STRAIN ISOLATED FROM INFANT FECES, AND PRODUCTION METHOD OF FERMENTED MILK HAVING ANTI-OXIDATIVE ACTIVITY AND IMPROVED IMMUNE FUNCTION USING SAME

Hereinafter, the present invention broadcast receiver through more detailed in the embodiment. The present invention is more specifically account for these in the embodiment is only, in the embodiment of the present invention according to the subject matter of invention number by one person with skill in the art that in these range-case is not in the art will.

[In the embodiment1] to pro bio tic [suAvailable Strain of selecting, identifying and said strain Characterization of using fermentation broth prepared by the number

1. Using the same number tank fermentation broth

It does not borrow the mulberry leaves or after freezing and drying the processed pre-bio tic [su[su] novel powder, milk or milk concentration 60 °C one warming to 0. Adding 2% so, about 1,000 rpm 15 minutes with agitator stored in the homogeneous. After this, about 15 minutes 95 °C in tank have a constant-temperature sterilization, then heated with (cooling) at room temperature, species concentration 3% 19 so that said strain is provided which reduces, in him as time 48 41 °C incubator. Said number prepared by the fermentation of a strain selection in the process of using the air.

2. Method for estimating the characteristic pH of fermentation broth

Microbiological fermentation characteristics for evaluating, said in the embodiment 1. 1 number in a high pressure liquid coolant by using a liquid fermentation pH were measured. More specifically, 1. 1 Kfcc before fermentation, and by using a pH of a Thermo yarn ORION STAR A211 pH meter, the results of table 1 is shown.

As shown in the table 1, expressed from recombinant yeasts using Leuconostoc citreum 505 to said fermentation broth prepared by the number adding probiotics material but is free and free bio tic [su fermentation time difference between large groups according to pH regulated her. However, material has been confirmed that the difference between the negative pre-bio tic [su[su] can nearly be neglected.

In addition, strain number 505 to expressed from recombinant yeasts, utilizing his then experiments.

[Table 1]

3. Fermentation according to confirm a degree hydrolysis

Hydrolysis of OPA method by using Leuconostoc citreum fermentation broth prepared by the number degree (Nielsen et al. , 2006) has been confirmed using. Also shown is the results of the 1.

As shown in the fig. 1, wherein the infant derived using Leuconostoc citreum 48 when fermentation time, the degree of hydrolysis increases upon addition of plant extract derived free bio tic [su[su] formate, in particular, it does not borrow the mulberry leaves extract has been confirmed by hydrolysis degree layer is significantly increased.

4. Identifying molecular biological molecules strain

Through 16s DNA sequencing, in the embodiment 1. 2 a method of selecting strains in identifying him. More specifically, after said strains cultured in MRS broth, separating DNA caused by him. Then, using a high pressure liquid coolant DNA stock number after PCR, PCR analysis (Analyzed PCR), via extraction of PCR product (purify PCR) and electrophoretic eastern phase identifying him. The results of table 2 is shown.

[Table 2]

As shown in the table 2, wherein the Lactobacillus strains isolated from start-infant 505 (Lactobacillus gasseri)Identifying and feeling to, said start-SRK505 Lactobacillus strains (Lactobacillus gasseri SRK545) termed the, user is deposited under the Korean degrading center 11 September 2015, a consignee number KCCM11766P impurities.

5. In peptide profiling fermentation broth

Using MALDI-a TOF/MS, wherein the plant extract derived from fermentation broth prepared by the number generated in analyzing peptide derived pro bio tic [su bio tic [su[su] for small pre to him. The results of table 3 is shown.

As shown in the table 3, two total 18 peptide therefrom. Strain generated by peptide to go room, mainly β-a casein derived in exactly that identified has been confirmed. Antioxidant peptides produced by fermentation is influence increasing activity much leakage and improving determined substrate.

[Table 3]

6. By using Leuconostoc citreum addition of fermentation broth prepared by the number of the present invention Free bio tic [su[su] By material Bacterium counting

Pre-strain of the present invention 505 bio tic [su[su] number by adding milk bacterium counting result table 4 shown to the material produced therewith.

As shown in the table 4, it does not borrow [ppong 505 lactic acid bacterium counting result fermentation extract of strain with sodium chloride, 8 germ possibility after fermentation. 94 ± 0. 1 02 log/CFU mL ferment than before. 56 log/CFU mL increases as the formate, sodium chloride lactic acid bacterium counting result fermentation extract of strain excellent flavor 505, 8 germ possibility after fermentation. 81 ± 0. The fermented 03 log/CFU mL 1 than before. The increase of 46 log/CFU mL has been confirmed. In addition, in the case of strain 505 fermenting milk fermentation, 7 germ possibility after fermentation. 30 ± 0. The fermented 02 log/CFU mL even though not difference has been confirmed.

[Table 4]

By using Leuconostoc citreum [in the embodiment 2] of the present invention identifying fermentation broth prepared by the number of antioxidant activity

1. ABTS assay

First, 7 mm to ethanol of ABTS (2, 2 a-azinobis (3 a-ethyl a-benzothiazoline provided 6 a-sulfonic acid) diammonium salt) solution A melts on the distilled water 2. 45 mm potassium solution (potassium persulfate) B melts it ladles, opinion pay [thu good after mixing, at room temperature 12 to 16 time leaving the active oxygen (radical) acid, 743 nm 1 same in absorbance. 4 dilution so the reservoir to a high pressure liquid coolant ABTS reagent his number. Then, 180 μl ABTS reagent reacting on a 20 μl sample (fermentation broth), have 6 minutes, in absorbance 734 nm were measured.

2. DPPH assay

0. 3 mm DPPH (2, 2 Diphenyl 1 picrylhydrazyl) visible light a number of plastic stand-alone after dissolving in methanol, in 517 nm absorbance of 0. 9 to 1. 0 absorbance using distilled water to a high pressure liquid coolant to DPPH reagent dilution of his number. On DPPH reagent 150 μl 50 μl sample solution after mixing herein, cancer conditions, 37 °C, 30 minutes by reacting, in absorbance 517 nm were measured.

3. FRAP assay

Means of FRAP (Ferric Reducing Ability of Plasma) reagent method such as high pressure liquid coolant to his number. First, 0. 31 g to 1 for preparing sodium trihydrate (sodium acetate trihydrate). After adding 6 ml of glacial (glacial acetic acid), 100 ml distilled water is added to adjust the volume to a high pressure liquid coolant solution A his number. In addition, 10 mm of TPTZ (2, 4, 6 a-tripyridyls non-triazine) was mixed with 10 ml 40 mm HCl solution by a number B of a high pressure liquid coolant. In addition, a distilled water 10 ml 20 mm FeCl36 H2O (Iron (III) Chloride hexahydrate) outermost C number was a high pressure liquid coolant. Then, solution A, B, C FRAP reagent mixing number was a high pressure liquid coolant. 6 μl sample mixing behind on FRAP reagent 180 μl, 37 °C 30 minutes reaction in the wafer, in absorbance 593 nm were measured. Entering the sample chuck of FRAP reagent blank value on contrast equal reacting filibuster. FeSO₄ 7H₂ (Iron (II) sulfate heptahydrate) O a 0 to 1. In measuring the concentration of 0 mm regulated so as to have same 593 nm wavelength absorbance, using straight calibrator curve corresponding absorbance creates an, anti-force FeSO sample₄ 7H₂ The concentration of O shown applied.

4. Total polyphenol content assay

Total polyphenol content by Folin provided Denis colorimetric quantitative him. 25 μl sample, folin-a ciocalteu's phenol reagent 62. 5 μl of a 20% sodium carbonate solution (sodium carbonate solution) 312. 5 μl are sequentially applied, been rapidly, 40 minutes left for him. The liquid reaction mixture using the spectrophotometer (spectrophotometer; UV-a 1601, SHIMADZU, Japan) in absorbance 765 nm were measured. Standard solution is gallic (gallic acid) solution (0 to 100 mg) as well as a.

5. Total flavonoid assay

Evaluate the flavonoid content, flavonoids (chelate) chelating metal ions formed from, using unique chromophoric reaction occurs. The liquid sample flavonoid qualitative and quantitative analyzing principle using the air. First, 0. 2g of AlCl₃ 6H₂ (Aluminum (III) chloride hexahydrate) 10 ml of ethanol for 1 to 2% of the AlCl O₃ 6H₂ O number after high pressure liquid coolant solution, said solution 100 μl 100 μl sample in 10 minutes on a reacting 430 nm were measured absorbance. Standard material is quercetin (Quercetin) solution (0 to 100 mg) by water.

6. Fermentation broth of polyphenol analysis using LC-a MS/MS added it does not borrow with the mulberry leaves mulberry leaf extract

Powder when it does not borrow the mulberry leaves or performance, by using Leuconostoc citreum fermenting time of polyphenol and process in the present invention 505 5 commercial fermentation using Leuconostoc citreum fermenting of polyphenol content of the fermentation time was 48.

Freeze-dried fermented liquid prepared by the number samples taken by using Leuconostoc citreum accurately 1g 7 ml, 50% ethanol 0. 05M H₃ P0₄ After the ethanol solution to the included spray, ultrasonic treatment is carried out at room temperature 20 minutes through sample extracted from the earth. After this, the previous frame was taken in 30 minutes centrifuging the liquid stream and 5000 rpm, 0 dissolves. 2 μm after filtering using a filter, LC-a MS was analyzed. A standard solution (Standard stock solution) (Neo-a chlorogenic acid) Neo - coffee, coffee (chlorogenic acid), at the time of draw it is delighted and it buys hydroxy 3, 4 - di (3, 4 a-Dihydroxyhydrocinnamic acid), caffeine acid (Caffeic acid), routine hydrate (Rutin hydrate), quercetin - 3 - galacto side (Quercetin provided 3 a-galactoside), quercetin - 3 - writing base nose side (Quercetin provided 3glucoside), galacto side (Kaempferol provided 3 a-galactoside) - 3 - [kham lung roll, - 3 - [kham lung roll base mote labor and management id (Kaempferol provided 3 a-rutinoside) - 3 - [kham lung roll or the output signal of 1 mg/mL after writing base nose side (Kaempferol provided 3 a-glucoside) number to bath, using 10% ACN (acetonitrile) of 10, 50, 100 and 1000 μg/mL to the dilution was used. LC-a MS Waters yarn is UPLC provided Acquity Binary pump, have experiment using ZEVO provided TQ, 0. Of 1% formic acid solution, 0. 1% of a solvent as well as formic acid included in the ACN A and B each. The Acquity UPLC BEH C18 1 column (Column). 7 μm (2. 1x100 mm) as well as a. And Gradient conditions such as table 5, table 6 LC-a MS detailed conditions such as disclosed. In addition, standard solutions for Tune result is said 10, such as conditions were the highest sensitivity to table 7. Through establishing overall MRM method have, quantitative seam using the air conditions.

[Table 5]

[Table 6]

[Table 7]

7. Integrated result

2 to 6 of the present invention addition of fermentation broth according to kind and also to strain free bio tic [su[su] material also showed anti-activating effect (also 2: ABTS result; also 3: DPPH result; 4: FRAP also result; also 5: polyphenol content; also 6: flavonoid content).

Also as shown in the 2 to 6 also, immediately after inoculation anti-highly active fermentation broth after fermentation has been confirmed that the fermentation broth than time 48. In particular, it does not borrow the mulberry leaves or powder added as pre-bio tic [su[su] material 4 to 7 times by high antioxidant activity appears to be Kfcc this milk fermentation broth than has been confirmed.

In addition, antioxidant activity difference between pre-bio tic [su[su] material e.g., ABTS, DPPH, FRAP experiments it does not borrow the mulberry leaves both adding high antioxidant activity than fermentation broth is excellent flavor added fermentation has been confirmed.

In addition, it does not borrow the mulberry leaves the results of experiment total polyphenol content fermentation broth and excellent flavor added to fermentation 144.3 polyphenol containing light clause added support, adding in particular it does not borrow the mulberry leaves fermentation having higher activity has been confirmed.

Figure 11 shows a concentration dilution according to 505 also strains also 7 strains to pre bio tic [su[su] efficacy experiments have shown the concentration of antioxidant activity by fermentation broth added result (also 7: ABTS; 8: DDPH also; 9: FRAP also; 10 also: polyphenol content; 11: flavonoid content).

7 also as shown in the variation, in the case of ABTS, it does not borrow the mulberry leaves or excellent flavor added antioxidant activity depending on concentration reducing both fermentation has been confirmed. In the case of adding it does not borrow the mulberry leaves fermentation, ultra antioxidant activating effect of milk but when, before fermentation, the difference between negative after has been confirmed.

As shown in the 8 also, in the case of DPPH, adding it does not borrow the mulberry leaves in the case of fermentation, 100 mg/mL concentration in 88% or more, 85% or more at a predetermined concentration 50 mg/mL anti-oxidation activity has been confirmed that the charge. In addition, in the case of fermentation broth powder, 100 mg/mL in 88%, in the case of 50 mg/mL, 70% or more antioxidant activity effect has been confirmed that the charge. It does not borrow the mulberry leaves by an activity of fermentation broth and fermentation broth powder layer is 24.4 difference, it does not borrow the mulberry leaves highly active as the fermentation broth is has been confirmed.

As shown in the 9 also, in the case of FRAP, it does not borrow the mulberry leaves than higher activity has been confirmed that the fermentation broth is 100 mg/mL concentration fermentation broth powder charge.

As shown in the 10 also, in the case of total polyphenol content, it does not borrow the mulberry leaves 6 times have higher total polyphenol content is also used for fermentation, in the case of fermentation broth powder, 2 times increases total polyphenol content identifying fermentation than before after fermentation has been confirmed.

As shown in the 11 also, in the case of total flavonoid content, it does not borrow the mulberry leaves more polyphenol content of 14 g/100g content after fermentation as compared to fermentation broth of fermentation broth powder charge 5 times the steam high content has been confirmed. In particular, in the case of fermentation broth powder, high total flavonoids has been confirmed.

12 to 16 also shown and analyzed with an MRM method to result (also 12: (A) standard solution TIC result; (B) it does not borrow the mulberry leaves adding fermentation of TIC result; (C) excellent flavor adding fermentation of TIC result: also 13: it does not borrow the mulberry leaves added baby feces derived using Leuconostoc citreum number prepared by the fermentation broth of Phenolic acid content of change indicates; also 14: excellent flavor added baby feces derived using Leuconostoc citreum number prepared by the fermentation broth of Phenolic aicid content of change indicates; also 15: it does not borrow the mulberry leaves added baby feces derived using Leuconostoc citreum number prepared by the fermentation broth of Rutin hydrate on polyphenol glycoside content variations indicates; also 16: excellent flavor added baby feces derived using Leuconostoc citreum Rutin hydrate of polyphenol glycoside content variations on fermentation broth prepared by the number difference)

Also as shown in the 12 to 16 also, the peaks of the standard solution Neo-a chlorogenic acid, Chlorogenic acid, 3, 4 a-Dihydroxyhydrocinnamic acid, Caffeic acid, Quercetin provided 3 a-galactoside, Quercetin provided 3 a-glucoside, Kaempferol provided 3 a-galactoside, Kaempferol provided 3 a-rutinoside, order Kamepferol provided 3 a-glucoside has been identified. In using Leuconostoc citreum Phenolic acid fermentation broth prepared by the number of Neo-a chlorogenic acid, Chlorogenic acid, have reduced the content of Caffeic acid, 3, 4 a-Dihydroxyhydro cinnamic acid by rotation the content of formate, on the glycoside-width after fermentation has been confirmed that the Rutin hydrate increases.

In addition, it does not borrow the mulberry leaves extract number of phenolic acid fermentation using high pressure liquid coolant, Rutin hydrate, polyphenol glycoside have higher than the content of mulberry leaf extract fermentation broth added confirm, thereby it does not borrow the mulberry leaves higher antioxidant activity of performance has been confirmed that the fermentation..

The use of a checking effect of the present invention [in the embodiment 3] by using Leuconostoc citreum fermentation broth prepared by the number

1. C. Elegans In the preparation of the immune

Experiment condition in order for experiments hereinafter 25 °C, survival rates in experiment fer-a 15; a fem-a 1 mutant was used. Said jl1506 15 °C or 25 °C laid in without laid in, in addition to the ability the phenotype of the mutant known as other sub surface of various experiments in particular, various immune activity and anti-aging experiments mutant used in gaskets, as well as said experiments. Various other than survival rates experiments C. ElegansActive change wild-type N2 by water. Said C. ElegansCulture of the NGM (Nematode growth medium; Breger 1976) and E. Coli OP50 him as using.

2. The preparation of new bio tic [su conditioning plate

18 hours in culture medium and said 505 strain MRS 37 °C, centrifugal separator (6,000 × g, 20 min) to, after retrieving the only cell pellets, M8 cleaning buffer solution was 5 times. Finally 5 times concentrated germ misfortune number after high pressure liquid coolant, after dividing the NGM agar (agar), 1 with respect to the drying time at room temperature. After drying, the installed 40 °C while, daytime conditioning plate as well as to pro bio tic [su 2. In the case of pre-bio tic [su[su]it does not borrow [ppong[ppong] or powder material, 0. The user who is pro bio tic [su conditioning plate number by adding his morning fair mixed extract of 2%.

3. High efficiency In Vivo C. Elegans Model using New bio tic [su Field immune ActivatorIdentifying small

In the embodiment 3. 2 method of by, new bio tic [su 24 hours the condition C. After washing 5 times in M9 buffer is a elegans intestinal inflammatory induced pathogens S. AureusRN6390 NGM dish and is less affected by the divided, 24 25 °C inducing pathogen infection in time. M9 buffer 5 times after infection is washed and, working medium (20% BHI + 80% M9 buffer) is divided by 6 well plate is infected C. Elegans 30 is carried out by a nematode embryo into next, survival of nematode 24 14 whether liver observing time intervals C. ElegansHis survival of pattern. When mobility is swung or brought into contact with the remaining living object C. ElegansEmbodiment 3 times and experiments was considered a minimum. The results of the 17 is also shown.

As shown in the variation also 17, new bio tic [su processing according to strain of the invention herein C. Elegansof S. Aureus The infection immunity activity measured, in the case of strain 505, it does not borrow the mulberry leaves extract and mixing treatment with significant capacity is remarkably increased immunization against pathogenic bacteria according to new bio tic [su has been confirmed.

4. Identifying immune gene expression of GFP-a promoter assay using qRT-a PCR and group

After selecting a population of synthesized per function been solved through immune gene, for small number it is under, qRT-a PCR primer, said primer 3 Input process (version 0. 4. 0) using small software specific primer-gate number. QRT-a PCR experiments on StepOneTM Real provided Time PCR System (Applied Biosystems) and has embodiment SuperScriptTMIII Platinum®®One provided Step qRT-a PCR Kit have, snb gene expression of mRNA for the internal control (internal control) change at night. In addition, CGC (Caenorhabditis Genetics Center) GFP fluorescent protein fused transgenic mutant worm promoter of immune related gene pmk provided 1 are stored in direct immunization using stored in the visual effect. The same conditions it does not borrow the mulberry leaves extract alone or mixed control it will roll new bio tic [su 505 immune activity experiments each object by extracting RNA strains tested in these C. Elegans 5 associated with important group of immune gene (cpr provided 1, thn provided 2, lys-a 5, clec-a 60, ilys provided 3) measuring changes in expression of fig. 18 has been shown, using GFP fluorescent protein fused transgenic mutant worm pmk provided 1 promoter of direct potentiating immune to 19 also for visualizing results have shown.

As shown in the variation also 18, qRT-a PCR result, high expression of a single processing 505 strain immune related gene have an excellent productivity increase, it does not borrow the mulberry leaves extract from affecting gene expression increase immune processing new bio tic [su mixing has been confirmed.

As shown in the variation also 19, qRT-a PCR results similarly, treatment by stimulating the expression of a single strain 505 pmk-a 1 path formate, it does not borrow the mulberry leaves extract when mixing new bio tic [su processing, pmk-a 1 part can affect the increased path of stimulation effect has been confirmed.

[In the embodiment 4] infant Feces Using the same number separating milk having antioxidative functionalbath

1. Suspension number bath

A high pressure liquid coolant by using Leuconostoc citreum number for milk of the present invention, lactic acid bacteria of the present invention wherein the lactic acid bacteria suspension number 18 days to him as time separating baby bath MRS broth. Culture nor post-incubation, 2 taken only once (pellet) pellet dissolves the use has surpassed the washed and, 1/10 pellets to concentrated suspension number was high pressure liquid coolant.

2. Number milk bath

According to table 8 of metering and stirring was blended in such a proportion (per the sugar-free which is instantly, flavor addition number). In the case of value per, followed by blended flavor-gate material in the subjected. After this, 95 °C, but 5 minutes, cooled and regulated so as to 43 °C 41 °C, after spawning culture liquid to been dried so, 41 °C after culture, pH 4. 4 to 4. 5 cultured in his terminate.

[Table 8]

3. Milk lactic acid bacteria counting

It does not borrow the mulberry leaves number using a lactic acid bacteria of the present invention strain and high pressure liquid coolant water milk were measured. The results of table 9 is shown.

As shown in the table 9, and a strain of the present invention added it does not borrow [ppong[ppong] concentrate milk after fermentation, lactic acid fermentation than before increases the number 2 to 3 log/CFU formate, this natural edible material acts as lactic acid concentrate is free bio tic [su[su]it does not borrow [ppong[ppong] determines that increasing with each other. In addition, due to the synergistic effect of total lactic acid bacteria strain derived from screening strains are commercial infant feces is incremented.

[Table 9]

Depository agency life: Korean degrading (abroad)

Consignee number: KCCM11766P

Consignee date: 2015911