10 mL of kimchi sample at room temperature. The results are shown in Table 6 below.

Kinds Thermal radish kimchi pH 0 days of fermentation 10 days of fermentation 20 days of fermentation Sample 1 5.30 4.91 4.30 Sample 2 5.38 5.19 4.45 Sample 3 5.42 4.56 4.40 Sample 4 5.40 5.05 4.39

Sample 1: glutinous rice paste + yeolmumul kimchi with sugar / sample 2: glutinous rice paste + yeolmumul kimchi with xylitol

Sample 3: Weissella spawn + hot radish kimchi with sugar / Sample 4: Weissella spawn + hot radish kimchi with xylitol

As a result of checking the pH during kimchi fermentation, the pH decrease was not large because the kimchi was stored at 5 ° C, but the change in spawn inoculation was found. From the 20th day of fermentation, all kimchi except Sample 2 was in the ripening season (pH 4.2 ~ 4.4) range. Sample 2 was found to be the slowest during fermentation with kimchi fermented by adding natural xylitol and xylitol. Samples 3 and 4 had faster fermentation rates than sample 2 and the pH, which was different on 10 days of fermentation, was similar after 20 days of fermentation. It was confirmed that the added kimchi was faster to fermentation speed than the kimchi added with xylitol only and slower than the fermentation rate of general kimchi without adding anything.

3-2-2. Microbial Changes During Kimchi Storage

The microbial measurement of kimchi was counted by smearing Kimchi samples diluted in MRS (Merck, Germany) agar and incubating at 30 ° C. The results are shown in Table 7 below.

Kinds Yeolmumul Kimchi Lactobacillus (CFU / g) 0 days of fermentation 10 days of fermentation 20 days of fermentation Sample 1 1.4 X 10 ⁴ 7.5X10 ⁷ 7.6X10 ⁸ Sample 2 4.0X10 ⁴ 2.5 X 10 ⁶ 8.4X10 ⁷ Sample 3 4.6 X10 ⁵ 6.3 X10 ⁷ 2.9 X10 ⁹ Sample 4 3.3 X10 ⁵ 3.8 X10 ⁷ 2.9 X10 ⁷

Sample 1: glutinous rice paste + yeolmumul kimchi with sugar / sample 2: glutinous rice paste + yeolmumul kimchi with xylitol

Sample 3: Weissella spawn + hot radish kimchi with sugar / Sample 4: Weissella spawn + hot radish kimchi with xylitol

As a result of the initial fermentation, it was confirmed that the total bacteria of Samples 1 and 2 which were not inoculated with spawn seed had 10 ¹ CFU / g less than those of Samples 3 and 4. In the case of sample 2 containing only xylitol, the total number of lactic acid bacteria was lower during fermentation compared to other kimchi, and sample 4 added by mixing xylitol and spawn showed no significant difference from sample 3, so it was confirmed that the use of xylitol was active. It was.

3-2-3. Viscosity Changes During Kimchi Storage

The viscosity of kimchi was measured by using a viscometer (DV-II pro, Brookfield, USA) using a S61 spindle at a speed of 20 ° C. and 100 rpm. The results are shown in Table 8 below.

Kinds Thermal waterless kimchi viscosity change (torque,%) 0 days of fermentation 10 days of fermentation 20 days of fermentation compare* Sample 1 10.8 11.5 15.4 1.43 times increase Sample 2 11.0 13.1 13.7 1.25 times increase Sample 3 12.5 15.7 18.5 1.48 times increase Sample 4 11.1 14.1 20.1 1.81 times increase

Sample 1: glutinous rice paste + yeolmumul kimchi with sugar / sample 2: glutinous rice paste + yeolmumul kimchi with xylitol

Sample 3: Weissella spawn + hot radish kimchi with sugar / Sample 4: Weissella spawn + hot radish kimchi with xylitol

* Viscosity of 20 days of fermentation / viscosity of 0 days of fermentation

As a result, the viscosity of the water kimchi broth at the beginning of fermentation was measured to 10-13 (torque,%). The viscosity of Sample 1 after 20 days of fermentation increased by about 1.43 times, about 2.25 times for Sample 2, about 1.48 times for Sample 3, and about 1.81 times for Sample 4, compared to the viscosity at the beginning of fermentation. In particular, the viscosity of the sample 3 and 4 added to the spawn was markedly increased, and among them, the viscosity of the sample 4 added to the spawn and the xylitol together was the highest.

While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. You will know. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention be construed as including all embodiments falling within the scope of the appended claims.

Agricultural Biotechnology Research Institute KACC91669 20110916 Agricultural Biotechnology Research Institute KACC91670 20110916 Agricultural Biotechnology Research Institute KACC91671 20110916 Agricultural Biotechnology Research Institute KACC91672 20110916

Claims (7)

Weissella with xylitol as carbon source Method of increasing the content of viscous polysaccharides by fermenting the genus microorganisms. The method of claim 1,
Weissella The genus microbe is Weissella confusa ) , Weissella cibaria ) , Weissella koreensis ) and bisella paramegenteroides ( Weissella paramesenteroides ) , the method of increasing the content of viscous polysaccharides, characterized in that any one selected from the group consisting of. The method of claim 1,
A method of increasing the content of viscous polysaccharides, characterized in that the microorganism of the genus Weissella is any one selected from the group consisting of KACC 91669, KACC 91670, KACC 91671 and KACC 91672. Weissella on xylitol A fermentation composition in which viscous polysaccharides are increased by fermentation with a genus microorganism. 5. The method of claim 4,
The fermentation composition is a fermentation composition, characterized in that kimchi. 5. The method of claim 4,
The fermentation composition is fermentation composition, characterized in that the water kimchi. Adding xylitol as a carbon source to kimchi, Weissella Fermentation of the genus microorganisms to produce a kimchi composition with increased viscosity polysaccharide.

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