KR20130063253A - The alcohol resistant strain of lactic acid bacteria, pediococcus acidilactici and its use - Google Patents

Abstract

PURPOSE: An lactic acid bacterium is provided to ensure strong resistance in 13% or more of alcohol, to grow in liquor of low alcohol degrees such as makkoli(Korean rice wine), and to ensure functionality without a change of product quality. CONSTITUTION: Pediococcus acidilactici AC-K3(KCTC 12039BP) has high alcohol resistance. The concentration of alcohol is 6-13%(v/v). A composition contains the strain and is selected from the group consisting of a composition for intestinal regulator, a probiotic composition, a feed composition, and a food additive composition. A method for making makkoli comprises a step of using the stain as a post additive. [Reference numerals] (AA) Growth(OD, A_600); (BB) Cultivation time(days)

Description

The alcohol resistant strain of lactic acid bacteria, Pediococcus acidilactici and its use

The present invention relates to a lactic acid bacterium pediococcus ACID-AC3 (KCTC 12039BP) having an alcohol resistance and a microbial agent comprising the same. The present invention relates to a pediococcus ashidilacti AC-K3 (KCTC 12039BP) and a microbial agent comprising the same, which are capable of being survived for a long time without growing at low temperature in the presence of alcohol.

As the interest and consumption of Korean traditional rice wine increases, the functionalities of lactic acid bacteria in rice wine are attracting attention. In the past, Makgeolli was a sterilized Makgeolli, which killed both lactic acid bacteria and yeast in order to increase the preservation period.

Lactic acid bacteria are known to act as probiotics by inhibiting the proliferation of harmful bacteria causing inflammation and cancer in the intestine, facilitating defecation and strengthening immunity.

However, in reality, live lactic acid bacteria are not in the 'sterilized rice wine' on the market at all, and the number of live bacteria is much smaller than what is known because many products are made through Japanese entry. Although some of the rice wine is made using traditional yeast, the number of lactobacillus of rice wine varies greatly according to the manufacturing method of rice wine.

Miyoung Seok et al. Reported that lactic acid bacteria in makgeolli are proliferated in the early stage of lactic acid bacteria, but are gradually affected by alcohol fermentation by yeast, and rapidly die when the alcohol concentration reaches 13% or higher (Korean Food Science Society, 2005). vol 37, No. 1, p61-66). Pediococcus Ashdi Lactici ( Pediococcus isolated from Andong Suzhou) acidilactici ) did not grow at all above 12% alcohol ( Kor . J. Microbiol) . Biotechnol . 2007. Vol. 35, No. 4, p310-315). Therefore, it is expected that lactic acid bacteria of high concentration alcohol resistance, which can be grown even at an alcohol concentration of 12-13%, can be usefully used for alcoholic fermentation such as makgeolli.

Korean Patent 10-0424659 discloses Lactobacillus plantarum ( Lactobacillus) with superior viability at an alcohol concentration of 8-32% than commercially available products commercially available as probiotics. plantarum ) was isolated and used as a probiotic, but it gave the property of alcohol resistance, and did not apply the property in particular. Republic of Korea Patent Publication 10-2006-0078894 alcohol concentration is 1-13% (v / v) in the alcohol and with acid at pH 2-7, psychrotrophic Lactobacillus yarn K which can grow at 5-40 ℃ (Lactobacillus sakei ) is used to separate fruit liquor and produce fruit liquor, but lactic acid bacteria may grow in liquor distributed at low temperature, such as makgeolli, resulting in a poor quality of liquor.

Disclosed is a prior art related to the present invention.

(i) Korean Patent No. 10-0424659 (Baek Hyun-dong, etc.) and Republic of Korea Patent Publication No. 10-2006-0078894 (Kim Il-yeon, etc.) exist as patent documents. changes in flora (Journal of the Korea Food, 2005, Vol 37, No. 1 p61-66) Identification and fermentation of lactic acid bacteria isolated from the fermentation broth Anton shochu ", and such background screen" (Kor. J. Microbiol Biotechnol . 2007, Vol. 35, No. 4, p310-315) ", but is essentially different from the technical specifics of the present invention.

An object of the present invention is to add or increase the functionality of lactic acid bacteria to various alcoholic beverages including makgeolli by separating alcohol-resistant lactic acid bacteria and using traditional liquor makgeolli and various alcoholic beverages. In addition, by only survival without growing at low temperatures, the purpose of minimizing the change in the quality of alcohol according to the application of alcohol-resistant lactic acid bacteria at low temperatures. In addition, the purpose is to apply to the non-alcoholic foods, dietary supplements, feed and pharmaceuticals.

The technical objects to be achieved by the present invention are not limited to the above-mentioned technical problems, and other technical subjects which are not mentioned can be clearly understood by those skilled in the art from the description of the present invention .

The present invention provides pediococcus ashidilactici AC-K3 (KCTC 12039BP) resistant to high concentrations of alcohol.

The high concentration alcohol may be characterized in that the alcohol of 6 to 13% (v / v).

The pediococcus ashidilacthy may be characterized in that it does not grow at low temperature, but only survives, and the low temperature may be 1 to 10 ° C.

In another aspect, the present invention (i) any one selected from the group consisting of (ii) a formal composition, a probiotic composition, a feed composition, a food additive composition and a probiotic composition containing (p) the Pediococcus ashidilacti AC-K3 (KCTC 12039BP). One composition is provided.

In addition, the present invention (i) fermentation of any one selected from the group consisting of (ii) makgeolli, wine, miso, red pepper paste, cheonggukjang, soy sauce and vinegar containing (Piecococcus) adi lacticitic AC-K3 (KCTC 12039BP) Provide the product.

In another aspect, the present invention provides a method for producing makgeolli using the pediococcus ashidilacti AC-K3 (KCTC 12039BP) as a post-additive of makgeolli.

In another aspect of the present invention, the pedicoccus ashidilacti AC-K3 (KCTC 12039BP) in a medium consisting of a carbon source, a nitrogen source, an organic acid and minerals incubated for 8 to 48 hours at 20 ~ 40 ℃ Provided is a method for culturing Okoccus ashidilacti AC-K3 (KCTC 12039BP).

Furthermore, the present invention recovers (i) the cells from the culture solution of pediococcus ashidilacti AC-K3 (KCTC 12039BP) obtained by the above culture method, and then comprises (ii) a protein protecting agent, a saccharide protecting agent and a pH adjusting agent. Mixing any one or more selected from the group, and (iii) provides a method for producing a lyophilized lyophilized product.

The present invention may be characterized in that it comprises the step of separating the alcohol resistant lactic acid bacteria, the alcohol resistant lactic acid bacteria to confirm the growth or viability at low temperature, and the step of culturing the alcohol-resistant lactic acid bacteria of alcohol resistant lactic acid bacteria in mass production. .

The present invention not only grows in lactic acid bacteria that have strong resistance to growth but also dies at more than 13% of alcohol, and can not only grow in low wines such as makgeolli, but also survive only without growing at 4-10 ° C., which is cold and cold temperature. By separating the lactic acid bacteria can be added to liquor containing liquor, such as makgeolli can give the functionality of the lactic acid bacteria without any quality change.

FIG. 1 is a growth curve during static culturing at 37 ° C. in MRS broth medium containing 13% alcohol of alcohol-resistant lactic acid bacteria selected first.
Figure 2 is a 16S rRNA gene sequence sequence of the selected AC-S1 and AC-K3 (KCTC 12039BP). AC-S1 and AC-K3 (KCTC 12039BP) are Pediococcud acidilactici was identified. DSM20336 in the Pediococcus 16S rRNA complete sequence (NCBI ACCESSION NR_042058) of DSM 20336, a type srtrain of pentosaceus , and DSM20284 is Pediococcus It represents the 16S rRNA complete sequence (NCBI ACCESSION NR_042057 ) of the reference strain DSM 20284 in acidilactici (type strain). K3 represents AC-K3 (KCTC 12039BP) strain and S1 represents AC-S1 strain.
FIG. 3 shows the selection of Pediococcus acidilactici AC after adding alcohol to MRS broth medium at 6% alcohol concentration of 12% and 13% alcohol concentration at the end of fermentation. Phedi the growth curve of the culture when inoculated -S1 and AC-K3 (KCTC 12039BP) to control bacteria Lactococcus o her CD rakti when (Pediococcus acidilactici ) compared with the KCTC 15064 strain, a type strain.
4 shows selected Pediococcus ashidilactici ( Pediococcus) acidilactici ) Variation in viable cell count when inoculated with commercial sterilized makgeolli (6% alcohol content) and AC-S1 and AC-K3 (KCTC 12039BP) at 4 ℃, 10 ℃ and 25 ℃.

The present invention relates to pediococcus ashidilactici AC-K3 (KCTC 12039BP) resistant to high alcohol concentrations.

The high concentration alcohol may be characterized in that the alcohol of 6 to 13% (v / v).

The pediococcus ashidilacthy may be characterized in that it does not grow at low temperature, but only survives, and the low temperature may be 1 to 10 ° C.

In another aspect, the present invention (i) any one selected from the group consisting of (ii) a formal composition, a probiotic composition, a feed composition, a food additive composition and a probiotic composition containing (p) the Pediococcus ashidilacti AC-K3 (KCTC 12039BP). It relates to one composition.

In addition, the present invention (i) fermentation of any one selected from the group consisting of (ii) makgeolli, wine, miso, red pepper paste, cheonggukjang, soy sauce and vinegar containing (Piecococcus) adi lacticitic AC-K3 (KCTC 12039BP) It is about a product.

In addition, the present invention relates to a method for preparing makgeolli using pediococcus ashidilacti AC-K3 (KCTC 12039BP) as a post-additive of makgeolli.

In another aspect of the present invention, the pedicoccus ashidilacti AC-K3 (KCTC 12039BP) is a pedi, characterized in that for 8 to 48 hours incubation at 20-40 ℃ in a medium consisting of carbon source, nitrogen source, organic acid and minerals The present invention relates to a method of culturing Okoccus ashidilactici AC-K3 (KCTC 12039BP).

Furthermore, the present invention recovers (i) the cells from the culture solution of pediococcus ashidilacti AC-K3 (KCTC 12039BP) obtained by the above culture method, and then comprises (ii) a protein protecting agent, a saccharide protecting agent and a pH adjusting agent. Mixing any one or more selected from the group, and (iii) provides a method for producing a lyophilized lyophilized product.

Hereinafter, the present inventors Pediococcus ashidilactici ( Pediococcus acidilactici ) AC-KC (KCTC 12039BP) is disclosed in more detail. Ashdilacthyse can be separated from fermented foods such as fresh makgeolli, yeast and kimchi, dairy products such as lactic acid bacteria fermented milk, and fish, animal and human intestines. It is preferable. As the separation medium of the present invention, lactic acid bacteria-related mediums, such as lactic acid bacteria differentiation medium (differentiation medium) such as BCP vegetation in food industry or selective medium, culture medium for lactic acid bacteria such as MRS medium, as well as lactic acid bacteria can be grown. General microorganisms can also be used, but it is advantageous to obtain a strain of the present invention by using a lactic acid bacteria-related medium and also a lactic acid bacteria selective medium or a differentiation medium.

In order to confirm the growth and viability at low temperature of the isolated strain can be measured by culturing in the section of 1 ~ 40 ℃ based on the mainstream and food to be applied to the isolated strain such as MRS and makgeolli. Preferably, it is practical to use objects such as alcohol and food to be applied.

For mass culture of the isolated strain, monosaccharides such as glucose, fructose, mannose and galactose, disaccharides such as maltose, and polysaccharides or acids or enzymatic products of these may be used alone or as a carbon source. For the purpose of nitrogen source and nutritional requirements, animal protein extracts such as yeast extract, various peptones and beef extracts can be used alone or in combination for mass culture. Potassium phosphates and sodium phosphates are commonly used to supply phosphorus, and other phosphates may also be used. Magnesium salt, manganese salt, iron compound, calcium salt, etc. can be used for the purpose of supplying an inorganic ion. In addition, organic acid salts such as acetate and citrate may be added as nutritional requirements or growth promoting substances, or fatty acids and vitamins such as oleic acid and polysorbate may be added. It may also be added.

For lyophilization of cultured cells, protein protecting agents such as skim milk, saccharide-based protecting agents such as trehalose, sugar, dextrin, starch, and pH adjusting agents such as phosphate may be used alone or in various ways.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Example 1 Isolation and Screening of Alcohol Resistant Strains

To detect alcohol-resistant lactic acid bacteria, take about 1 g of sample from commercial Makgeolli, Nuruk, Kimchi, and lactic acid bacteria fermented milk, make a suspension in physiological saline, and dilute by serial dilution method. Yeast extract 2.5g / l, peptone 5g / l, glucose 1g / l, Tween 80 1g / l, L-cysteine 0.1g / l, Agar 15 g / l, pH6.8-7.0, bromocresol purple (bromocresol purple 0.004-0.006%, 121 ° C. x 15 min sterilization) was plated on agar plate after colonization at 37 ° C. for 24-48 hours. Colony was separated from the colony with yellow color and purified by MRS agar medium [USA Difco product: Proteose peptone No. 3 10g / l.Beef extract 10g / l , Yeast extract 5g / l, glucose 20g / l, polysorbate 80 1g / l, ammonium ci trate) 2 g / l, sodium acetate 5 g / l, magnesium sulfate 0.1 g / l, manganese sulfate 0.05 g / l, dipotassium phosphate 2 g / l, Agar 15 g / l, 121 ℃ x 15 minutes sterilization] plate was implanted and incubated for at least 24 hours at 37 ℃ was used for the alcohol resistance test.

Lactic acid strain 1 platinum isolated as described above MRS broth medium containing alcohol 0% (v / v), 10% (v / v), 15% (v / v) (Difco, USA, MRS agar Inoculated in 10ml medium except agar and incubated at 37 ℃ for 24 hours. The OD ₆₀₀ of the culture was measured using a spectrophotometer, and strains with increased OD in MRS broth medium containing 10% (v / v) and 15% (v / v) of alcohol were selected first. Primary screening back to 0% of a strain (v / v), a 10% (v / v), 13% (v / v) and the alcohol has one platinum inoculate contained MRS broth medium OD ₆₀₀ in 48 hours As a result, two strains showing the highest growth in the medium containing 13% alcohol, AC-K3 (KCTC 12039BP) and AC-S1 were selected (FIG. 1).

Example 2 Identification of Alcohol Resistant Screening Strains

Two kinds of lactic acid bacteria AC-K3 (KCTC 12039BP) and AC-S1 were cultured in MRS broth medium, and then genomic DNA was isolated and phylogenetic identification was performed through sequencing of 16S rRNA gene. As a result of sequencing of 16S rRNA gene using bacterial 27F primer and 1492R primer, both strains of lactic acid bacteria were Pediococcus. It was found to be closest to acidilactici (FIG. 2).

Example 3 Confirmation of Alcohol Resistance of Isolated Alcohol-Resistant Lactic Acid Bacteria

Selected 2 kinds of lactic acid bacteria, Pediococcus ashidilactici ( Pediococcus acidilactici ) AC-K3 (KCTC 12039BP) and Pediococcus ashidilactici ( Pediococcus acidilactici ) 10 ml of MRS broth medium containing 0% (v / v), 6% (v / v), 12% (v / v) and 13% (v / v) of alcohol at 37 ° C. in MRS agar , Inoculated with platinum 1 cells incubated for 24 hours, the culture medium sample was taken in the middle of 144 hours incubation at 37 ℃, diluted 10-fold, and the growth degree was measured by measuring the OD660 (Fig. 3). Pediococcus acidilactici type strain KCTC 15064 was cultured and compared as a control strain. The addition concentration of alcohol was determined with reference to 6% (v / v) of alcohol of commercial makgeolli, 12% (v / v) and 13% (v / v) of alcohol at the end of makgeolli fermentation. As a result, the separated two alcohol resistant strains were identified as alcohol resistant strains by growing in 12% and 13% of the non-grown alcohol.

Example 4 Stability in Rice Wine

Two kinds of lactic acid bacteria, Pediococcus ashidilactici, which were obtained by dispensing commercially available sterile rice wine in aseptically sterilized test tubes acidilactici ) AC-K3 (KCTC 12039BP) and Pediococcus acidilactici AC-S1 were inoculated to live cell number 10 ⁶ ~ 10 ⁷ at 37 ℃ for 24 hours in MRS agar The change in viable cell count was measured while standing at 4 ° C, 10 ° C and 25 ° C. The viable cell count was measured by the pour plate method using BCP agar medium as shown in the health functional food code. As a result, they did not grow at 4-10 ° C., but survived, while growing at 25 ° C.

Example 5 Mass Culture of Separated Strains

Pediococcus acidilactici ) AC-K3 (KCTC 12039BP) was smeared on MRSm agar and amplified at 37 ° C for 48hrs.Then, 1 loop of culture was taken and inoculated into a 30 ml test tube containing 10 ml of MRSm broth. Incubated for hrs time. 0.5 ml of the culture is mixed with sterile 5% (w / v) skim milk and 0.5 ml of 50% (v / v) glycerol aqueous suspension, and the seed is stored by freezing in a deep freezer at -70 ° C. The medium composition of MRSm agar and broth was casein peptone 1% (w / v), yeast extract 0.4% (w / v), beef extract 0.8% (w / v), dibasic potassium phosphate 0.2% (w / v), Sodium acetate 0.5% (w / v), ammonium citrate 0.2% (w / v), magnesium sulfate 0.02% (w / v), manganese sulfate 0.01% (w / v), polysorbate 80 (twin 80) 0.1% (w / v), 1.5% (w / v) when agar is added and sterilized at 121 ℃ for 15 minutes.

1 ml of the spawn stored at -70 ° C was thawed and inoculated into a sterile MRSm broth medium prepared in a 30 ml test tube, and was incubated at 37 ° C for 12 hours to activate culture. 1 ml of the activated culture was inoculated into a 35 ml sterile MRSm broth culture prepared in a 50 ml serum bottle using a sterile syringe and left incubated at 37 ° C. for 12 hours for seed culture.

In a 5L fermenter, the main culture medium (4% (w / v) glucose, 1% (w / v) soy peptone, 1% (w / v) yeast extract, 0.1% (w / v) potassium diphosphate, citric acid Potassium 0.1% (w / v), magnesium sulfate 0.01% (w / v), manganese sulfate 0.005% (w / v)) were added 3.5L and sterilized for 15 minutes at 121 ℃. After sterilization, 35 ml of seed culture was inoculated to inoculate 1% (v / v). Culture conditions were incubated at 37 ℃, pH 5.5, 0.5M NaOH was used as a pH regulator, and stirred at a low 100 rpm to prevent the settling of the cells. In order to maintain anaerobic until the completion of culture after sterilization of the medium, N ² gas was charged, and the internal pressure of about 0.2 kg / cm ² was lowered to reduce the risk of contamination, and when internal pressure dropped, N ₂ gas was supplemented at that time. The OD ₆₆₀ of the 12-hour culture was diluted 10-fold to 0.727 and the viable cell count was 5.7 x 10 ⁹ cfu / ml.

Example 6 Cell Harvesting and Freeze-Drying of Bulk Culture Solution

The 5L culture was centrifuged at 6,000 rpm at 4 ° C. using a refrigerated centrifuge to obtain 32 g of wet cells. After drying for 32 hours in a freeze dryer, 31 g of a freeze protection agent solution (trehalose 20% (w / v), maltodextrin 10% (w / v), skim milk powder 1% (w / v)) was mixed with 32 g of wet cells. 16g of dried raw powder was obtained, and the number of viable cells of lyophilized raw powder was measured by pour plate method using BCP agar medium, which was found in the health functional foods, and was determined to be 4.0 x 10 ¹¹ cfu / g.

The present invention has been described above in connection with specific embodiments of the present invention, but this is only an example and the present invention is not limited thereto. Those skilled in the art can change or modify the described embodiments without departing from the scope of the present invention, and such changes or modifications are within the scope of the present invention. In addition, the materials of each component described herein can be readily selected and substituted for various materials known to those skilled in the art. Those skilled in the art will also appreciate that some of the components described herein can be omitted without degrading performance or adding components to improve performance. In addition, those skilled in the art may change the order of the method steps described herein depending on the process environment or equipment. Therefore, the scope of the present invention should be determined by the appended claims and equivalents thereof, not by the embodiments described.

The present invention can be used in the food industry, such as liquor by providing a microorganism including the Pediococcus acelacthysis AC-K3 (KCTC 12039BP) derived from makgeolli and the same. In particular, it is particularly useful as a lactic acid bacterium post-additive of makgeolli fermentation distributed at low temperature by using a property that does not grow at low temperature in addition to alcohol resistance. In addition, the lactic acid bacteria of the present invention can be used in various preparations for food additives, probiotics, suits, probiotics, feed additives, pharmaceuticals and other fermented products.

Korea Biotechnology Research Institute KCTC12039BP 20111025

Claims (10)

Pediococcus ashidilacti AC-K3 (KCTC 12039BP).
The pediococcus ashidilacti AC-K3 (KCTC 12039BP) according to claim 1, wherein the pediococcus ashidilacthysis is resistant to a high concentration of alcohol.
The pediococcus ashidilacti AC-K3 (KCTC 12039BP) according to claim 2, wherein the high concentration alcohol is 6-13% (v / v) alcohol.
The pediococcus ashidilacti AC-K3 (KCTC 12039BP) according to claim 1, wherein the pediococcus ashidilacti does not grow at low temperature, but only survives.
The pediococcus ashidilacti AC-K3 (KCTC 12039BP) according to claim 4, wherein the low temperature is 1 to 10 ° C.
any one selected from the group consisting of (i) a formal composition, a probiotic composition, a feed composition, a food additive composition and a probiotic composition comprising (i) the pediococcus ashidilacti AC-K3 (KCTC 12039BP) of claim 1 Composition.
(i) any fermentation product selected from the group consisting of (ii) makgeolli, wine, soybean paste, red pepper paste, cheonggukjang, soy sauce and vinegar, containing (1) pediococcus ashidilacti AC-K3 (KCTC 12039BP).
Method for producing makgeolli using pediococcus ashidilacti AC-K3 (KCTC 12039BP) of claim 1 as a post-additive of makgeolli.
Pediococcus ash of the pediococcus AC-K3 (KCTC 12039BP) of claim 1 in the medium consisting of carbon source, nitrogen source, organic acid and minerals for 20 to 40 ℃ incubated for 8 to 48 hours Cultivation method of lactish AC-K3 (KCTC 12039BP).
After recovering (i) the cells from the culture solution of pediococcus ashidilacti AC-K3 (KCTC 12039BP) obtained by the culture method of claim 9, Mixing any one or more selected, and (iii) a method for producing a lyophilized lyophilized product.

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