JP2017012163A - Lactobacillus plantarum llp5193 with high acid- and bile-resistance and high cell attaching ability and products containing it as the effective ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vegetable lactic acid bacterium Lactobacillus plantarum isolated from kimutsi, which is highly adaptable to the Korean intestinal environment.SOLUTION: The invention relates to a novel lactic acid bacterium, Lactobacillus plantarum LLP5193, isolated from kimutsi, and a product comprising it as an effective ingredient. Lactobacillus plantarum LLP5193 is a vegetable lactic acid bacterium, suitable for the Korean intestinal environment, excellent in acid resistance and bile resistance, capable of reaching the intestine, excellent in attaching ability to the intestinal cells for stable habitation in the intestine. Foodstuff produced by adding an excipient at a determined concentration to Lactobacillus plantarum LLP5193 is good in texture and abrasion resistant, advantageous for formulation without generating much powder, and easy to eat.SELECTED DRAWING: Figure 3

Description

  The present invention relates to Lactobacillus plantarum LLP5193, which is a novel lactic acid bacterium isolated from kimchi, and a product containing this as an active ingredient. Lactobacillus plantarum LLP5193 is a plant lactic acid bacterium that is suitable for the Korean intestinal environment, has excellent acid resistance and bile resistance, can safely reach the intestine, and has excellent intestinal cell adhesion. It can settle stably in the intestine. In addition, a food produced by adding an excipient to Lactobacillus plantarum LLP5193 at a constant concentration has good texture and wear resistance, is advantageous for formulation, does not generate much powder, and is easy to eat.

  Humans have used lactic acid bacteria as food since BC. Lactic acid bacteria are rich in nutrients and contain a variety of physiologically active substances. Lactic acid bacteria have an intestinal action that prevents diarrhea and constipation, suppresses the growth of harmful bacteria, prevents intestinal cancer and aging, generates vitamins to promote growth, regulates cholesterol, and controls adult diseases It has the effect of preventing and enhancing immunity.

  In addition, foods made from lactic acid bacteria contain lactic acid, so it has a long shelf life, has a unique refreshing acidity, suppresses the growth of harmful microorganisms, increases the digestibility of milk proteins, calcium, iron, There are many advantages such as promoting absorption of phosphorus.

  Currently, most of the lactic acid bacteria used in Korea are imported from abroad, and are animal lactic acid bacteria isolated from the intestines of animals and raw milk. Unlike Westerners, Koreans use vegetable foods as their staple food, so the intestinal environment is different and the length of the intestines is long. Therefore, it is better in terms of effectiveness to ingest plant lactic acid bacteria separated from foods obtained by fermenting vegetables, fruits and other plants, and fruit vegetables such as kimchi, rather than animal lactic acid bacteria.

  Korean Patent No. 10-2001-0075577 relates to a Lactobacillus plantarum strain isolated from kimchi. Lactobacillus plantarum (Lactobacillus Plantarum) according to this prior art is a plant lactic acid bacterium, which can be successfully applied to Koreans, has excellent acid resistance and bile resistance, and can safely reach the intestines. . However, there is a problem that there is no knowledge about the ability of lactic acid bacteria to adhere to the intestinal cells. Moreover, in order to increase the survival rate of lactic acid bacteria, it is necessary to further improve acid resistance and bile resistance.

Korean Published Patent No. 10-2001-0075577

  An object of the present invention is to provide Lactobacillus plantarum, which is a plant lactic acid bacterium isolated from kimchi and highly adaptable to the Korean intestinal environment.

  Another object of the present invention is to provide a Lactobacillus plantarum that has excellent acid resistance and bile resistance and can reliably reach the intestines.

  Another object of the present invention is to provide Lactobacillus plantarum that is excellent in intestinal cell adhesion ability and can sufficiently exert the effect of lactic acid bacteria.

  Another object of the present invention is to provide a food having excellent texture and wear resistance by adding an excipient to Lactobacillus plantarum.

  The object of the present invention is not limited to the object mentioned above. Objects of the present invention will become more apparent from the following description, and may be realized by the means described in the claims and combinations thereof.

  In order to achieve the above object, the present invention includes the following configurations.

  The present invention provides Lactobacillus plantarum LLP5193 (KCCM11598P), which is a lactic acid bacterium isolated from kimchi and excellent in acid resistance, bile resistance and intestinal cell adhesion.

Moreover, this invention provides the fermented milk and lyophilizate which contain the said Lactobacillus plantarum LLP5193 as an active ingredient.
The lyophilized product of the present invention may further contain one or more excipients selected from the group consisting of dextrin, rice flour and tapioca starch.
As this excipient, one added with 3 to 5% by weight of tapioca starch can also be used.

  Since the Lactobacillus plantarum according to the present invention is a plant lactic acid bacterium isolated from kimchi, it is suitable for the Korean intestinal environment.

  In addition, the Lactobacillus plantarum according to the present invention is excellent in acid resistance and bile resistance and can reach the intestine with certainty.

  In addition, Lactobacillus plantarum according to the present invention is excellent in intestinal cell adhesion ability and can be stably settled in the intestine, so that the effect as a lactic acid bacterium can be sufficiently exerted.

  In addition, the product manufactured from Lactobacillus plantarum according to the present invention has good texture and wear resistance, is advantageous for formulation, and is easy to eat.

It is a sugar utilization measurement result of Lactobacillus plantarum LLP5193. It is a sequence homology analysis result of Lactobacillus plantarum LLP5193. It is a cell attachment ability result with respect to Caco-2 cell of Lactobacillus plantarum LLP5193. This is a lyophilized product produced by adding an excipient to Lactobacillus plantarum LLP5193.

  Hereinafter, the present invention will be described in detail based on examples. The embodiments of the present invention can be modified into various forms as long as the gist of the invention is not changed. However, the scope of rights of the present invention is not limited to the following examples.

  If it is determined that the gist of the present invention can be obscured, descriptions of known configurations and functions are omitted. In this specification, “including” means that other components can be further included unless otherwise specified.

  The present invention provides Lactobacillus plantarum LLP5193 (deposit number KCCM11598P) which is a novel plant lactic acid bacterium isolated from kimchi.

  In recent years, research results have been published that plant lactic acid bacteria isolated from fermented foods such as vegetables and fruits and fruit vegetables such as kimchi have superior effects compared to animal lactic acid bacteria.

  Animal lactic acid bacteria (fermented milk lactic acid bacteria) use protein, fat, etc. along with carbohydrates as nutrients in the medium. Therefore, there is a drawback that the number of strains that are spoiled by protein and fat increases during a long-term fermentation process. On the other hand, plant lactic acid bacteria do not rot during fermentation because they use only carbohydrates as nutrients in the medium.

  In addition, plant lactic acid bacteria survive in places with less nutrients than animal lactic acid bacteria, so they have strong viability in harsh environments such as low pH and high salt concentration, are easy to culture, and are stable in the intestines. There is an advantage that it can be arrived at. Furthermore, since Koreans eat vegetable foods as a staple food, they have an intestinal environment suitable for plant lactic acid bacteria.

  Lactobacillus plantarum LLP5193 according to the present invention has excellent acid resistance and bile resistance.

  In order for lactic acid bacteria to be useful for intestinal action and bacteriostatic action, it must reach the intestine reliably. Therefore, in order to pass through the stomach without being killed by gastric acid, it must have excellent acid resistance and must be resistant to bile acids secreted from the upper part of the duodenum.

  Lactobacillus plantarum LLP5193 according to the present invention has excellent cell adhesion ability. In order for lactic acid bacteria to exert their effects in the intestine, they must adhere to intestinal cells (Intestinal cells), and thus the cell adhesion ability is important.

  The present invention provides a freeze-dried product that has a good texture and is suitable for eating by adding an excipient to Lactobacillus plantarum LLP5193 at a constant content and has a low friability and is advantageous for formulation.

  In particular, since infants and infants are hard to eat foods that are powdery and hard foods, the freeze-dried product according to the present invention can be very useful for producing lactic acid bacteria-containing products for infants and infants.

  The present invention provides Lactobacillus plantarum LLP5193, which is a lactic acid bacterium selected by a process of collecting a kimchi sample and selecting a strain excellent in acid resistance, bile resistance and cell adhesion ability from the sample.

  In the present invention, Lactobacillus plantarum LLP5193 is cultured under specific conditions, and then fermented under specific conditions to produce fermented milk. In addition, a certain amount of excipient is added to the fermented milk and freeze-dried under specific conditions to produce a freeze-dried product.

  The Lactobacillus plantarum LLP5193 can be cultured at 25-42 ° C. for 18-36 hours in a commercial milk medium containing 2-10% by weight of MRS medium or skim milk powder (Skim milk 20-100 g / L).

  Fermented milk obtained by adding 0.5-2% by weight of the culture solution of Lactobacillus plantarum LLP5193 to a commercial milk medium containing 2-10% by weight of skim milk and fermenting at 25-42 ° C. for 12-24 hours Can be manufactured.

  Tapioca starch was added to the fermented milk in an amount of 1 to 5% by weight, frozen at 70 ° C. for 2 to 6 hours, and then freeze-dried for 18 to 36 hours in a freeze dryer maintained at 50 mtorr and 80 ° C. Can be manufactured.

  Hereinafter, the present invention will be described in more detail based on examples. However, these examples are for the purpose of illustrating the present invention, and the scope of the present invention is not limited by these examples.

  Example 1-Selection of Lactobacillus plantarum LLP5193

  (1) Sample collection

  As shown in [Table 1] below, about 150 kinds of kimchi samples were collected at traditional traditional markets throughout the country.

  Sterile distilled water was added to the collected sample to prepare a diluted solution, and then the inside of the sample was homogenized with a pulverization homogenizer (Storcher, trade name: Pro-Media SH-001, manufactured by ELMEX).

  The diluted solution was diluted stepwise with sterile saline, and 0.1 ml was taken. This was smeared on an MRS solid medium supplemented with 0.002% by weight of BCP (Bromocresol purple) and 1.5% by weight of agar (Agar), and cultured in an incubator maintained at 37 ° C. for 48 hours.

  After the culture, colonies showing a yellow ring were selected, and each strain was purely isolated by subculturing 2-3 times in the same medium for each individual.

  The above strains were individually inoculated into an MRS liquid medium, re-cultured at 37 ° C. for 48 hours, and then used while refrigerated at 4 ° C.

  (2) Selection of strains

  A strain having excellent intestinal cell adhesion ability was selected from the strains using Caco-2 cells.

Caco-2 cells are inoculated in a 6-well plate at a concentration of 2 × 10 ⁵ cells / ml, and then cultured in an incubator that maintains conditions of 37 ° C. and 5% CO ₂ for about 15 days. What formed the monolayer (monolayer) was used.

  The strain is inoculated into an MRS liquid medium, cultured at 37 ° C. for 24 hours, centrifuged (8,000 rpm, 10 minutes), and the cells are collected, and then a phosphate buffer (Potassium phosphate buffer) is added. A strain suspension was prepared.

1 ml of the strain suspension is added to a 6-well plate containing Caco-2 cells, reacted for 2 hours at 37 ° C. and 5% CO ₂ , and then washed three times with a phosphate buffer. did.

  After adding 2 ml of MRS liquid medium to the washed 6-well plate, Caco-2 cells and strains were scraped with a sterilized scraper. This was transferred to a 10 ml tube and then diluted in multiple stages with sterile saline. After inoculating an MRS plate medium with 0.1 ml of an appropriate multiple dilution, the cells were cultured at 37 ° C. for 48 hours. The number of colonies cultured on the MRS plate medium was confirmed and the number of bacteria was measured.

  The density | concentration of the microbial cell added at the beginning of the strain and the microbial cell concentration after washing | cleaning were measured, respectively, and the adhesion rate (microbe concentration after washing | cleaning / initial microbial cell concentration) was confirmed. The strain that showed the best adherence rate was finally selected.

  The finally selected strain was inoculated into MRS liquid medium, cultured at 37 ° C. for 48 hours to grow, and centrifuged (10,000 g, 10 minutes) to obtain bacterial cells. 1 ml of the above microbial cells was added to a freezing solution containing MRS liquid medium: glycerol (Glycerol) in a ratio of 4: 1, and then added to a cryotube (freezing at −70 ° C.). saved. The cryopreserved strain was used as a starter for subsequent testing.

  Example 2-Strain characterization and identification

  (1) Feature analysis

  The morphological and biochemical characteristics of the selected strains were analyzed. With reference to the following [Table 2], it was confirmed that the strain was Gram positive, Neisseria gonorrhoeae, spore-forming ability and catalase were negative.

  (2) Strain identification

  The sugar utilization of the strain was analyzed with API 50 CHL kit (Biomerieux, France). Referring to [Table 3] and FIG. 1 below, it was confirmed that the utilization against trehalose was 96% different from the standard strain of Lactobacillus plantarum (Lactobacillus plantarum 1), which was 99.7%. .

  The 16S rRNA gene base sequence of the strain is as shown in SEQ ID NO: 1 shown in [Sequence Listing]. As a result of nucleotide sequence analysis, the strain has a homology of up to 99% with the Lactobacillus strain. This can be confirmed from FIG.

  Therefore, the present inventor identified the above strain as a novel Lactobacillus plantarum strain, named it Lactobacillus plantarum LLP5193, and deposited it with the Korean Preservation Center on November 3, 2014 (deposit number KCCM11598P).

  Example 3 Measurement of Acid Resistance and Bile Resistance of Lactobacillus plantarum LLP5193

  When lactic acid bacteria are ingested from the oral cavity, a considerable number are killed by gastric acid and bile. Therefore, in order to increase the number of lactic acid bacteria reaching the small and large intestines, excellent acid resistance and bile resistance are required.

  Lactobacillus plantarum LLP5193 was inoculated into MRS plate medium and cultured at 37 ° C. for 24 hours. The cultured colonies were collected and allowed to stand at 37 ° C. for 24 hours in an MRS liquid medium to produce an inoculum culture solution.

  (1) Acid resistance test

1% of the inoculum culture solution was inoculated into MRS liquid medium and cultured at 37 ° C. for 24 hours. To the obtained culture broth, 10% H ₂ SO ₄ was added to make it a strongly acidic condition of pH 3.0 and left for 2 hours. Thereafter, the diluted solution obtained by multi-stage dilution of the sample was smeared on the MRS plate medium and cultured at 37 ° C. for 24 hours, and the initial viable cell count and the viable cell count after pH 3.0 treatment were measured. In the present invention, acid resistance is expressed as a ratio of (initial viable cell count / viable cell count after pH treatment).

  (2) Bile resistance test

  An MRS plate medium produced by adding bile at 0.1 wt%, 0.3 wt%, and 0.5 wt% was prepared. Each MRS plate medium was inoculated with an inoculum culture solution and then cultured at 37 ° C. for 24 hours to confirm the growth state.

  (3) Test results

  A Lactobacillus plantarum standard strain (KTCTC21004, ATCC14917) was used as a comparative group for each test. The results of acid resistance test and bile resistance test are as shown in [Table 4] below.

  Lactobacillus plantarum LLP5193 showed a survival rate of 99.6% even after treatment for about 2 hours under strongly acidic conditions at pH 3.0. It was also confirmed that growth was possible when 0.5% by weight of bile was added.

  Therefore, since Lactobacillus plantarum LLP5193 has excellent acid resistance and bile resistance and has a high probability of reaching the intestine, when it is ingested as a food or health functional food, it effectively exhibits various effects of lactic acid bacteria. be able to.

  Example 4-Measurement of intestinal cell adhesion ability of Lactobacillus plantarum LLP5193

  From the above Example 3, Lactobacillus plantarum LLP5193 was excellent in acid resistance and bile resistance, and it was confirmed that the rate of reaching the intestine was higher than other lactic acid bacteria.

  However, even if the lactic acid bacteria reach the intestines, they will be lost if they do not adhere to the intestinal cells, so that the effect cannot be exhibited normally. Therefore, it can be said that cell adhesion ability is a more important characteristic than acid resistance and bile resistance.

  The intestinal cell adhesion ability of Lactobacillus plantarum LLP5193 was quantitatively measured, and the adhesion form was observed with a microscope.

  (1) Quantitative measurement

  Intestinal cell adhesion ability of Lactobacillus plantarum LLP5193 was measured in the same manner as in the intestinal cell adhesion ability test using Caco-2 cells in Example 1. The results are as shown in [Table 5] below.

A total of 6 tests were repeated. Examining the average value, when Lactobacillus plantarum LLP5193 was administered at about 4.5 × 10 ⁹ CFU / ml, about 5.1 × 10 ⁸ CFU / ml adhered after the adhesion reaction. From this, a log value indicating the concentration of microorganisms was generally taken and the adhesion rate was calculated. As a result, it was very high, about 90.2% [(8.71 log / 9.65 log) × 100].

Considering that the intake standard of a general probiotic lactic acid bacteria product is 1 × 10 ⁸ CFU / ml (8.0 log), it can be seen that the ability of Lactobacillus plantarum LLP5193 to adhere to intestinal cells is very excellent.

  (2) Microscopic observation

  In the adhesion test using Caco-2 cells, a part of the culture solution was collected after completion of the adhesion reaction. An appropriate amount of the culture solution was transferred to a slide glass, and then Lactobacillus plantarum LLP5193 was stained with Gram staining. The result is as shown in FIG.

  It can be confirmed that a large amount of stained Lactobacillus plantarum LLP5193 was attached around Caco-2 cells.

  Since Lactobacillus plantarum LLP5193 is excellent in acid resistance and bile resistance, it can stably reach the intestine and has excellent cell adhesion ability, so that a large number can adhere to intestinal cells. Therefore, Lactobacillus plantarum LLP5193 can exhibit the effect as a lactic acid bacterium very well.

  Example 5-Production of a product containing Lactobacillus plantarum LLP5193 as an active ingredient

(1) Production of fermented milk Lactobacillus plantarum LLP5193 was inoculated into MS solid (agar) medium and then activated by culturing at 37 ° C for 24 hours. Activated Lactobacillus plantarum LLP5193 colonies were inoculated into a medium in which 5% by weight of nonfat dry milk was added to 100 ml of milk, and cultured at 37 ° C. for about 12 hours until a curd was produced. After mixing 1% by weight of the inoculum culture with 1 L of milk to which 5% by weight of skim milk powder was added, fermented milk was produced by culturing at 37 ° C. for about 12 hours until a curd was formed.

  (2) Production of freeze-dried product

  The viable cell count, texture and erosion rate of the freeze-dried product prepared by adding a certain concentration of excipient to the fermented milk were measured.

  Dextrin, rice flour and tapioca starch were added to the fermented milk at 1 wt%, 3 wt% and 5 wt%. The mixture was put into a mold for molding and rapidly frozen in a cryogenic refrigerator (maintained at 70 ° C. below zero) for 2 hours. This was transferred to a lyophilizer (Ilsin Biobase, MCFD Series, South Korea), and then lyophilized at 50 mtorr and 80 ° C. for 18 hours to produce a lyophilized product.

  The viable count of the lyophilized product was measured. After the lyophilized product was diluted with sterile saline, an appropriate dilution was taken. This was smeared on an MRS solid medium, cultured at 37 ° C. for 48 hours, and then colonies were counted.

  The texture of the lyophilized product was measured. After arbitrarily selecting 5 persons and taking a lyophilized product, the hardness was evaluated.

  The friability of the lyophilizate was measured. The lyophilized product was put into a rolling machine and then rotated 50 times. The weight of the lyophilized product before rotation and the weight after rotation were measured. The wear rate was expressed as (weight before rotation−weight after rotation) / weight before rotation × 100.

  The results of the viable cell count, texture, and abrasion rate of the lyophilized product to which the excipient was added are shown in Table 6 below.

* 1: Hard → 5: Soft

  It was found that the number of viable cells was increased by adding the excipient compared to the case of no addition. However, it was confirmed that the difference depending on the type of excipient was not significant.

  As a result of examining the wear rate and the texture when the excipient was added, the texture was soft when no excipient was added, but the wear rate was as high as 66.7% and the lyophilized form should be preserved. Was almost impossible.

  When dextrin and rice flour were used, the texture was almost the same as when tapioca starch of the same concentration was used, but the wear rate was high. The lyophilized product to which 3% by weight of tapioca starch was added showed the most excellent abrasion resistance and texture.

  Therefore, when 3% by weight of tapioca starch is added, there is an advantage that foods for infants and infants can be manufactured because the wear resistance is good, a large amount of powder is not generated, and the texture is good.

  The actual product obtained by adding 3% by weight of tapioca starch to the lyophilized product is shown in FIG.

  The Lactobacillus plantarum according to the present invention is suitable for the Korean intestinal environment as a plant lactic acid bacterium isolated from kimchi.

  In addition, Lactobacillus plantarum according to the present invention is excellent in acid resistance and bile resistance and can reach the intestine with certainty.

  In addition, the Lactobacillus plantarum according to the present invention is excellent in intestinal cell adhesion ability and can be stably settled in the intestine, so that the effect as a lactic acid bacterium can be sufficiently exerted.

  In addition, since the product produced from Lactobacillus plantarum according to the present invention has good texture and abrasion resistance, it is advantageous for formulation, does not generate much powder, and is easy to eat.

  The present invention has been described in detail above. However, the scope of rights of the present invention is not limited to this, and is determined by the following claims.

KCCM11598P

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Claims (4)

  Lactobacillus plantarum LLP5193 (KCCM11598P) excellent in acid resistance, bile resistance and intestinal cell adhesion ability isolated from kimchi.   A freeze-dried product comprising Lactobacillus plantarum LLP5193 of claim 1 as an active ingredient.   The lyophilizate according to claim 2, further comprising one or more excipients selected from the group consisting of dextrin, rice flour and tapioca starch.   The freeze-dried product according to claim 3, wherein the excipient is added with 3 to 5% by weight of tapioca starch.