KR20070035154A - Probiotic lactic acid bacteria and composition comprising the same - Google Patents

Abstract

The present invention relates to a probiotic lactic acid bacteria and a probiotic composition comprising the same that can effectively inhibit various enteric pathogenic bacteria, Lactobacillus crispatus G19 strains of the present invention and Bifidobacte The Bifidobacterium boum R5 strain exhibits excellent antibacterial activity against various pathogenic bacteria, as well as excellent acid and bile resistance. Terium bow R5 shows excellent resistance to oxygen resistance and dry heat, and thus can be usefully used for the manufacture of feed, food, medicine and cosmetics.

Description

PROBIOTIC LACTIC ACID BACTERIA AND COMPOSITION COMPRISING THE SAME}

1 is a group of probiotic agents including L19 , S21, and B47, Lactobacillus rhamnosus GG, and Lactobacillus pentosus K34, a comparative strain, Lactobacillus pentosus K34. We confirmed the effect of killing the mouse against Salmonella typhimurium LT2 infection of test strains in the administration group and the single administration group.

FIG. 2 shows Salmonella of test strains for the Bifidobacterium longphil (BL) and Bifidobacterium thermophilum (BT) administration groups, and the non-administration group, the Bifido test strain R5, and the comparative strain Bifidobacterium longum (BL) and Bifidobacterium thermophilum (BT). We confirmed the effect of lethal suppression of mice following Typhimurium LT2 infection.

3 is a dendogram comparing the 16S rDNA sequences of the Lactobacillus crispatus G19 strain of the present invention with the sequences on the GenBank.

4 is a Bifidobacterium bom of the present invention ( Bifidobacterium boum ) is a dendogram comparing the 16S rDNA nucleotide sequence of the R5 strain with the nucleotide sequences on the gene bank.

The present invention relates to probiotic lactic acid bacteria that effectively inhibit various enteric pathogenic bacteria, and probiotic compositions comprising the same.

Lactobacillus is a microorganism that produces lactic acid that is beneficial to humans without producing harmful substances such as indole, sketol, phenol, amine and ammonia. It is present at the highest density in the intestine of animals, especially in the small intestine where digestive absorption occurs. It is established as an slime (Barnes, FM et al. , Amer. J. Clin. Nutr. , 33, 2426-2433, 1980; and Salanitro, JP et al. , Appl. Environ.Microbiol . , 33, 79-84, 1977).

Such lactic acid bacteria are typically Carnobacterium , Enterococcus genus (Enterococcus), Lactobacillus genus (Lactobacillus), Lactococcus genus (Lactococcus), flow Pocono stock in (Leuconostoc), Phedi O Rhodococcus genus (Pediococcus), Streptococcus genus (Streptococcus), tetra-gen Rhodococcus genus (Tetragenococcus ), Vagococcus and Weissella strains (Stiles, ME et al. , Int. J. Food Microbiol. , 36, 1-29, 1997), in addition to the stable distribution of Lactobacillus in the human intestine and abundant in breast-fed children, the genus Bifidus genus, an amorphous anaerobic bacterium known to function to prevent diarrhea during breastfeeding. ( Bifidobacterium ) strains and the like (Stark, PL and A. Lee., J. Med. Microbiol. 15: 189-203, 1982; Ballongue, J., In Lactic acid bacteria: Microbiology and functional aspects.Series: Food science and technology, Marcel Dekker, Inc., pp. 519-587, 1998), among which the Lactobacillus and Bifidus strains have excellent safety and are used in various studies (Havenaar, R. et al., In Probiotics: The scientific basis , Chapmann & Hall, 209-224, 1992).

In addition to lactic acid production ability, lactic acid bacteria have attracted attention in various pharmaceutical and food science fields because they exhibit various functions such as pathogenic bacteria inhibition, antimutagenicity, anticancer and antitumor activity, immune enhancement and cholesterol lowering (Sanders, ME, J. Nutr. , 130, 384S-390S, 2000; and Rolfe, RD, J. Nutr. , 130, 396S-402S, 2000). Recently, it is possible to develop probiotic formulations using excellent antimicrobial conditions such as low pH, organic acid, bacteriocin, carbon dioxide, ethanol, diacetyl, low molecular weight antimicrobial substance, low reduction potential, nutrient depletion and dominance formed with the growth of lactic acid bacteria. (Sanders, ME et al., In Biotechnology and Food Ingredient , Van Nostrand Reinhold., 433-459, 1991; and Adams, MR et al . , Food Control. , 8, 227-239, 1997). In addition, the amount of supplementary probiotics added to health foods such as fermented milk or livestock feed using lactic acid bacteria as a probiotic formulation is gradually increasing.

Probiotic lactobacillus requires the following characteristics, specifically, it should be advantageous to separate from the host to be applied in relation to intestinal dominant competition and fixation, and to endure various substances secreted from the intestinal digestive organs after ingestion. It should be excellent in intestinal viability by showing acid resistance and bile resistance, and in addition to low pH, nutrient consumption, potential difference, hydrogen peroxide production and antimicrobial production as well as intestinal adhesion, the first stage of colonization. For example, bacteriocin) must be excellent in inhibiting pathogenic bacteria, and must be resistant to environmental stresses such as temperature and humidity while producing probiotic formulations, and excellent in oxygen resistance and shelf life stability (Salminen, S., etc.). , In Lactic acid bacteria: Microbiology and functional aspects.Series : Food science and technology , Marcel Dekker Inc., 211-253 , 1998).

The antimicrobial effects of probiotic agents on pathogenic microorganisms can be explained by several mechanisms, such as the production of inhibitors that can kill pathogens (Vanderberg, PA, FEMS Microbiol. Rev. , 12, 221). 238, 1993), nutrient competition with pathogens in settlements (Bernet, MF et al . , Gut. , 35, 483-489, 1994; Majamaa, H. et al . , J. Pediatr. Gastroenterol. Nutr. , 20, 333- 338, 1995; and Sato, K., et al ., Microbiol. Immunol ., 32, 1189-1200, 1988), enhancing host immunity (Perdigon, ME et al., Immunology , 63, 17-23, 1988; Hatcher, GE et al., J. Dairy Sci. , 76, 2485-2492, 1993; And Paubert, B. et al. , Int. J. Immunother. , 11, 153-161, 1995), and inhibition of bacterial toxin production and activity (Czerucka, D., et al ., Gastroenterol , 106, 65-72, 1994; Brandzo, RL et al ., Appl. Environ.Microbiol . 64: 564-568, 1998).

Meanwhile, the common infectious bacteria include Salmonella spp., Staphylococcus aureus , Enterotoxigenic E. coli , Listeria monocytogenes , and Campylobacter, which cause food poisoning. Campylobacter jejuni and Clostridium perfringens have been used, and antibiotics have been used so far to suppress the common infectious bacteria.

However, the use of these antibiotics has a strict regulation on the amount used due to problems such as resistance and retention, and also destroys the beneficial flora in the intestine, thus losing the preventive effect against the pathogenic bacterial infection caused by the normal flora. Therefore, probiotic preparations using beneficial bacteria, such as lactic acid bacteria, can inhibit the pathogenic bacteria and at the same time reduce the amount of antibiotics exhibiting various problems, and can be useful in the production of livestock products and foods because they are safe for human body.

Accordingly, the present inventors have studied the lactic acid bacteria strains that can be developed as probiotic preparations that are excellent in the antibacterial effect against various intestinal pathogenic bacteria and beneficial to the human body, and thus, Lactobacillus crispatus G19 and Bifidobacterium bom R5 strains are various. The antimicrobial effect of the common intestinal pathogenic bacteria, but also excellent in acid resistance and bile resistance, and when used in combination of these two strains were confirmed to exhibit a better lethal inhibitory effect than the case of each single bacteria to complete the present invention It was.

An object of the present invention is to provide a probiotic lactic acid bacteria excellent in acid resistance and bile resistance while excellent in ability to inhibit various enteric pathogenic bacteria.

Another object of the present invention is to provide a probiotic composition containing the probiotic lactic acid bacteria or its culture as an active ingredient.

In accordance with one aspect, the present invention provides a novel Lactobacillus Cri Spa tooth (Lactobacillus crispatus) G19 strain (KCCM 10668P) and Bifidobacterium boum (Bifidobacterium boum) R5 strain (KCCM 10669P).

According to another object, the present invention provides a probiotic composition comprising the Lactobacillus crispatus G19 strain, Bifidobacterium bom R5 strain, a mixed strain of the two, or a culture thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

The two strains of the present invention are Raffinose-containing Albi medium (Raffinose-Bifidobacterium; Hartemink, R. et al . , J. Microbiol.Methods. , 27, 33-43, 1996) [0.75% D-Raffinose, 0.5% yeast extract , 0.5% casein sodium salt, 1.5% sodium propionate, 0.3% lithium chloride, 0.05% sodium thioglycolate, 0.05% cysteine hydrochloride, 0.4% salt solution (0.2% magnesium sulfate, 0.2% calcium chloride, 1% potassium diphosphate, 1 % Monobasic potassium phosphate, 10% sodium bicarbonate, 2% calcium chloride), 0.2% promocresol purple 1% solution]. It removes the raffinose family oligosaccharides, which are anti-nutritive factors such as os and bopascose, so as to promote the absorption of nutrients, and thus can be used in substitute oils and baby foods.

Two strains of the present invention are Salmonella gallinarum , Salmonella enteritidis , Salmonella typhimurium , Staphylococcus aureus, Enterocoxigenic E. coli , It can inhibit several enteric pathogenic bacteria, such as Listeria monocytogenes , Campylobacter jejuni , and Clostridium perfringens , and is particularly effective against Salmonella bacteria in mice. Excellent anti-lethal effect, and shows excellent acid resistance and bile resistance can be useful as a probiotic lactic acid bacteria.

Looking specifically at each strain, Lactobacillus crispatus G19 strain of the present invention is an anaerobic gram-positive bacillus isolated from pig feces, has a 16S rDNA represented by the nucleotide sequence of SEQ ID NO: 5, Lactobacillus crispatus ( Lactobacillus crispatus ) ATCC 33820 showing a homology of 99% and has a flexible relationship of the dendogram of FIG. In addition, the size ranges from 1.2 to 2.0 μm in diameter, grows on MLS (MRS, Difco) solid medium to form a gray colony, in particular, it is characterized by excellent adhesion to pig small intestinal epithelial cells.

Such Lactobacillus crispatus G19 strains can grow under temperatures ranging from 30 to 50 ° C., preferably from 30 to 40 ° C., and from pH conditions ranging from 3.0 to 11.0, preferably from 4.5 to 10, and include galactose, glucose, and fructose. It can be cultured using sugars such as toss, mannose, N-acetylglucosamine, esculin, cellobiose, maltose, lactose, sucrose and raffinose, and β-gentiobioses.

Bifidobacterium boum R5 strain of the present invention is an absolute anaerobic Gram-positive amorphous form isolated from the cow's first contents. As a bacterium, it has the 16S rDNA nucleotide sequence represented by the nucleotide sequence of SEQ ID NO: 6, and exhibits 97% homology with Bifidobacterium boum JCM 1211 and has a flexible relationship with the dendogram of FIG. 2. In addition, the size is 1.0 diameter It is in the range of 1.5 to 1.5 μm, and anaerobicly grows on Siemal (MSL containing 0.05% cysteine hydrochloride) agar medium to form white colonies, and in particular, it is characterized by excellent oxygen resistance and dry heat resistance.

Such Bifidobacterium bom R5 strains can be grown under temperatures ranging from 30 to 50 ° C., preferably from 30 to 40 ° C., and from pH conditions ranging from 3.0 to 11.0, preferably from 4.5 to 10, including glucose, fructose, It can be cultured using sugars such as esculin, maltose, melibiose, sucrose, raffinose, amidone and glycogen.

In addition, the present invention provides a probiotic composition comprising the Lactobacillus crispatus G19 strain, Bifidobacterium bom R5 strain, a mixed strain of the two, or a culture thereof as an active ingredient. At this time, the culture medium of the two microorganisms include a variety of antimicrobial organic acids and non-protein antimicrobial substances produced by the microorganisms can have the same effect as the composition containing the strain when included as an active ingredient in the probiotic composition.

The composition of the present invention may further include conventional pharmaceutical carriers and excipients in addition to the active ingredient, and such compositions may be prepared by use in a probiotic form by heat drying or freeze-drying according to a conventional probiotic composition preparation method. Can be.

The composition of the present invention is based on the total weight of the composition, when the active ingredient is Lactobacillus crispatus G19 strain, Bifidobacterium bom R5 strain or a mixture of the two, respectively, 10 ⁵ to 10 ¹² cfu / g, preferably May be contained in a content of 10 ⁷ to 10 ¹¹ cfu / g, and the active ingredient is a culture solution of Lactobacillus crispatus G19 strain, Bifidobacterium bom R5 strain or a mixture of the two 10 ⁶ to 10 ¹² ml / g, preferably 10 ⁷ to 10 ¹¹ ml / g.

The composition of the present invention includes probiotic lactic acid bacteria having excellent intestinal pathogenic bacteria, acid resistance and biliary resistance, or a culture medium thereof, which is excellent in inhibiting and treating various intestinal pathogenic bacteria and inhibiting bacterial disease infection. In addition to preventing bacterial diseases by normalizing the intestinal flora of the host as well as additionally exhibiting intestinal action, anti-diabetic activity, digestion and anti-cancer activity, intestinal pathogenic bacterial inhibitors, fungicides, digestive agents, formal drugs and anti-diabetic agents As a probiotic composition, such as can be usefully used for feed, food, cosmetics and pharmaceuticals.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation of Probiotic Lactic Acid Bacteria

In order to select probiotic lactic acid bacteria that suppress various intestinal pathogenic bacteria, contents such as infant feces, swine small intestine, pig feces and cow No. 1 were collected and spread on Albi plate medium. of Separated. The isolated lactic acid bacteria were incubated anaerobicly or aerobicly for 2 days at 37 ° C. on Albi medium. As a result, a turbid ring was formed around the colonies using casein sodium salt, a medium component of lactic acid bacteria, and grown anaerobicly. While strains that do not grow aerobically were selected as Bifidobacterium strains, strains that grow both aerobicly or anaerobicly without forming a cloudy ring around the colonies were selected as Lactobacillus strains.

The selected lactic acid bacteria were anaerobicly cultured in a liquid medium of MMS (MRS, Difco Co., Ltd.) containing 0.05% cysteine hydrochloride at 37 ° C. for 3 days, respectively, and the obtained culture solution was centrifuged to take a supernatant. It was. Salmonella gallinarum , Salmonella enteritidis , Salmonella typhimurium , enterotoxigenic E. coli , Staphylococcus aureus , Listeria In the agar plate medium containing Listeria monocytogenes , Campylobacter jejuni or Clostridium perfringens , the supernatant was 30 μl each for Salmonella, and In case of other bacteria, 100 μl was added dropwise by passing into a peniciller tube (Fisher scientific, USA, Cat. No. 07-907-5), and a medium containing lactic acid bacteria culture supernatant was dropped at 37 ° C. for 12 hours or more. After incubation, the diameter (mm) of the inhibited transparent ring formed was measured. At this time, Lactobacillus pentosus K34 (KCCM 10331), Bifidobacterium thermophilum (KCCM 19097T) and Bifidobacterium longgum ( Bifidobacterium ), which are known to be excellent in inhibiting intestinal pathogenic bacteria as comparative strains longum ) (KCCM 11853T) was used, and the results showed that 7 strains (Lactobacillus strains B2, G12, G19, S21 and B47, and bifidus strains R5 and R9) showed the same pathogenic bacterial inhibitory activity as the comparison group. The results are shown in Table 1 below.

As a result, as shown in Table 1, it was confirmed that the seven strains showed a better or equivalent pathogenic bacteria inhibitory ability compared to the comparative strain.

Example 2: Acid and Bile Resistance Measurements

A total of seven kinds of lactic acid bacteria whose inhibitory ability against intestinal pathogenic bacteria was verified in Example 1 were siems liquid medium and 0.3% (v / v) hydrochloric acid or 0.3% (w / v) bile salt (Difco, USA), respectively. Inoculated with 1 × 10 ⁸ bacteria / ml each of the liquid containing medium CM culture / ml and incubated for 6 hours at 37 ℃, each was smeared onto a plate of Siem ALS agar cultured anaerobicly. At this time, Lactobacillus rhamnosus GG (obtained from Ildong Pharmaceutical Co., Ltd.), L. pentosus K34, B. thermofilum and B. long gum were used as comparative strains. After measuring the number of viable cells, the survival rate (%) was calculated according to the following formula and is shown in Table 2 below.

% Survival = (number of viable cells in medium containing 0.3% hydrochloric acid or bile salt / viable cell in medium free) × 100

As a result, it was confirmed that all the lactic acid bacteria except L2 and G12, Lactobacillus strains showed relatively excellent acid resistance and bile resistance.

Example 3: Confirmation of Adhesion Ability of Pig Small Intestine Epithelial Cells of Lactobacillus Strains

In Example 2, it was confirmed that the small intestine epithelial cell adhesion ability of the Lactobacillus strains G19, S21, and B47, which have been tested for excellent acid resistance and bile resistance, and as a comparative strain, L. rhamnosus GG, L Planta Room (KFCC 11322) and L. Pentosus K34 were used.

Immediately after the slaughter of the pig, the middle part of the upper intestine was incised and the contents were removed by applying pressure to the incision, and the mucous surface was washed with 0.01 M phosphate buffered saline (PBS, pH 7.4) and carefully scraped to obtain a mucus layer. Washed with HEPES-Hank's buffer. In order to separate epidermal cells and intestinal membrane from the obtained mucus layer, centrifugation was carried out twice for 15 minutes at 4 ° C. and 12,000 × g, followed by further centrifugation at 4 ° C. and 27,000 × g for 15 minutes to remove the mucus layer. After treating the enzyme solution (Sigma No. H-3506) containing 1 mg / ml of hyaluronidase in the buffer, it was reacted at 37 ° C for 30 minutes, and then carefully cut with a sterile spoon to separate epidermal cells. The separated epidermal cell solution was filtered through a sterile gauze to remove lumps and debris, and the supernatant was discarded after centrifugation of the epidermal cell suspension twice at 100 × g for 1 minute, and then, 5 × Herpes Hanks buffer solution was added to 120 × g. The cells were washed by centrifugation twice for 10 minutes at, and finally, 15 ml Herpes Hanks buffer was added, and centrifuged once at 120 x g for 10 minutes to obtain epidermal cells.

To 0.5 ml of the obtained epidermal cell solution (1 × 10 ⁴ cell number / ml), 0.5 ml of the culture solution of each of the Lactobacillus strains (number of bacteria: 1 × 10 ⁹ cell number / ml) was added thereto, and reacted at 4 ° C. for 40 minutes. The obtained cell suspension was centrifuged at 200 x g for 15 minutes, and then 1.0 ml of Herpes Hanks buffer was added three times to remove unattached lactic acid bacteria. Obtained cells With 0.5 ml Herpes Hanks Buffer The suspension was fixed to the slide glass with methanol, and after Gram staining, the number of bacteria attached was measured by observing with an optical microscope. At this time, the number of bacteria attached to the 10 cells was measured and then the number of bacteria attached to the average value was calculated.

As a result, as shown in Table 3, it was confirmed that the Lactobacillus G19 strain isolated from pig feces had the best porcine epithelial cell adhesion.

Example 4: Mouse Test of Lactobacillus Strains

A mouse test was carried out on the L19 Bacillus strains G19, S21, and B47, which were tested for excellent acid resistance and bile resistance in Example 2, wherein L. rhamnosus GG and L. pentosus K34 were compared. Was used.

The strains were prepared using conventional lactic acid bacteria industrial medium (composition: 1% yeast extract, 1% glucose, 2% corn immersion, 0.2% potassium phosphate, 0.1% magnesium sulfate, 7 water and 0.05% manganese sulfate, 7 water). The cells were incubated anaerobicly at 37 ° C. for 3 days, and each of the cultured strains was mixed with an excipient degreasing steel, followed by hot air drying at 40 ° C. for 1 day to obtain respective lactic acid bacteria dried products.

On the other hand, Clostridium butyricum M7, a strain for producing probiotic formulations, which is commonly used in the manufacture of feed additives, has 2% corn starch, 1% yeast extract, 1% molasses, 0.1% potassium phosphate, 0.05% It was cultured in a conventional industrial medium containing cysteine hydrochloride, 0.05% magnesium sulfate -7 water and 0.05% zinc sulfate, and Bacillus subtilis V35 and Saccharomyces cerevisiae . STV89 was incubated in conventional industrial media (composition: 1% glucose, 0.5% yeast extract, 1% corn immersion and 0.1% potassium monophosphate). At this time, Bacillus subtilis V35 was incubated at 37 ° C. for 2 days in Saccharomyces cerevisiae. STV89 was shaken at 30 ° C. for 2 days, and Clostridium butycomb M7 was anaerobicly cultured at 37 ° C. for 2 days. Each cultured strain was mixed with excipient degreasing steel and hot-air-dried at 40 ° C. for 1 day to obtain dried strains of each strain.

A feed additive was prepared by mixing yeast culture, enzyme, and mineral so that the three dried strains were 1 × 10 ⁸ bacteria / ml or more, respectively. Inclusion in weight percent produced a probiotic formulation of each Lactobacillus strain.

Each prepared probiotic formulation was mixed with gamma sterile mouse powder feed (Samtaco, Korea) at 0.3% concentration and freely administered to 4 week old BALB / c mice.On day 7, Salmonella typhimurium LT2 was only 2.5 × once. 0.3 ml were infected by oral administration with mice by diluting in 0.85% saline at a concentration of 10 ⁶ cells / ml (LD ₅₀ × 10). After the Salmonella typhimurium infection was continuously administered freely administered mouse powder feed mixed with lactic acid bacteria-containing feed additives, the survival rate was measured in 21 days after infection, and the survival rate after 21 days and 1 week after infection was measured. Feed intake and weight gain were measured and shown in Table 4 below. At this time, the control group (None), antibiotic administration group (Anti; 0.05% antibiotic oraquindox) and feed additive single administration group (FA; Feed Additive without LAB) was also measured together.

As a result, compared to the control group, the survival rate of the mice in the probiotic-added group was higher than 12.5% to 75%, and it was confirmed that the G19 strain showed superior mouse survival rate of 75%. In addition, the probiotic formulation group containing the G19 strain also showed excellent feed intake and weight gain for 7 days after Salmonella infection.

Example 5: Confirm the oxygen resistance of the bifidus strain

Oxygen resistance was confirmed in the following examples of the bifidus strains R5 and R9 of which excellent acid resistance and bile resistance were verified in Example 2, and B. long gum and B. thermofilum were used as comparative strains.

After incubating each of the bifidus strains anaerobicly for 2 days in 10 ml of the Siem RS liquid medium, 300 μl of the obtained culture solution was boiled. 30 ml sterilized vials containing 10 ml of Siems liquid medium prepared by anaerobic conditions by replacing with carbon dioxide gas or 10 ml of Siems liquid medium prepared by aerobic conditions. Each was inoculated at and incubated at 37 ° C. for 24 hours. In order to confirm the cell growth of each strain, the absorbance was measured with a spectrophotometer (Shimazu, Japan) at 600 nm of the obtained culture solution, and the oxygen resistance was measured according to the following formula, and the results are shown in Table 5 below. .

Oxygen Resistance (%) = [Aerobic Growth (A ₆₀₀ ) / Aerobic Growth (A ₆₀₀ )] × 100

As a result, as shown in Table 5, R5, a bifidus strain isolated from the cow first place, showed the best oxygen resistance.

Example 6: Confirmation of dry heat stability of bifidus strain

remind The R5 strain and the comparative group Bifidobacterium thermofilum, which have been tested for excellent oxygen resistance in Example 5, were incubated anaerobicly for 3 days in 10 ml of SiMSL liquid medium, and then the production conditions of the conventional probiotic preparation The same was mixed with the excipient degreasing steel. 1 g of each of the obtained strain samples was taken, and the rest was dried for 1 day with 40 ° C. hot air, and then 1 g of dried samples were taken. Each of the following conventional Hungate methods (Hungate, RE, Methods in Microbiology, Academic Press Inc. , New York, Vol 3B, p 117-132, 1969) was used to determine the number of viable cells in the sample before or after drying.

After diluting 1 g of the sample with anaerobic diluent (0.2% yeast extract, 0.2% potassium diphosphate, 0.05% cysteine hydrochloride), 0.1 ml of each diluent contained 2% agar (solidification) at 60 ° C. Injections were made in 10 ml of alb or siemals medium. This was placed in a 35 × 35 cm square container containing cold water, rolled and solidified by hand, and then cultured anaerobicly for 2 days according to a conventional method to measure the number of viable cells.

The survival rate (%) was calculated according to the following formula based on the measured number of viable cells before drying and the number of viable cells after drying, and is shown in Table 6 below.

Survival rate (%) = (number of viable cells after drying / number of live cells before drying) × 100

As a result, as shown in Table 6, it was confirmed that the viability of the R5 strain was higher than the Bifidobacterium thermofilum of the comparative strain, and thus it can be seen that it has excellent dry heat stability.

Example 7: Mouse Test of Bifidus Strain

remind R5 strains having excellent oxygen resistance and dry heat stability in Examples 5 and 6, and comparative strains Bifidobacterium thermofilum and Bifidobacterium long gum were used for 3 days at 37 ° C. using SIEMS liquid medium. Ten milliliters of the culture solution obtained by anaerobic culture were centrifuged at 4,000 rpm to remove the supernatant and only cells were recovered. The obtained cells were resuspended in physiological saline (0.85% NaCl) at a concentration of 1 × 10 ¹⁰ cells / ml and orally administered 0.3 ml once daily to 4 week old mice. On the 7th day, highly infectious Salmonella typhimurium LT2 ( S. typhimurium LT2 NCIMB 10248) bacteria were diluted in saline at a concentration of LD ₅₀ × 10 and infected with 0.3 ml of single oral dose. After infection, the saline suspension of bifidus lactobacillus cells was orally administered once daily to measure the survival rate for 21 days after infection, and the survival rate and feed intake and body weight for 7 days after infection were observed in 21 days after infection. The increase was measured and shown in Table 7 below. At this time, the non-administered group (None) was also measured as a control.

As a result, as shown in FIG. 2, the mortality rate of mice in the control group and the control group increased rapidly from 7 days after Salmonella infection, whereas the R5 strain had a survival rate of 62.5% from the 10th to the 21st day after the Salmonella oral administration. And it was confirmed that the excellent mouse lethal inhibitory effect compared to the comparison group. In addition, as shown in Table 7, it was confirmed that the R5 strain of the present invention is also excellent intake and weight gain for 7 days after infection.

Example 8: for complex lactic acid strains Mouse test

 Example 4 and Example 7 except for using a mixed lactobacillus strain of the strain of L19 bacterium G19 and Bifidobacterium strain R5 showed excellent lethal inhibitory effect, the same as in Example 4 The mouse test was performed by the method, and the results are shown in Table 8 below.

As a result, as shown in Table 8, the survival rate (87.5%) of the complex strain was superior to the survival rate (75.0%) of the G19 strain administration group as well as the survival rate (11.0%) of the feed additive single administration group without the addition of lactic acid bacteria. The weekly total feed intake and weekly weight gain were higher than the G19 strain group.

These results Salmonella typhimurium Head Stadium (S. typhimurium) It is similar to the introduction test (Timmerman et al. , Int. J. Food Microbiol. , 96, 219-233, 2004) that the use of lactic acid bacteria multispecies shows better mouse survival than with single bacteria. Therefore, Lactobacillus crispatus G19 and Bifidobacterium bom R5 of the present invention not only show excellent antimicrobial activity when used with each single bacterium, but also show synergistic inhibitory effect against infection when used together. Able to know.

Example 9 Identification of Probiotic Lactic Acid Bacteria of the Invention

In order to identify Lactobacillus G19 and Bifidobacterium R5 strains which were judged to be the most probiotic lactic acid bacteria among the lactic acid bacteria investigated, their 16S rDNA sequencing was performed.

First, the selected strains were anaerobicly cultured in an MS plate containing 0.05% cysteine, and then each single colony generated was taken to accucu genomic DNA Extraction kit (BIONEER). Genomic DNA was isolated. After the isolated genomic DNA was confirmed by electrophoresis, it was subjected to a polymerase chain reaction (PCR) as a template and using primer pairs of SEQ ID NOs: 1 and 2. In this case, PCR was performed 35 times at 95 ° C. for 30 seconds (denature), 55 ° C. for 1 minute (annealing), and 72 ° C. for 1 minute (extension) using a GeneAmp PCR System 2700, and the obtained PCR product was subjected to electrophoresis. As a result, it was confirmed that 16S rDNA having a size of about 1,500 bp was amplified. The amplified 16S rDNA was purified by AccuPrep PCR Purification kit (BIONEER), and then sequenced using an automated DNA Sequencer (ABI 3100, Applied Biosystem). In this case, primers of SEQ ID NOS: 1 to 4 were used as primers for sequence analysis. The sequences analyzed therefrom were combined using Cluster X software and then advanced blast search (NCBI) provided by The National Center for Biotechnology Information (http: //www.ncbi.nlm.nih). Advanced Blast search, Altschul et al., Nucleic Acids Research 25: 3389-3402, 1997) were used to compare gene sequences with genbanks, and finally by dendritic-joining analysis. A gram (dendrogram) was created and shown in FIGS. 3 and 4.

As a result, the Lactobacillus G19 strain has 16S rDNA represented by the nucleotide sequence of SEQ ID NO: 5 and shows 99% homology with Lactobacillus crispatus ATCC 33820 , which shows the flexibility of the dendogram of FIG. It was confirmed that there was a relationship. In addition, the Bifidobacterium R5 strain has a 16S rDNA sequence represented by the nucleotide sequence of SEQ ID NO: 6, and shows the dendogram of FIG. 4 showing 97% homology with Bifidobacterium boum JCM 1211. It has been confirmed that it has a flexible relationship with.

Thus, the naming of the strains in each of Lactobacillus Creative Spa tooth (Lactobacillus crispatus) G19 and Bifidobacterium boum (Bifidobacterium boum) R5, and each August 9, 2005 in Korea microbial preservation center (KCCM) KCCM 10668P and Deposited with accession number of KCCM 10669P.

Example 10 Carbon Source Availability Measurements

Lactobacillus crispatus G19 and Bifidobacterium bom R5 strains identified in Example 9 were inoculated in MLS plate medium containing 0.05% cysteine and incubated anaerobicly for 48 hours at 37 ° C. The colonies formed were observed.

As a result, it was confirmed that both strains formed a circular colony of 0.5-4 mm in diameter with Gram-positive bacillus, Lactobacillus crispatus G19 was gray colony, and Bifidobacterium bom R5 was white colony. It was confirmed to form. In addition, the strains were found to exhibit optimal culture conditions at temperatures ranging from 30 to 40 ° C., preferably from 35 to 40 ° C., and pH conditions ranging from 3.0 to 11.0, preferably from 6 to 8.

In order to confirm the biochemical properties of the strain, the carbon source availability of Lactobacillus crispatus G19 and Bifidobacterium bom R5 strains was investigated as follows.

After inoculating each strain on MLS agar medium containing 0.05% cysteine and anaerobic culture at 37 ° C. for 2 days, the colonies formed were suspended in 5 ml of 0.85% NaCl saline at high concentration. This 5 ml Suspended in 0.85% NaCl saline to McF (McFarland standard, BioMerieux) turbidity 2 was confirmed, based on this, 0.05% cysteine, a medium contained in the API 50 CHL kit (BioMerieux), a lactic acid bacteria identification kit CHL medium contained [1% polypeptone, 0.5% yeast extract, 0.1% Tween 80, 0.2% potassium diphosphate, 0.5% sodium acetate, 0.2% diammonium citrate, 0.02% MgSO ₄ 7H ₂ O , 0.005% MnSO ₄ 4H ₂ O, 0.017% bromocresol purple] was inoculated to suspension obtained after suspension to McF turbidity 2. CHL medium inoculated with each strain was dispensed into each well of CH50 strips (20 strips) by 140 μl and mineral oil was dispensed until the scale of each well was convex. While culturing anaerobicly at 37 ° C., the color change of the phenolphthalein indicator contained in the medium was observed at 24 and 48 hours to confirm sugar availability, and the results are shown in Tables 9 (G19) and 10 (R5). Shown in

As a result, as shown in Table 9, the Lactobacillus crispatus G19 strain of the present invention is galactose, D-glucose, D-fructose, D-mannose, N-acetyl-glucosamine, esculin, cellobiose, maltose , Lactose, sucrose, raffinose and β-gentiobioses were positive. As shown in Table 10, the Bifidobacterium bom R5 strain of the present invention is glucose, fructose, esculin, maltose, It was positive for melibiose, sucrose, raffinose, amidone and glycogen.

Preparation Example 1 Preparation of Probiotic Composition (Feed Probiotic Composition)

Lactobacillus crispatus G19, Bifidobacterium bom R5 strains or mixed strains thereof of the present invention were respectively prepared with 2.0% glucose, 1.0% peptone, 1.0% yeast extract, 0.2% diphosphate, 0.05% magnesium sulfate and 0.05% cysteine. The medium was inoculated and incubated anaerobicly for 3 days at 37 ℃. The culture medium and the excipient degreasing steel of each strain were simply mixed in a weight ratio of 1: 1, and dried at 40 ° C. or less, and the dried mixture was pulverized to prepare a probiotic composition for feed addition. At this time, the prepared probiotic composition was to include each strain 1 × 10 ⁸ cfu / g or more.

Preparation Example 2 Preparation of Probiotic Composition (Feed Probiotic Composition)

Lactobacillus crispatus G19, Bifidobacterium bom R5 strains or mixed strains thereof of the present invention were respectively prepared with 2.0% glucose, 1.0% peptone, 1.0% yeast extract, 0.2% diphosphate, 0.05% magnesium sulfate and 0.05% cysteine. The medium was inoculated and incubated anaerobicly for 3 days at 37 ℃. After inoculating 10% by weight of the culture solution of each strain in a solid incubator containing skim steel, soybean meal, jade powder and molasses in a weight ratio of 3: 1: 1: 1, respectively, 45-60% by weight of water was added thereto. Was added. This was carried out anaerobic solid fermentation while stirring at 40 ℃ for 3 days to prepare a live feed composition for feed addition, wherein the prepared live bacteria composition was to be included in each strain 1 × 10 ⁷ cfu / g or more.

Preparation Example 3 Preparation of Probiotic Composition (Probiotic Composition for Health Functional Food)

Lactobacillus crispatus G19, Bifidobacterium bom R5 strains or mixed strains thereof of the present invention were respectively prepared with 2.0% glucose, 1.0% peptone, 1.0% yeast extract, 0.2% diphosphate and 0.05% magnesium sulfate, 0.05% cysteine. It was inoculated in a medium containing the back and incubated anaerobically for 3 days at 37 ℃. 60% by weight of the culture solution was added to a mixture of milk powder or skim milk and starch or lactose in a 1: 1 weight ratio, which was then rapidly frozen at -70 ° C or lower and then -36 ° C for 36 hours. Freeze-drying to prepare a probiotic composition for health functional equipment. At this time, the temperature of the hot plate to increase the water evaporation efficiency during lyophilization was 30 ℃, and the prepared live cell composition was to include each strain at 1 × 10 ¹⁰ cfu / g or more.

Preparation Example 4 Preparation of Probiotic Composition (Substitute Oil Composition)

Lactobacillus crispatus G19, Bifidobacterium bom R5 strains or mixed strains thereof of the present invention were respectively prepared with 2.0% glucose, 1.0% peptone, 1.0% yeast extract, 0.2% diphosphate, 0.05% magnesium sulfate and 0.05% cysteine. This medium was inoculated and incubated anaerobicly at 37 ° C. for 2 days. After inoculating 1 wt% of each strain culture solution obtained in soybean oil which has undergone conventional washing, crushing, filtration and sterilization, it was incubated at 37 ° C. for 2 days to prepare a surrogate oil composition. At least 1 × 10 ⁷ cfu / g was included.

Preparation Example 5 Preparation of Probiotic Composition (Pharmaceutical Composition)

The probiotic composition powders obtained in Preparation Examples 1, 2 and 3, respectively, were molded into tablets, pills, and capsules according to a conventional method to prepare pharmaceutical compositions, wherein the prepared compositions were each strain of 1 × 10 ⁷ cfu / g or more. It was included as.

As described above, the Lactobacillus of the present invention, the Bacillus Cri Spa tooth (Lactobacillus crispatus) G19 strain and the Bifidobacterium boum (Bifidobacterium boum) R5 strain exhibits excellent antimicrobial activity as well as acid resistance and bile in different pathogenic bacteria St. Lactobacillus crispatus G19 has excellent intestinal epithelial cell adhesion, and Bifidobacterium bore R5 shows excellent resistance to oxygen resistance and dry heat. It can be usefully used in feed, food, cosmetics and medicines by preparing a probiotic composition such as a digestive agent, a suit and a branch.

<110> Biotopia Co. Ltd. <120> PROBIOTIC LACTIC ACID BACTERIA AND COMPOSITION COMPRISING THE          SAME <130> FPD200508-0085 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tacggytacc ttgttacgac tt 22 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gtgccagcmg ccgcgg 16 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gggttgcgct cgttg 15 <210> 5 <211> 1468 <212> DNA <213> Lactobacillus crispatus G19 <400> 5 gctggcggcg tgcctaatac atgcaagtcg agcgagcgga accaacagat ttacttcggt 60 aatgacgttg ggaaagcgag cggcggatgg gtgagtaaca cgtggggaac ctgcccctaa 120 gtctgggata ccatttggaa acaggtgcta ataccggata ataaagcaga tcgcatgatc 180 agcttttgaa aggcggcgta agctgtcgct aagggatggc cccgcggtgc attagctagt 240 tggtaaggta acggcttacc aaggcgacga tgcatagccg agttgagaga ctgatcggcc 300 acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttccac 360 aatggacgca agtctgatgg agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa 420 gctctgttgt tggtgaagaa ggatagaggt agtaactggc ctttatttga cggtaatcaa 480 ccagaaagtc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540 gtccggattt attgggcgta aagcgagcgc aggcggaaaa ataagtctaa tgtgaaagcc 600 ctcggcttaa ccgaggaact gcatcggaaa ctgtttttct tgagtgcaga agaggagagt 660 ggaactccat gtgtagcggt ggaatgcgta gatatatgga agaacaccag tggcgaaggc 720 ggctctctgg tctgcaactg acgctgaggc tcgaaagcat gggtagcgaa caggattaga 780 taccctggta gtccatgccg taaacgatga gtgctaagtg ttgggaggtt tccgcctctc 840 agtgctgcag ctaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact 900 caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 960 cgaagaacct taccaggtct tgacatctag tgcaatctgt agagatacgg agttcccttc 1020 ggggacgcta agacaggtgg tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt 1080 aagtcccgca acgagcgcaa cccttgttat tagttgccag cattaagttg ggcactctaa 1140 tgagactgcc ggtgacaaac cggaggaagg tggggatgag gtcaagtcat catgcccctt 1200 atgacctggg ctacacacgt gctacaatgg gcagtacaac gagaagcaag cctgcgaagg 1260 caagcgaatc tctgaaagct gttctcagtt cggactgcag tctgcaactc gactgcacga 1320 agctggaatc gctagtaatc gcggatcagc acgccgcggt gaatacgttc ccgggccttg 1380 tacacaccgc ccgtcacacc atgggagtct gcaatgccca aagccggtgg cctaaccttc 1440 gggaaggagc cgtctaaggc agggcaga 1468 <210> 6 <211> 1434 <212> DNA <213> Bifidobacterium boum R5 <400> 6 tacactggcg gcgtgcttac acatgcaagt ctaacgggat cctgcaagct tgcttgcggg 60 tgagagtggc gaacgggtga gtaatgcgtg accaacctgc cccatgctcc ggaatagctc 120 ctggaaacgg gtggtaatgc cggatgtgcc gggctcctgc atgggggtcc gggaaagctc 180 tggcggcgtg ggatggggtc gcgtcctatc agcttgttgg cggggtgagg gcccaccaag 240 gcttcgacgg gtagccggcc tgagagggcg accggccaca ttgggactga gatacggccc 300 agactcctac gggaggcagc agtggggaat attgcacaat gggcgcaagc ctgatgcagc 360 gacgccgcgt gcgggatgga ggccttcggg ttgtaaaccg cttttgtttg ggagcaagcc 420 cttcggggtg agtgtacctt tcgaataagc accggctaac tacgtgccag cagccgcggt 480 aatacgtagg gtgcgagcgt tatccggatt tattgggcgt aaagggctcg taggcggttc 540 gtcgcgtccg gtgtgaaagt ccatcgccta acggtggatc tgcgccgggt acgggcgggc 600 tggagtgcgg taggggagac tggaattccc ggtgtaacgg tggaatgtgt agatatcggg 660 aagaacacca atggcgaagg caggtctctg ggccgttact gacgctgagg agcgaaagcg 720 tggggagcga acaggattag ataccctggt agtccacgcc gtaaacggtg gatgctggat 780 gtggggccct tccacgggtc ccgtgtcgga gccaacgcgt taagcatccc gcctggggag 840 tacggccgca aggctaaaac tcaaagaaat tgacgggggc ccgcacaagc ggcggagcat 900 gcggattaat tcgatgcaac gcgaagaacc ttacctgggc ttgacatgtt cccggcgacg 960 gcggagacgt cgtttccctt cggggcgggt tcacaggtgg tgcatggtcg tcgtcagctc 1020 gtgtcgtgag atgttgggtc aagtcccgca acgagcgcaa ccctcgcccc gtgttgccag 1080 cgcgtcatgg cgggaactca cgggggaccg ccggggttaa cccggaggaa ggtggggatg 1140 acgtcagatc atcatgcccc ttacgtccag ggcttcacgc atgctacaat ggccggtaca 1200 gcgggatgcg acatggtgac atggagcgga tccctgaaaa ccggtctcag ttcggatcgg 1260 agcctgcaac ccggctccgt gaaggcggag tcgctagtaa tcgcggatca gcaacgccgc 1320 ggtgaatgcg ttcccgggcc ttgtacacac cgcccgtcaa gtcatgaaag tgggcagcac 1380 ccgaagccgg tggcctgacc agttctgctg gaagagcctt taagggaggc tcgg 1434

Claims (9)

Lactobacillus crispatus G19 lactic acid bacteria having intestinal pathogenic bacteria inhibitory ability (Accession No .: KCCM 10668P). Bifidobacterium boum R5 lactic acid bacterium having an intestinal pathogenic bactericidal activity (Accession No .: KCCM 10669P). The method according to claim 1 or 2, The enteric pathogenic bacteria Salmonella Galina room (Salmonella gallinarum), Salmonella Entebbe utility disk (Salmonella enteritidis), Salmonella tie blood head Titanium (Salmonella typhimurium), Staphylococcus aureus (Staphylococcus aureus), Chapter toxic E. coli (enterotoxigenic E. coli), L. monocytogenes Lactobacillus, characterized in that selected from the group consisting of Listeria monocytogenes , Campylobacter jejuni , Clostridium perfringens and combinations thereof. The probiotic composition comprising the lactic acid bacteria of claim 1 or 2, a mixed strain of the two, or a culture solution thereof as an active ingredient. The method of claim 4, wherein Salmonella Galina room (Salmonella gallinarum), Salmonella Entebbe utility disk (Salmonella enteritidis), Salmonella tie blood head Titanium (Salmonella typhimurium), Staphylococcus aureus (Staphylococcus aureus), Chapter toxic E. coli (enterotoxigenic E. coli), Listeria monocytogenes jeneseu (Listeria monocytogenes ), Campylobacter jejuni , Clostridium perfringens , and combinations thereof, which are used for the treatment or prevention of infectious diseases of enteric pathogenic bacteria selected from the group consisting of Biotic composition. The method of claim 4, wherein A probiotic composition characterized in that the pharmaceutical composition further comprises a pharmaceutical carrier and an excipient. The method of claim 4, wherein Probiotic composition, characterized in that used as additives for feed or food. The method of claim 4, wherein A probiotic composition characterized in that it is prepared in the form of a probiotic by heat drying or freeze-drying. The method of claim 4, wherein Regarding the total weight of the composition, Lactobacillus crispatus G19 strain, Bifidobacterium bom R5 strain, or a mixed strain of the two are included as an active ingredient in an amount of 10 ⁵ to 10 ¹² cfu / g, respectively, or a culture thereof Probiotic composition, characterized in that it comprises a content of 10 ⁶ to 10 ¹² ml / g each.

Images