KR101783672B1 - Strain isolated from infant feces and method to produce fermented milk having antioxidative activity and improving immunsystem using the same - Google Patents

Abstract

To a method for producing fermented milk having antioxidant ability and immunizing ability by using the strain isolated from infant feces. When the strain of the present invention is used as a probiotic, the antioxidative effect and the immunity increasing effect are excellent in the fermentation broth and can be usefully used for the production of health foods including fermented milk and the like.

Description

[0001] The present invention relates to a strain isolated from infant feces and a method for producing antioxidant and fermented milk having immunity-enhancing function using the same,

The present invention relates to a strain isolated from infant feces and a method for producing fermented milk having antioxidant and immunity-enhancing functions.

Probiotics are used to maintain beneficial microorganisms in the intestinal microflora, and are used as formulases, lactic acid bacteria, feed additives, and health functional foods. In general, the microorganisms used as probiotics are safe, and they are resistant to the in vivo inhibitory environment such as gastric acid in the stomach, gallbladder bile and small intestine in ingestion, and have ability to proliferate and settle by reaching the colon and rectum (FAO / WHO, 2002; In addition, the probiotics are effective in normalizing the intestinal microflora, reducing the production of decay products due to inhibition of harmful bacteria, preventing disease, anticancer activity, activation of immunity, lowering of cholesterol and antioxidant effect of blood, Useful functions are premised.

Prebiotics is a generic term for non-digestible food ingredients that act selectively on the growth and vitality of microorganisms in the intestines and which has a beneficial effect on the host. It acts as a substrate for the probiotic agent and ultimately, . Until recently, dietary fibers such as cellulose, hemicellulose, lignin and pectin, and oligosaccharides such as non-degradable carbohydrates, etc., have been shown to promote selective growth of certain strains and participate in the growth and proliferation of intestinal microflora. Prebiotics, in the sense of the agreement, are mainly non-reducing sugars. However, since oligosaccharides can affect nonbiobiotic microorganisms including foodborne pathogens, the concept of prebiotics has recently been developed in a broader sense. Prebiotics is known to promote intestinal proliferation of probiotics. In recent years, research results have been reported confirming that intestinal microflora are improved in rats after addition of new biotics in food. In the case of inflammatory bowel disease such as IBD, treatment of probiotics capable of metabolizing certain prebiotics has been found to reduce inflammatory cytokines, and active research on synbiotics has been under way. The approach of synbiotics in diseases is being done. However, most of the synbiotics studies still use non-digestible oligosaccharides, and no studies have evaluated the synergistic effects of non-carbohydrate-based prebiotics and probiotics.

Thus, the present inventors have isolated the strain from infant feces and confirmed that the fermentation broth prepared by using the isolated strain and the plant extract as a prebiotic material is excellent in antioxidative and immuno-enhancing effects, By confirming the synergy effect, the present invention has been completed.

No. 10-2014-0055583 Novel strains of Bacillus licheniformis and uses thereof No. 1020120042030 Novel Lactobacillus Arizononensis BCNU 9200 strain and a biocidal composition comprising the same as an active ingredient No. 1020120037400 A novel strain of Bacillus subtilis BCNU 9169 and a biocidal composition comprising the same as an active ingredient

It is an object of the present invention to provide a strain isolated from infant feces.

Another object of the present invention is to provide a culture product of the strain.

Another object of the present invention is to provide a biocidal composition comprising the strain or a culture product thereof as an active ingredient.

It is still another object of the present invention to provide a food composition for improving intestinal function comprising the strain or a culture product thereof as an active ingredient.

Another object of the present invention is to provide a method for producing fermented milk for antioxidation using the strain or a culture product thereof.

Accordingly, the present invention provides a strain of Lactobacillus gasseri SRK505 (Accession No. KCCM11766P) isolated from infant feces.

The present invention also provides a method for producing a prodrug, comprising the step of culturing the above-mentioned strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, a fermented product of the strain, to provide.

The present invention also relates to a method for improving the intestinal function comprising an effective ingredient selected from the group consisting of the strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, the fermented product of the strain, ≪ / RTI >

The present invention also provides an antioxidant fermented milk using the strain.

Hereinafter, the present invention will be described in detail.

The strain of the present invention was isolated from the feces of an infant and the molecular biological characteristics of the isolated strain were analyzed by 16S rDNA gene sequence to find that the isolated strain exhibited high homology with Lactobacillus gasseri Respectively. These strains were named as Lactobacillus gasseri SRK505 strain and deposited on Sep. 11, 2015 at the Korean Microorganism Conservation Center (overseas) to receive the deposit number KCCM11766P.

When the strain of the present invention is used as a probiotic, there is a high antioxidative effect in a fermentation broth, which can be usefully used for the production of a health food for improving bowel function including fermented milk and the like.

According to another aspect of the present invention, there is provided a cultured product selected from the group consisting of a culture of the strain, a fermentation product of the strain, a concentrate of the culture, a dried product of the culture, and a combination thereof. The cultured product may be the one obtained by removing the strain of Lactobacillus gasseri SRK505 or the supernatant obtained by centrifuging the culture. In addition, the cultured product may include both a concentrated product of the cultured product and a dried product of the cultured product. The fermentation product of the present invention is fermented using the strain, and includes liquid and solid, and is used in a broad concept including a fermentation broth.

According to another aspect of the present invention, there is provided a method for producing a microorganism, which comprises culturing a strain of the present invention, a culture of the strain, a fermentation product of the strain, a concentrate of the culture, a dried product of the culture, Lt; / RTI >

As used herein, the term " probiotic "or" probiotics "refers to a living organism that improves intestinal microbial environment by enhancing resistance to harmful microorganisms in the gastrointestinal tract of an animal, including humans, Means microorganisms. Probiotics may be fed in human or animal-derived cell or fermented product form in the form of single or complex strains. When the strain of the present invention is used as a probiotic agent, a synergistic effect with the prebiotic material can be caused. The prebiotic material is not limited, but a plant extract is preferable. More preferably, it is a leaf extract or mulberry leaf extract.

The extract of the present invention can be used by processing to be suitable for fermentation. For example, the extract of Cucumis japonica or Mulberry leaf can be used. When the extract of the present invention is obtained by treating the leaves or mulberry leaves with an extraction solvent, various extraction solvents may be used. Preferably, a polar solvent or a non-polar solvent can be used. Suitable polar solvents are (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, 1-pentanol, Or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethyl sulfoxide (DMSO). Suitable nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- But are not limited to, pentane, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2- chloropropane, toluene, 1- chloropropane, chlorobenzene, benzene, diethyl ether, diethylsulfide, Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF. More preferably, the extraction solvent used in the present invention is (a) water, (b) anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.) (E) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane and (j) diethyl ether. . Most preferably, the extract of the present invention is a water extract. As used herein, the term " extract " means that it is used in the art as a crude extract as described above, but broadly includes fractions obtained by further fractionating the extract. That is, the extract of the present invention includes not only those obtained by using the above-mentioned extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, and a separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The fraction obtained by the purification method is also included in the extract of the present invention. The extract used in the present invention can be produced in powder form by an additional process such as vacuum distillation and lyophilization or spray drying

The term "prebiotics" refers to a substance collectively referred to as non-degradable food ingredients that selectively affect the growth of microorganisms in the intestines and have a beneficial effect on the host.

The culture product according to the present invention, which is selected from the group consisting of a strain, a culture thereof, a fermentation product thereof, a concentrate of the culture, a dried product of the culture, and a combination thereof, is excellent in antioxidant ability and immunity, It can be usefully used as a non-toxic probiotic. Accordingly, the culture product according to the present invention, which is any one selected from the group consisting of a strain, a culture thereof, a fermentation product thereof, a concentrate of the culture, a dried product of the culture, and a combination thereof is used as a probiotic agent, And can be usefully used in formulations, lactic acid bacteria, feed additives, and health foods.

According to another aspect of the present invention, there is provided a method for producing Lactobacillus gasseri SRK505 ( Lactobacillus gasseri SRK505), a culture of the strain, a fermentation product of the strain, a concentrate of the culture, a dried product of the culture, A food composition, a food or a drink, or a fermented milk.

In addition to the above-mentioned ingredients, known additives include natural flavors such as plum, lemon flavor, pineapple flavor or herb flavor, natural flavors such as natural fruit juice, chlorpillin and flavonoid, sweeteners such as fructose, honey, sugar alcohol , Sugar or the like and an acid agent such as citric acid or sodium citrate may be mixed and used. Also. Lactobacillus biasing Li SRK505 (Lactobacillus gasseri SRK505) may be processed in various forms such as a strain or difference in food, including its culture product, jelly, juice, extract, beverages, food of the present invention taking eopeumyeonseo adverse effect on the human body This is easy and long-term storage is possible. When a strain of Lactobacillus gasseri SRK505 or a culture product of Lactobacillus gasseri SRK505 is used as a food additive, each of the above strains or a culture product thereof may be directly added or used together with other food or food ingredients, Can be suitably used. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, a strain of Lactobacillus gasseri SRK505 or a culture product thereof is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on 100 parts by weight of the whole food. However, in the case of long-term consumption intended for health and hygiene purposes or health control purposes, the amount may be less than the above range, and there is no problem in terms of safety, so that the active ingredient may be used in an amount exceeding the above range. There is no particular limitation on the kind of the food. Examples of the food to which the Lactobacillus gasseri SRK505 strain of the present invention or a culture product thereof can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, Dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense. The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau Martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. Is generally 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the Lactobacillus gasseri SRK505 strain or culture product thereof. The Lactobacillus gasseri SRK505 strain of the present invention or a culture product thereof can be used for various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the probiotics of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the proportion of such additives is not critical, the probiotics of the present invention are generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight

The present invention can provide a fermented milk comprising a strain of Lactobacillus gasseri SRK505 or a culture product thereof. The Lactobacillus gasseri SRK505 strain or the culture product thereof is preferably 0.01 to 50 parts by weight per 100 parts by weight of the fermented milk, but is not limited thereto, and a person skilled in the art can adjust the weight of the product.

The present invention also provides a method for producing fermented milk using a strain of Lactobacillus gasseri SRK505 or a culture product thereof. Hereinafter, a method for producing fermented milk will be described in detail.

The first step is a step of adding the prebiotic material to the milk. It is preferable that the prebiotic material is used as a prebiotic material, but it is preferably a plant extract, more preferably a leaf or mulberry leaf extract, The extract is preferably lyophilized. In addition, when added to milk, the concentration of the extract may be 0.1 to 10% by weight, preferably 0.1 to 1% by weight. Also, after step 1), a homogenization step can be carried out.

The second step is a step of sterilizing the mixture, wherein the preferred temperature for the pasteurization is 70 ° C to 90 ° C and the time is 10 minutes to 20 minutes.

The third step is a step of inoculating and inoculating a strain of Lactobacillus gasseri SRK505 (Accession No. KCCM11766P) or a culture thereof, wherein the strain is used in an amount of 1 to 10% by weight, 2 to 5% by weight. Thereafter, the mixture can be fermented at 30 ° C to 50 ° C for 24 to 100 hours.

When the strain of the present invention is used as a probiotic, the antioxidative effect and the immunity increasing effect are excellent in the fermentation broth and can be usefully used for the production of health foods including fermented milk and the like.

FIG. 1 is a graph showing the degree of hydrolysis of a fermentation broth prepared using a strain.
FIG. 2 is a graph showing the antioxidative activity effect of the fermentation broth according to the addition and kinds of the strains and prebiotics of the present invention using ABTS.
FIG. 3 is a graph showing the antioxidative activity effect of the fermentation broth according to the addition and kind of the strains and prebiotics of the present invention using DPPH.
FIG. 4 is a graph showing the antioxidative activity effect of the fermentation broth according to the addition and kind of the strain and prebiotic material of the present invention using FRAP.
FIG. 5 is a graph showing the antioxidative activity effect of the fermentation broth according to the addition and type of the strain and prebiotic material of the present invention, using polyphenol content results.
FIG. 6 is a graph showing the antioxidative activity effect of the fermentation broth according to the addition and type of the strains and prebiotics of the present invention, using the results of the flavonoid contents.
FIG. 7 is a graph showing the antioxidative activity effect of the prebiotic-added fermentation broth by ABTS when the strain of the present invention is diluted by concentration.
FIG. 8 is a graph showing the antioxidative activity of DDP according to the concentration of fermentation broth containing prebiotics when the strain of the present invention is diluted by concentration.
FIG. 9 is a graph showing the antioxidative activity of FRI according to the concentration of the fermentation broth added with prebiotics when the strain of the present invention is diluted by concentration, using FRAP.
FIG. 10 shows the antioxidative activity of the strain of the present invention when the concentration of the strain is diluted according to the concentration of the fermentation broth with prebiotics.
FIG. 11 is a graph showing the antioxidative activity of prebiotic-added fermentation broth according to the concentration of flavonoid when the strain of the present invention is diluted by concentration.
12 is an analysis of the content in the fermentation broth using the MRM method. ((A) TIC results of the standard solution (B) Cocoa addition fermentation broth (C) Mulberry leaf fermentation broth
13 is a graph showing changes in the content of phenolic acid in a fermentation broth with cucumber leaf prepared using the strain of the present invention.
FIG. 14 is a graph showing changes in content of phenolic aicid in a fermentation broth containing mulberry leaves prepared using the strain of the present invention. FIG.
15 is a graph showing changes in the contents of Rutin hydrate and polyphenol glycoside in a fermentation broth containing cucumber leaf prepared using the strain of the present invention
FIG. 16 is a graph showing changes in the content of Rutin hydrate and polyphenol glycoside in a fermentation broth containing mulberry leaves prepared using the strain of the present invention. FIG.
FIG. 17 is a graph showing the immune enhancing activity against S. aureus infection of C. elegans according to the strain or the synbiotics treatment of the present invention. FIG.
18 is a view for confirming the expression of the immune gene group of C. elegans by the strain or the synbiotics treatment of the present invention using qRT-PCR.
FIG. 19 is a graph showing the effect of the strain or the synbiotics treatment of the present invention It is the limit for confirming the expression of the immune gene group of C. elegans .
20 is a view illustrating a process for producing fermented milk using the strain of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

[ Embodiment 1] with probiotics Selection and identification of available strains and characterization of fermentation broths prepared using the strains

1. Preparation of fermentation broth using strain

The new material of prebiotics, cucumber leaf or mulberry leaf, was lyophilized, added to milk at a temperature of 60 ° C to a concentration of 0.2%, and homogenized at 1,000 rpm for about 15 minutes with a stirrer. Thereafter, the cells were sterilized in a constant temperature water bath at 95 ° C for about 15 minutes, cooled at room temperature, inoculated with 19 strains at a concentration of 3%, and cultured in a 41 ° C incubator for 48 hours. The fermentation broth prepared through the above procedure was used for the selection of strains.

2. Evaluation of pH of fermentation broth

In order to evaluate the microbiological fermentation characteristics, the pH was measured using the fermentation liquid prepared in Example 1.1 above. More specifically, the pH of the fermentation broth of 1.1 before and after fermentation was measured using a Thermo ORION STAR A211 pH meter, and the results are shown in Table 1.

As shown in Table 1, 505 strains were selected, and the fermentation broth prepared using the strain showed a large difference in pH depending on the fermentation time between the control group without the prebiotic material and the groups added with the prebiotics It looked. However, it was confirmed that there is almost no difference between the prebiotics materials.

In addition, strain No. 505 was selected and used in the experiment thereafter.

[Table 1]

3. Determination of degree of hydrolysis by fermentation

The degree of hydrolysis of the fermentation broth was determined using the OPA method (Nielsen et al., 2006). The results are shown in Fig.

As shown in FIG. 1, when the fermentation broth was fermented for 48 hours using the strain derived from infant feces, the degree of hydrolysis was increased upon addition of the prebiotics derived from plant extracts. In particular, when hydrolyzed Of the total number of patients.

4. Molecular biology of the strain

16s DNA sequence analysis, the strains selected in Example 1.2 were identified. More specifically, the strain was cultured in MRS broth and then harvested to isolate DNA. Then, DNA stocks were prepared using PCR, and then analyzed by PCR, analyzed by purify PCR, and electrophoresis. The results are shown in Table 2.

[Table 2]

As shown in Table 2, 505 strains isolated from infant feces were identified as Lactobacillus gasseri , and the strain was named Lactobacillus gasseri SRK545 ( Lactobacillus gasseri SRK545) Deposited on September 11, 2015, and granted accession number KCCM11766P.

 5. Peptide profiling in fermentation broth

MALDI-TOF / MS was used to analyze the peptides produced in the fermentation broth prepared from plant extract-derived prebiotics and infant feces-derived probiotics. The results are shown in Table 3.

As shown in Table 3, a total of 18 peptides were identified. There was no significant difference in the peptides produced by each strain, and it was confirmed that many peptides derived mainly from β-casein were found. Peptides produced by fermentation may be expected to influence the antioxidant activity.

[Table 3]

6. Measurement of the number of bacteria in the fermentation broth prepared by using the strain of the present invention by prebiotics

Table 4 shows the results of measurement of the number of bacteria in the fermented milk prepared by adding the strain 505 of the present invention and the prebiotics.

As shown in Table 4, the fermentation broth fermented with 505 strains and cucurbitan showed 8.69 ± 0.02 log / CFU mL after fermentation and increased to 1.56 log / CFU mL before fermentation. As a result of lactic acid bacteria count after fermentation, fermented broth was found to have a bacterial count of 8.81 ± 0.03 log / CFU mL, which was 1.46 log / CFU mL before fermentation. In the case of fermented broth fermented with 505 strains, the number of bacteria after fermentation was 7.30 ± 0.02 log / CFU mL.

[Table 4]

[Example 2] Confirmation of antioxidant activity of a fermentation broth prepared using the strain of the present invention

1.ABTS assay

First, a solution A in which 7 mM ABTS (2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt) was dissolved in ethanol and a solution B in which 2.45 mM potassium persulfate was dissolved in distilled water After mixing well, the mixture was allowed to stand at room temperature for 12 to 16 hours to produce active radicals. The ABTS reagent was prepared by diluting it with distilled water to an absorbance of 1.4 at 743 nm. Subsequently, 20 μL of the sample (fermentation broth) was reacted with 180 μL ABTS reagent, allowed to stand for 6 minutes, and absorbance was measured at 734 nm.

2. DPPH assay

The DPPH reagent was prepared by dissolving 0.3 mM DPPH (2,2 Diphenyl 1 picrylhydrazyl) in methanol with a visible light for a long time and then diluting it with distilled water to have an absorbance of 0.9 to 1.0 at an absorbance of 517 nm. 50 μL of the sample solution and 150 μL of the DPPH reagent were mixed and reacted at 37 ° C. for 30 minutes under dark condition, and the absorbance was measured at 517 nm.

3.FRAP assay

Ferric Reducing Ability of Plasma (FRAP) reagent was prepared as follows. First, 1.6 mL of glacial acetic acid was added to 0.31 g of sodium acetate trihydrate, and then distilled water was added thereto, and the volume was adjusted to 100 mL to prepare Solution A. In addition, Solution B was prepared by mixing 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) with 10 mL of 40 mM HCl. In addition, a solution C in which 20 mM of FeCl 36H 2 O (Iron (III) Chloride hexahydrate) was dissolved in 10 mL of distilled water was prepared. After that, FRAP reagent was prepared by mixing solutions A, B, and C. After mixing 6 μL of the sample and 180 μL of the FRAP reagent, the reaction was carried out at 37 ° C. for 30 minutes and the absorbance was measured at 593 nm. As a control, samples and the same amount of distilled water were reacted with FRAP reagent to make a blank value. FeSO ₄ 7H ₂ O (iron (II) sulfate heptahydrate) was adjusted to a concentration of 0-1.0 mM, and the absorbance was measured at a wavelength of 593 nm. A calibration curves were prepared using the absorbance and the antioxidant activity of FeSO ₄ 7H are shown against the concentration of O _2.

4. Total polyphenol content assay

Total polyphenol content was determined by colorimetry by the Folin-Denis method. 25 μL of the sample and 62.5 μL of the folin-ciocalteu's phenol reagent were sequentially added to 312.5 μL of a 20% sodium carbonate solution, and the mixture was left to stand for 40 minutes. The absorbance of the reaction solution was measured at 765 nm using a spectrophotometer (UV-1601, SHIMADZU, Japan). The standard solution was a gallic acid solution (0-100 mg).

5. Total flavonoid assay

In order to evaluate the content of flavonoid, flavonoid, metal ion and chelate are formed, and specific color reaction occurs. The principle of analyzing the qualities and quantities of flavonoids in the sample was used. First, 0.2 g of AlCl ₃ 6H ₂ O (Aluminum (III) chloride hexahydrate) was dissolved in 10 ml of ethanol to prepare a 2% AlCl ₃ 6H ₂ O solution. Then, 100 μL of the sample and 100 μL of the solution were reacted for 10 minutes And the absorbance was measured at 430 nm. Quercetin solution (0-100 mg) was used as a standard substance.

6. Polyphenol analysis of fermentation broth containing cucumber leaf and mulberry leaf extract by LC-MS / MS

When polyglycerol leaves or mulberry leaf extract were added, the content of polyphenols in the fermentation broth fermented for 5 hours using commercial strains and fermented broth fermented for 48 hours using 505 strains in the present invention were analyzed.

Take 1 g of the lyophilized sample of the fermentation broth prepared using the strain, add 7 mL of ethanol solution containing 50% ethanol and 0.05MH ₃ PO ₄ , and mix the sample with ultrasonic treatment for 20 minutes at room temperature. Respectively. The supernatant was then centrifuged at 5000 rpm for 30 minutes, and the supernatant was filtered through a 0.2 μm filter and analyzed by LC-MS. Standard stock solution is a mixture of neo-chlorogenic acid, chlorogenic acid, 3,4-dihydroxyhydrocinnamic acid, caffeic acid, Rutin hydrate, Quercetin-3-galactoside, Quercetin-3-glucoside, Kaempferol-3-galactoside, , Kaempferol-3-rutinoside, or Kaempferol-3-glucoside were mixed to prepare 1 mg / mL of the solution. Then, 10% ACN (acetonitrile) 10, 50, 100, and 1000 μg / mL, respectively. The LC-MS was tested with UPLC-Acquity Binary pump, ZEVO-TQ from Waters, and ACN containing 0.1% formic acid solution and 0.1% formic acid was used as A and B solvents, respectively. Acquity UPLC BEH C18 1.7 mu m (2.1 x 100 mm) was used as a column. Gradient conditions are shown in Table 5, and LC-MS detailed conditions are shown in Table 6. As a result of the tune for the above 10 standard solutions, the highest sensitivity was obtained under the conditions shown in Table 7 below. Through this, the whole MRM method was established and this condition was used for quantification.

[Table 5]

[Table 6]

[Table 7]

7. Results synthesis

2 to 6 show the antioxidant activity of the fermentation broth according to the addition and type of the strain and prebiotics of the present invention (Fig. 2: ABTS results; Fig. 3: DPPH results; Fig. 4: FRAP results; : Polyphenol content; Fig. 6: flavonoid content).

As shown in FIG. 2 to FIG. 6, it was confirmed that the antioxidant activity of the fermentation broth after 48 hours fermentation was higher than that of the fermentation broth immediately after inoculation. Especially, it was confirmed that the fermentation broth of cucumber leaves or mulberry leaves added as prebiotic material showed antioxidative activity about 4 to 7 times higher than that of milk fermentation broth.

The results of ABTS, DPPH and FRAP showed that the fermentation broth added with cilantro leaves had higher antioxidant activity than the fermented broth containing mulberry leaves.

The results of the total polyphenol contents showed that the fermented broth containing mulberry leaf and mulberry leaf contained polyphenol in a similar manner. Especially, fermented broth with culm leaves showed higher activity.

FIGS. 7 to 11 show the results of an experiment on antioxidant activity of prebiotics-added fermentation broth by diluting 505 strains of selected strains by concentration (FIG. 7: ABTS; FIG. 8: DDPH; Figure 10: polyphenol content; Figure 11: flavonoid content).

 As shown in FIG. 7, in the case of ABTS, the antioxidative activity of the fermented broth added with cucumber leaf or mulberry leaf was decreased in a concentration-dependent manner. Antioxidant activity of the fermented broth added with cilantro leaves was the greatest, but milk showed no difference before and after fermentation.

As shown in FIG. 8, in the case of DPPH, it was confirmed that the fermentation broth containing cucurbit leaf showed antioxidative activities of 88% or more at a concentration of 100 mg / mL and 85% or more at a concentration of 50 mg / mL. In addition, the mulberry leaf fermentation broth showed an antioxidant activity of 88% at 100 mg / mL and 70% at 50 mg / mL. As the concentration was lowered, the activity of leaf - fermented broth and mulberry leaf - fermented broth were different from each other.

As shown in FIG. 9, in the case of FRAP, it was confirmed that cucurbit leaf fermentation broth had higher activity at a concentration of 100 mg / mL than that of the mulberry leaf fermentation broth.

As shown in Fig. 10, it was confirmed that the total polyphenol content of the fermented broth was 6 times higher than that of the fermented broth. In the case of the fermented broth of mulberry leaf, the total polyphenol content after fermentation increased more than 2 times .

As shown in FIG. 11, the content of total flavonoids was found to be higher than that of the mulberry leaf fermentation broth by about 5 times as much as that of the fermented broth of fermented leaves of 14 g / 100 g or more. Especially, the content of total flavonoid was higher in the mulberry leaf fermentation broth.

12 to 16 show results of the analysis using the MRM method (Fig. 12: (A) TIC results of the standard solution; (B) TIC results of the fermented broth added with curd leaf; (C) TIC Result: Fig. 13: Changes in Phenolic acid content of fermentation broth prepared from infant feces derived from cucurbit leaf; Fig. 14: Phenolic acid content of fermented broth obtained from infant feces derived from mulberry leaf Figure 15: Changes in the content of Rutin hydrate and polyphenol glycoside in fermented broth prepared from infant feces derived from cucurbit leaf; Fig. 16: Infant fecal bacterial strain with mulberry leaf added Changes in the content of Rutin hydrate and polyphenol glycoside in the prepared fermentation broth.

As shown in FIG. 12 to FIG. 16, peaks of the standard solutions were measured by using Neo-chlorogenic acid, Chlorogenic acid, 3,4-Dihydroxyhydrocinnamic acid, Caffeic acid, Quercetin-3-galactoside, Quercetin-3-glucoside, Kaempferol- , Kaempferol-3-rutinoside, and Kamepferol-3-glucoside. The content of neo-chlorogenic acid, chlorogenic acid, and caffeic acid in phenolic acid decreased and the content of 3,4-dihydroxyhydro cinnamic acid was significantly increased in fermented broth using fermented broth. Rutin hydrate and glycosides were fermented And then increased slightly.

The content of phenolic acid, rutin hydrate and polyphenol glycosides in the fermented broth was higher than that of the fermented broth containing mulberry leaf extract. Therefore, the antioxidant activity .

[Example 3] Confirmation of immunity enhancing effect of a fermentation broth prepared using the strain of the present invention

One. C. elegans  Preparation of Immune Models

To facilitate the experiment at the experimental condition of 25 ° C, the fer-15; fem-1 mutant was used in the survival experiment. The mutant is known to be a mutant which hatches at 15 ° C but does not hatch at 25 ° C and has no phenotypic change other than the hatching ability. Therefore, it is used in various experiments, in particular, mutants used for immunological activity and aging experiments . The survival rate of C. elegans The activity changes were N2 wild type. The C. elegans was cultured using NGM (Nematode growth medium; Breger 1976) and E. coli OP50.

2. Preparation of a synbiotics conditioning plate

The 505 strain was cultivated in MRS medium at 37 ° C for 18 hours, centrifuged (6,000 × g, 20 min), and the cell pellet was collected and washed 5 times with M8 buffer solution. Finally, a 5-fold concentrated bacterium was prepared, and the mixture was dispensed into an NGM agar, followed by drying at room temperature for 1 hour. After drying, it was stored at 40 ° C and used for 2 weeks with a probiotic conditioning plate. For prebiotics, 0.2% extract was prepared and added to the probiotics conditioning plate.

3. High-efficiency in vivo C. elegans model for identification of synoviotics long- acting activator

C. elegans conditioned with synbiotics for 24 hours was washed five times with M9 buffer according to the method of Example 3.2, and S. aureus RN6390 was transferred to a dispensed NGM dish and induced a bacterial infection at 25 ° C for 24 hours. After infection, the cells were washed five times with M9 buffer, and 30 C. elegans nematodes infected in a 6 well plate in a working medium (20% BHI + 80% M9 buffer) were added. After 24 hours, The survival rate of C. elegans was investigated for 14 days. Subjects who had mobility when shaken or touched were regarded as living C. elegans , and the experiment was repeated at least 3 times. The results are shown in Fig.

As shown in FIG. 17, the immune enhancement activity of C. elegans against S. aureus infection according to the present invention strain or a synbiotics treatment was measured. In the case of 505 strains, the mixture of leaf extract and synbiotics , The immunological activity against pathogenic bacteria was significantly increased.

4. Expression of immunologic gene groups using qRT-PCR and GFP-promoter assay

After selection of functional gene groups identified by the existing literature, qRT-PCR primers were prepared. Specific primers were prepared using Primer3 Input (version 0.4.0) software. qRT-PCR was performed using the SuperScriptTMIII Platinum®One-Step qRT-PCR Kit and the StepOne ™ Real-Time PCR System (Applied Biosystems). To correct for changes in mRNA expression, the snb gene was amplified by internal control ). In addition, the direct immune enhancement effect was visualized using a transgenic mutant worm fused with the GFP fluorescent protein to the promoter of the immune-related gene pmk-1 purchased from the CGC (Caenorhabditis Genetics Center). The same conditions as the immune activity test as 505 strain alone treatment or kkujippong leaf extract and SynBio vertices to target them to extract the RNA, respectively intended for mixing treatment C. elegans immunity and related significant five genes (cpr-1, thn that 18, and the transgenic mutant worm in which the GFP fluorescent protein was fused to the promoter of pmk-1 was used as a direct immunization enhancer. The visualization result is shown in Fig.

As shown in FIG. 18, qRT-PCR showed that the expression of the high immunity-related gene was increased by the single treatment of 505 strains, and the mixed neurotic treatment of the extract of Cryptomeria japonica influenced the increase of the immunogen gene expression Respectively.

As shown in Fig. 19, similar to the results of qRT-PCR, it was confirmed that the expression of the pmk-1 pathway was stimulated by the single treatment of 505 strains. In the case of the mixed neurotoxic treatment of the cilantro leaf extract, pmk-1 Pathway stimulation was found to have some effect on the increase of pathway stimulation.

[ Example 4] Production of fermented milk having antioxidative function using infant feces isolate strain

1. Manufacture of suspension

To produce fermented milk using the strain of the present invention, the lactic acid bacteria isolated from infant feces of the present invention was inoculated into MRS broth of lactic acid bacterium suspension preparation and cultured for 18 hours. After the incubation, the supernatant was discarded by centrifugation, and the pellet was washed twice by taking only the pellet, and the pellet was concentrated to 1/10 to prepare a suspension.

2. Production of fermented milk

And were weighed and stirred according to the blending ratio shown in Table 8 below. In case of oil sugar, fragrance was added, and the mixture was stirred to mix the materials. Thereafter, the mixture was sterilized at 95 DEG C for 5 minutes, adjusted to 41 DEG C to 43 DEG C, cooled, and incubated at 41 DEG C after inoculation with the suspension, followed by termination of the culture at pH 4.4 to 4.5.

[Table 8]

3. Measurement of the number of lactic acid bacteria in fermented milk

The number of lactic acid bacteria in the fermented milk prepared using the strain of the present invention and cucumber leaves was measured. The results are shown in Table 9.

As shown in Table 9, it was confirmed that the number of lactic acid bacteria was increased by 2 to 3 log / CFU more than before the fermentation of the fermented milk to which the strain of the present invention and the concentrate of coconut juice were added, And the number of lactic acid bacteria was increased. In addition, the total number of lactic acid bacteria is increased due to the synergistic effect of the commercial strains and the screening strains derived from infant feces.

[Table 9]

Name of depository: Korea Microorganism Conservation Center (overseas)

Accession number: KCCM11766P

Checked on: 2015911

Claims (5)

A mixture of 0.1 to 10% by weight of leaf extract or mulberry leaf extract in milk; And
To the mixture, 1 to 10% by weight of Lactobacillus gasseri SRK505 SRK505) (Accession No. KCCM11766P).
The method according to claim 1,
Wherein the strain further comprises any one selected from the group consisting of a culture of the strain, a concentrate of the culture, a fermentation product of the strain, and a dried product of the culture.
1) adding 0.1 to 10% by weight of leaf extract or mulberry leaf extract to milk to obtain a mixture;
2) pasteurizing the mixture obtained in the step 1) at 70 to 90 ° C for 10 to 20 minutes; And
3) Inoculating 1 to 10% by weight of Lactobacillus gasseri SRK505 strain (Accession No. KCCM11766P) into the mixture obtained in the step 2) and fermenting at 30 to 50 ° C for 24 to 100 hours; Wherein the strain further comprises any one selected from the group consisting of a culture of the strain, a concentrate of the culture, a fermentation product of the strain, and a dried product of the culture. Way.
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