KR20160078688A - Manufacturing method of probiotics powder containing goat milk - Google Patents

Abstract

The present invention relates to immune-enhancing probiotics powder containing goat milk and, more specifically, to immune-enhancing probiotics powder containing goat milk, which is composed of 16 wt% of fermented goat milk powder, 48 wt% of mango powder, 0.8 wt% of vitamin C, 15.2 wt% of polydextrose, and 20 wt% of maltodextrin.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for manufacturing an immunostimulatory probiotic powder containing a goat milk,

The present invention relates to an immune enhancing probiotic powder containing a goat milk, and more particularly, to a fermented probiotic powder containing 16 wt% of fermented goat milk powder, 48 wt% of mango powder, 0.8 wt% of vitamin C, 15.9 wt% of polydextrose 15.2 And 20% by weight of maltodextrin. The present invention relates to a probiotic powder for immune enhancement containing a goat milk.

Lactic acid bacteria are the most beneficial species among microorganisms and have a long history and have a close and direct relationship with the lives of human beings, mainly fermented dairy products.

 Currently, Lactobacillus is a representative probiotics strain that has beneficial effects on the host by improving the balance of intestinal microorganisms. It is a generic Recognized As Safe (GRAS) and has a long history as fermented foods (dairy products, Fermented sausage), etc., and the probiotic strain is widely applied to human life.

Probiotics strains have been proven to have various effects, such as antibiotics, cholesterol-lowering substances and osteoporosis prevention, which are produced by lactic acid bacteria. There is a lot of emphasis on probiotics efficacy studies.

In addition, specific probiotics strains contain various immune substances that enhance immune function, and studies are underway. In recent years, functions related to disease prevention and bio-defense functions such as obesity and allergy prevention have been observed.

It is an object of the present invention to provide a probiotic powder product for immunostimulation comprising a fermented goat milk powder inoculated with a lactic acid bacterium having excellent immunity.

For this purpose, the present invention provides a fermented goat milk powder comprising 16% by weight of powdered fermented goat milk, 48% by weight of mango powder, 0.8% by weight of vitamin C, 15.2% by weight of polydextrose and 20% by weight of maltodextrin, . ≪ / RTI >

In addition, in the present invention, the fermented goat milk powder is prepared by mixing 1 part by weight of goat milk powder with 100 parts by weight of distilled water, sterilizing the mixture at 90 DEG C for 10 minutes, cooling the mixture to 42 DEG C, and inoculating 1 part by weight of lactic acid bacterium Followed by incubation at 42 DEG C for 8 hours, followed by lyophilization.

In the present invention, the lactic acid bacteria may be selected from the group consisting of Bifidobacterium bifidum , L. casei , L. helveticus , and L. rhamnosus . And one or two or more of them are mixed .

In the present invention, the lactic acid bacterium may be selected from the group consisting of B. bifidum , L. casei , L. helveticus , and L. rhamnosus , Are mixed at a ratio of 1: 1: 1: 1.

The present invention as described above is characterized in that one of Bifidobacterium bifidum , L. casei , L. helveticus and L. rhamnosus Or fermented goat milk powder inoculated with two or more kinds of lactic acid bacteria is added, so that it is excellent in immunity, and mango powder and vitamin C are added, so that color taste and taste are excellent.

FIGS. 1A to 1C show the viability of Raw 264.7 cells in the culture medium of lactic acid bacteria cultured on MRS broth medium.
FIG. 2 shows the viability of Raw 264.7 cells in heat-treated extracts of lactic acid bacteria.
FIG. 3 is a graph showing the inhibitory effect on the production of NO (nitric oxide) by Raw 264.7 cells in the culture medium of lactic acid bacteria cultured in MRS broth medium.
4 is a graph showing the inhibitory effect of the extract of the lactic acid bacteria on NO production in Raw 264.7 cells.
FIG. 5 shows changes in viable counts of selected strains depending on the concentration of goat milk powder.
6 shows the survival rate of Raw 264.7 cells according to the concentration of the goat milk powder.
FIG. 7 shows the results of measurement of inhibitory effect of NO production on Raw 264.7 cells in the treatment of goat milk powder at different concentrations.
FIG. 8 shows changes in the number of viable cells and pH of the lactic acid bacteria in the 1% goat milk powder according to the incubation time.
FIG. 9 shows changes in viable counts of lactic acid bacteria in the goat milk powder supplemented with growth factor.
10 shows the cell proliferation (P < 0.05) of Raw 264.7 cells treated with the goat milk powder of lactic acid bacteria and the MRS broth culture,
11 shows the cell proliferative activity of Raw 264.7 cells treated with the goat milk powder and the MRS broth culture of lactic acid bacteria.

Hereinafter, a method of producing a probiotic powder for an immunity enhancement containing a goat's milk according to the present invention will be described with reference to the accompanying drawings.

1. Research method.

1) Selection of immune enhancing lactic acid bacteria.

(1) Culture of lactic acid bacteria and macrophages used in the experiment.

Bifidobacterium bifidum, B. breve, B. longum, Lactobacillus acidophilus, L. delbrueckii subsp. Bulgaricus, L. casei, L. helveticus, L. plantarum, L. rhamnosus, Leuconostoc lactis, and Streptococcus thermophilus were purchased from the Korean Microorganism Conservation Center (Korea) and used for experiments. To investigate the effect of RAW 264.7 cells on the immune enhancement, (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 100 U / ml streptomycin / penicillin (Gibco, USA) (Dulbecco's Modified Eagle Medium) (Gibco, USA) in a 5% CO ₂ incubator at 37 ° C for 2-3 days. Lactic acid bacteria were cultured in MRS broth (Oxoid, England) for 48 hours at 37 ℃ for activation.

(2) Pretreatment of strain.

Lactic acid bacteria were inoculated on MRS broth medium, cultured at 37 ° C for 48 hours, diluted to 1.0 ⁸ CFU / ml, centrifuged at 10,000 rpm for 10 minutes, and separated into supernatants and pellets. The supernatant was diluted with distilled water after filtration with a 0.45 μm syringe filter and used as a sample. The pellet was washed twice with sterilized distilled water, suspended in 1 ml of distilled water, heat-treated at 100 ° C for 30 minutes, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was filtered with a 0.45 μm syringe filter After storage at -80 캜, it was used as a sample.

(3) Measurement of cell viability.

Effects of lactic acid bacteria culture medium and cell lysate on the growth of Raw 264.7 cells were measured. The cells were subcultured in a 96-well plate at ⁴ × 10 ⁴ cells / well. After stabilization for 24 hours, each sample was treated and reacted for 24 hours. Then, 10 μl of PrestoBlue ^™ reagent (Invitrogen, USA) was added and reacted at 37 ° C. for 20 minutes. Then, the absorbance at 570 nm (reference 600 nm) was measured using an enzyme-linked immunosorbent assay reader (DYEX, USA) Were measured. The absorbance of the control group without treatment of the sample was 100%, indicating the degree of cell proliferation.

(4) Measuring ability to inhibit nitric oxide (NO) production.

Effects of lactic acid bacteria culture medium and cell lysate on NO production were measured in Raw 264.7 cells stimulated with lipopolysaccharide (LPS) (Sigma, USA). The cells were subcultured in 96-well plates at ⁴ × 10 ⁴ cells / well and stabilized for 24 hours. Each sample was treated and 2 hours later, lipopolysaccharide (LPS) was treated at a concentration of 1 / / mL and cultured for 24 hours. After 24 hours, the same amount of a Griess reagent (Promega, USA) was added to 100 μl of the supernatant, and the mixture was allowed to stand at room temperature for 20 minutes, and the absorbance at 530 nm was measured with an enzyme-linked immunosorbent assay reader (ELISA reader). The concentration of NO was calculated by preparing a standard curve using sodium nitrate.

2) Determination of acid resistance and bile acidity of selected lactic acid bacteria.

(1) Acid resistance measurement.

The acid resistance test was carried out by adding 1% pepsin to the MRS broth medium adjusted to pH 2.5 with 1N HCl and inoculating 2% each of the four strains cultured at 37 ° C for 48 hours in the MRS broth medium And cultured at 37 DEG C for 3 hours. After 3 hours, the cells were diluted with 0.85% physiological saline by decidual dilution method and cultured at 37 ° C for 48 hours using BCP agar medium. The viable cell counts were compared with the number of viable cells before culturing.

(2) Measurement of biliary properties.

Biliary tests were performed by inoculating 0.3% bile salt (Sigma, USA) into MRS broth medium and then inoculating 2% each of 4 strains cultured at 37 ° C for 48 hours in MRS broth medium And cultured at 37 ° C for 24 hours. Thereafter, the cells were diluted by decidual dilution method using 0.85% physiological saline, and then cultured at 37 ° C for 48 hours using BCP agar medium. The viable cell counts were measured and compared with the number of viable cells before the incubation.

3) Examination of culture medium for improving immunological activity.

(1) Culture of lactic acid bacteria according to the concentration of goat milk powder.

1 part by weight of goat milk powder was added to 100 parts by weight of distilled water, sterilized at 90 DEG C for 10 minutes, cooled to 42 DEG C, and 1% each of lactic acid bacteria activated in an MRS broth medium was inoculated at 42 DEG C for 24 hours. The number of lactic acid bacteria was measured using an agar medium.

(2) Effects of goat milk powder on survival and NO production inhibition of Raw 264.7 cells.

The goat milk powder was diluted with distilled water to make 1%, sterilized at 90 ° C for 10 minutes, centrifuged at 8,500 rpm for 10 minutes, filtered through a 0.45 μm filter with supernatant, and added to Raw 264.7 cells at 6.25-100 μl / ml And the cell viability and NO production inhibition ability were measured.

4) Studies on high concentration culture of lactic acid bacteria in goat milk powder.

(1) Characterization of Lactic Acid Bacteria Culture in Goat Milk Powder.

To 100 parts by weight of distilled water, 1 part by weight of goat milk powder was added, followed by sterilization at 90 DEG C for 10 minutes, cooling to 42 DEG C, and cultivation in MRS broth medium for 48 hours to activate and isolate 1% Were inoculated at 0.25% in total to 1%), and then incubated at 42 ° C. for 48 hours to measure viable cell count and pH.

(2) Examination of conditions for high concentration cultivation of lactic acid bacteria.

1%, 2% and 3% of glucose and fructo-oligosaccharide were added as a carbon source to 1% goat milk powder, followed by sterilization at 90 ° C for 10 minutes and cooling. The total number of viable cells was measured by inoculating 4% of the lactic acid bacteria activated in MRS broth medium at 37 ° C for 48 hours in 0.25% total amount of 1% and incubating at 42 ° C for 24 hours.

5) Effect of Fermented Goat Milk Powder on the Immune Activity Enhancement.

1 part of the goat milk powder was added to 100 parts of distilled water, sterilized at 90 ° C for 10 minutes, cooled to 42 ° C, and cultured for 48 hours in an MRS broth medium. The activated lactic acid bacteria were individually and mixed with 1% 0.25% of each strain was inoculated so as to have a total of 1%), and the cells were inoculated and cultured at 42 ° C so that the number of lactic acid bacteria was 10 ⁸ CFU / ml. After centrifugation at 8,500 rpm for 10 minutes, the supernatant was filtered through a 0.45 μm filter and treated with 100 μl / ml of Raw 264.7 cells. Cell viability and NO production inhibition were measured.

6) Statistics.

The data were expressed as mean standard deviation. Statistical analysis was performed by one-way ANOVA using the Statistics Base 18 (SPSS 18) program followed by Duncan's multiple range test. The significance level was expressed as p-value <0.05. Data were also analyzed for significance level p-value <0.05 for indepentent t-test. (Here, the p value is a statistical term, meaning the probability that the statistical value is greater than the actual observed value, assuming that the null hypothesis is correct.)

Results.

1) Selection of immune enhancing lactic acid bacteria.

(1) Measurement of Raw 264.7 cell viability by culture medium and cells of lactic acid bacteria.

Immunoreactivity was measured in 11 strains of standard microorganisms in order to select immune enhancing lactobacillus strains. Standard microbial strains were purchased from the Korean Center for Microbiological Conservation (KCCM). Standard microbial strains were identified as Bifidobacterium bifidum , B. breve , B. longum , Lactobacillus acidophillus , L. delbrueckii subsp. bulgaricus , L. casei , L. helveticus , L. plantarum , L. rhamnosus , Leuconostoc lactis and Streptococcus thermophilus were preferentially used. Immunoactivity measurements were performed using Raw 264.7 cells to determine cell viability and / NO production inhibitory activity was measured. Lactic acid bacteria were cultivated primarily on MRS broth medium, and the culture broth and lactic acid bacteria were used as experimental raw materials. In the culture medium, Raw 264.7 cells were inhibited by 10 times dilution in all experimental groups, whereas Raw cell cultures were not inhibited by dilution more than 1000 times. Therefore, we decided to measure the effect of Raw 264.7 cells on the inhibition of NO production by using a 1000-fold diluted culture medium that does not show toxicity.

Cell viability was measured by treatment of Raw cells with the heat - treated cell extracts to confirm the immune activity against the lactic acid bacteria. L. helveticus and Bifidobacterium bifidum did not inhibit the growth of Raw 264.7 cells in all experimental groups, and about 13% and 11% of Raw 264.7 cells. &Lt; / RTI &gt; Therefore, the effect of Lactobacillus extract isolated from Raw 264.7 cells on NO production inhibition was measured.

(2) Measurement of inhibitory effect of Raw 264.7 cells on NO production by culture medium and cells of lactic acid bacteria.

Macrophages digest foreign substances in the body or generate NO to destroy foreign bacteria or tumor cells. However, overexpression of NO, a cytotoxic substance due to excessive inflammatory reaction, can negatively affect the human body such as tissue damage, genetic mutation, damage to nerves, and vascular permeability to cause edema. Therefore, we investigated the inhibition of NO production by cultured Lactobacillus acidophilus and mycelial cells after treatment of lipopolysaccharide (LPS) with macrophage Raw 264.7 cells.

Lactobacillus cultures cultured in MRS broth medium were significantly inhibited NO production in all experimental groups. However, the MRS broth medium itself was similar to that of the MRS broth medium. Since the MRS broth medium is difficult to use directly as a food material, we tried to use goat milk as an immunity enhancing base material that can be used as a food material.

As a result of measuring the NO production ability of the heat-treated extract of the lactic acid bacteria, all the groups showed a significant inhibition of NO production, Lactobacillus L. rhamnosus and L. casei tended to inhibit NO production by more than 30% compared to the lipopolysaccharide (LPS) positive control.

Cell viability was the highest concentration that did not inhibit the growth of Raw cells in the culture medium diluted 1000 times or more. In the heat - treated cell extract, L. helveticus and Bifidobacterium bifidum ( B bifidum ) promoted the growth of Raw 264.7 cells by about 13% and 11%, respectively.

In addition, inhibition of NO production is thought to inhibit NO production in the MRS broth medium itself, which is the basic medium for culture of lactic acid bacteria in the culture medium. Lactobacillus L. rhamnosus and L. casei were found to inhibit NO production by more than 30% compared to the lipopolysaccharide (LPS) positive control.

Therefore, it has been found that Bifidobacterium bifidum , L. casei , L. helveticus and Lactobacillus lambosus , which have been shown to have an effect on cell survival rate and inhibition of NO production, L. rhamnosus ) were selected as the lactic acid bacteria that enhance the immunological activity.

2) Determination of acid resistance and bile acidity of selected lactic acid bacteria.

Survival rates in gastric acid and bile acid were measured for the four selected strains of lactic acid bacteria that promote immunological activity (see Tables 1 and 2). As a result, all strains were found to survive at least 95% and 84% or more in gastric acid and bile acid All four species showed excellent acid resistance and durability.

Table 1 shows acid resistance of selected lactic acid bacteria. Strain
Before culture
(log CFU / mL) After incubation
(log CFU / mL) Survival rate (%)
pH 7.0 pH 2.5 Bifidus Bert Terim Bifidum
( B. bifidum) 7.92 7.65 96.59 Lactobacillus casei
( L. casei) 7.46 7.06 94.64 Lactobacillus helveticus
( L. helveticus) 7.82 7.77 99.36 Lactobacillus lambosus
( L. rhamnosus) 7.80 7.70 98.72

Table 2 shows the bacteriostatic properties of the selected lactic acid bacteria Strain
Before culture
(log CFU / mL) After incubation
(log CFU / mL) Survival rate (%)
Bifidus Bert Terim Bifidum
( B. bifidum) 7.45 7.32 98.26 Lactobacillus casei
( L. casei) 7.10 6.82 96.06 Lactobacillus helveticus
( L. helveticus) 7.68 6.53 85.03 Lactobacillus lambosus
( L. rhamnosus) 7.88 6.60 83.76

3) Examination of culture medium of lactic acid bacteria for improving immunological activity.

(1) Examination of culture medium of lactic acid bacteria of goat milk powder.

In order to increase the concentration and selectivity of the selected lactic acid bacteria, a culture medium other than the MRS broth medium was examined. To use a natural culture medium, a standard strain was cultured using a goat milk powder. When the selected strain was cultured in the 1% and 5% concentration of goat milk powder, the number of viable cells was about 10 ⁸ CFU / ml and the number of lactic acid bacteria increased as the concentration of the goat milk powder increased. 1% goat milk powder did not significantly inhibit the growth of lactic acid bacteria. In order to reduce the cost of cultivation of lactic acid bacteria during industrialization, 1% of goat milk powder was selected as a culture medium, and immunological activity confirmation test was conducted using this.

(2) Effect of Raw 264.7 cells on the enhancement of immunological activity of goat milk powder.

1% goat milk powder was treated with 6.25, 12.5, 25, 50 and 100 μL / mL of Raw 264.7 cells and the MRS broth medium was treated with 10 ^-3 , which was not toxic to the cell survival rate, And NO production inhibiting ability were measured. The cell viability increased with increasing the concentration of goat milk powder. Especially, the cell survival rate of 12.5μl / mL of goat milk powder was significantly increased to 9.7% ~ 16.5% compared to MRS.

NO production inhibition was significantly inhibited in all experimental groups compared to lipopolysaccharide (LPS) positive control. The NO production inhibitory activity tended to be slightly increased as the goat milk powder concentration increased, The inhibition of NO production was about 2.5 ~ 10.1% compared to the MRS broth medium. Therefore, the goat milk powder was expected to have immunological activity.

4) Studies on high concentration culture of lactic acid bacteria in goat milk powder.

(1) Culture characteristics of lactic acid bacteria in goat milk powder.

To determine the growth characteristics of 1% goat milk powder of selected lactic acid bacterium, the viable cell count and pH were measured according to the incubation time of lactic acid bacterium in 1% goat milk powder. Bifidobacterium bifidum, L. rhamnosus and four mixed strains reached 10 ⁸ CFU / mL at 6 hours of culture, and L. helveticus , Reached 10 ⁸ CFU / ml during 12 h incubation and then decreased to 10 ⁷ CFU / ml at 24 h. The pH rapidly decreased in all experimental groups to 4.0 at 6 hours of incubation and thereafter a slight decrease in pH was observed.

(2) Examination of conditions for high concentration cultivation of lactic acid bacteria.

In order to cultivate high concentration of lactic acid bacteria in 1% goat milk powder, glucose and prebiotics fructo-oligosaccharide, which are used as an energy source of lactic acid bacteria, Respectively. When glucose was added to goat milk powder, it grew to 10 ⁹ CFU / ml at 12 hours of culture, but there was no difference according to the concentration. In the addition of fructo-oligosaccharide, the number of viable cells reached 10 ⁹ CFU / ml at 12 hours of incubation in 1% of the medium, and 10 ⁸ CFU / ml of the 2% and 3% And the number of viable cells tended to decrease as the concentration of fructooligosaccharide was increased.

5) Measurement of the effect of the fermented goat milk powder on the enhancement of the immune activity of selected lactic acid bacteria.

The concentration of goat milk powder and fermented goat milk powder, which do not show toxicity in Raw 264.7 cells, was 100 μl / ml in the case of 1% goat milk powder fermentation and was used as a test sample for the immune activity when the number of lactic acid bacteria reached 10 ⁸ CFU / And cell viability was measured by treating the cells. Cell survival rates of L. casei and L. helveticus were significantly higher than those of non - fermented goat milk powder, especially about 11.4% and 15.2% Growth. The cell viability of L. casei was significantly higher in the L. casei compared to the MRS culture and the goat milk powder compared to the MRS culture, .

Inhibition of NO production by inoculation of 1% goat milk powder with lactic acid bacteria was significantly inhibited in all groups compared with lipopolysaccharide (LPS) positive control. Lactobacillus helveticus , L. rhamnosus , and four kinds of mixed goat milk powder showed significantly higher NO production inhibitory activity. The NO production inhibitory activity of the MRS broth culture and the goat milk powder culture was compared with that of the goat milk powder culture. In particular, the inhibitory effect of the B. bifidum Lactobacillus L. rhamnosus showed 5.6% and 17.4% inhibition of NO production, respectively.

The immune enhancing probiotic powder containing the goat milk according to the present invention was prepared by mixing 16 wt% of the fermented goat milk powder, 48 wt% of mango powder, 0.8 wt% of vitamin C, 15.2 wt% of polydextrose, and 20% by weight of maltodextrin.

Mangoes have carotenoids with antioxidant activity, ascorbic acid and quercetin, and polyphenols such as camphorol, and are especially rich in beta-carotene, the most active vitamin A. It removes free radicals and helps to improve vision, skin regeneration and immune function. Therefore, by adding the mango powder, the synergy effect for enhancing the immune function and the preference of the color and sweetness of the mango itself are improved.

Next, vitamin C has the advantage of preventing scurvy, promoting collagen formation, promoting iron absorption, and strengthening the immune system, as well as preventing oxidative stress-related diseases. The present invention enhances the immunity enhancing activity and sour taste by adding vitamin C having such effect.

Next, polydextrose is an insoluble polysaccharide, which is a water-soluble dietary fiber giving a low energy, and has an advantage of facilitating the defecation activity smoothly. Maltodextrin, which is produced mainly from corn, is used in foods that are low in hygroscopicity and in which water absorption is undesirable. The polydextrose and maltodextrin are used as an extender in combination with their own advantages.

Examples.

100 parts by weight of distilled water was mixed with 1 part by weight of goat milk powder, sterilized at 90 DEG C for 10 minutes, and cooled to 42 DEG C to prepare a goat milk. Then, 100 parts by weight of the goat milk was inoculated with 1 part by weight of lactic acid bacteria, followed by incubation at 42 DEG C for 8 hours, followed by lyophilization to form a fermented goat milk powder.

Herein, the lactic acid bacteria are selected from the group consisting of Bifidobacterium bifidum , L. casei , L. helveticus and L. rhamnosus at 0.25 By weight.

Next, 16% by weight of the fermented goat milk powder, 48% by weight of mango powder, 0.8% by weight of vitamin C, 15.2% by weight of polydextrose and 20% by weight of maltodextrin were mixed, To prepare an immunostimulatory probiotic powder containing a goat milk.

Claims (4)

Characterized in that it comprises 16% by weight of fermented goat milk powder, 48% by weight of mango powder, 0.8% by weight of vitamin C, 15.2% by weight of polydextrose and 20% by weight of maltodextrin. Containing probiotic powder for immunity enhancement.
The fermented goat milk powder according to claim 1, wherein 1 part by weight of the goat milk powder is mixed with 100 parts by weight of distilled water, sterilized at 90 ° C for 10 minutes, and cooled to 42 ° C. , Followed by incubation at 42 ° C for 8 hours, followed by lyophilization. The immunostimulatory probiotic powder containing the goat milk.
The method of claim 2, wherein the lactic acid bacteria are selected from the group consisting of Bifidobacterium bifidum , L. casei , L. helveticus , and L. rhamnosus . Or a mixture of two or more of the probiotics for immune enhancement.
4. The method according to claim 3, wherein the lactic acid bacteria are selected from the group consisting of Bifidobacterium bifidum , L. casei , L. helveticus , and L. rhamnosus (Lactobacillus sp. ) Is mixed at a ratio of 1: 1: 1: 1.

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