KR20200007386A - A milk and fermented milk having anti-inflammatory effects on intestinal epithelial cells and having excellent ability to reach to intestinal epithelial cells - Google Patents

Abstract

The present invention relates to milk and fermented milk with excellent intestinal reaching power and improved intestinal inflammation prevention effect. According to the present invention, milk for preventing or mitigating intestinal inflammation comprises composite LB-9 lactic acid bacteria as an active ingredient, and the LB-9 lactic acid bacteria comprises a Lactobacillus plantarum LLP5193 strain and a Lactobacillus plantarum LLP5273 strain. Fermented milk for preventing or mitigating intestinal inflammation comprises composite LB-9 lactic acid bacteria as an active ingredient, and the LB-9 lactic acid bacteria comprises a Lactobacillus plantarum LLP5193 strain and a Lactobacillus plantarum LLP5273 strain.

Description

A milk and fermented milk having anti-inflammatory effects on intestinal epithelial cells and having excellent ability to reach to intestinal epithelial cells}

The present invention relates to milk and fermented milk with excellent intestinal reachability and improved intestinal inflammation prevention effect.

In modern times, the rapid westernization of diet and excessive stress increased the consumption of fast foods and high-calorie foods that can solve one meal quickly, while constipation due to poor eating habits and irregular lifestyles such as reduced intake of vegetables and fiber. And the population that complains of diseases such as adult diseases is increasing trend. In particular, inflammatory bowel disease is a disease that causes chronic inflammation of the intestine and can be divided into ulcerative colitis and Crohn's disease. The cause and pathophysiology of inflammatory bowel disease are not known yet, but genetic factors, environmental factors such as intestinal bacteria and foods, and immunological factors are thought to be involved in the complex developmental mechanism.

Recently, many studies have been made on the prevention and improvement using probiotics in the treatment of inflammatory bowel disease. Probiotics, according to the definition of WHO / FAO, are known as "living microorganisms that have a beneficial effect on the host when ingested." Probiotics are mainly composed of lactic acid bacteria according to the Korean dietary supplement standard. Herias MV et al. Reported that ulcerative colitis (UC) with DSS (Dextran sulfate sodium) caused inflammation and decreased inflammation when Lactobacillus case (Lactobacillus case) was administered, and Ghouri YA et al. And reviewed a variety of studies in which prebiotics and neobiotics were administered.

When the immune cells in the intestine are stimulated or stressed, they secrete various inflammatory cytokines (cytokines), which trigger an inflammatory response. Many probiotics represented by lactic acid bacteria are known to relieve inflammation through the inhibition of expression of these inflammatory cytokines.

However, many probiotics for preventing intestinal inflammation known to date are mainly known as lactic acid bacteria derived from traditional foods or feces, most of them correspond to animal lactic acid bacteria. A representative example is a fermented lactic acid bacterium, and animal lactic acid bacteria (milk fermented lactic acid bacteria) use protein, fat, etc. together with carbohydrate as a medium nutrient. Therefore, there is a disadvantage in that the number of strains rotted by proteins and fats during a long fermentation process increases.

In addition, in the case of products for the health of the intestine were often added mainly as a fermentation product or powder for health functional food.

However, studies on maximizing the effect of preventing intestinal inflammation by adding vegetable lactic acid bacteria isolated from kimchi, a traditional fermented food of Korea, to foods that are easily accessible to the general consumer is insufficient.

Korean Patent Registration No. 10-1683686 Korea Patent Registration No. 10-1696670 Korean Laid-Open Patent No. 2012-0039381 Korean Unexamined Patent Publication No. 2013-0033709

1. M.V. Herias, J.F. Koninkx, J.G. Vos, J.H. Huis in't Veld, J.E. van Dijk., Probiotic effects of Lactobacillus casei on DSS-induced ulcerative colitis in mice, Int J Food Microbiol 103 (2005) 143-155. 2. Y.A. Ghouri, D.M. Richards, E.F. Rahimi, J.T. Krill, K.A. Jelinek, A.W. DuPont, Systematic review of randomized controlled trials of probiotics, prebiotics, and synbiotics in inflammatory bowel disease, Clin Exp Gastroenterol 7 (2014) 473-487.

Therefore, the inventors of the present invention have studied to apply the lactic acid bacteria product suitable for Koreans using vegetable lactic acid bacteria isolated from kimchi, which is a traditional fermented food.

The present inventors intend to verify the intestinal inflammation prevention effect when two patented vegetable lactic acid bacteria are added together with milk and fermented milk, and provide the lactic acid bacteria mixed milk and fermented milk.

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

One aspect of the invention, the LB-9 complex lactic acid bacteria as an active ingredient, the LB-9 complex lactic acid bacteria include Lactobacillus plantarum LLP5193 strain and Lactobacillus plantarum (Lactobacillus plantarum) LLP5273 strain Provides milk for preventing or reducing intestinal inflammation.

In one aspect of the present invention, the Lactobacillus plantarum LLP5193 strain is a strain having a deposit number of KCCM11598P, and the Lactobacillus plantarum LLP5273 strain is a strain having a deposit number of KCCM11696P, intestinal inflammation prevention Or provide relief milk.

In one aspect of the present invention, the LB-9 complex lactic acid bacteria, LLP5193 and LLP5273 strain provides a live bacteria of 10: 1 to 1:10, the intestinal inflammation prevention or alleviating milk.

In one aspect of the present invention, the intestinal inflammation prevention or alleviation milk contains LB-9 complex lactic acid bacteria 1.0x10 ^ 6 CFU / ml or more with respect to the total volume (ml) of the intestinal inflammation prevention or alleviation milk To provide.

Another aspect of the present invention, LB-9 complex lactic acid bacteria as an active ingredient, the LB-9 complex lactic acid bacteria Lactobacillus plantarum (Lactobacillus plantarum) LLP5193 strain and Lactobacillus plantarum (Lactobacillus plantarum) LLP5273 strain comprising Providing fermented milk for preventing or alleviating intestinal inflammation.

In another aspect of the present invention, the Lactobacillus plantarum LLP5193 strain is a strain having a deposit number of KCCM11598P, and the Lactobacillus plantarum LLP5273 strain is a strain having a deposit number of KCCM11696P, intestinal inflammation prevention Or reducing fermented milk.

In another aspect of the present invention, the fermented milk provides LB-9 complex lactic acid bacteria powder at least 0.1% by weight based on the total weight of the fermented milk, provides a fermented milk for preventing or reducing intestinal inflammation.

Another aspect of the present invention, the method for producing any one of the fermented milk for preventing or alleviating intestinal inflammation, comprising the steps of adding a starter bacteria for producing fermented milk to milk; Fermenting the milk to which the starter bacteria have been added; And adding and mixing the LB-9 complex lactic acid bacteria to the fermented milk.

In another aspect of the invention, the step of fermentation is to ferment for 4 hours to 12 hours at a temperature of 40 ℃ to 45 ℃, provides a method for producing fermented milk for preventing or reducing intestinal inflammation.

In another aspect of the present invention, the step of fermentation is a final pH of 3.5 to 4.5, the fermentation until the starter bacteria number is 1.0x10 ^ 8 CFU / ml or more of the intestinal inflammation prevention or alleviation of fermented milk It provides a manufacturing method.

Milk according to one aspect of the present invention or fermented milk according to another aspect of the present invention may include a vegetable complex lactic acid bacteria suitable for the constitution of the Korean as an active ingredient, it can provide milk or fermented milk suitable for the Korean intestinal environment.

Milk according to one aspect of the present invention or fermented milk according to another aspect of the present invention is excellent in the intestinal reach of vegetable lactic acid bacteria.

Milk according to one aspect of the present invention or fermented milk according to another aspect of the present invention is excellent in the ability to adhere to the intestinal cells has an effect that can be stably settled in the intestine.

Milk according to one aspect of the present invention or fermented milk according to another aspect of the present invention is excellent in preventing or reducing intestinal inflammation.

The effect of this invention is not limited to the effect mentioned above. It is to be understood that the effects of the present invention include all the effects deduced from the description below.

1 is a result of measuring the adhesion rate of the gastric juice, artificial intestinal fluid and intestinal cells of LB-9 complex lactic acid bacteria mixed with two kinds of lactic acid bacteria used in the present invention.
2 is mixed with LB-9 lactic acid bacteria and Rhodamine 123, a fluorescent staining solution, fed to rats, and observed by fluorescence microscopy.
Figure 3 is a diagram showing the animal experiment progress method for verifying the colitis prevention effect of LB-9 complex lactic acid bacteria and milk and fermented milk products containing the lactic acid bacteria.
Figure 4 is the result of showing the weight loss rate in the initial ratio of the animal experiment to verify the colitis prevention effect of LB-9 complex lactic acid bacteria and milk and fermented milk products containing the lactic acid bacteria.
Figure 5 is a result of measuring the intestinal length for each experimental group dissected after the animal experiment for verifying the colitis prevention effect of LB-9 complex lactic acid bacteria and lactic acid bacteria and milk and fermented milk products.
Figure 6 is a histological observation of colon tissue for each experimental group dissected after the animal experiment to verify the colitis prevention effect of LB-9 complex lactic acid bacteria and lactic acid bacteria and milk and fermented milk products.
7 is a result of measuring the inflammatory cell infiltration in each experimental group after the animal experiment to verify the colitis prevention effect of the LB-9 complex lactic acid bacteria and the lactic acid bacteria and milk and fermented milk products, and the result of analysis for Ly6.
8 is a result of measuring the inflammatory cell infiltration in each experimental group after the animal test for verifying the colitis prevention effect of LB-9 complex lactic acid bacteria and the lactic acid bacteria and milk and fermented milk products, and the analysis results for CD11b.
9 is a result of performing a TUNEL assay to measure the degree of cell death in each experimental group after the animal experiment for verifying the colitis prevention effect of LB-9 complex lactic acid bacteria and lactic acid bacteria and milk and fermented milk products.
10 is a result of measuring the amount of expression by protein type in the experimental group after the animal experiment for verifying the colitis prevention effect of LB-9 complex lactic acid bacteria and lactic acid bacteria and milk and fermented milk products.
11 is an objective numerical comparison of the results measured by Western blot after the animal experiment for verifying the colitis prevention effect of LB-9 complex lactic acid bacteria and lactic acid bacteria and milk and fermented milk products One result.
12 is a cell death based on the results of Western blot measurement and quantification of the expression level for each protein type in the experimental group after the animal experiment for verifying the colitis prevention effect of LB-9 complex lactic acid bacteria and milk and fermented milk products containing the lactic acid bacteria It is a schematic diagram inferring mechanism.

Unless otherwise stated, all numbers, values, and / or expressions used herein to express components, reaction conditions, content of components, and the like, the various values of the measurements that occur in obtaining these values, among other things, are inherently different. Since they are approximations of uncertainty, they should be understood as being modified in all cases by the term "about." In addition, where numerical ranges are disclosed herein, these ranges are continuous and include all values from the minimum to the maximum including the maximum, unless otherwise indicated. Furthermore, where such ranges refer to integers, all integers are included, including the minimum to the maximum including the maximum unless otherwise indicated.

In the present specification, when a range is described for a variable, it will be understood that the variable includes all values within the described range including the listed endpoints of the range. For example, the range "5 to 10" includes any subrange such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, as well as values of 5, 6, 7, 8, 9, and 10. And any value between integers that fall within the scope of the described ranges such as 5.5, 6.5, 7.5, 5.5-8.5, 6.5-9, and the like. Also for example, the range of “10% to 30%” ranges from 10% to 15%, 12% to 10%, 11%, 12%, 13%, etc. as well as all integers including up to 30%. It will be understood to include any subranges such as 18%, 20% to 30%, and any value between reasonable integers within the range of the stated range, such as 10.5%, 15.5%, 25.5% and the like.

Hereinafter, the present invention will be described in detail.

In general, the addition of strains derived from milk or strains derived from fermented milk to dairy foods, which are animal foods, can be expected. However, when such lactic acid bacteria are added to milk, the fermentation process takes place. A separate process must be added. However, in the case of vegetable lactic acid bacteria, in particular, Lactobacillus plantarum LLP5193 strain and Lactobacillus plantarum LLP5273 strain, which is a vegetable lactic acid bacterium used in the present invention, were not fermented even when stored in milk for about 4 weeks. This is believed to be the reason for the low availability of carbon sources (such as lactose) in milk.

In addition, the Lactobacillus plantarum LLP5193 strain, a plant lactic acid bacterium used in the present invention, is a slightly superior strain having acid resistance, bile resistance, and intestinal cell adhesion.

However, the Lactobacillus plantarum LLP5273 strain, a plant lactic acid bacterium used in the present invention, has no known effect on the resistance to acid resistance, tolerability and intestinal cell adhesion, and in the patent 10-1683686, bile acid resolution and hepatocyte HMG- It is only disclosed that the CoA reductase activity inhibitory ability is excellent. There is no disclosure about the intestinal reachability and intestinal inflammation prevention effect of the Lactobacillus plantarum LLP5273 strain as described above, and a person of ordinary skill in the art may use the Lactobacillus plantarum LLP5273 for intestinal reachability and intestinal inflammation prevention effect. It will be hard to anticipate using it at all. In addition, it would be difficult to anticipate dairy products with improved lactobacillus plantarum LLP5273, which is a vegetable lactic acid bacterium, added to animal food dairy products and improved intestinal delivery and intestinal inflammation.

The inventors of the present invention add the lactobacillus plantarum LLP5193 strain and Lactobacillus plantarum LLP5273 strain, which is a specific lactic acid bacterium, to milk or fermented milk, so that the specific physiologically active intestinal reachability is improved and the intestinal inflammation prevention effect is improved. And research on fermented milk has led to the present invention.

The present invention is to add the lactobacillus plantarum LLP5193 strain and Lactobacillus plantarum LLP5273 strain, which is a vegetable lactic acid bacterium, to milk or fermented milk, to provide milk and fermented milk with excellent intestinal reachability and improved intestinal inflammation.

Hereinafter, various aspects of the present invention will be described.

One aspect of the invention, the LB-9 complex lactic acid bacteria as an active ingredient, the LB-9 complex lactic acid bacteria include Lactobacillus plantarum LLP5193 strain and Lactobacillus plantarum (Lactobacillus plantarum) LLP5273 strain Provides milk for preventing or reducing intestinal inflammation.

In one aspect of the present invention, the Lactobacillus plantarum LLP5193 strain is a strain having a deposit number of KCCM11598P, and the Lactobacillus plantarum LLP5273 strain is a strain having a deposit number of KCCM11696P, intestinal inflammation prevention Or provide relief milk.

In one aspect of the present invention, the LB-9 complex lactic acid bacteria, LLP5193 and LLP5273 strain provides a live bacteria of 10: 1 to 1:10, the intestinal inflammation prevention or alleviating milk.

In one aspect of the present invention, the intestinal inflammation prevention or alleviation milk contains LB-9 complex lactic acid bacteria 1.0x10 ^ 6 CFU / ml or more with respect to the total volume (ml) of the intestinal inflammation prevention or alleviation milk To provide.

Another aspect of the present invention, LB-9 complex lactic acid bacteria as an active ingredient, the LB-9 complex lactic acid bacteria Lactobacillus plantarum (Lactobacillus plantarum) LLP5193 strain and Lactobacillus plantarum (Lactobacillus plantarum) LLP5273 strain comprising Providing fermented milk for preventing or alleviating intestinal inflammation.

In another aspect of the present invention, the Lactobacillus plantarum LLP5193 strain is a strain having a deposit number of KCCM11598P, and the Lactobacillus plantarum LLP5273 strain is a strain having a deposit number of KCCM11696P, intestinal inflammation prevention Or reducing fermented milk.

In another aspect of the present invention, the fermented milk provides LB-9 complex lactic acid bacteria powder at least 0.1% by weight based on the total weight of the fermented milk, provides a fermented milk for preventing or reducing intestinal inflammation.

Another aspect of the present invention, the method for producing any one of the fermented milk for preventing or alleviating intestinal inflammation, comprising the steps of adding a starter bacteria for producing fermented milk to milk; Fermenting the milk to which the starter bacteria have been added; And adding and mixing the LB-9 complex lactic acid bacteria to the fermented milk.

In another aspect of the invention, the step of fermentation is to ferment for 4 hours to 12 hours at a temperature of 40 ℃ to 45 ℃, provides a method for producing fermented milk for preventing or reducing intestinal inflammation.

In another aspect of the present invention, the step of fermentation is a final pH of 3.5 to 4.5, the fermentation until the starter bacteria number is 1.0x10 ^ 8 CFU / ml or more of the intestinal inflammation prevention or alleviation of fermented milk It provides a manufacturing method.

Hereinafter, the present invention will be described in detail through examples.

Example 1 Preparation of Elbinin (LB-9) Complex Lactic Acid Bacteria

After the inventors have invented two kinds of Lactobacillus plant tarum LLP5193 and LLP5273, which were invented and patented, to Mediogen Co., Ltd. (Jecheon, Chungcheongbuk-do, Korea), and then fermented and lyophilized. Prepared as a powder. Each lactic acid bacteria powder was prepared at 1.5 x 10 ^ 11 CFU / g based on the number of viable cells per g, and then mixed based on the analyzed bioburden number to prepare 1.0 x 10 ^ 11 CFU / g of complex lactic acid powder, which was elbe It was named Nine (LB-9) complex lactic acid bacteria.

Example 2 Determination of Artificial Digestion Stability of Elbinin (LB-9) Complex Lactic Acid Bacteria

The purpose of this study was to measure the stability of two types of lactic acid bacteria after digestion, namely gastric and intestinal fluid, and to predict whether they could reach the intestines when LB-9 lactic acid was actually ingested. The artificial gastric juice was adjusted to pH of 2.5 with 0.1M sodium phosphate buffer using 10% sulfuric acid (H2SO4), and then autoclave (121 ℃, 20min), cooled to below 60 ℃, and then pepsin (Pepsin). from porcine gastric mucosa, Sigma, USA) was added to 180U / ml. Similar to artificial gastric juice, artificial intestinal fluid was used as 0.1M sodium phosphate buffer, and bovine-derived bile acid (Oxgall, Sigma, USA) was added to 0.3% and adjusted to pH 6.8 ± 0.2 to adjust autoclave ((121 ℃). After autoclave, after cooling to 60 ℃ or less, pancreatin (Pancreatin from porcine pancreas, Sigma, USA) was added to 180U / ml to complete the artificial intestinal fluid.

Two LLP5193 and LLP5273 lactic acid bacteria and 1 g of LB-9 complex lactic acid bacteria powder mixed with the lactic acid bacteria were collected and then administered to 20 ml of artificial gastric juice and intestinal fluid, respectively. Based on the time of administration, 1ml of samples were taken at intervals of 1 hour up to 3 hours, and the number of viable cells was analyzed by multi-stage dilution method and smearing method. Lactobacillus rhamnosus ATCC53103 was distributed from ATCC to be used as a control for survival test for artificial gastric juice and intestinal fluid, and the same strain was also made with powders of 1.5 x 10 ^ 11 CFU / g by requesting to Mediogen. Survival was measured according to the method. In the present invention, the survival rate for artificial gastric juice and artificial intestinal fluid was calculated as (viable cell number / initial viable cell number x 100 after pH treatment).

Survival measurement results for artificial gastric juice are shown in Table 1, and survival measurement results for artificial intestinal fluid are shown in Table 2.

time ATCC53103 LLP5193 LLP5273 LB-9 CFU / ml Survival rate (%) CFU / ml Survival rate (%) CFU / ml Survival rate (%) CFU / ml Survival rate (%) 0 1.18 x ^{10 11} - 1.31 x ^{10 11} - 1.28 x ^{10 11} - 1.18 x ^{10 11} - One 1.16 x ^{10 11} 98.3 1.29 x ^{10 11} 98.5 1.08 x ^{10 11} 84.4 1.16 x ^{10 11} 97.2 2 1.13 x ^{10 11} 95.8 1.24 x ^{10 11} 94.7 1.06 x ^{10 11} 82.8 1.13 x ^{10 11} 91.7 3 1.13 x ^{10 11} 95.7 1.22 x ^{10 11} 92.7 1.02 x ^{10 11} 79.1 1.13 x ^{10 11} 87.7

time ATCC53103 LLP5193 LLP5273 LB-9 CFU / ml Survival rate (%) CFU / ml Survival rate (%) CFU / ml Survival rate (%) CFU / ml Survival rate (%) 0 1.24 x ^{10 11} - 1.19 x ^{10 11} - 1.31 x ^{10 11} - 1.15 x ^{10 11} - One 1.01 x ^{10 11} 81.5 1.15 x ^{10 11} 96.6 1.32 x ^{10 11} 100.8 1.14 x ^{10 11} 99.1 2 9.20 x ^{10 10} 74.2 1.17 x ^{10 11} 98.3 1.27 x ^{10 11} 96.9 1.12 x ^{10 11} 97.4 3 6.60 x ^{10 10} 53.2 1.16 x ^{10 11} 97.5 1.25 x ^{10 11} 95.4 1.11 x ^{10 11} 96.5

As shown in the above results, L. rhamnosus ATCC53101 showed the highest survival rate of 95.7% in artificial gastric juice, but very low survival rate of 53.2% compared to other strains in artificial intestinal fluid.

LLP5193 and LLP5273 showed different survival rates of 92.7% and 79.1%, respectively, but there was no significant difference in survival rate in artificial serous fluid.

The LB-9 complex lactic acid bacteria prepared by mixing the two lactic acid bacteria showed survival rate of gastric juice 87.7% and survival rate of artificial serous 96.5%.

Example 3 Measurement of Intestinal Cell Adhesion of Elbinin (LB-9) Complex Lactic Acid Bacteria

Even if lactic acid bacteria are delivered to the intestine, they will be lost as they are not attached to the intestinal cells, so they will not work properly. Therefore, cell adhesion is also known to be very important as well as acid and bile resistance.

Therefore, L. rhamnosus ATCC53103 control group, L. plantarum LLP5193 and LLP5273, and LB-9 complex lactic acid bacteria mixed with these two species were measured to measure the intestinal cell adhesion.

Intestinal cells were prepared using Caco-2 (30030.1) cells, which were obtained from the Korean Cell Line Bank (KCLB), and inoculated at a concentration of 2x10 ⁵ cells / ml in a 6-well plate. After that, the cells were cultured for about 15 days in an incubator maintained at 37 ° C. and 5% CO ₂ to form a monolayer.

Four kinds of lactic acid bacteria were inoculated in MRS liquid medium and incubated at 37 ° C. for 24 hours, and the cells were recovered by centrifugation (8,000 rpm, 10 minutes), and then, a strain suspension was prepared by adding phosphate buffer (Potassium phosphate buffer). It was. The lactic acid bacteria suspension was added to 1 ml 6-well plate containing Caco-2 cells and reacted for 2 hours at 37 ° C. and 5% CO ₂ , followed by washing three times with phosphate buffer.

After completion of the reaction, 2 ml of MRS medium was added to the 6-well plate, and then the caco-2 cells and strains were scraped off using a sterile scraper. It was transferred to a 10 ml tube and diluted in multiple stages using sterile saline. 0.1 ml of the appropriate dilution was inoculated into MRS plate medium, and then incubated at 37 ° C. for 48 hours. The number of colonies cultured on MRS plate medium was checked to determine the number of bacteria.

The initial cell concentration of the strain and the cell concentration after the intestinal attachment reaction were measured, respectively, and the adhesion rate (cell concentration after the reaction / initial cell concentration) was confirmed. Table 3 shows the measurement results for four kinds of lactic acid bacteria.

Lactobacillus Viable Count (CFU / ml) Adhesion Rate (%) Strain administration Strain Attachment L. rhamnosus ATCC53103 2.81 x ^{10 9} 2.13 x ^{10 9} 75.8 L. plantarum LLP5193 3.30 x ^{10 9} 2.20 x ^{10 9} 66.7 L. plantarum LLP5273 2.94 x ^{10 9} 2.21 x ^{10 9} 75.2 LB-9 Complex Lactic Acid Bacteria 1.31 x ^{10 9} 9.30 x ^{10 8} 71.0

As a result of measuring the intestinal cell adhesion rate to three kinds of lactic acid bacteria, L. rhamnosus ATCC53103, L. plantarum LLP5193 and LLP5273, no significant difference was found among the three species, but only LLP5193 showed slightly lower adhesion rate than other lactic acid bacteria. Seemed.

In Example 2, the final in vivo transfer rate was predicted as shown in FIG.

As shown in Figure 1, the final in vivo transfer rate of the three strains and LB-9 complex lactic acid bacteria were predicted to be 38.6% in ATCC53103, 60.2% in LLP5193, 56.8% in LLP5273 and 60.2% in LB-9.

In particular, ATCC53103 had the highest survival rate in gastric juice but the lowest in vivo delivery rate due to low survival rate in artificial intestinal fluid. On the other hand, LLP5193 and LLP5273 showed delivery rates of 56.8% to 60.2%.

Example 4 Intestinal Reach Observation of Elbinin (LB-9) Complex Lactic Acid Bacteria

Fluorescence images were taken to visually confirm that LB-9 lactic acid bacteria actually reached the intestines. LB-9 complex lactic acid bacteria were cultured and mixed in MRS broth, and then Rhodamine 123 (Sigma-Aldrich, USA), a membrane permeable fluorescent dye solution, was administered at a concentration of 0.05 μg / ml and allowed to react for 1 hour. After the reaction, the culture solution was administered to 12-week-old rats, and 4 hours after the administration, LB-9 lactic acid bacteria were delivered to the inside of the rats by fluorescence microscopy.

The observation results are shown in FIG. 2, photographed before and after laparotomy and visualized by LB-9 lactobacillus concentrations distributed in the intestine, and the higher the concentration, the darker red color. As shown in FIG. 2, LB-9 lactic acid bacteria of high concentration were seen to be delivered to the body both before and after dissection. Particularly, in the case of fluorescence image after dissection, it was confirmed that it was evenly distributed in the intestinal tissue of the rat.

Example 5-Confirmation of Fermentation of Milk Added LLP5193, LLP5273, and LB-9 Complex Lactic Acid Bacteria

If lactic acid bacteria are to be provided as a milk product, the fermentation process should not occur according to the addition of lactic acid bacteria. If the fermentation process occurs, a problem arises that a separate process must be added because it must remain milk. Therefore, it was confirmed whether curd was formed or not when the lactic acid bacteria strain of the present invention was added to milk.

LLP5193, LLP5273 and LR-9 strains of the present invention were identified as curd formation when added to milk. The strain was added to 750 ml of milk produced from Lotte Food Pasteur in an amount of 10 ^ 7 CFU / ml, and stored at 10 ° C. to confirm curd formation for 21 days. As a control, the strain as shown in Table 4 below was used as a control to determine whether curd was formed in the same manner as above.

Experimental results were as shown in Table 4 below.

Table 4 shows the presence of curd according to storage period. (O: Curd is formed, X: Curd is not formed, 10 ° C storage)

Strain Retention period (days) 0 2 3 5 7 14 21 L. plantarum LLP5193 X X X X X X X L. plantarum LLP5273 X X X X X X X LB-9 (LLP5193 + 5273) X X X X X X X L. rhamnosus ATCC53103 X X X O O O O S. thermophilus MG510 X X O O O O O L. acidophilus ATCC314 X X X O O O O

As shown in Table 4, the strain of the present invention did not form a curd when added to the milk, it was confirmed that the excellent lactic acid bacteria to provide a milk product added.

Example 6 Preparation of Milk and Fermented Milk Added LB-9 Complex Lactic Acid Bacteria

In the case of milk containing LB-9 complex lactic acid bacteria, LB-9 complex lactic acid bacteria was added to 750 ml of milk produced from Lotte Food Pasteur with 1 billion CFU / 750 ml of viable bacteria per ml or more than 1.3x10 ^ 6 CFU / ml. Prepared.

In the case of LB-9 complex lactic acid bacteria fermented milk, fermented milk was first made using a starter for producing fermented milk, and then LB-9 complex lactic acid bacteria powder was administered. That is, fermented milk was prepared by adding 0.01 wt% of Christian Hansen ABT-5 to milk weight and fermenting at 42 ° C. for 8 hours. The final pH was 3.9 and the number of lactic acid bacteria was 3.5x10 ^ 8 CFU / ml. Confirmed. LB-9 composite lactobacillus powder was added to the prepared fermented milk by 0.2% by weight, followed by mixing to complete LB-9 complex lactic acid bacteria fermented milk.

Example 7 Intestinal Health and Immune Function Verification Using a Colitis Induction Model

In order to verify the colitis functionality of LB-9 complex lactic acid bacteria and the product added to the lactic acid bacteria, the experimental group was configured as shown in Table 4 according to the experimental process as shown in FIG.

Experimental animals were carried out 12 animals for each experimental group, and after the initial experimental group was organized for about 1 week, the same age, weight, disease symptoms and so on. During the first week, only PBS and LB-9 lactobacillus samples were administered, and from day 8, DSS (Dextran sulfate sodium) was mixed with drinking water to 3% and then artificially induced colitis. After 14 days of feeding, stool status and post-dissection histological examination were used to compare the degree of colitis symptoms between the experimental groups. In particular, colonic length measurement, histological examination, inflammatory cell infiltration and apoptosis were confirmed.

Experimental group Dosing Sample Colitis Treatment Sample General control X PBs colitis Control Dextran sulfate sodium 3%
(Drinking water mixed salary) PBS Plain milk Lactobacillus LB-9 Lactobacillus Powder
(1.0 x 10 ⁹ CFU / 1 day) LB-9 Milk
(1.0 x 10 ⁶ CFU / 1 day) LB-9 Fermented Milk (1.0 x 10 ⁶ CFU / day)

(1) weight loss after colitis induction

The body weight of the experimental animals was measured once daily after colitis induction by 3% DSS, and the weight loss of the measurement period is shown in FIG. 4.

As shown in Figure 4, the weight control did not occur in the control group treated nothing, it was confirmed that the weight gain of about 0.5 to 4% daily during the experiment.

On the other hand, when only DSS was treated, body weight was lost after 5 days, and on day 6, weight loss was more than 9%. In addition, the weight loss of about 5% was confirmed on the 6th day, although it was somewhat lower than the DSS-treated and non-DSS-induced group. This result is similar to previous studies that can prevent some colitis with normal milk alone.

LB-9 lactobacillus products, namely powder, milk, and fermented milk, showed a lower weight loss than the experimental group containing only DSS and the experimental group containing only milk, with a weight loss amount of about 2 to 3% on the 6th. Although high, there was no significant difference.

(2) colon length comparison

After 7 days of induction of colitis, experimental animals were dissected to measure intestinal length. In general, the longer the intestine is formed, the more favorable it is for digestion and absorption.

The comparison of the length of the intestines of the control group and the experimental group administered with LB-9 lactic acid bacteria is shown in FIG. As shown in the results, the control group, that is, the longest intestinal length was formed in the experimental group that did not induce colitis, and no significant difference was found in the other five experimental groups. However, when comparing only the trend, the control group which received only DSS was the shortest, followed by the length of the intestine in the order of control-milk, LB-9 powder, LB-9 milk and Lb-9 fermented milk.

(3) histological observation

After the end of the feeding experiment, the experimental animals were dissected to prepare sections of the colon tissue, and then the structural changes in the intestines were shown in FIG. 6.

In the case of the control group, there are not many empty spaces and the internal villi are well formed in a dense manner, whereas in the control DSS, the villi are destroyed and the internal space is also empty. On the other hand, LB-9 lactobacillus and LB-9 fermented milk maintained similar levels as the control group, and in particular, LB-9 fermented milk showed better tissue morphology.

(4) inflammatory cell infiltration

Leukocyte and macrophage data were used to determine the extent of inflammatory cell invasion following DSS administration and colitis induction. Ly6 and CD11b were measured as macrophage markers, and fluorescence staining using DAPI was used to compare the degree of luminescence stained by fluorescence microscopy.

The measurement results are shown in FIGS. 7 and 8, and in the case of FIG. 7, when ingested LB-9 milk, white blood cell infiltration was less likely to occur than other experimental groups. FIG. 8 shows the results obtained by staining CD11b and confirmed the least macrophage invasion in LB-9 powder and LB-9 fermented milk.

(5) cell death mechanism

DNA debris (Terminal deoxy-nucleotidyl transferase dUTP nick end labeling, TUNEL) analysis to determine the degree of cell death is shown in Figure 9 shown.

As shown in the results of Figure 9, it was confirmed that significantly less apoptosis in LB-9 lactobacillus powder and LB-9 milk, LB-9 fermented milk was also confirmed that a slightly lower level of cell death compared to the DSS control group It became.

Based on the results of FIG. 9, cell death mechanisms were determined, and expression levels of proteins involved in cell death mechanisms were measured through Western blot.

10 is a result of measuring the amount of expression for each type of protein involved in cell death through Western blot, Figure 11 is an objective numerical representation of the protein measured in FIG.

As shown in Figures 10 and 11, it was confirmed that the expression of p-ERK in the experimental group ingested LB-9 lactic acid bacteria and LB-9 milk. -IkB, It is believed that by reducing the expression of p-ERK involved in the inflammatory cytokine pathway leading to p-p65, consequently induces p-p65 reduction. Reduced p-p65 may eventually reduce cell death by decreasing FAS / FADD or Bax, Bcl-2.

As a result, treatment with DSS is thought to increase the induction of p-IKK or IkB degradation in intestinal epithelial cells, eventually leading to capsase-3, resulting in increased cell death, but LB-9 lactobacillus or LB-9 milk is involved. Inhibition of p-ERK expression involved in the pathway eventually appears to inhibit cell death.

Korea Microbial Conservation Center (overseas) KCCM11598P 20141103 Korea Microbial Conservation Center (overseas) KCCM11696P 20150507

Claims (10)

LB-9 complex lactic acid bacteria as an active ingredient,
The LB-9 complex lactic acid bacteria include Lactobacillus plantarum LLP5193 strain and Lactobacillus plantarum LLP5273 strain, intestinal inflammation prevention or reduction milk.
The method of claim 1,
The Lactobacillus plantarum LLP5193 strain is a strain having a deposit number of KCCM11598P,
The Lactobacillus plantarum (Lactobacillus plantarum) LLP5273 strain is a strain having a deposit number of KCCM11696P, intestinal inflammation prevention or reduction milk.
The method of claim 1,
The LB-9 complex lactic acid bacteria is LLP5193 and LLP5273 strains of the number of viable bacteria 10: 1 to 1:10, intestinal inflammation prevention or reduction milk.
The method of claim 1,
The intestinal inflammation prevention or reduction milk contains LB-9 complex lactic acid bacteria 1.0x10 ^ 6 CFU / ml or more with respect to the total volume of milk (ml), enteric inflammation prevention or reduction milk.
LB-9 complex lactic acid bacteria as an active ingredient,
The LB-9 complex lactic acid bacteria include Lactobacillus plantarum LLP5193 strain and Lactobacillus plantarum LLP5273 strain, intestinal inflammation prevention or reduction fermented milk.
The method of claim 5,
The Lactobacillus plantarum LLP5193 strain is a strain having a deposit number of KCCM11598P,
The Lactobacillus plantarum LLP5273 strain is a strain having a deposit number of KCCM11696P, fermented milk for preventing or reducing intestinal inflammation.
The method of claim 5,
The fermented milk is LB-9 complex lactic acid bacteria powder containing 0.1% by weight or more based on the total weight of fermented milk, fermented milk for preventing or reducing intestinal inflammation.
In the manufacturing method of the fermented milk for preventing or alleviating intestinal inflammation of any one of Claims 5-7,
Adding starter bacteria for the production of fermented milk to milk;
Fermenting the milk to which the starter bacteria have been added; And
LB-9 complex lactic acid bacteria are added to the fermented milk and mixed.
The method of claim 8,
The fermentation step is to ferment for 4 hours to 12 hours at a temperature condition of 40 ℃ to 45 ℃, the method of producing fermented milk for preventing or reducing intestinal inflammation.
The method of claim 9,
The fermentation step is a final pH is 3.5 to 4.5, and the fermentation until the starter bacteria number is 1.0x10 ^ 8 CFU / ml or more, the method for producing fermented milk for preventing or reducing intestinal inflammation.

Images