KR100813637B1 - Bifidobacterium infantis MAEIL-K9-derived peptidoglycan-containing extract, and a composition for preventing or treating rotaviral infection containing the same - Google Patents

Abstract

The present invention relates to a peptidoglycan-containing extract derived from Bifidobacterium infantis MAEIL-K9 and a composition for preventing or treating rotavirus infectious diarrhea containing the same, and more specifically, the peptido according to the present invention. The glycan-containing extract acts as an intestinal fixation promoting factor to enhance the intestinal fixation ability of the bifidus bacteria, thereby making the colony of the bifidus bacteria more superior to the rotavirus in the intestine, thereby inhibiting the settlement and proliferation of the rotavirus. Rotavirus infectious diarrhea.

Rotavirus Infectious Diarrhea, Bifidobacterium Infantis, Bifitus, Probiotic Lactobacillus, Peptidoglycan Extract

Description

Bifidobacterium infantis MAEIL-K9-derived peptidoglycan-containing extract, and a composition for Bifidobacterium infantis MAIEL-Ｋ９-derived peptidoglycan-containing extract and rotavirus infectious diarrhea preventing or treating rotaviral infection containing the same}

1 is a view showing a change in the fixation capacity of human-derived rotavirus (S2) according to the simultaneous treatment of peptidoglycan-containing extract and bifidus live bacteria of the present invention (in FIG. 1, the inhibitor is a peptidoglycan-containing extract Means).

Figure 2 is a diagram showing the effect of inhibiting rotavirus according to the concentration of bifidus bacteria-derived material (in Figure 2, DCM shows the heat treated bifidus cells (OD 1.0), PGN is a peptidoglycan containing extract (0.1% solution) ).

3 is a diagram showing the inhibitory effect of the peptidoglycan-containing extract against various rotaviruses (in FIG. 3, PGN represents the peptidoglycan-containing extract (0.1% solution), and JBR and NCDV are rota derived from bovine) Virus, and S2 represents rotavirus from human).

Figure 4 is a view showing the particle size distribution according to the extraction method and pH of the peptidoglycan-containing extract.

The present invention relates to a peptidoglycan-containing extract derived from Bifidobacterium infantis MAEIL-K9 and a composition for the treatment or prevention of rotavirus infectious diarrhea containing the same, and more specifically, the peptido according to the present invention. The glycan-containing extract acts as an intestinal fixation promoting factor to enhance the intestinal fixation ability of the bifidus bacteria, thereby making the colony of the bifidus bacteria more superior to the rotavirus in the intestine, thereby inhibiting the settlement and proliferation of the rotavirus. Rotavirus infectious diarrhea can be suppressed.

Like pathogenic bacteria, some viruses infect the intestinal cells and cause diseases such as diarrhea. In particular, acute diarrhea is a major symptom occurring in most infants in developing countries, and the most important cause of infant acute diarrhea is known as rotavirus. The number of infants dying from rotavirus infectious enteritis reaches more than 10,000 in developing countries every year, and 1.5 million children worldwide suffer from rotavirus infectious diarrhea and vomiting. In the past, bacterial diarrhea was very high at 80% among summer diarrhea, but today, viral infectious diarrhea accounts for 70-80%, and the rate of diarrhea due to viral infection is much higher. In addition to vomiting and diarrhea, young children with rotavirus infection also have symptoms of fever and respiratory tract infections, sometimes requiring blood transfusions because they cause dehydration. Rotavirus infectious enteritis appears to be milder than that caused by bacterial enteritis, but may also cause more severe diarrhea.

Rotavirus infection, which causes diarrhea in infants, is caused by two external proteins, VP7 and VP4, which produce neutralizing antibodies, and are degraded and aggregated by VP7 and protease, a glycoprotein on the surface of the virus. VP4, a protease-cleaved hemagglutamin cleaved by proteases (eg proteases), is known to play an important role in the neutralization and protection of viruses. Electron microscopic studies of infected cells in the gut have shown that rotavirus particles enter the cell by intracellular disintegration and direct cell membrane penetration. Rotavirus infection weakens the immune system of the infant and plays a harmful role in health because it is involved in the composition of the intestinal flora.

Efforts have been made to improve acute rotavirus infectious diarrhea and to suppress rotavirus infection, but to date no new medicines are available for the treatment of acute diarrhea and only minimal improvement by oral administration of some inhibitors This is recognized.

For example, as a method for treating rotavirus infectious diarrhea, Guarino et al. (Guarino, A., A. Casola, E. Bruzzese, M. Saini, L. Niitsch, and A. Rubino. 1996. Human serum immunoglobulin counteracts rotaviral infection Pediatr. Res. 40 (6): 881-887) studied the inhibitory effects of antibodies such as antibodies in a study model in which rotavirus was infected with human intestinal epithelial cells. As a result, it was reported that the immunoprotein has an inhibitory effect on the infection of Caco-2 cells by rotavirus. However, these results are different depending on the timing and content of the administration of the inhibitory immunoprotein, and it is expensive to obtain the immunoprotein as a purified form, and its administration may cause immune side effects such as inflammation.

Meanwhile, vaccines have been developed to prevent the infection of rotavirus. However, due to the various serotypes of rotavirus, there is a difficulty in defending the disease sufficiently and broadly. In addition, there is also a folk treatment method by the food ingredients such as colostrum, egg yolk, green tea extract, lactoferrin in the treatment of rotavirus infection, there may be a problem in terms of reproducibility of the effect.

Therefore, there is a need in the art for the development of more effective methods of inhibiting rotavirus infection and preventing or treating rotavirus infectious diarrhea.

Therefore, the present inventors also conducted a number of studies to find a method for improving the rotavirus infectious diarrhea by inhibiting the infection of rotavirus, as a result, the extract containing peptidoglycan derived from Bifidobacterium inpantis MAEIL-K9 strain By acting as an intestinal fixation promoting factor to enhance the intestinal fixation ability of the bifidus bacteria, and to make the colony of the bifidus bacteria more superior to the rotavirus in the intestine, by inhibiting the settlement and proliferation of the rotavirus to prevent rotavirus infectious diarrhea It has been found that it can be suppressed and the present invention has been completed.                         

Accordingly, an object of the present invention is to provide a peptidoglycan-containing extract derived from Bifidobacterium infantis MAEIL-K9 strain contained as an active ingredient of the composition.

Another object of the present invention is to provide a composition for treating or preventing rotavirus infectious diarrhea.

In order to achieve the above object, the peptidoglycan-containing extract derived from the Bifidobacterium infantis MAEIL-K9 strain according to the present invention is a Bifidobacterium inpantis MAEIL-K9 strain to lysozyme or protease And, optionally, by physical crushing or ultrasonic crushing prior to the enzyme treatment.

In addition, the composition for the treatment or prevention of rotavirus infectious diarrhea according to the present invention is characterized in that it contains a peptidoglycan-containing extract derived from Bifidobacterium infantis MAEIL-K9 strain as an active ingredient.

Hereinafter, the present invention will be described in more detail.

Recently, with increasing knowledge of mesenteric immunity over the last few years, a method of improving acute rotavirus infectious diarrhea in infants by oral immunization has been proposed to suppress viral and bacterial infections in the intestinal mucosa and respiratory tract of humans or animals. That is, the therapeutic effect of the method of taking an infection inhibitor and probiotic lactic acid bacteria, preferably Lactobacillus and bifidus lactic acid bacteria, for the prevention and treatment of rotavirus infectious diarrhea has been found. For example, Saavedra et al. (Saavedra, JM, NA Bauman, I. Oung, JAPerman, and RHYolken. 1994. Feeding of Bifidobacteirum bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus.The Lancet. 344: 1046-1049) reported that oral administration of Bifidobacterium bifidum and Streptococcus thermophilus to infants treated with rotavirus infectious diarrhea resulted in a shortened duration of acute diarrhea. The function of these Lactobacillus GG lactobacillus has been reported to strengthen the intestinal immunity to rotavirus locally, stabilize the intestinal permeability to macromolecules, and reduce the infection and diarrhea of infants caused by rotavirus.

Yasui et al. (Yasui, H., J. Kiyoshima, and H. Ushijima. 1995. Passive protection against rotavirus-induced diarrhea of mouse pups born to and nursed by dams fed Bifidobacterium breve YIT4064. J. Infect. Dis. 172: 403-409) showed that when orally administered Bifidobacterium breve YIT4064 in rats, the secretion of anti-rotavirus infectivity-specific IgA was significantly increased, which had a protective effect against rotavirus infection, and the rotavirus inhibition of these immunoproteins. It is reported that the effect depends on the concentration of Bifidus bacteria and the reaction time.

However, until now, the mechanism of preventing viral diarrhea through the intake of probiotic strains is not known, but according to the results of the research in the industry, the intestinal pathogenic bacteria and rotavirus infectious diarrhea of probiotic lactic acid bacteria The improvement and inhibitory effect of the probiotic lactic acid bacteria ① protective effect by increasing the organic acid in the intestine; ② protective effect by inhibiting the settlement of pathogenic bacteria by intestinal epithelial cell fixation and inhibition of macromolecular permeation; ③ growth inhibitory effect of pathogenic bacteria and rotavirus by inhibitors derived from lactic acid bacteria; ④ It is due to the inhibitory effect of immune enhancing effects such as the increase of phagocytosis of macrophage (macrophage) by the lactic acid bacteria, the increase of immune hormones, the increase of specific IgA by isotype switching of immune proteins, etc. Shown.

However, ingesting the probiotic microorganisms as described above may have a problem for storing the microorganisms before ingestion, and also when the bifidus bacteria die under the above conditions when the microorganism reaches the intestine, the activity There may be a problem such as reduced.

On the other hand, the present inventors confirmed that the prevention of the viral diarrhea by the bifidus lactic acid bacteria can be obtained by the peptidoglycan containing extract derived from the Bifidobacterium infantis MAEIL-K9 strain.

Bifidobacterium infantis MAEIL-K9 strain (KCTC 10294BP) is a strain found by the present inventors, a novel strain having excellent intestinal fixation ability and comprehensively excellent basic activity of probiotic lactic acid bacteria, By Bifidobacterium infantis MAEIL-K9 bifidus lactic acid bacteria and the health functional food containing the same filed by the same application.

According to the present invention, the peptidoglycan-containing extract derived from the Bifidobacterium infantis MAEIL-K9 strain may act as an intestinal fixation promoting factor for improving the intestinal fixation ability of the Bifidobacterial strain. As described above, in order for the rotavirus to function in the body, prerequisites for invading after settling into intestinal epithelial cells are required. On the other hand, in the intestine, bifidus bacteria and these pathogenic microorganisms form an intestinal flora and have mutually inhibitory activity. Therefore, if bifidus bacteria settle in the intestine and form superior intestinal flora, pathogenic microorganisms such as Escherichia coli and rotavirus are reduced in the intestinal colony by bifidus bacteria settled in the intestine, and thus these pathogenic microorganisms Can significantly reduce the expression rate of infectious diseases. Therefore, the peptidoglycan-containing extract according to the present invention acts as an intestinal fixation promoting factor of bifidus bacteria, thereby inhibiting the intestinal fixation of rotavirus relatively, thereby suppressing rotavirus infectious diarrhea.

Meanwhile, those skilled in the art, in addition to using the peptidoglycan-containing extract according to the present invention for the rotavirus infectious diarrhea described above, inhibit the intestinal settlement of any microorganisms (e.g., E. coli O157: H7) that settle in the intestine and cause infection. Thus, it can be readily appreciated that it can also be used to prevent or treat diseases caused by infection of these microorganisms.

According to the present invention, the higher the added amount of the peptidoglycan-containing extract of the present invention increases the fixing ability of the bifidus bacteria, but this effect provides more increased fixing ability to the bifidus bacteria having intestinal fixing ability, Poor bifidus strains show no enhancement effect. Therefore, when orally administering bifidus bacteria and the peptidoglycan-containing extract according to the present invention, which are known to have excellent intestinal fixation ability at the same time, rotavirus infectious diarrhea can be more markedly suppressed.

On the other hand, peptidoglycan (peptidoglycan) is one of the important components of the cell wall, muramic acid (muramic acid) (glucosamine) (glucosamine) (glucosamine), alanine (alanine), glutamic acid (glutamic acid), ornithine (ornithine), Or lysine. In addition, sugars constituting the cell wall include glucose, galactose, rhamnose, and the like. The composition of the peptidoglycan constituting the cell wall of Bifidus bacillus shows some difference depending on the subspecies of Bifidus bacillus. For example, Bifidobacterium bifiderm is a tetrapeptide peptide consisting of L-alanine, D-glutamic acid-amide, L-ornithine, and D-alanine. Bifidobacterium adolescents contain lysine or ornithine in a tetrapeptide. In addition, Bifidobacterium infantis and Bifidobacterium breve have lysine in the tetrapeptide (tetrapeptide), and Bifidobacterium long gum contains ornithine in the tetrapeptide (tetrapeptide) Doing.

According to the present invention, the peptidoglycan-containing extract according to the present invention has the above characteristics, and thus can be obtained by enzymatic treatment with a suitable protease or lysozyme. In addition, the peptidoglycan-containing extract may be subjected to selective physical disruption (eg, "Polytron" or "French press" to destroy cell walls to remove cell internals) or ultrasonic disruption before the enzyme treatment. . The present invention showed that the extracted peptidoglycan-containing extracts had very good activity when the strains were treated with enzyme treatment and selective physical force. However, according to the present invention, the peptidoglycan-containing extract extracted by performing only physical disruption did not have any activity as an intestinal fixation promoting factor. In addition, lyophilization may be carried out to facilitate storage of the peptidoglycan-containing extract according to the present invention.

On the other hand, the present invention provides a composition for preventing or treating rotavirus infectious diarrhea, which contains a peptidoglycan-containing extract derived from Bifidobacterium infantis MAEIL-K9 as an active ingredient.

In the composition for preventing or treating rotavirus infectious diarrhea of the present invention, it is preferable to orally administer the peptidoglycan-containing extract derived from Bifidobacterium infantis MAEIL-K9 as an active ingredient.

In addition, the composition according to the present invention may further comprise bifidus lactic acid bacteria commonly known to have excellent intestinal fixation ability.

On the other hand, the preparation for administering the peptidoglycan-containing extract of the present invention within the above range may have a conventional form, for example, can be used in the form of pills, capsules or drinks, medicines and the like.

Henriksson et al. (Henriksson, A., R. Szewzyk, and PLConway. 1991. Characteristics of the adhesive determinants of Lactobacillus fermentum 104. Appl. Environ. Microbiol. 57: 499-502) And Greene and Klaenhammer (Greene, JD and TRKlaenhammer. 1994. Factors involved in adherence of lactobacilli to human Caco-2 cells.Appl.Environ.Microbiol. 60 (12) (4487-4494) reported that when Lactobacillus cells were treated with proteolytic enzymes, they showed different effects on fixation ability according to the types of the enzymes. This fact means that the peptidoglycan-containing extract, which is a kind of cell wall component, acts as a promoter for fixation in the settlement of microorganisms against intestinal epithelial cells. Therefore, the peptidoglycan-containing extract of Bifidobacteria can act as a primary protective barrier in the intestine of the human body, and has no side effects compared to any vaccine and many chemicals, and thus, natural supplements in addition to the prevention and treatment of diarrhea. As can be seen that sufficient functionality can be exhibited.

It is also well known that extracts containing peptidoglycan derived from the cell walls of Gram positive microorganisms are immune enhancers, and peptidoglycan containing extracts are commonly used to treat various immune deficiencies in humans. . Namioka et al. (Namioka, S., T. Sasaki, and Y. Maede. 1991. Immunopotentiation of the small intestine of weaning piglets by peptidoglycan derived from Bifidobacterium thermophilum.Bifidobacteria Microflora. 10 (1): 1-9). Peptidoglycan-containing extract derived from Leeum thermofilum was investigated to show that the peptidoglycan-containing extract is effective in the treatment of postnatal diarrhea caused by Escherichia coli and bifidus lactobacillus. Reported.                     

In addition, Namba et al. (Namba, Y., Y. Hidaka, K. Taki, and T. Morimoto. 1981. Effect of oral administration of lysozyme or digested bacterial cell walls on immunostimulation in guinea pigs.Infect. Immun. 31 (2) : 580-583) studied the immune enhancement of guinea pigs following oral administration of microbial enzymes and microbial cell wall lysates. As a result, cellular immunity is increased in the treatment group orally administered with lysozyme and pronase together, and the extract containing peptidoglycan derived from Bifidobacterium longgum is included. It has been found that the oral administration of cell wall lysates increases the immune response in body fluids. Sasaki et al. (Sasaki, T., S. Fukami, and S. Namioka. 1994. Enhanced resistance of mice to Escherichia coli infection induced by administration of peptidoglycan derived from Bifidobacterium thermophilum. J. Vet. Med. Sci. 56 (3): 433-437) showed that the survival rate against E. coli was reduced when 500 μg of pepidoglycan-containing extract derived from Bifidobacterium thermofilum was applied to rats. It is said to be enhanced by glycan-containing extract supplements.

Hereinafter, the present invention will be described in more detail based on Examples. The following examples are provided to facilitate understanding of the present invention, and the scope of the present invention is not limited to these examples.

Example 1 Preparation of Experimental Strains and Peptidoglycan-Containing Extracts

The bifidus lactic acid bacteria used in this example were Bifidobacterium bifidum MAEIL-M3, Bifidobacterium longgum MAEIL-M8, a isolate derived from Korean, Bifidobacterium adolescents MAEIL-K8 and Bifi separated at the clinical site. Gamble bacterium Infantis MAEIL-K9 was used. On the other hand, rotavirus was used in the experiment of one species derived from human (S2) and two species derived from cow (NCDV, JBR) stored in the dairy processing laboratory of Konkuk University.

The separation method of the peptidoglycan-containing extract derived from bifidus lactic acid bacteria is as follows. 100 ml of Bifidus bacteria (0.1% inoculation of Bifidus bacteria) were inoculated into mMRS broth, anaerobic incubation at 37 ° C for 24 hours, followed by centrifugation (3,500 rpm / 15 min) to separate only the cells, and then PBS (pH 6.6) was removed. Adjusted to 5 ml. After grinding at 12,000 rpm / 5 minutes using Polytron (PT3000, Swiss), centrifugation (3,500 rpm / 15 minutes), the precipitate was washed twice and the volume was readjusted to 2 ml (recalibration buffer). Is, for example, PBS (pH 6.6). 0.1% lysozyme and 0.2% proteinase (pronase) were added to 2 ml of the bacterial solution, incubated at 37 ° C. for 48 hours, and stored by lyophilization. In subsequent experiments, the lyophilized peptidoglycan-containing extracts were diluted with PBS, stirred for 30 minutes, sonicated for 15 minutes and then used in the experiment.

Example 2 Fixation Enhancement Effect of Peptidoglycan-Containing Extract from Bifidus Bacteria

When explaining the specific experimental method of the present Example, incubate Caco-2 cells inoculated 100ml (1 × 107 cfu / well) of bacterial suspension (OD 1.0) in a 24 well plate formed with a monolayer (5 × 1) CO2 incubator After the reaction was carried out for 1 hour and 30 minutes, the fixing ability was measured by the plate method (Konishi et al., 1996). Bifidobacteria and E. coli O157: H7 were suspended in PBS suspension of the maximum vitality strain was corrected to OD 1.0, the bacterial count was measured using BLHB agar (containing 5% horse blood).

The difference between the Bifidobacterium infantis MAEIL-K9 strain and the E. coli O157: H7 strain alone or mixed settle bacteria according to the concentration of peptidoglycan-containing extract derived from Bifidobacterium infantis MAEIL-K9 Table 1 shows.

Reaction method Experimental strain Reaction bacteria count (cfu / well) Non-PGN ^a PGN500 ^b Mounting bacteria (cfu / well) Settlement Rate (%) Settlement bacteria (cfu / well) Settlement Rate (%) A Bifidobacterium infant MAEIL-K9 1.1 × 10 ⁷ 1.9 × 10 ⁵ 1.73 5.4 × 10 ⁵ 4.91 Escherichia coli O157: H7 3.7 × 10 ⁷ 9.8 × 10 ⁵ 2.65 9.9 × 10 ⁵ 2.68 B Bifidobacterium infant MAEIL-K9 1.1 × 10 ⁷ 1.8 × 10 ⁵ 1.64 6.1 × 10 ⁵ 5.55 Escherichia coli O157: H7 3.7 × 10 ⁷ 6.9 × 10 ⁵ 1.86 6.1 × 10 ⁵ 1.65 C Bifidobacterium infant MAEIL-K9 1.1 × 10 ⁷ 1.6 × 10 ⁵ 1.45 5.9 × 10 ⁵ 5.36 Escherichia coli O157: H7 3.7 × 10 ⁷ 1.7 × 10 ⁵ 0.46 3.2 × 10 ⁵ 0.86 A: reacting a Bifidobacterium and Escherichia coli simultaneously in Caco-2 cells, B: After the reaction for the bifidobacteria two hours of E. coli reaction C: After the reaction of E. coli for two hours bifidobacteria reaction ^a peptidoglycan-containing extract with no additives ^b peptidoglycan glycinate Add Can extract containing (500㎍ / well)

In single or mixed settling experiments, bifidus bacteria showed a rapid increase in the concentration of extract containing peptidoglycan to 100 μg / well. This is because Bock et al. (Bock, K., MEBreimer, A.Brignole, GCHansson, KAKarlss, G. Larson, who reported that oligosaccharide-containing glycoproteins and glycolipids in the cell membrane components of Bifidobacteria can be microbial fixation receptors). , H.Leffler, BCSamuelsson, C. Stromberg Svanborg-Eden, and J. Thurin. 1985. Specificity of binding of strain of uropathogenic E. coli to αGal (1,4) Gal containing glycosphingolipids.J.Biol.Chem.260 (8545-8551), Chadee et al. (Chadee, K., MLJohnson, E.Orozco, WAPetri, and J. Ravdin. 1988. Binding and internalization of rat colonic mucins by the galactose / N-acetyl-D-galactosamine adherence lectin of Entomoeba histolytica.J.Infect.Dis. 158: 398-406), Krivan et al. (Krivan, HC, GFClark, DFSmith, and TDWilkins. 1986.Cell surface binding site for Clostridium difficile enterotoxin: Evidence for a glycoconjugate containing the sequence αGal (1,3) βGal (1,4) GlcNAc.Infect. Immun. 53: 573-581). Meanwhile, while the number of bifidus bacteria increased significantly, the number of E. coli bacteria rapidly decreased. In particular, the decrease in the number of bacteria at 1,000 μg / well seems to be because the monolayer in Caco-2 cells was slightly damaged by osmotic pressure as the concentration of the peptidoglycan containing extract was increased.

From these results, it is not clear whether the peptidoglycan-containing extract according to the present invention affects the settlement of E. coli O157: H7. Can be.

On the other hand, when lactic acid bacteria are pre-established in the intestine, the protective effect against pathogenic bacteria becomes greater, and Gibson and Wang (Gibson) show that a large number of Bifidobacteria act as a barrier against the growth of foreign pathogens. GR and X. Wang. 1994. Regulatory effects of bifidobacteria on the growth of other colonic bacteria. J. Appl. Bacteriol. 77: 412-420), considering the results of the peptidoglycan-containing extract according to the present invention, It can be seen that the effect of enhancing the fixation of bifidus bacteria by the addition can provide a primary protective barrier of the intestinal lining, thereby increasing the inhibition of the settlement of pathogenic bacteria.

In addition, Sasaki et al. (Sasaki, T., S. Fukami, and S. Namioka. 1994. Enhancement of mice to Escherichia coli infection induced by E. coli infection increased by feeding peptidoglycan-containing extracts. by administration of peptidoglycan derived from Bifidobacterium thermophilum.J.Vet.Med.Sci.56 (3): 433-437) and in vivo experiments to select lactic acid bacteria that inhibit the growth of E. coli O157: H7. Zhao et al. (Zhao, T., MPDoyle, BGHarmon, CABrown, POEMueller, and AH) reported that when administered to calves prior to E. coli O157: H7 infection, infection with E. coli O157: H7 could be inhibited. Parks. 1998. Reduction of carriage of enterohemorrhagic Escherichia coli O157: H7 in cattle by inoculation with probiotic bacteria.J.Clin.Microbiol. 36 (3): 641-647), according to the present invention, peptidogly The extract containing cans simultaneously enhances the anchoring effect of Bifidobacteria and the immune enhancing effect of the host. I can see that I am losing.

Example 3 Changes in Fixation Capacity of Peptidoglycan-Containing Extracts According to Bifidus Species

Specific experimental method of the present Example is the same as in Example 2, and was added and reacted with the addition of the same amount of 0.1% peptidoglycan extract at 100 ㎍ / well concentration at the time of inoculation of the bacteria suspension.

As shown above, it can be seen that the peptidoglycan-containing extract is effective as a factor for promoting the establishment of bifidus bacteria. Therefore, in the present embodiment was performed to determine the degree of promoting the settlement of the peptidoglycan-containing extract according to the origin of the bifidus species.

Settle each peptidoglycan containing extract extracted according to the method of Example 1 from Bifidobacterium long gum MAEIL-M8, Bifidobacterium adolescents MAEIL-K8, Bifidobacterium infantis MAEIL-K9 Settlement crossover experiments were performed with Bifidobacterium longgum MAEIL-M8, Bifidobacterium adolescents MAEIL-K8, and Bifidobacterium inpantis MAEIL-K9 strains. The results are shown in Table 2 below.

Sources Bifidobacterium long gum MAEIL-M8 Bifidobacterium adolescentes MAEIL-K8 Bifidobacterium infant MAEIL-K9 cfu / well % ^* cfu / well % cfu / well % Non-PGN ^a 9.5 × 10 ³ 0.04 4.5 × 10 ⁵ 2.50 4.1 × 10 ⁵ 3.72 M8-PGN100 ^b 7.5 × 10 ³ 0.03 9.6 × 10 ⁵ 5.33 1.8 × 10 ⁶ 16.36 K8-PGN100 ^c 5.0 × 10 ³ 0.02 1.0 × 10 ⁶ 5.55 1.4 × 10 ⁶ 12.72 K9-PGN100 ^d 5.0 × 10 ³ 0.02 8.6 × 10 ⁵ 4.77 1.3 × 10 ⁶ 11.81 ^* Jeongchakyul ^a peptidoglycan-free extracts containing ^b Bifidobacterium ronggeom peptidoglycan-containing extract derived from the MAEIL-M8 (100㎍ / well) ^c Bifidobacterium aedol sentiseu also containing glycans of peptides derived MAEIL-K8 Extract (100 µg / well) ^d Bifidobacterium infantis Peptidoglycan containing extract from MAEIL-K9 (100 µg / well)

As shown in Table 2, all strains except for Bifidobacterium longgum MAEIL-M8 strain showed an increase in fixation, and the extract containing peptidoglycan derived from Bifidobacterium longgum MAEIL-M8 with low fixation ability In addition, all strains except Bifidobacterium longgum MAEIL-M8 showed an increase in fixation. As shown in the results, it was found that there is no difference in activity of the peptidoglycan-containing extracts according to the bifidus species, but it was found that the strains with low fixation ability did not show an intestinal fixation enhancing function. In addition, the Bifidobacterium infantis MAEIL-K9 strain was found to have a significantly higher intestinal fixation rate than the peptidoglycan-containing extract addition group compared to the no addition group. From these results, it was found that the peptidoglycan-containing extract derived from the bifidus bacteria did not act as an anchorage promoting factor of the Bifidobacterium longgum MAEIL-M8 strain, but acted as a more reliable anchorage factor for other strains having good anchorage ability. Can be.

Example 4 Rotavirus Inhibitory Effect According to Concentration of Bifidus-derived Material

Referring to the specific experimental method of the present embodiment, Kaljot et al. (Kaljot, KT, RDShaw, DH, etc.), in which inoculated and cultured MA-104 cells were added to Bifidobacterium microorganism and PGN in a 96 well plate in which a monolayer was formed. Rubin, and HB Greenberg, 1988. Infecrious rotavirus enters cells by direct cell membrane penetration, not by endocytosis.J. Virol. 62 (4): 1136-1144) was applied and measured by AEC staining.

Fig. 3 shows the inhibitory effect of rotavirus according to the concentrations of the peptidoglycan-containing extract derived from Bifidobacterium infantis MAEIL-K9 and the dead cell mass (DCM, dead cell mass). The inhibitory effect of the peptidoglycan containing extract derived from Dobacterium infantis MAEIL-K9 is shown in FIG. 2. The reason why the experiment was carried out for the dead cells was because dead dead cells could settle in intestinal epithelial cells and play a role of defense against rotavirus infection.

When 25.6% of the peptidoglycan-containing extract and the mycelium component suspension of the present invention were added to the total M199 medium, the rotavirus inhibitory effects were 99.1% and 85.4%, respectively. In addition, the inhibitory effect when 0.2% was added was 41.8% and 35.8%, respectively. From these results, the rotavirus inhibitory effect was clearly seen in both test substances, but the peptidoglycan-containing extract was found to be superior to the rotavirus in comparison to the mycelial component.

This phenomenon is described by Matsumoto et al. (Matsumoto, S., H. Ogawa, T. Kusama, O. Nagase, N. Sawaki, M. Inabe, S. Kusumoto, and T. Shiva. 1981. Stimulation of nonspecific resistance to infection induced by infection 6-o-acyl muramyl dipeptide analogs in mice.Infect. Immun. 32: 748-758) reported that Peptidoglycan-containing extracts have therapeutic effects according to immunopotentiation in vivo. The rotavirus inhibitory effect of the derived peptidoglycan-containing extract is expected to be further weighted in vivo.

On the other hand, it can be seen from the results in Table 3 that the inhibitory effect of rotavirus infection by the bacteriostatic component is similarly recognized in the ability of the bacteriostatic component and live cells of lactic acid bacteria.

Experimental strain Reputation Direct microscopy LCM ^a (cfu / well) LCM (/ 100 cell count ^* ) DCM ^b (/ 100 cell count)  Bifidobacterium Bifidum MAEIL-M3 1.6 × 10 ⁴ 71 35  Bifidobacterium long gum MAEIL-M8 1.0 × 10 ³ 19 4  Bifidobacterium adolescentes MAEIL-K8 1.8 × 10 ⁵ 114 57  Bifidobacterium infant MAEIL-K9 2.4 × 10 ⁵ 188 79 ^a living organism of bifidus ^b heat treated bifidus cells ^* Number of settled bacteria per 100 MA-104 cells

From FIG. 3, the inhibitory effect on the rotavirus S2 derived from the human body and the JBR and NCDV rotavirus derived from the animal were excellent at 99.0%, 99.0%, and 99.1%, respectively, when the concentration of the extract containing peptidoglycan was 25.6%. When the concentration of the extract containing peptidoglycan was 0.2%, 21.1%, 22.0%, and 41.8%, respectively, were found to have inhibitory effects, depending on the type of rotavirus.

Example 5 Effect of Inhibiting Rotavirus Infection According to Bifidobacteria

Referring to the specific experimental method of this embodiment, inoculating and incubating MA-104 cells inoculated and settled by inoculating live cells of bifidus bacteria in 96 well plate formed with a monolayer (monolayer), and after 2 hours 3 with serum-free M199 medium 3 Washed twice and stained for AEC. In addition, the inhibitory effect on rotavirus was observed by adding live cells of cPGN and bifidus.                     

Various rotavirus infection inhibitory effects by bifidus bacteria are shown in FIG. 3. In all the rotaviruses used in the experiment, the effect of probiotic bacteria was recognized, but NCDV showed a low inhibitory range according to the concentration change. This phenomenon seems to be characteristic of rotavirus itself. In addition, the synergistic effect of the rotavirus inhibitory effect and the peptidoglycan-containing extract as the inhibitory material according to the number of bifidus microbial cells is shown in FIG.

As shown in FIG. 1, it can be seen that the higher the number of the settled bacteria of Bifidobacterium infantis MAEIL-K9 strain, the greater the inhibitory effect on rotavirus. In addition, the mixed use of the peptidoglycan-containing extract and the bifidus microbial bacteria was shown to have a better rotavirus inhibitory effect.

These results indicate that the infectious virus or bacterium competitively binds to the probiotic lactic acid bacteria and the anchoring site of epithelial cells. It can be seen that it prevents the infection of viruses and bacteria.

Example 6 Change in Enhancement of Settlement of Bifidus Bacteria by Extraction Method of Peptidoglycan-Containing Extract

The specific experimental method of this embodiment is the same as in Example 4.

In this example, in order to confirm whether the extraction conditions of the peptidoglycan-containing extract is related to the fixing enhancement effect of Bifidus bacteria, seven samples were extracted under the conditions shown in Table 4 below. Where PGN-1 is the control, PGN-2 is the sonication only, PGN-3 is the physical destruction and sonication, PGN-4 and 5 are the enzyme treatment only, and PGN-6 and 7 are the physical destruction and enzyme treatment and sonication. Means a group.

PGN-1 PGN-2 PGN-3 PGN-4 PGN-5 PGN-6 PGN-7 Physical shredding - - + - - + + Enzyme treatment Lysozyme - - - 0.05% 0.01% 0.05% 0.01% Protease - - - 0.15% 0.05% 0.15% 0.05% Ultrasonic fracturing - + + - - + + +: Treatment,-: no treatment

The extraction method of the extracted peptidoglycan-containing extract and the particle size change according to pH are shown in FIG. 4. The enzyme-free group (PGN-1, 2, 3) showed little change in solubility according to the pH change, while the enzyme-treated samples (PGN-4, 5, 6, 7) had a particle size at pH 7 or higher. It was found that the solubility was increased with decreasing the.

Table 5 shows the results of confirming the influence on the enhancement of fixation of the Bifidobacterium infantis MAEIL-K9 strain between the groups having a difference in particle size and solubility.

sample pH 6.0 pH 7.2 % Settlement Settlement growth rate (%) % Settlement Settlement growth rate (%) Non-PGN 1.00 0 1.31 0 PGN-01 1.05 +5 1.17 -11 PGN-02 0.97 -3 1.09 -17 PGN-03 0.99 -One 1.11 -15 PGN-04 3.54 +354 7.08 +540 PGN-05 3.54 +354 6.08 +464 PGN-06 3.23 +323 3.62 +276 PGN-07 3.54 +354 3.46 +264 Non-PGN: No extract containing peptidoglycan PGN-01 ~ 07: Samples with extract containing peptidoglycan according to various extraction methods

From the results in Table 5, it was shown that the enzyme enhancer effect is higher in the enzyme treatment group than the non-enzyme group, and the solubility of the peptidoglycan-containing extract is increased under the conditions of pH 7.0 or higher, and only the enzyme is treated under such conditions. When one peptidoglycan-containing extract (PGN-4 and 5) is used, it can be seen that fixation enhancement is the highest.

Comparing these conditions, it is preferable that the enzymatic method be used in mass production because the enzymatic method is superior to the physical bombardment method as the processing condition necessary for producing the peptidoglycan-containing extract from the microbial cells. It is judged that the conditions for mass production are set.

 As described above, the peptidoglycan-containing extract derived from Bifidobacterium infantis MAEIL-K9 according to the present invention acts as an intestinal fixation promoting factor, thereby enhancing the intestinal fixation ability of Bifidobacterium in the intestine. It can be seen that the population of Bifidobacterium is more superior to the rotavirus, thereby suppressing the infection of rotavirus. Therefore, the composition according to the present invention can be usefully used for preventing or treating rotavirus infectious diarrhea.

Claims (4)

Peptido derived from Bifidobacterium infantis MAEIL-K9 strain (KCTC 10294BP), characterized in that it is extracted by a method comprising treating the Bifidobacterium infantis MAEIL-K9 strain with lysozyme or protease. Glycan containing extract. The method of claim 1, Peptidoglycan-containing extract, characterized in that the extraction by the method further comprising the treatment by physical disruption or ultrasonic disruption before the enzyme treatment. A composition for preventing or treating rotavirus infectious diarrhea, characterized in that it contains the peptidoglycan-containing extract of claim 1 or 2 as an active ingredient. 4. The composition according to claim 3, wherein the composition further contains bifidus bacteria having enteric fixing ability.

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