KR20220099914A - Composition for Activating Probiotics through Inhibition of Harmful Bacteria - Google Patents

Abstract

The present invention relates to a prebiotics composition that activates probiotics and inhibits the growth of harmful bacteria, wherein the prebiotics composition inhibits the proliferation of intestinal harmful or neutral bacteria to create an environment in which the probiotics can dominate in intestines, can inhibit the proliferation of the harmful bacteria, thereby preventing or treating diseases caused by food poisoning or infection with pathogenic bacteria, helps the intestinal dominance of the probiotics to be able to expect excellent effects in consideration of effects exhibited with the same dosage or can reduce administration cost by controlling the number of administered lactic acid bacteria.

Description

Composition for Activating Probiotics through Inhibition of Harmful Bacteria

The present invention relates to a prebiotic composition that activates probiotics and inhibits the growth of harmful bacteria, and more particularly, contains glucose oxidase as an active ingredient. It relates to a composition and a synbiotics composition containing the prebiotics and lactic acid bacteria.

Probiotics are live bacteria that improve the balance of microbes in the gut and promote the health of the host. Probiotics are known to enhance the availability of several minerals, synthesize vitamins, prevent pathogens that cause diseases in the intestine, boost immunity, and aid in protein digestion and absorption. Prebiotics are small-molecular fibers that are not broken down by digestive enzymes in the stomach and small intestine. A substance that improves the health of the host by selectively using useful microorganisms in the large intestine. Raffinose, oligosaccharides, fructooligosaccharides, inulin, oligosaccharides such as galactooligosaccharides are representative (Gibson GR et al., Nature Reviews. Gastroenterology & Hepatology . 14:491, 2017).

Probiotics and prebiotics are known to affect human health, and the combination of probiotics and prebiotics creates synergy, and this combination is called synbiotics. Probiotics are live bacteria that improve the balance of microbes in the gut and promote the health of the host. Probiotics are known to enhance the availability of several minerals, synthesize vitamins, prevent pathogens that cause diseases in the intestine, boost immunity, and aid in protein digestion and absorption. In general, probiotics are ingested by yogurt or fermented food. Recently, they are being sold in various forms such as capsules and powder. Prebiotics are indigestible ingredients that help the growth of beneficial strains in the intestine, and are substances that help improve the intestinal environment by becoming a nutrient source for probiotics. Typical prebiotics include dietary fibers such as inulin, fructooligosaccharides, and galactooligosaccharides.

Most of the currently used representative probiotics strains have high production costs, and in order to dominate the intestinal conditions, a sufficient amount of probiotics and prebiotics that feed the beneficial bacteria are supplied and the growth of beneficial bacteria The composition of the intestinal environment suitable for In the case of lactic acid bacteria, a representative probiotic, a high production cost occurs due to the limitation of high-concentration culture and the application of a coating formulation to impart resistance to gastric and bile acids.

Therefore, in the present invention, as a result of diligent efforts to develop a prebiotic composition that regulates the intestinal environment to be suitable for the growth of beneficial bacteria, intestinal bacteria (E. coli) and lactobacillus are cultured together under the condition of adding glucose oxidase In this case, it was confirmed that the growth of lactic acid bacteria is superior to intestinal bacteria, and the present invention has been completed.

It is an object of the present invention to provide a prebiotic composition that inhibits the growth of harmful bacteria in the intestine and activates lactic acid bacteria.

Another object of the present invention is to provide a synbiotic composition containing the prebiotics and lactic acid bacteria.

In order to achieve the above object, the present invention provides a prebiotic composition for inhibiting the growth of harmful intestinal bacteria and activating lactic acid bacteria comprising glucose oxidase as an active ingredient.

The present invention also provides a synbiotics composition containing the prebiotic composition and lactic acid bacteria.

The present invention also provides a food containing the prebiotic composition and lactic acid bacteria as active ingredients.

The present invention also provides a feed additive containing the prebiotic composition and lactic acid bacteria as active ingredients.

The prebiotic composition according to the present invention creates an environment in which probiotics can dominate the intestine by inhibiting the proliferation of harmful bacteria or neutral bacteria, and can inhibit the proliferation of harmful bacteria, thereby preventing diseases caused by food poisoning or infection of pathogenic bacteria Or it can help in treatment, and by helping the intestinal dominance of probiotics, an excellent effect can be expected compared to the same dose, or it has the advantage of reducing the administration cost by controlling the number of administered lactic acid bacteria.

1 shows the results of confirming the growth effect on each prebiotic lactic acid bacteria ( L. gasseri BNR17 strain and L. plantarum ).
Figure 2 shows the results of confirming the growth rate change by the addition of galactooligosaccharide of harmful intestinal bacteria ( E. coli and S. flexneri ).
Figure 3 shows the results of comparing the survival rate for each concentration of hydrogen peroxide (H ₂ O ₂ ) for lactic acid bacteria ( L. gasseri BNR17 strain and L. plantarum ) and intestinal harmful bacteria ( E. coli and S. flexneri ).
Figure 4 is a result of comparing the survival rate (survival rate) after 48 hours according to the glucose oxidase treatment concentration for lactic acid bacteria ( L. gasseri BNR17 strain and L. plantarum ) and intestinal harmful bacteria ( E. coli and S. flexneri ) it has been shown
Figure 5 shows the results of confirming the changes in the intestinal microflora of obese dogs fed the composition according to the present invention.

Most of the currently used probiotics strains have high production costs, and in order to dominate the intestinal conditions, it is necessary to supply a sufficient amount of probiotics and prebiotics to feed the beneficial bacteria and the growth of beneficial bacteria. The composition of an appropriate intestinal environment should be considered. In the case of lactic acid bacteria, which are probiotics, high production cost occurs due to the limitation of high-concentration culture and the application of coating formulations to impart resistance to gastric and bile acids.

_In the present invention, it was attempted to develop a material that can change _the intestinal environment to be suitable for beneficial bacteria to grow. In the case of treatment with glucose oxidase, which can generate

Accordingly, the present invention, in one aspect, relates to a prebiotic composition for inhibiting the growth of harmful intestinal bacteria and activating lactic acid bacteria comprising glucose oxidase as an active ingredient.

In the present invention, the glucose oxidase (glucose oxidase) can be used without limitation as long as it is an enzyme capable of generating hydrogen peroxide by glucose oxidation, preferably Aspergillus (Aspergillus) sp. strain, Penicillium (Penicillium) Enzymes derived from a strain selected from the group consisting of sp. strains and Streptomyces sp. strains or a combination thereof may be used.

In the present invention, the glucose oxidase (glucose oxidase) may be preferably included to act as 0.01 ~ 1 unit in 1 ml of the intestinal fluid.

The prebiotics of the present invention may further include galactooligosaccharide.

In one aspect of the present invention. The addition of prebiotics such as galactooligosaccharide (GOS), fructooligosaccharide (FOS), polydextrose and indigestible maltodextrin The effect on growth was confirmed, and both the L. gasseri BNR17 strain and the L. plantarum strain used in the experiment had the best use of GOS among prebiotics, and showed higher growth compared to the control group (FIG. 1).

The Lactobacillus gasseri BMR17 strain, a probiotic strain used in the examples of the present invention, is a representative strain identified in breast milk. It verified the body fat reduction, irritable bowel syndrome (IBS) treatment effect, and blood sugar strengthening effect, and published it through a number of papers and patents. It has already been recognized as a functional ingredient for health functional food.

In the present invention, the lactic acid bacteria may be characterized as lactic acid bacteria having resistance to hydrogen peroxide, preferably Bifidobacterium, Lactobacillus, Lactococcus or Streptococcus. It may be a caucus (Streptococcus).

Hydrogen peroxide (H ₂ O ₂ ) is a substance produced during energy metabolism and is typically produced by pyruvate oxidase, lactate oxidase, and glucose oxidase. Hydrogen peroxide has strong oxidizing properties, so it is mainly used for bleaching cotton, other textiles, wood pulp, etc., or for making fuel, cosmetics, and pharmaceuticals. Hydrogen peroxide is also synthesized and used by probiotic strains. In the intestinal environment, probiotic strains synthesize and secrete hydrogen peroxide to inhibit the growth of other microorganisms, thereby improving the intestinal environment.

In the present invention, the harmful bacteria Shigella flexneri ( Shigella flexneri ), Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Enterococcus faecalis ( Enterococcus faecalis ), Clostridium buty Likum ( Clostridium butyricum ), Prevotella intermedia ( Prevotella intermedia ) or Clostridium ramosum ( Clostridium ramosum ) It may be characterized as being.

Many harmful or opportunistic bacteria have lower resistance to hydrogen peroxide than lactic acid bacteria.

The present invention is for promoting the growth of lactic acid bacteria through the use of prebiotics of Lactobacillus lactic acid bacteria. In this application, for example, Lactobacillus lactobacilli and prebiotics, which are probiotics, were added to enhance growth, and a dominant effect due to competitive exclusion using enzymes was confirmed.

Accordingly, the present invention relates to a synbiotics composition containing the prebiotic composition and lactic acid bacteria from another aspect.

In one aspect of the present invention, the L. gasseri BNR17 strain and suitable prebiotics were identified, and synbiotics were made together to help the growth of the L. gasseri BNR17 strain. In addition, by using a glucose oxidase enzyme, H ₂ O ₂ was synthesized in the intestine to prevent the growth of strains other than L. gasseri BNR17, which has high H ₂ O ₂ resistance compared to other strains, thereby increasing the relative dominance to promote growth.

In the present invention, the lactic acid bacteria may be characterized as lactic acid bacteria having resistance to hydrogen peroxide, preferably, the lactic acid bacteria are Bifidobacterium, Lactobacillus, Lactococcus ) and lactic acid bacteria selected from the group consisting of Streptococcus or a combination thereof may be used.

More preferably, the lactic acid bacteria of the present invention is Lactobacillus gasseri ( Lactobacillus gasseri ), Lactobacillus pullantarum ( Lactobacillus plantarum ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus delbrueckii ( Lactobacillus delbrueckii ) And Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) Lactobacillus selected from the group consisting of or a combination thereof may be used.

In another aspect of the present invention, after ingesting a composition containing glucose oxidase and galactooligosaccharide as active ingredients, and L. gasseri BNR17 and L. plantarum as lactic acid bacteria to obese dogs for 10 weeks, weight and body fullness index and It was confirmed that all fat areas were significantly reduced.

Accordingly, in another aspect, the present invention relates to a food containing the prebiotic composition and lactic acid bacteria as active ingredients.

In the present invention, the lactic acid bacteria are Lactobacillus gasseri ( Lactobacillus gasseri ), Lactobacillus pullantarum ( Lactobacillus plantarum ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus delbruckii ( Lactobacillus delbrueckii ) and lactobacillus delbrueckii Rhamnosus ( Lactobacillus rhamnosus ) It may be characterized in that the lactic acid bacteria selected from the group consisting of or a combination thereof.

In the present invention, the food may be a food for human or animal intake, and may be a food for a human or animal diet supplement.

In another aspect, the present invention relates to a feed additive containing the prebiotic composition and lactic acid bacteria as active ingredients.

As the companion animal industry grows, the problem of companion animal obesity is caused, and the treatment cost of companion animals due to obesity-related diseases is increasing every year. Accordingly, it is necessary to manage the weight of companion animals.

In the present invention, the feed additive may be a health functional feed additive for animals or a feed additive for diet supplementation.

Hereinafter, the present invention will be described in more detail by way of Examples. These examples are merely for illustrating the present invention in more detail, and thereby it will be apparent to those skilled in the art that the technical scope of the present invention is not limited to these examples.

Example 1: Comparison of Lactobacillus Lactobacillus Growth Effect by Prebiotics

Lactobacillus by the addition of prebiotics galactooligosaccharide (GOS), fructooligosaccharide (FOS), polydextrose and indigestible maltodextrin . gasseri BNR17 (KCTC 109202BP) and Lactobacillus. plantarum (LALLEMAND, Canada) was confirmed to have an effect on growth.

As a medium for culturing lactic acid bacteria, modified MRS (mMRS) broth not containing glucose was used. mMRS medium is peptone 10 g, yeast extract 2.5 g, tryptose 3 g, Tween 80 1 ml, K ₂ HPO ₄ 3 g, KH ₂ PO ₄ 3 g, ammonium citrate 0.2 g, pyruvic acid sodium salt 0.2 g, 0.575 g of MgSO ₄ 7H ₂ O, 0.12 g of MnSO ₄ H ₂ O, and 0.034 g of FeSO ₄ 7H ₂ O were dissolved in distilled water, adjusted to 1 liter, and prepared by autoclaving (121° C., 15 min). . Cys-Hcl 0.5 g/L was added to the medium after filtration with a syringe filter (0.45 μm), and the pH of the final medium was adjusted to 6.8 and used as a culture medium. Cultivation was performed by adjusting the concentration of each carbon source to 0.5% during culturing. Before performing the main culture, each strain was selected from a single cultured agar plate, and 3ml liquid culture was performed at 24 hr, 37°C, 100 rpm. Thereafter, culture was performed in a 24-well plate (SPL life science) using a plate reader, SYNERGY H1 microplate reader. At this time, the plate film (Diversified biotech, Cas. BEM-1) was used. Culture conditions were 500 μl, 37° C., slow shaking, interval 30 min, and 1% seeding conditions.

As a result, as shown in Figure 1, the L. gasseri BNR17 strain showed a similar growth rate in all prebiotics added conditions used in the experiment. However, the carbon sources that showed better growth than the only mMRS condition without a carbon source were glucose and GOS, and growth similar to the only mMRS condition was confirmed under other prebiotic conditions. When comparing the control group, glucose condition and GOS condition, growth was gradually decreased under glucose condition, and continued growth under GOS condition. The growth rate was increased by about 20% or more in the GOS condition compared to the control group.

The L. plantarum strain showed insignificant growth in only mMRS conditions, and increased OD values in FOS, polydextrose and indigestible maltodextrin conditions than in only mMRS conditions. L. plantarum strain was found to be difficult to grow in the absence of a carbon source. The L. plantarum strain had an OD value about 1.5 to 2 times lower in FOS, polydextrose, and indigestible maltodextrin conditions when compared to glucose as a control. In contrast, the GOS condition showed a slower growth rate than the glucose condition, but the maximum OD was about 10% higher (Fig. 1). Both strains showed the highest availability of GOS among prebiotics, and showed high growth compared to the control group.

Example 2: Use of galactooligosaccharide (GOS) of harmful bacteria

In order to compare the growth promoting effect of Lactobacillus lactic acid bacteria, the growth effect was confirmed using intestinal microorganisms Escherichia coli (KCTC 1039) and Shigellar flexneri (KCTC 12073). The experimental method confirmed the growth rate in the same manner as in Example 1. E. coli and S. flexneri were studied using LB broth as a culture medium.

As a result, as shown in FIG. 2 , the E. coli and S. flexneri strains exhibited higher OD values under the glucose condition than under the GOS-added condition, and the growth rate under the GOS condition compared to the glucose condition was about the same as that of E. coli . 95% and S. flexneri showed growth rates of 92%. Both strains showed similar growth rates under glucose and GOS conditions, but the final growth rate was lower under GOS conditions.

In conclusion, when GOS , a prebiotic, is used as a carbon source, the growth of E. coli and S. flexneri strains, which are intestinal microbes, is lower than that of Lactobacillus . expected.

Example 3: Hydrogen peroxidase (H) of lactic acid bacteria and harmful bacteria ₂ O ₂ ) resistance check

As an experiment to increase the dominance due to the competitive exclusion effect of Lactobacillus, hydrogen peroxide (H ₂ O ₂ ) resistance was measured for each strain.

L. gasseri BNR17, L. plantarum, E. coli and S. flexneri strains were each purely cultured on an agar plate, inoculated in 3 ml broth, and cultured with shaking at 37 °C and 100 rpm for 24 hr. After that, the cultured strain was inoculated 5% in a new 20 ml broth and cultured under the same conditions. After 2 to 3 hours of incubation, when the OD of the culture medium was 0.8, the culture medium was centrifuged (4000 rpm, 5 min) to recover the bacteria. After discarding the supernatant, 2X broth was added to half of the culture medium to re-suspend the bacteria (resuspension). Each of the suspended bacteria was aliquoted in 1 ml each in a 2 ml tube.

To create an intestinal environment, simulated intestinal fluid was dissolved in DW to 2X, and filtration was performed using a syringe filter (0.45μm), and then 2X SSIF was added to each tube with 1X. was added as much as possible. The composition of artificial small intestine SSIF is g/L: 0.87 g NaOH, NaH ₂ PO ₄ 2H ₂ O 4.46 g, NaCl 3.48 g. H ₂ O ₂ concentration was added to 0-20 mM.

The hydrogen peroxide treatment concentration was set at a concentration of 0 to 10 mM. All experimental groups were reacted at 37 °C and 100 rpm, and at 0 min and 30 min of the experiment, each sample was plated on an agar plate. measured. The survival rate was calculated by comparing the number of viable cells at 30 min to the number of viable cells at 0 min of the reaction.

As a result, as shown in FIG. 3 , the L. gasseri BNR17 strain showed a gradual decrease in the survival rate from the H ₂ O ₂ concentration of about 8 mM, and showed a survival rate of 80% at the concentration of about 10 mM. Lb. gasseri BNR17 strain did not show a tendency to rapidly decrease the survival rate by concentration. Lb. The plantarum strain showed a similar number of viable cells to the control until the concentration of 6 mM, but as the concentration increased to 6 mM to 8 mM, the survival rate was found to drop sharply. Lb. gasseri BNR17 strain Lb. It was found that the resistance to hydrogen peroxide was higher than that of the plantarum strain.

The E. coli strain showed a survival rate of about 10% or less from a concentration of 2mM, and it was found that the strain did not survive from a concentration of 6mM or more. The S. flexneri strain showed a survival rate of 50% at a concentration of 0 to 2 mM, a survival rate of less than 10% at a concentration of 4 mM, and did not survive at a concentration of 8 mM or more.

As a result, the hydrogen peroxide resistance of Lactobacillus was higher than that of E. coli and S flexneri strains, suggesting that the dominant effect of Lactobacillus can be seen due to the competitive exclusion effect of intestinal microorganisms using hydrogen peroxide.

Example 4: Comparison of survival rates for each strain according to glucose oxidase treatment

Growth inhibitory effect using glucose oxidase to produce H ₂ O ₂ using glucose was confirmed.

L. gasseri BNR17, L. plantarum, E. coli and S. flexneri strains were cultured in broth at 24 hr, 37°C, 100 rpm. This culture was performed in a 24-well plate, and the culture conditions were adjusted to a total of 500 μl by adding 2X broth, 2X SSIF, glucose oxidase and glucose. Culture conditions were 24 hr, 37 °C, slow shaking, and culture was performed at 30 min interval. After dissolving glucose oxidase (sunson) at a concentration of 1 U/ml, filtration was performed using a syringe filter (0.45 μm). The amount of glucose oxidase added was in the range of 0.01~0.05 U/ml, and the OD value was measured using a Synergy H1 instrument. compared.

As a result, as shown in FIG. 4, Lb. gasseri BNR17 strain showed no effect on the growth rate when the enzyme concentration was 0~0.02 U/ml, and the growth rate decreased from 89% to 70%, respectively, as the concentration increased from 0.03 to 0.05 U/ml. Lb. plantarum strain showed a tendency to continuously decrease from the concentration of 0.01 U/ml when compared to the control condition, and showed a growth rate of about 80% at the concentration of 0.03 to 0.05 U/ml. E. coli strain was shown to have little effect at a concentration of 0 ~ 0.02 U / ml, but it was found that it did not grow at a concentration of 0.03 U / ml or more. S. flexneri strain showed a similar growth rate at a concentration of 0 to 0.03 U/ml, but it was found that the growth rate dropped to 90% at a concentration of 0.04 U/ml, and it did not grow at a concentration of 0.05 U/ml or more.

As a result, when the concentration of glucose oxidase is 0.05 U/ml, it was found that the intestinal microbes, E. coli and S. flexneri strains, did not grow, and on the contrary, it partially affects the growth of Lactobacillus strains, but is resistant to other strains. It is believed that this is excellent and will help dominance of the intestine.

Example 5: Confirmation of the effect of intake of the composition of the present invention in obese dogs

After ingesting a mixed composition of lactic acid bacteria and prebiotics for healthy obese dogs without disease, body changes were confirmed. The mixing ratio of the prepared composition is shown in Table 1.

composition ratio Raw material mixing ratio standard manufacturing company country of manufacture L. gasseri BNR17 3.00% ≥ 1.0 X 10 ¹⁰ cfu/g Bioneer korea L. plantarum 1.70% ≥ 1.7 X 10 ¹¹ cfu/g Lallemand canada Galactooligosaccharide 6.00% ≥ 90% Quantum Hi-Tech Biological co., ltd china fructooligosaccharide 6.00% ≥ 95% Quantum Hi-Tech Biological co., ltd china indigestible maltodextrin 23.15% ≥ 85% Daesang korea polydextrin 60.00% ≥ 90% Samyang korea glucose oxidase 0.15% > 850 U/g Sunson china

Experimental animals were conducted on healthy obese dogs regardless of age and sex. From week 0 to week 10, 1 g of a composition for activating probiotics containing probiotics was mixed with the feed and fed to confirm the change of microorganisms.

Body weight was measured using the same digital scale before and after treatment with the composition, and at the time points at 0 weeks and 10 weeks respectively. The evaluation of the body condition score (BCS) was conducted by the same observer before and after the composition treatment at 0 weeks and 10 weeks respectively. In puppies of various body types, the body fidelity index can systematically evaluate the degree of obesity. There are two body fidelity index systems, ranging from 1 to 5 and 1 to 9, but the 1 to 9 scale system is preferred to identify subtle changes in body weight. This scoring requires visualization and facilitation. The degree of visual obesity was evaluated along the line of the waist bent behind the rib cage. In dogs of normal weight, the ribs are easily palpable. This evaluation was conducted by placing the thumb on the spine, palpation of the rib cage with the fingers, and palpation of the fat layer just below the ribs. The body fidelity index was determined by synthesizing the evaluations made in the same way as above. In order to determine whether the dog has reached the ideal weight during obesity management, both the body fidelity index and the actual weight were considered.

Complete blood counts (leukocytes, neutrophils, etc.) were performed with blood collected from the jugular vein, and serum was separated, alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), blood urea nitrogen (BUN), cholesterol, C-reactive protein ( CRP), etc. were measured, and the same measuring machine was used at the time of the 0th week and the 10th week respectively.

Right abdominal radiography was performed using a digital radiography system. Subcutaneous fat thickness (ST) was measured at the level of the third lumbar vertebrae (L3) and sixth lumbar vertebrae (L6) on lateral radiographs. To explain the different body shapes of each test subject, the thickness ratio of subcutaneous fat (rST) was calculated as the ratio of the thickness of subcutaneous fat to the body length of each lumbar vertebrae (L3, L6). Radiography was performed with the same machine at weeks 0 and 10.

Scans were performed using a 32-row multi-detector CT scanner. Body fat content regions were determined by analyzing digital image data sets obtained from individual subjects. The total area (TA) and subcutaneous fat area (SA) were estimated by manually drawing the region of interest around the body from the CT transverse cross-sectional images of L3 and L6. To evaluate the visceral area (VA) of the abdominal cavity, the area of interest surrounded by the peritoneal cavity was drawn and SA was calculated by subtracting VA from TA. The ratio of total fat area to body area was calculated. Computed tomography was performed with the same machine at weeks 0 and 10.

Additionally, paired t-test and Wilcoxon signed-rank test were performed to test the effect of composition administration on body fat reduction. At this time, the program for statistical analysis was IBM SPSS Statistics 23.0.

The difference in the body changes of dogs before and after the composition is shown in Table 2 of the results.

As a result, there were significant differences in body weight and body fidelity index (BCS), fat area at the third lumbar (L3) level (SA3), and fat area ratio at the third lumbar level (pSA3). The median body weight significantly decreased from 14.0 kg at week 0 to 13.5 kg at week 10 (p<0.05). The body fidelity index significantly decreased from 9.0 at week 0 to 7.5 at week 10 (p<0.05). Also, the average of the fat area at the 3rd lumbar vertebrae level significantly decreased from 99.86±34.81 at week 0 to 82.70±31.13 at week 10 (p<0.05), and the average of the fat area at the 3rd lumbar level was also at 36.14±5.47% at week 0. At week 10, it was significantly reduced to 31.05±7.24%. On the other hand, the radiographic ratio of lumbar to subcutaneous fat at the level of the third lumbar vertebrae (rST3), the radiographic ratio of the lumbar vertebrae and subcutaneous fat at the level of the sixth lumbar vertebrae (rST6), the fat area at the level of the sixth lumbar (SA6), and the sixth Other variables such as fat area ratio at the lumbar level (pSA6) were not significant.

Example 6: Analysis of changes in the intestinal microflora of dogs before and after feeding the composition according to the present invention

In Example 5, the change of intestinal microflora before and after ingestion of the composition according to the present invention was confirmed in obese dogs who performed the experiment.

For the analysis of microorganisms, total DNA of the feces of obese dogs was extracted, and a 16S rRNA gene amplicon was prepared using primers targeting the V5-V6 region of the 16S rRNA gene from the extracted DNA. For the prepared amplicon, sequencing was performed using the Illumina MiSeq method to obtain raw data. For raw data, after QC using QIIME and mothur, sequences that are not the same as PCR primers and sequences less than 100bp that are difficult to determine or cause random errors in sequencing were removed. In addition, after sorting the sequences through trimming, chemera detection and removing, OTU clustering was used to analyze bacterial flora distribution and diversity.

The results of comparing microorganisms before and after feeding in genus units are shown in FIG. 5 .

Enterococcus , which was the most dominant species before feeding, decreased from 50% to 10%, whereas Bacteroides increased from about 7% to more than 20%. As a result, the pathogenic species, Enterococcus , decreased significantly after feeding, and Bacteriodes , which is involved in the generation of volatile delayed phase (VFA), which has the potential to utilize energy through carbohydrate fermentation, showed a tendency to increase. In addition, Prevotella , which is involved in fibrin decomposition and prevention of intestinal inflammation, showed a tendency to increase by about 3%. Through the above results, it was confirmed that the composition of the intestinal flora was positively changed as the composition according to the present invention was fed.

Table 3 shows changes in the composition of the flora through the diversity analysis of intestinal microflora before and after composition feeding. After application of the composition, the number of species increased by about 1.46 times, the species abundance increased by 1.08 times, the species diversity by 1.32 times, and the species concentration increased by 1.21 times. Overall, it was confirmed that species diversity and concentration increased after feeding.

Table 4 shows data on strains that were reduced compared to before and after ingestion among the results of analyzing microorganisms at the species level.

The most decreased strain was Enterococcus hirae , which decreased by 12.07%. This strain is known as a pathogen as a strain involved in bacteremia and sepsis in the intestine. The next most decreased strain was Enterococcus saigonensis , which was 10.04%, and although it is not yet known what disease it is related to, it is thought that most Enterococcus strains are related to urinary tract infection. In addition to Enterococcus strains, strains of genera such as Mediterraneibacter, Bacteroides, Klebsiella, Peptacetobacter and Escherichia showed a decreasing trend. Most of these strains are known to cause disease in the intestine.

In contrast to the decreased strains, the strains increased after ingestion of the composition are shown in Table 5.

The strain that increased the most was the strain Fusobacterium mortiferum , which increased by 8.38%. The strain that increased in the next order was Kineothrix alysoides strain, which is known as a strain that produces organic acids such as butyrate production. In addition to the increased strains, the content of strains such as phocaeicola, Megamonas, Colinesella, Blautia and lactobacilus increased. Among the increased strains, some strains were beneficial strains that produced organic acids such as butyrate. Additionally, it was found that the content of two strains of Lactobacillus, the target lactic acid bacteria, was increased.

As a result, when checking the microbial change due to ingestion of the composition, most of the decreased microorganisms were confirmed as pathogen-related microorganisms, and the increased microorganisms were mostly beneficial bacteria.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications or changes of the present invention can be easily used by those of ordinary skill in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (13)

A prebiotic composition for inhibiting the growth of harmful intestinal bacteria and activating lactic acid bacteria, comprising glucose oxidase as an active ingredient.
The method according to claim 1, wherein the glucose oxidase is derived from a strain selected from the group consisting of Aspergillus sp. strain, Penicillium sp. strain and Streptomyces sp. strain. Prebiotic composition, characterized in that the enzyme or a combination thereof.
The prebiotic composition according to claim 1, further comprising galactooligosaccharide.
The prebiotic composition according to claim 1, wherein the lactic acid bacteria are lactic acid bacteria resistant to hydrogen peroxide.
According to claim 1, wherein the harmful bacteria Shigella flexneri ( Shigella flexneri ), Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Enterococcus faecalis ), Clostridium Butyricum ( Clostridium butyricum ), Prevotella intermedia ( Prevotella intermedia ) and Clostridium ramosum ( Clostridium ramosum ) Prebiotic composition, characterized in that selected from the group consisting of.
A synbiotics composition comprising the prebiotic composition of claim 1 and lactic acid bacteria.
The synbiotics composition according to claim 6, wherein the lactic acid bacteria are lactic acid bacteria resistant to hydrogen peroxide.
According to claim 6, wherein the lactic acid bacteria is a lactic acid bacteria selected from the group consisting of Bifidobacterium, Lactobacillus, Lactococcus and Streptococcus or a combination thereof, characterized in that A synbiotics composition.
According to claim 6, wherein the lactic acid bacteria is Lactobacillus gasseri ( Lactobacillus gasseri ), Lactobacillus pullantarum ( Lactobacillus plantarum ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus acidophilus ( Lactobacillus delbrueckii ) and Lactobacillus delbrueckii (Lactobacillus delbrueckii) Bacillus rhamnosus ( Lactobacillus rhamnosus ) Synbiotics (synbiotics) composition, characterized in that the lactic acid bacteria or a combination thereof selected from the group consisting of.
A food containing the prebiotic composition of claim 1 and lactic acid bacteria as active ingredients.
11. The method of claim 10, wherein the lactic acid bacteria is Lactobacillus gasseri ( Lactobacillus gasseri ), Lactobacillus pullantarum ( Lactobacillus plantarum ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus acidophilus ( Lactobacillus delbrueckii ) and Lactobacillus delbrueckii (Lactobacillus delbrueckii) Bacillus rhamnosus ( Lactobacillus rhamnosus ) Food, characterized in that the lactic acid bacteria or a combination thereof selected from the group consisting of.
A feed additive comprising the prebiotic composition of claim 1 and lactic acid bacteria as active ingredients.
The method of claim 12, wherein the lactic acid bacteria is Lactobacillus gasseri ( Lactobacillus gasseri ), Lactobacillus pullantarum ( Lactobacillus plantarum ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus acidophilus ( Lactobacillus delbrueckii ) and Lactobacillus delbrueckii Bacillus rhamnosus ( Lactobacillus rhamnosus ) Feed additive, characterized in that the lactic acid bacteria or a combination thereof selected from the group consisting of.

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