WO2018111023A1 - Lactic acid bacteria having increased stability, and method for producing same - Google Patents

Abstract

The present invention relates to lactic acid bacteria having increased stability, and a method for producing same, more specifically to lactic acid bacteria having enhanced storage and thermal stability, acid resistance, bile resistance, and stability against digestive enzymes due to a sequential triple-coating of xylitol, gelatin or collagen, and dextrin, and a method for producing the lactic acid bacteria. By sequentially triple-coating with xylitol, gelatin or collagen, and dextrin, the lactic acid bacteria produced by means of the coating method according to the present invention has, in comparison to conventional lactic acid bacteria with no coating, experimentally confirmed greater storage temperature and thermal stability, acid resistance, bile resistance, and stability against digestive enzymes, and thus resistance to external stresses, increased viability of the strain when passing through the gastrointestinal track, and the ability to maintain the original bioactivity in the intestine have been confirmed, and therefore the coating method according to the present invention is anticipated to be effectively applied in related industries using lactic acid bacteria, such as fermented milk, fermented food, functional food, general food, cosmetics and medicine.

Description

Lactic acid bacteria with enhanced stability and preparation method thereof

The present invention relates to a lactic acid bacterium having improved stability and a method for preparing the same, more specifically xylitol; Gelatin or collagen; And the present invention relates to a lactic acid bacterium and a method for producing the same which is enhanced by storage and thermal stability, acid resistance, bile resistance, and digestive enzymes by triple coating of dextrin sequentially.

Lactic acid bacteria, also known as lactic acid bacteria or lactic acid bacteria, are gram-positive bacteria that break down sugars such as glucose to produce lactic acid. They are found in the digestive tract, oral cavity, and vagina of humans and mammals. Lactic acid bacteria are one of the most widely used microorganisms for a long time, and do not produce harmful substances to the intestines of humans or animals, and are useful as a formal preparation by preventing abnormal fermentation by harmful bacteria in the intestines. In addition, the lactic acid produced by the lactic acid fermentation has been used to produce foods such as dairy products, kimchi, brewed foods, etc., which inhibit the growth of pathogens and harmful bacteria.

On the other hand, probiotics are living bacteria that enter the body and give a healthy effect. Most probiotics known to date are Lactobacillus sp . , Lactococcus sp . ), Streptococcus sp . , Leuconostoc sp . ), And O Phedi deulyimyeo lactic acid bacteria belonging to genus Lactococcus such as (Pedicoccus sp.), Includes a part of Bacillus (Bacillus) and the like. The function of lactic acid bacteria and probiotics has been studied for a long time since it was found by Ilya Mechinnikov that Bulgarians enjoy longevity due to the consumption of fermented milk fermented with Lactobacillus.

Clinically, probiotics are reported to be effective in preventing lactose intolerance and colon cancer, lowering cholesterol and blood pressure, improving immune function, preventing infection, improving mineral absorption, growing harmful bacteria due to stress, and improving irritable bowel syndrome and colitis. It has also been recommended as a treatment and antibiotic for strengthening the immune system and for treating intestinal diseases associated with candidiasis. In order to be recognized as probiotics, microorganisms including lactic acid bacteria must survive in gastric acid and bile acids, reach the small intestine, proliferate and settle in the intestine, exhibit useful effects in the intestine, be nontoxic and non-pathogenic.

Lactic acid bacteria food is made of powder, granules, tablets, capsules, etc. to cultivate live bacteria such as lactobacillus, lactobacillus, bifidus and other foods in a stable and easy way. Say that. Lactic acid bacteria production process as described above is largely divided into lactic acid bacteria culture, cell recovery, lyophilization, grinding, commercialization, etc. In this process, lactic acid bacteria are exposed to various physicochemical stresses. In other words, the cell recovery is affected by osmotic pressure due to concentration, and during freeze-drying process, ice crystals in the cytoplasm are formed due to rapid temperature change, or ice crystals and dehydration generated outside the cell affect the temperature and osmotic pressure. At the same time. In addition, the stability of lactic acid bacteria is reduced when exposed to high temperature, high pressure or hydrated by moisture in the air during grinding and commercialization, and liquid products such as lactic acid bacteria fermented foods, lactic acid bacteria fermented milk, lactic acid bacteria beverages stored for a short period of time, as well as long-term Even in products manufactured in powder form for the purpose of storage, it has been pointed out that when exposed to oxygen, the fatty acids constituting the cell membrane are oxidized to reduce the survival rate.

In addition, because probiotics products utilize the live bacteria themselves, they are exposed to various stresses in the human body before reaching the intestines after ingestion. For example, in the gastrointestinal tract, the survival rate may be greatly reduced by being exposed to an acidic environment falling below pH 3 and affected by digestive enzymes and bile acids secreted in the small intestine. Even when it reaches the intestine, it competes with the microorganisms that have settled in the intestine, and at the same time, the growth is inhibited by various harmful components and active oxygen.

Until recently, in order to solve the above problems, various studies have been made on the method of coating lactic acid bacteria. For example, there have been enteric coatings using capsules and microencapsulation processes using gelatin, sugars, gums, etc., but problems of using expensive coatings or adding processes have been pointed out.

In order to solve the above problems, a high concentration of lactic acid bacteria is introduced a method of producing a double-structured jelly or double coating with protein and polysaccharides, or direct reaction with air, water is suppressed and heat resistance, acid resistance and A method of preparing tri-coated lactic acid bacteria (BK Patent Application No. 10-2008-10397) with enhanced bile properties and enhanced viability and processing stability has been developed. However, the coating technique of the conventional lactic acid bacteria also can not completely coat the surface of the lactic acid bacteria, it is still pointed out that the lactic acid bacteria prepared by the above method is not sufficiently excellent in heat resistance, acid resistance and bile resistance.

Therefore, it is necessary to develop a coating method that can significantly improve storage stability, heat resistance, acid resistance, and bile resistance so that lactic acid bacteria can fully function in the body.

The present inventors earnestly researched to develop a method capable of improving the storage and lactic acid bacteria stability in the body, as a result, the lactic acid bacteria, preferably Lactobacillus pentosus strain Xylitol; Gelatin or collagen; And the result of the three-coating sequentially using the dextrin was confirmed that the storage stability, thermal stability, acid resistance, bile resistance, and the stability of the digestive enzymes of the strain is improved, the present invention was completed based on this.

Accordingly, an object of the present invention is to provide a method for producing lactic acid bacteria with improved stability.

It is another object of the present invention to provide a method for enhancing the stability of lactic acid bacteria.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a method for producing lactic acid bacteria with improved stability, including the following steps.

(a) adding a xylitol to the lactic acid bacteria and homogenizing the first coating; (b) adding a gelatin or collagen to the first coated lactic acid bacteria and homogenizing the second coating to homogenize; And (c) adding a dextrin to the second coated lactic acid bacterium and homogenizing the third coating.

In addition, the present invention provides a method for enhancing the stability of lactic acid bacteria, comprising the following steps.

(a) adding a xylitol to the lactic acid bacteria and homogenizing the first coating; (b) adding a gelatin or collagen to the first coated lactic acid bacteria and homogenizing the second coating to homogenize; And (c) adding a dextrin to the second coated lactic acid bacterium and homogenizing the third coating.

In one embodiment of the present invention, xylitol in the step (a) may be 1% to 30% concentration.

In another embodiment of the present invention, the gelatin or collagen in step (b) may be 1% to 30% concentration.

In another embodiment of the present invention, the dextrin in step (c) may be a concentration of 10% to 50%.

In another embodiment of the present invention, in the step (a), the xylitol is mixed with the primary coating in a ratio of 10 parts by weight to 100 parts by weight with respect to 100 parts by weight of lactic acid bacteria cells; In the step (b), the gelatin or collagen is mixed with a secondary coating in a ratio of 0.5 parts by weight to 50 parts by weight with respect to 100 parts by weight of lactic acid bacteria cells; And in the step (c) the dextrin may comprise a third coating by mixing in a ratio of 0.5 parts by weight to 50 parts by weight with respect to 100 parts by weight of lactic acid bacteria cells.

In another embodiment of the present invention, the lactic acid bacteria is Lactobacillus sp. , Genus Bifidobacterium ( Bifidobacterium) sp . ), Genus Streptococcus sp. , Genus Lactococcus sp . ) Enterococcus sp . ), Pediococcus sp . , Genus Leuconostoc sp . ), And Weissella sp. ) May be one or more strains selected from the group consisting of.

In another embodiment of the present invention, the lactic acid bacteria may be Lactobacillus pentosus .

Lactic acid bacteria prepared by the coating method according to the present invention is xylitol; Gelatin or collagen; And triple-coating sequentially with dextrin, to experimentally confirm that the storage temperature and heat stability, acid resistance, bile resistance, and digestive enzyme stability are improved compared to lactic acid bacteria which have not been conventionally coated, thereby resisting external stress. And it was confirmed that the viability of the strain when passing through the gastrointestinal tract and can maintain the original physiological activity function in the intestine, the coating method according to the present invention is fermented milk, fermented food, functional food, general food, cosmetics, and using lactic acid bacteria It is expected to be usefully used in related industries such as pharmaceuticals.

1 is a photograph of the final raw material after freeze-drying the Lactobacillus pentosus KF340 strain sequentially coated with xylitol, gelatin, and dextrin triple.

Figure 2 is a photograph of the final raw material after lyophilization of Lactobacillus pentosus KF340 strain sequentially coated with xylitol, collagen, and dextrin in threefold.

Figure 3 is a photograph taken with a scanning electron microscope of the Lactobacillus pentosus KF340 strain sequentially coated with xylitol, gelatin, and dextrin triple.

4 is a photograph taken with a scanning electron microscope of the Lactobacillus pentosus KF340 strain sequentially coated with xylitol, collagen, and dextrin in a triple.

The present inventors have studied diligently to develop a coating method that can enhance the storage and stability of the lactic acid bacteria in the body, and as a result, the storage and thermal stability of the strain, acid resistance, bile resistance, and lactic acid bacteria with enhanced stability to digestive enzymes The present invention has been completed by developing a coating method.

Therefore, the present invention (a) adding xylitol to the lactic acid bacteria and homogenizing the first coating; (b) adding a gelatin or collagen to the first coated lactic acid bacteria and homogenizing the second coating to homogenize; And (c) adding a dextrin (dextrin) to the secondary coated lactic acid bacteria and provides a method of producing a lactic acid bacteria with improved stability, comprising the step of homogeneous tertiary coating.

In addition, the present invention (a) adding xylitol to the lactic acid bacteria and homogenizing the first coating; (b) adding a gelatin or collagen to the first coated lactic acid bacteria and homogenizing the second coating to homogenize; And (c) adding a dextrin to the secondary coated lactic acid bacteria and homogenizing the third coating to provide a method for enhancing stability of the lactic acid bacteria.

Hereinafter, the present invention will be described in detail step by step.

The process of culturing the lactic acid bacteria before performing the lactic acid bacteria coating according to the production method, the lactic acid bacteria culture can be carried out in conventional lactic acid culture medium and culture conditions according to the type of lactic acid bacteria to be used, and is not particularly limited.

In step (a), xylitol is dissolved in distilled water and may be in a concentration of 1% to 30%, preferably 1% to 15%, more preferably 3% to 7%, even more preferably 5%, and use. Pre-sterilized, cooled to room temperature and available for coating.

The xylitol is added to 10 parts by weight to 100 parts by weight, preferably 50 parts by weight to 100 parts by weight, more preferably 100 parts by weight, which is the same amount as that of recovered cells, and homogenized, and room temperature. The cells can be first coated by standing at room temperature for 0.5 to 3 hours, more preferably 1 hour to 2 hours.

In the step (b), gelatin may be dissolved in distilled water at a concentration of 1% to 30%, preferably 1% to 15%, more preferably 4% to 7%, even more preferably 6%, and used. Pre-sterilized, cooled to room temperature and available for coating.

The gelatin is added to 0.5 parts by weight to 50 parts by weight, preferably 5 parts by weight to 50 parts by weight, more preferably 10 parts by weight to 30 parts by weight, based on 100 parts by weight of the cells recovered after the culture, and homogenized at room temperature. The cells can be secondary coated by standing at room temperature for 0.5 to 3 hours, more preferably 1 to 2 hours.

In the step (b), the collagen may be dissolved in distilled water at a concentration of 1% to 30%, preferably 5% to 20%, more preferably 10% to 15%, even more preferably 12%, and used. Pre-sterilized, cooled to room temperature and available for coating.

The collagen is homogenized after adding 0.5 parts by weight to 50 parts by weight, preferably 5 parts by weight to 50 parts by weight, more preferably 10 parts by weight to 30 parts by weight, based on 100 parts by weight of the cells recovered after the culture. The cells can be secondary coated by standing at room temperature for 0.5 to 3 hours, more preferably 1 to 2 hours.

In the step (c), the dextrin may be dissolved in distilled water at a concentration of 10% to 50%, preferably 10% to 30%, more preferably 15% to 25%, even more preferably 20%, and used. Pre-sterilized, cooled to room temperature and available for coating.

The dextrin is homogenized after adding 0.5 parts by weight to 50 parts by weight, preferably 5 parts by weight to 50 parts by weight, more preferably 10 parts by weight to 30 parts by weight, based on 100 parts by weight of the cells recovered after the culture. The cells may be tertiary coated by standing at room temperature for 0.5 to 3 hours, more preferably 1 to 2 hours.

According to the present invention, it may include a process of lyophilizing the coated lactic acid bacteria after the lactic acid bacteria coating process as described above, it can be carried out by those skilled in the art according to a conventional method.

In the present invention, the stability is meant to include both in vitro stability and in vivo stability to lactic acid bacteria, more specifically, storage and heat resistance to lactic acid bacteria, bile acid, acid resistance, and digestive enzymes Stability, and stability in intestinal conditions.

In the embodiment of the present invention, the stability of the strain was evaluated in various aspects as compared to the case where the Lactobacillus pentosus strain was not coated after the triple coating by the above production method.

In one embodiment of the present invention, the viable cell count and viability were measured at two-week intervals while storing the lactic acid bacteria samples for 4 weeks at 4, 25, and 40 ° C. conditions to evaluate storage stability enhancement. As a result, as the storage temperature increases, the rate of decrease of the cells increases, and it was confirmed that the survival rate of the tri-coated lactic acid bacteria was significantly increased as compared with the case without the coating treatment (see Example 3).

In another embodiment of the present invention, in order to evaluate the acid resistance of the lactic acid bacteria, the lactic acid bacteria samples were treated with artificial gastric juice of pH 2.0 and 2.5, respectively, and reacted for 30 minutes, 60 minutes, and 120 minutes, and then the viable cell count and survival rate. Measured. As a result, it was found that the survival rate was higher in the triple coated sample according to the present invention compared to the case where the coating was not performed (see Example 4).

In another embodiment of the present invention, to evaluate the bile resistance of the lactic acid bacteria to the lactic acid bacteria samples treated with 0.3% and 2.0% bile solution, respectively, and reacted for 30 minutes, 60 minutes, and 120 minutes As a result of measuring the number and survival rate, it was observed that the bile resistance significantly increased in the tri-coated lactic acid bacteria compared with the case without the coating treatment (see Example 5).

In another embodiment of the present invention, the lactic acid bacteria were treated with 0.2% pancreatin and 100 unit / mL of alpha-amylase, respectively, for 30 minutes, 60 minutes, and to evaluate the stability of the lactic acid bacteria for digestive enzymes. After reacting for 120 minutes, viable cell count and viability were measured. As a result, it was possible to confirm the high digestive enzyme stability of the Lactobacillus pentosus strain itself used in this example, it was confirmed that the stability of the digestive enzymes of the lactic acid bacteria by the triple coating treatment according to the present invention is more enhanced. (See Example 6).

In another embodiment of the present invention, to evaluate the heat stability of the lactic acid bacteria to the lactic acid bacteria samples were added sterilized saline at a temperature of 40 ℃, 50 ℃, and 70 ℃ respectively and 10 minutes and 30 minutes in a constant temperature bath The viable cell count and viability were measured after maintaining the temperature for a while. As a result, the survival rate of the tri-coated lactic acid bacteria was higher than that of the non-coated sample (see Example 7).

Through the results of the above examples, it is found that the triple coating method according to the present invention generally improves the storage stability, acid resistance, bile resistance, stability against digestive enzymes, and thermal stability according to storage temperature, compared to the non-coated lactic acid bacteria. Could.

Thus, the present invention provides a tri-coated lactic acid bacteria prepared by the above method.

In the present invention, the lactic acid bacteria are Lactobacillus bacteria (Lactobacillus sp.) Genus, Bifidobacterium (Bifidobacterium sp.), A Streptococcus (Streptococcus sp.) Genus Lactococcus (Lactococcus sp.) Genus Enterococcus (Enterococcus sp . ), Pediococcus sp . , Genus Leuconostoc sp . ), And Weissella sp. Genus may be one or more strains selected from the group consisting of, more preferably Lactobacillus pentosus ( Lactobacillus pentosus ), more preferably Lactobacillus pentosus KF340 (Accession number KCCM 11675P).

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example  One. Lactobacillus Pentosus  Culture of KF340 Strain

Lactobacillus pentosus KF340 strain was incubated with stirring for 12 ± 3 hours at 32 ℃, pH 6.0 conditions using 360 L modified-MRS medium. The cultured Lactobacillus pentosus KF340 cells were separated using a centrifuge, and 5.2 Kg of cells were recovered through this process.

Example  2. Lactobacillus Pentosus  KF340 strain Triple  Coating and Lyophilization

In order to improve the stability of the Lactobacillus pentosus KF340 strain cultured in accordance with the method of Example 1 to coat the strain using a triple coating method to evaluate the stability enhancement by various methods. Experimental group 1 is a triple coating strain using xylitol, gelatin, and dextrin, experimental group 2 is a triple coating strain using xylitol, collagen, and dextrin, and the control group is an uncoated strain.

Specific triple coating material composition and control composition according to the present invention is shown in Table 1 below.

Sample no. One 2 3 Coating Raw Material 5% Xylitol 5% Xylitol No treatment 6% gelatin 12% collagen 20% Dextrin 20% Dextrin

In order to evaluate the stability enhancing effect of the Lactobacillus pentosus KF340 according to the triple coating of the composition, Experimental Group 1 dissolved each raw material in distilled water so as to have a concentration of 5% xylitol, 6% gelatin, and 20% dextrin. Sterilized at 121 ° C. for 15 minutes and then cooled at room temperature. Next, the same amount of 5% xylitol aqueous solution was added to the recovered cells, and the cells were homogenized and allowed to stand for 1 hour to be first coated. 6% gelatin aqueous solution was added to 25% of the recovered cells, homogenized and left to rest for 1 hour, followed by secondary coating, and 20% dextrin aqueous solution was added to 25% of the recovered cells. After homogenization, the mixture was allowed to stand for 1 hour and subjected to tertiary coating. In Experiment 2, each raw material was dissolved in distilled water so as to have a concentration of 5% xylitol, 12% collagen, and 20% dextrin, sterilized at 121 ° C. for 15 minutes, and then cooled at room temperature. Next, the same amount of 5% xylitol aqueous solution was added to the recovered cells, and the cells were homogenized and allowed to stand for 1 hour to be first coated. After adding 12% collagen aqueous solution to 25% of the recovered cells and homogenizing the cells, the mixture was left to stand for 1 hour and then coated with 20% dextrin solution to 25% of the recovered cells. After homogenization, the mixture was allowed to stand for 1 hour and subjected to tertiary coating. In this case, the same cells without coating treatment were used as the control (sample 3) without any raw material.

Cells treated with the above materials were triple-coated or uncoated cells were lyophilized for about 66 hours under the following conditions. More specifically, pre-freezing was maintained at −38 ° C. for at least 5 hours, and lyophilization was performed at −38 ° C. and at 10 ° C., followed by final termination at 25 ° C. The final raw material photograph of Lactobacillus pentosus KF340 triple-coated and lyophilized according to the above method is shown in FIGS. 1 (Experimental Group 1) and 2 (Experimental Group 2).

 Then, the stability of the Lactobacillus pentosus KF340 strain in the samples was evaluated in various aspects.

Example  3. Triple  Coated Lactobacillus Pentosus  Storage Stability Evaluation of KF340 Strain

In order to evaluate the storage stability of the three-coated Lactobacillus pentosus KF340 with the raw material according to the present invention, the samples of Table 1 were prepared according to the method of Example 2, and then 4 weeks at 4, 25, and 40 ° C., respectively. The viable cell counts were measured at two week intervals during storage. In order to measure the number of viable cells, the cells stored at each storage temperature were suspended in sterile saline solution, aliquoted and smeared according to dilution ratio in MRS (Difco) solid medium, and then cultured for 48 hours at 37 ° C. to measure colony numbers. . The survival rate (%) is expressed as [(Log index value at Week 4 / Log index value at Week 0) X 100].

As a result of storing the samples at 4, 25, and 40 ° C., respectively, as shown in Tables 2 to 4, the control group without coating treatment was compared with the cells of Experimental groups 1 and 2, which were triple coated under all temperature conditions. Low survival rate was shown. In addition, as the storage temperature was increased tendency of the reduction rate of the cells showed a tendency, which is similar to the existing reports, the higher the storage temperature, the survival rate of the experimental group significantly increased compared to the control group treated nothing. Triple coating process according to it was confirmed that greatly improve the storage stability of the strain.

Experiment group 1 Experiment group 2 Control Week 0 4.6 X 10 ⁸ (CFU / mg) 2.8 X 10 ⁸ (CFU / mg) 1.2 X 10 ⁸ (CFU / mg) 2 weeks 2.0 X 10 ⁸ (CFU / mg) 2.4 X 10 ⁸ (CFU / mg) 2.5 X 10 ⁷ (CFU / mg) 4 Weeks 2.0 X 10 ⁸ (CFU / mg) 2.2 X 10 ⁸ (CFU / mg) 3.1 X 10 ⁷ (CFU / mg) Survival Rate after Week 4 (%) 95.8 (%) 98.8 (%) 92.6 (%)

Experiment group 1 Experiment group 2 Control Week 0 4.6 X 10 ⁸ (CFU / mg) 2.8 X 10 ⁸ (CFU / mg) 1.2 X 10 ⁸ (CFU / mg) 2 weeks 8.3 X 10 ⁷ (CFU / mg) 9.3 X 10 ⁷ (CFU / mg) 1.3 X 10 ⁶ (CFU / mg) 4 Weeks 8.9 X 10 ⁷ (CFU / mg) 5.0 X 10 ⁷ (CFU / mg) 5.1 X 10 ⁶ (CFU / mg) Survival Rate after Week 4 (%) 91.8 (%) 91.2 (%) 82.9 (%)

Experiment group 1 Experiment group 2 Control Week 0 4.6 X 10 ⁸ (CFU / mg) 2.8 X 10 ⁸ (CFU / mg) 1.2 X 10 ⁸ (CFU / mg) 2 weeks 1.6 X 10 ⁷ (CFU / mg) 1.5 X 10 ⁷ (CFU / mg) 1.7 X 10 ³ (CFU / mg) 4 Weeks 4.5 X 10 ⁶ (CFU / mg) 1.2 X 10 ⁷ (CFU / mg) 4.5 X 10 ³ (CFU / mg) Survival Rate after Week 4 (%) 76.8 (%) 83.9 (%) 45.1 (%)

Example  4. Triple  Coated Lactobacillus Pentosus  KF340 strain Acid resistance  evaluation

In order to evaluate the acid resistance of the three-coated lactobacillus pentosus KF340 with the raw material according to the present invention, the samples of Table 1 were prepared according to the method of Example 2 and then added with artificial gastric juice of pH 2.0 and 2.5, respectively, for 30 minutes, After reacting for 60 minutes and 120 minutes, the solution was diluted 10-fold with sterile saline solution and dispensed and plated according to the dilution ratio in MRS (Difco) solid medium. Thereafter, the colonies were measured by incubating at 37 ° C. for 48 hours, and the survival rate (%) was expressed as [(Log index value after initial gastric fluid exposure / initial Log index value) × 100].

As a result, as shown in Table 5 and Table 6, the survival rate of the cells showed a tendency to decrease as the time of exposure to gastric juice (pH solution) increased, and coating treatment was performed at both pH 2.0 and pH 2.5 conditions. Compared with the control group did not show a high survival rate in the three-coated experiment groups 1 and 2. In addition, it was confirmed that the triple coating method significantly increased acid resistance compared to the case where no treatment was observed when maintaining a survival rate of about 95% or more in an acidic environment of 98% or more and pH 2.0 in a pH 2.5 condition.

Experiment group 1 Experiment group 2 Control 0 min 2.0 X 10 ⁸ (CFU / mg) 2.1 X 10 ⁸ (CFU / mg) 1.7 X 10 ⁷ (CFU / mg) 30 minutes 2.8 X 10 ⁸ (CFU / mg) 9.4 X 10 ⁷ (CFU / mg) 2.1 X 10 ⁷ (CFU / mg) 60 minutes 1.0 X 10 ⁸ (CFU / mg) 1.1 X 10 ⁸ (CFU / mg) 6.1 X 10 ⁶ (CFU / mg) 120 minutes 7.9 X 10 ⁷ (CFU / mg) 7.4 X 10 ⁷ (CFU / mg) 4.8 X 10 ⁶ (CFU / mg) Survival rate after 120 minutes (%) 95.0 (%) 94.5 (%) 92.5 (%)

Experiment group 1 Experiment group 2 Control 0 min 2.0 X 10 ⁸ (CFU / mg) 2.1 X 10 ⁸ (CFU / mg) 1.4 X 10 ⁷ (CFU / mg) 30 minutes 2.3 X 10 ⁸ (CFU / mg) 1.7 X 10 ⁸ (CFU / mg) 1.4 X 10 ⁷ (CFU / mg) 60 minutes 2.2 X 10 ⁸ (CFU / mg) 1.7 X 10 ⁸ (CFU / mg) 1.5 X 10 ⁷ (CFU / mg) 120 minutes 1.6 X 10 ⁸ (CFU / mg) 1.6 X 10 ⁸ (CFU / mg) 6.7 X 10 ⁶ (CFU / mg) Survival rate after 120 minutes (%) 98.8 (%) 98.4 (%) 94.4 (%)

Example  5. Triple  Coated Lactobacillus Pentosus  KF340 strain Bile resistance  evaluation

In order to evaluate the bile resistance of Lactobacillus pentosus KF340 three-coated with the raw material according to the present invention, the samples of Table 1 were prepared according to the method of Example 2 and then bile solution (Bile extract 0.3%, 2.0%) The reaction was carried out for 30 minutes, 60 minutes and 120 minutes, and then diluted 10-fold with sterile saline, and then dispensed and plated according to the dilution ratio in MRS (Difco) solid medium. After culturing at 37 ° C. for 48 hours to determine colony numbers, the survival rate (%) was expressed as [(Log index value after initial bile acid exposure / initial Log index value) × 100].

As a result of the reaction with 0.3% and 2.0% bile solution, as shown in Tables 7 and 8, the viability of the cells showed a tendency to decrease as the concentration and exposure time of the bile solution increased. Tri-coated groups 1 and 2 showed relatively high survival rates when compared to untreated controls. The results showed that the triple coating method significantly increased the bile resistance compared to the case where nothing was treated.

Experiment group 1 Experiment group 2 Control 0 min 1.9 X 10 ⁸ (CFU / mg) 1.4 X 10 ⁸ (CFU / mg) 3.1 X 10 ⁷ (CFU / mg) 30 minutes 2.9 X 10 ⁷ (CFU / mg) 3.6 X 10 ⁷ (CFU / mg) 3.0 X 10 ⁶ (CFU / mg) 60 minutes 3.3 X 10 ⁷ (CFU / mg) 3.8 X 10 ⁷ (CFU / mg) 5.8 X 10 ⁶ (CFU / mg) 120 minutes 6.3 X 10 ⁷ (CFU / mg) 3.7 X 10 ⁷ (CFU / mg) 2.6 X 10 ⁶ (CFU / mg) Survival rate after 120 minutes (%) 94.3 (%) 92.8 (%) 85.7 (%)

Experiment group 1 Experiment group 2 Control 0 min 1.9 X 10 ⁸ (CFU / mg) 1.4 X 10 ⁸ (CFU / mg) 3.1 X 10 ⁷ (CFU / mg) 30 minutes 2.3 X 10 ⁷ (CFU / mg) 2.7 X 10 ⁷ (CFU / mg) 4.0 X 10 ⁶ (CFU / mg) 60 minutes 2.6 X 10 ⁷ (CFU / mg) 3.1 X 10 ⁷ (CFU / mg) 2.4 X 10 ⁶ (CFU / mg) 120 minutes 4.1 X 10 ⁷ (CFU / mg) 5.0 X 10 ⁷ (CFU / mg) 3.3 X 10 ⁶ (CFU / mg) Survival rate after 120 minutes (%) 92.0 (%) 94.4 (%) 87.1 (%)

Example  6. Triple  Coated Lactobacillus Pentosus  Evaluation of Stability of Digestive Enzymes of KF340 Strains

In order to evaluate the stability of the three-coated lactobacillus pentosus KF340 with the raw material according to the invention for the digestive enzymes, the samples of Table 1 were prepared according to the method of Example 2 after digestion enzyme solution, that is, 0.2% pancreatin (pancreatin) and 100 unit / mL of alpha-amylase were added, respectively, and reacted for 30 minutes, 60 minutes, and 120 minutes, and then diluted 10-fold with sterile saline solution to dilute the MRS (Difco) solid medium. Dispensed and smeared accordingly. Colonies were measured after incubation at 37 ° C. for 48 hours. The survival rate (%) was expressed as [(Log index value / initial Log index value after digestive enzyme solution exposure) × 100].

As a result of reacting the samples with pancreatin and amylase, respectively, as shown in Tables 9 and 10, the survival rates of the cells were high in samples 1 to 3, and 95 even in the control group without coating treatment. Survival rate of more than% was confirmed that the Lactobacillus pentosus KF340 strain itself was very resistant to pancreatin and amylase. However, the tri-coating treatment improved the stability of the Lactobacillus pentosus KF340 strain against digestive enzymes in that the survival rate of the triple coated groups 1 and 2 was maintained at about 100% than the uncoated control group. there was.

Experiment group 1 Experiment group 2 Control 0 min 2.2 X 10 ⁸ (CFU / mg) 1.2 X 10 ⁸ (CFU / mg) 2.8 X 10 ⁷ (CFU / mg) 30 minutes 3.0 X 10 ⁸ (CFU / mg) 2.2 X 10 ⁸ (CFU / mg) 2.7 X 10 ⁷ (CFU / mg) 60 minutes 3.5 X 10 ⁸ (CFU / mg) 2.4 X 10 ⁸ (CFU / mg) 3.2 X 10 ⁷ (CFU / mg) 120 minutes 2.4 X 10 ⁸ (CFU / mg) 2.0 X 10 ⁸ (CFU / mg) 1.1 X 10 ⁷ (CFU / mg) Survival rate after 120 minutes (%) 100.4 (%) 102.6 (%) 94.8 (%)

Experiment group 1 Experiment group 2 Control 0 min 1.5 X 10 ⁸ (CFU / mg) 1.5 X 10 ⁸ (CFU / mg) 2.2 X 10 ⁷ (CFU / mg) 30 minutes 3.3 X 10 ⁸ (CFU / mg) 2.4 X 10 ⁸ (CFU / mg) 1.5 X 10 ⁷ (CFU / mg) 60 minutes 3.6 X 10 ⁸ (CFU / mg) 2.5 X 10 ⁸ (CFU / mg) 2.1 X 10 ⁷ (CFU / mg) 120 minutes 2.9 X 10 ⁸ (CFU / mg) 3.1 X 10 ⁸ (CFU / mg) 1.5 X 10 ⁷ (CFU / mg) Survival rate after 120 minutes (%) 103.4 (%) 104.1 (%) 97.9 (%)

Example  7. Triple  Coated Lactobacillus Pentosus  KF340 strain Thermal stability  evaluation

In order to evaluate the heat stability of the three-coated lactobacillus pentosus KF340 with the raw material according to the invention, the samples of Table 1 were prepared according to the method of Example 2 and then the temperature at 40 ℃, 50 ℃, and 70 ℃ Sterile saline solution was added to each sample and maintained at the same temperature for 10 minutes and 30 minutes in a constant temperature bath, and then diluted 10 times with sterile saline solution and dispensed and plated according to the dilution ratio in MRS (Difco) solid medium. After 48 hours of incubation at 37 ℃ colony number was measured, the survival rate (%) is expressed as [(Log index value / initial Log index value after heat treatment) X 100].

As a result of evaluation of thermal stability at 40 ° C., 50 ° C., and 70 ° C., respectively, as shown in Tables 11 to 13 below, at 40 ° C., at least 96% of the control group not coated with the test group was coated in triplicate 1 And the high thermal stability of Lactobacillus pentosus KF340 at 40 ° C. in that the survival rate of 2 and 2 was maintained at about 100%. The stability of the LPKF340 strain against heat was improved by the fact that the uncoated control group maintained the survival rate of about 85% at 50 ° C, whereas the experimental groups 1 and 2, which were triple coated, improved the survival rate by 90% or more. It can be seen that to improve more. The difference in thermal stability by coating treatment was more prominent at 70 ℃ high temperature condition, whereas the control group without coating was not identified after living treatment for 30 minutes. The experimental group 2, which is greatly improved to the above and triple-coated, has great significance in that the cell survival rate is improved to 20% or more. Therefore, in view of the previous results, the triple-coated sample of Lactobacillus pentosus KF340 protects the strain from the external environment, thereby increasing the storage stability, acid resistance, bile resistance, stability of digestive enzymes, and thermal stability according to storage temperature. It was found to improve.

Experiment group 1 Experiment group 2 Control 0 min 1.4 X 10 ⁸ (CFU / mg) 1.4 X 10 ⁸ (CFU / mg) 1.9 X 10 ⁷ (CFU / mg) 10 minutes 2.4 X 10 ⁸ (CFU / mg) 2.1 X 10 ⁸ (CFU / mg) 1.2 X 10 ⁷ (CFU / mg) 30 minutes 2.2 X 10 ⁸ (CFU / mg) 1.7 X 10 ⁸ (CFU / mg) 1.0 X 10 ⁷ (CFU / mg) Survival rate after 30 minutes (%) 102.4 (%) 101.2 (%) 96.4 (%)

Experiment group 1 Experiment group 2 Control 0 min 1.4 X 10 ⁸ (CFU / mg) 1.4 X 10 ⁸ (CFU / mg) 1.9 X 10 ⁷ (CFU / mg) 10 minutes 4.8 X 10 ⁷ (CFU / mg) 4.2 X 10 ⁷ (CFU / mg) 3.6 X 10 ⁶ (CFU / mg) 30 minutes 2.8 X 10 ⁷ (CFU / mg) 2.2 X 10 ⁷ (CFU / mg) 1.6 X 10 ⁶ (CFU / mg) Survival rate after 30 minutes (%) 91.4 (%) 90.3 (%) 85.1 (%)

Experiment group 1 Experiment group 2 Control 0 min 2.1 X 10 ⁸ (CFU / mg) 2.1 X 10 ⁸ (CFU / mg) 1.1 X 10 ⁷ (CFU / mg) 10 minutes 4.3 X 10 ⁴ (CFU / mg) 9.0 X 10 ⁴ (CFU / mg) 7.3 X 10 ³ (CFU / mg) 30 minutes 5.5 X 10 ² (CFU / mg) 5.0 X 10 ¹ (CFU / mg) - Survival rate after 30 minutes (%) 33.0 (%) 20.4 (%) 0.0 (%)

The above description of the present invention is intended for illustration, and those of ordinary skill in the art can understand that the present invention can be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

The present invention is xylitol; Gelatin or collagen; And by sequentially coating the lactic acid bacteria with dextrin in triplicate, it was experimentally confirmed that the stability to storage temperature and heat, acid resistance, bile resistance, and digestive enzymes were improved more than lactic acid bacteria which were not conventionally coated. Thus, the present invention can be maintained in the intestinal physiological activity through the gastrointestinal tract without killing the lactic acid bacteria enhanced stability by triple coating, fermented milk, fermented foods, functional foods, general foods, cosmetics, and pharmaceuticals It is expected to be usefully used in related industries.

Claims (14)

A method for preparing lactic acid bacteria having enhanced stability, comprising the following steps: (a) adding a xylitol to the lactic acid bacteria and homogenizing the first coating; (b) adding a gelatin or collagen to the first coated lactic acid bacteria and homogenizing the second coating to homogenize; And (c) adding a dextrin to the second coated lactic acid bacteria and homogenizing the third coating. The method of claim 1, In the step (a) xylitol is characterized in that the concentration of 1% to 30%, the manufacturing method. The method of claim 1, Gelatin or collagen in the step (b) is characterized in that the concentration of 1% to 30%. The method of claim 1, Dextrin in step (c) is characterized in that the concentration of 10% to 50%, the preparation method. The method of claim 1, The lactic acid bacteria are Lactobacillus sp . , Bifidobacterium sp . , Streptococcus sp . , Lactococcus sp . , Enterococcus sp . ), Pediococcus sp . , Genus Leuconostoc sp .) genus, and Bisella ( Weissella sp. ) genus, characterized in that at least one strain selected from the group consisting of. The method of claim 1, The lactic acid bacteria is characterized in that Lactobacillus pentosus ( Lactobacillus pentosus ), the production method. The method of claim 1, In the step (a), the xylitol is mixed with a primary coating of 10 parts by weight to 100 parts by weight with respect to 100 parts by weight of lactic acid bacteria cells; In the step (b), the gelatin or collagen is mixed with a secondary coating in a ratio of 0.5 parts by weight to 50 parts by weight with respect to 100 parts by weight of lactic acid bacteria cells; And In the step (c), the dextrin is mixed with a ratio of 0.5 parts by weight to 50 parts by weight with respect to 100 parts by weight of lactic acid bacteria cells, characterized in that it comprises the step of tertiary coating. Method for improving the stability of lactic acid bacteria, comprising the following steps: (a) adding a xylitol to the lactic acid bacteria and homogenizing the first coating; (b) adding a gelatin or collagen to the first coated lactic acid bacteria and homogenizing the second coating to homogenize; And (c) adding a dextrin to the second coated lactic acid bacteria and homogenizing the third coating. The method of claim 8, In the step (a) xylitol is characterized in that the concentration of 1% to 30%, the enhancement method. The method of claim 8, Gelatin or collagen in the step (b) is characterized in that the concentration of 1% to 30%, the enhancement method. The method of claim 8, Dextrin in step (c) is characterized in that the concentration of 10% to 50%, the enhancement method. The method of claim 8, The lactic acid bacteria are Lactobacillus sp . , Bifidobacterium sp . , Streptococcus sp . , Lactococcus sp . , Enterococcus sp . ), Pediococcus sp . , Genus Leuconostoc sp .) genus, and Weissella sp. genus, characterized in that at least one strain selected from the group consisting of. The method of claim 8, The lactic acid bacteria is Lactobacillus pentosus ( Lactobacillus pentosus ), characterized in that, the enhancement method. The method of claim 8, In the step (a), the xylitol is mixed with a primary coating of 10 parts by weight to 100 parts by weight with respect to 100 parts by weight of lactic acid bacteria cells; In the step (b), the gelatin or collagen is mixed with a secondary coating in a ratio of 0.5 parts by weight to 50 parts by weight with respect to 100 parts by weight of lactic acid bacteria cells; And In the step (c), the dextrin is characterized in that the mixing step of mixing 0.5 to 50 parts by weight to 100 parts by weight of the lactic acid bacteria cells, characterized in that the step of tertiary coating.

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