KR102098655B1 - Lactobacillus fermented drink mixture for preventing virus disease - Google Patents

Abstract

The present invention is a bakery powder obtained by drying and crushing a bakery product prepared by fermenting a dough containing whey with a mixed probiotic of two types of yeast strains of lactic acid bacteria and Saccharomyces of the genus Leuconostock and Lactobacillus. Purified water, whey powder, and added sugar to control the sugar content of the lactobacillus nhs221 strain (Accession No. KCTC-11749BP) culture broth fermentation and fermentation lactic acid bacteria fermentation broth having a protective effect.

Description

Lactobacillus fermented drink mixture for preventing virus disease}

The present invention relates to a lactic acid bacteria fermentation broth having a virus disease prevention effect. More specifically, the present invention is obtained by drying and crushing a bakery product prepared by fermenting a dough containing whey with a mixed probiotic of two yeast strains of the genus Leuconostock and Lactobacillus and Saccharomyces. Purified water, whey powder, saccharides are added to the bakery powder and the sugar content is adjusted, and then a culture medium of the strain Lactobacillus nhs221 (Accession No. KCTC-11749BP) is fermented and fermented.

Virus means a toxic substance in Latin and means infectious pathogenic particles passing through a bacterial filter (0.22㎛). Viruses are classified into bacteriophage, plant virus, and animal virus according to the type of host cell, and also classified into DNA virus and RNA virus according to the type of nucleic acid. Recently, various viral diseases such as swine flu, AI, and foot and mouth disease are causing social problems, and accordingly, prevention of viral diseases is urgently required.

Currently, the best way to prevent viral disease is vaccination. Nevertheless, in the case of diseases caused by viruses, the effectiveness of the vaccine is raised as many virus serotypes (subtypes) are generated.

Representative antiviral agents that inhibit the proliferation of influenza virus include amantadine and rimantadine, but these two antiviral agents are effective only for serotype A influenza virus and serotype B without M2 protein. It was found to have no effect on the influenza virus. On the other hand, in order to compensate for the disadvantages of the antiviral agent, zanamivir and oseltamivir have been developed as antiviral agents effective against all 16 serotype A influenza viruses and serotype B influenza viruses. However, zanamivir has the disadvantage of inhalation and intravenous administration, and oseltamivir can be orally administered, but it has been pointed out that side effects of vomiting and dizziness occur during oral administration and reports of the emergence of resistant viruses.

Herpes simplex virus (HSV) is a DNA virus belonging to the genus Herpes virus and is a relatively large virus of about 175 nm in size. Herpes simplex infection is an acute vesicular disease that affects the mucous membranes or skin, and is an infection commonly seen in people with atopy. HSV-1 mainly forms around the mouth and eyes, and HSV-2 forms blisters around the genitals. It is reported that the infection of HSV progresses severely in patients with reduced immune function.

HSV begins to replicate in the skin or mucosal epidermal cells after the initial infection, moves along the neural tissue, and remains active in the vertebral ganglion throughout life. As a therapeutic agent for HSV infectious diseases, the nucleoside derivative Acyclovir is known to be very effective in initial infection, but it is necessary to continuously administer the drug and if the administration is discontinued, the infectious diseases caused by HSV are likely to recur. It is known.

Recently, various health functional preparations using a probiotic (probiotic), which is a living microbial preparation containing a Bifidobacteria strain, have been developed.

Accordingly, the present inventor put the purified water and sugar into the bread powder through the patent application of Korean Patent Application No. 10-2017-0017318, 'Lactic Acid Bacteria Fermentation Solution with Weight Control Effect', and after controlling sugar content, the strain nhs221 of the genus Lactobacillus (Accession No. KCTC -11749BP) It has been disclosed that the fermented lactic acid bacteria fermented by adding the culture broth as a functional beverage having obesity prevention and weight control effect.

The functional beverage disclosed in the above-mentioned patent document is the Republic of Korea Patent Registration No. 10-923051 'Functional probiotic preparation and bread produced using the same' as the main material, which has been developed and commercialized by the present inventor, and after adding purified water to prepare a mixed solution, click here Lactobacillus nhs221 strain (Accession No. KCTC-11749BP) is a lactic acid bacteria fermentation broth prepared by fermentation and aging.

However, the present inventor tested lactobacillus nhs221 strain (Accession No. KCTC-11749BP) while lactic acid bacteria fermentation broth was tested for the health and functional properties of whey-containing dough, Leuconostock and Lactobacillus lactic acid bacteria and Saccharomyces genus. After drying the bakery product prepared by fermentation with a mixed probiotic of a yeast strain and then pulverizing, put purified water, whey powder, and saccharides in the obtained bakery powder to control the sugar content, and then control the sugar content of the nhs221 strain (Accession No. KCTC-11749BP) The present invention was completed by confirming that the lactic acid bacteria fermentation broth fermented by fermentation and fermentation exhibited a virus disease prevention effect such as influenza virus and herpes simplex virus (HSV), and thus developed a lactic acid bacteria fermentation liquid having a virus disease prevention effect.

The problem to be solved by the present invention is to dry the bakery product prepared by fermenting the dough containing whey with a mixed probiotic of two yeast strains of the genus Leuconostock and Lactobacillus and the Saccharomyces genus. Purified water, whey powder, and saccharides were added to the obtained bakery powder, and after controlling the sugar content, a culture medium of Lactobacillus nhs221 strain (Accession No. KCTC-11749BP) was added to fermentation and fermentation of lactic acid bacteria fermentation broth, herpes simplex virus (HSV) It was intended to measure the effectiveness of preventing such viral diseases. In addition, the intention was to develop a lactic acid bacterium fermentation broth having an effect of preventing viral diseases such as influenza virus and herpes simplex virus (HSV).

The object of the present invention is ⅰ) adding 100 to 10 parts by weight of whey powder to 100 parts by weight of the raw material dough to prepare a dough by a medium method; Ii) adding 0.01 to 1.0 parts by weight of a mixed probiotic mixture of two yeast strains of the genus Lactococcus and Lactobacillus and Saccharomyces, followed by fermentation and aging to prepare a bakery product; Iv) 300 to 500 parts by weight of purified water is added to 100 parts by weight of the bakery powder obtained by drying and pulverizing the bakery product, and 0.1 to 10 parts by weight of whey powder, sugar and / or fructose are added to a concentration of 10 to 15 ° Brix. Adjusting sugar content; And iv) 0.5 to 2.0 parts by weight (1.0 to 8.0 × 10 ⁸ CFU) of Lactobacillus nhs221 strain (Accession No. KCTC-11749BP) culture medium was added and fermented for 40 to 70 days at room temperature to obtain a lactic acid bacteria fermentation broth; In the method for producing a lactic acid bacteria fermentation broth having a virus disease prevention effect consisting of, the mixed probiotic is Leukonostock genus nhs210 strain (Accession No: KCTC-11226BP), Lactobacillus genus nhs221 strain (Accession No: KCTC-11749BP), Saccharomyces CCRG321 yeast strain (Accession No .: KCTC-13300BP) and Saccharomyces genus nhs321 yeast strain (Accession No .: KCTC-11227BP) provide a method for producing a lactic acid bacteria fermentation broth with virus disease prevention effect Is to do.

At this time, the lactic acid bacteria fermentation broth is characterized by having a virus disease prevention effect, such as influenza virus and herpes simplex virus.

In addition, the Saccharomyces genus CCRG321 yeast strain (Accession No .: KCTC-13300BP) was passaged through a plate for separation of PDA (Potato dextrose agar) separation medium with 3% by weight of whey powder, followed by wild-type baking yeast. It is characterized by being a strain obtained by separating and culturing the yeast strain colonies having excellent growth potential by inoculation culture three times.

Another object of the present invention is to provide an animal feed having a viral disease preventing effect, including 1 part by weight of the lactic acid bacteria fermentation broth and 100 to 10,000 parts by weight of the feed.

The effect of the present invention is a bakery obtained by drying and crushing a bakery product prepared by fermenting a dough containing whey with a mixed probiotic of two types of yeast strains of the genus Leuconostock and Lactobacillus and Saccharomyces. After adding purified water, whey powder and saccharides to the powder, and controlling the sugar content, the fermentation and maturation of influenza virus, herpes simplex virus (HSV), etc. by fermentation of nhs221 strain (Accession No. KCTC-11749BP) culture medium It is to provide a lactic acid bacteria fermentation broth.

1 is a diagram showing the antiviral activity test results of the lactic acid bacteria fermentation broth prepared in Preparation Example 1 of the present invention for PR8-GFP (influenza virus-green fluorescent protein). 1A is a diagram showing cell viability and FIG. 1B is a diagram showing GFP (green fluorescent protein) absorption.
Figure 2 is a view showing the results of antiviral activity of the lactic acid bacteria fermentation broth prepared in Preparation Example 1 of the present invention for HSV-GFP (herpes simplex virus-green fluorescent protein). Figure 2A is a diagram showing cell viability and Figure 2B is a diagram showing GFP uptake.

The present invention comprises the steps of: iii) adding 1.0 to 10 parts by weight of whey powder to 100 parts by weight of the raw material dough to prepare a dough by a medium method; Ii) adding 0.01 to 1.0 parts by weight of a mixed probiotic mixture of two yeast strains of the genus Lactococcus and Lactobacillus and Saccharomyces, followed by fermentation and aging to prepare a bakery product; Iv) 300 to 500 parts by weight of purified water is added to 100 parts by weight of the bakery powder obtained by drying and pulverizing the bakery product, and 0.1 to 10 parts by weight of whey powder, sugar and / or fructose are added to a concentration of 10 to 15 ° Brix. Adjusting sugar content; And iv) 0.5 to 2.0 parts by weight (1.0 to 8.0 × 10 ⁸ CFU) of Lactobacillus nhs221 strain (Accession No. KCTC-11749BP) culture medium was added and fermented for 40 to 70 days at room temperature to obtain a lactic acid bacteria fermentation broth; In the method for producing a lactic acid bacteria fermentation broth having a virus disease prevention effect consisting of, the mixed probiotic is Leukonostock genus nhs210 strain (Accession No: KCTC-11226BP), Lactobacillus genus nhs221 strain (Accession No: KCTC-11749BP), Saccharomyces genus CCRG321 yeast strain (Accession No .: KCTC-13300BP) and Saccharomyces genus nhs321 yeast strain (Accession No .: KCTC-11227BP) with respect to a method for producing a lactic acid bacteria fermentation broth having a virus disease prevention effect will be.

In addition, the present invention relates to an animal feed having a virus disease prevention effect, including 1 part by weight of the fermentation broth of the lactic acid bacteria and 100 to 10,000 parts by weight of the feed.

Hereinafter, a method for preparing a lactic acid bacteria fermentation broth having a virus disease preventing effect of the present invention will be described in more detail.

(Step 1) It is a step of preparing dough by intermediate method by adding 1.0 to 10 parts by weight of whey powder to 100 parts by weight of raw material dough.

(Step 2) Fermentation by adding 0.01 to 1.0 parts by weight of a mixed probiotic of lactic acid bacteria of the genus Leuconostock and Lactobacillus and two yeast strains of Saccharomyces to the dough prepared by the intermediate method prepared in step 1 above After aging, bake in an oven to prepare bakery products.

At this time, the mixed probiotic preparations include Leukonostock genus nhs210 strain (Accession number: KCTC-11226BP), Lactobacillus genus nhs221 strain (Accession number: KCTC-11749BP), Saccharomyces genus CCRG321 yeast strain (Accession number: KCTC-13300BP) ) And Saccharomyces nhs321 yeast strain (Accession No .: KCTC-11227BP).

(Step 3) 300 to 500 parts by weight of purified water is added to 100 parts by weight of the bakery powder obtained by drying and crushing the bakery product prepared in step 2, and 0.1 to 10 parts by weight of whey powder, sugar, and / or fructose are added. This step is to control the sugar content to 10 ~ 15 ° Brix concentration.

(Step 4) 0.5 to 2.0 parts by weight (1.0 to 8.0 × 10 ⁸ CFU) of culture medium of nhs221 strain (Accession No. KCTC-11749BP) of the genus Lactobacillus in the mixture prepared in step 3 was fermented at room temperature for 40 to 70 days This is a step of aging to obtain a lactic acid bacteria fermentation broth.

At this time, the method for separating the probiotic strain is as follows.

1. Live strain of Ryukonostock (Accession No. KCTC-11226BP) and Saccharomyces

    Separation of yeast strains (Accession No. KCTC-11227BP)

The method of separation and identification of live strains of genus Leukonostock (accession number KCTC-11226BP) and yeast strains of genus Saccharomyces (accession number KCTC-11227BP) is described in Korean Patent Registration No. 10-923051 previously invented by the present inventors It is disclosed in the specification of 'functional probiotic and bread made using it'.

2. Isolation of the strain Lactobacillus nhs221 (Accession No .: KCTC-11749BP)

When natural fruit juice is added to the sponge for bread production, and left at room temperature for 1 week, microorganisms in the air are in contact with the sponge and fermented and cultured. After drying the fermented cultured sponge, the growth of microorganisms is promoted at room temperature for 2 to 3 days under anaerobic conditions. After crushing the dry sponge containing microorganisms into a flat plate, 5 mg of dry sponges crushed in a flat plate are separately obtained at 10 locations. The obtained 10 crushed dry sponge 5 mg each was divided into 10 culture dishes, 5 g of starch medium was added to the culture dish, and cultured for 48 hours at 20 ° C. and 75% humidity conditions to give the most lactic acid among the 10 culture dishes. The fermentation-produced culture dish was selectively passaged and the strain was isolated. At this time, the isolated strain existed in a mixed state of the yeast strain and the lactic acid bacteria strain, but only the lactic acid bacteria strain was identified.

3. Isolation of Saccharomyces genus CCRG321 yeast strain (Accession No .: KCTC-13300BP)

Saccharomyces CCRG321 yeast strain (Accession No .: KCTC-13300BP) is a PDA (Potato dextrose agar) medium in which 3% by weight of whey powder is added to a plate plate using wild-type baker's yeast strain used for bakery as a parent strain. It is a strain obtained by separating and subculturing the yeast strain having excellent growth potential in the culture medium after passage through passage.

In addition, the whey powder used at this time was spray dried by concentrating the remaining whey after cheese production, and has a composition of whey protein 18 wt%, carbohydrate 57 wt%, crude fat 8 wt%, and moisture 5 wt%.

The wild type baker's yeast strain was used as a parent strain to isolate the strain colonies having the optimal growth in the separation medium, and then again cultured three times in the same manner to isolate the yeast strains optimally grown in whey powder-containing PDA medium. The present inventor named this strain Saccharomyces CCRG321 and carried out international deposits of microorganisms under the Budapest Treaty with the accession number KCTC-13300BP as of July 18, 2017 to the Korean Collection for Type Cultures.

Saccharomyces genus CCRG321 yeast strain of the present invention (Accession No .: KCTC-13300BP) The general mycological properties were generally consistent with those of Saccharomyces baker's yeast strain. However, unlike the conventional Saccharomyces spp., It was found to be a new strain that grows optimally by releasing a large amount of carbon dioxide gas in a PDA medium containing whey powder.

To confirm this, after adding 3 parts by weight of whey powder to 100 parts by weight of PDA medium, the yeast strain of the present invention was inoculated at 1 × 10 ³ CFU / ml per 1 ml of PDA medium to measure the amount of carbon dioxide generated in 24 hours and 36 hours. Did. As a control group, baker's commercial yeast sold by Zenico was used. Table 1 shows the amount of carbon dioxide generated.

Carbon dioxide generation (ml) 24 hours 36 hours Yeast strain of the present invention (KCTC-13300BP) 6 14 Zeniko's yeast 3 9

In addition, YPD basal medium (yeast extract 1%, bactopeptone 1%, glucose 2%) to measure the assimilation of sugar and whey powders of the Saccharomyces genus CCRG321 yeast strain (Accession No .: KCTC-13300BP) of the present invention ), 20 g / L of fructose, glucose, and whey powders were added, and assimilation of fructose, glucose, and whey powders was measured.

Saccharomyces CCRG321 yeast strain (Accession No .: KCTC-13300BP) grown in YPD liquid medium to which sugar or whey powder was added after 0, 12, 24, and 36 hours of inoculation of yeast strains was subjected to cell sample. After collection, it was centrifuged and washed to measure with a spectrophotometer. Table 2 shows the dry cell mass after culturing the yeast strain of the present invention.

0 hours 12 hours 24 hours 36 hours Fructose 0.05 2.0 3.3 4.6 glucose 0.05 1.8 3.2 4.5 Whey powder 0.05 1.4 3.8 5.1

                                                        (Unit: g / L)

As shown in Table 2, whey powder assimilation of the Saccharomyces genus CCRG321 yeast strain (Accession No .: KCTC-13300BP) of the present invention was lower than that of fructose or glucose after 12 hours of cultivation, but 24 After time, it showed higher anabolic properties than fructose or glucose. Through this, using the CCRG321 yeast strain of Saccharomyces genus (Accession No .: KCTC-13300BP) of the present invention, it was confirmed that a bakery product can be prepared when the paste containing whey powder is aged for 24 hours or more at low temperature fermentation.

In order to measure the anti-viral effect of the lactic acid bacteria fermentation broth of the present invention, antiviral effects on influenza virus and herpes simplex virus were measured through the mouse macrophage cell line RAW 264.7. That is, the viability of the macrophage cell line was observed and measured through a green fluorescent protein. The antiviral effect of the lactic acid bacteria fermentation broth of the present invention showed an increase in the viability of the macrophage cell line of 15% or more than the lactic acid bacteria fermentation broth prepared in Production Comparative Example 1.

The lactic acid bacteria fermentation broth of the present invention may be consumed as it is, but for example, other foods such as bread, chocolate, confectionery, ramen, ice cream, meat products, dairy products, or other quantitative sweeteners, flavors, vitamins, such as oligosaccharides, fructose or sugar, It is also possible to develop and drink lactic acid bacteria fermented beverages by adding minerals and pigments.

In addition, the lactic acid bacteria fermentation broth of the present invention can be developed, administered, and taken in the form of pharmaceutical preparations by including a pharmaceutically acceptable carrier, excipient, and diluent.

The dosage amount of the lactic acid bacteria fermentation broth of the present invention may be determined according to various factors such as the condition, age, sex and disease level of the user, but is usually 0.5 to 20 ml per 1 kg / day for adults, preferably 5 to 10 ml, and at this time, per 1 ml The lactic acid bacteria content is 0.1 to 200 × 10 ⁸ CFU.

On the other hand, the lactic acid bacteria fermentation broth of the present invention can be added to the feed to produce an animal feed having a virus disease preventing effect. Through this, an animal feed having a virus disease prevention effect including 1 part by weight of the lactic acid bacteria fermentation broth and 100 to 10,000 parts by weight of the feed can be prepared.

Hereinafter, the present invention will be described in more detail through preparation examples, manufacturing examples, manufacturing comparative examples, and examples.

(Preparation Example 1) Separation of live strains of genus Leukonostock (Accession No. KCTC-11226BP) and yeast strains of Saccharomyces (Accession No. KCTC-11227BP)

The method of separation and identification of live strains of genus Leukonostock (accession number KCTC-11226BP) and yeast strains of genus Saccharomyces (accession number KCTC-11227BP) is described in Korean Patent Registration No. 10-923051 previously invented by the present inventors It is disclosed in the specification of 'functional probiotic and bread made using it'.

(Preparation Example 2) Isolation of nhs221 strain (Accession No .: KCTC-11749BP) of the genus Lactobacillus

When natural fruit juice is added to the sponge for bread production, and left at room temperature for 1 week, microorganisms in the air are in contact with the sponge and fermented and cultured. After drying the fermented cultured sponge, the growth of microorganisms is promoted at room temperature for 2 to 3 days under anaerobic conditions. After crushing the dry sponge containing microorganisms into a flat plate, 5 mg of dry sponges crushed in a flat plate are separately obtained at 10 locations. The obtained 10 crushed dry sponge 5 mg each was divided into 10 culture dishes, 5 g of starch medium was added to the culture dish, and cultured for 48 hours at 20 ° C. and 75% humidity conditions to give the most lactic acid among the 10 culture dishes. The fermentation-produced culture dish was selectively passaged and the strain was isolated. At this time, the isolated strain existed in a mixed state of the yeast strain and the lactic acid bacteria strain, but only the lactic acid bacteria strain was identified.

(Preparation Example 3) Isolation of Saccharomyces genus CCRG321 yeast strain (Accession No .: KCTC-13300BP)

After passing the PDA (Potato dextrose agar) medium to which 3% by weight of whey powder was added to the plate plate by using the wild-type bakery yeast strain used in the bakery as a parent strain, and separating the yeast strain having excellent growth in the medium, 3 It was obtained by separating from passage culture. That is, after separating the strain colonies having the optimum growth in the separation medium, the cells were subcultured three times in the same manner, and the yeast strains optimally growing in the whey powder-containing PDA medium were separately identified.

(Production Example) Preparation of lactic acid bacteria fermentation broth of the present invention

2 g of whey powder is added to 100 g of the raw material dough to prepare the dough by intermediate method. To the prepared dough, 0.5 g of a mixed probiotic mixture of two yeast strains of Lactococcus and Lactobacillus and Saccharomyces is added to fermentation and aging to prepare a bakery product. At this time, the mixed probiotic preparations include Leukonostock genus nhs210 strain (Accession number: KCTC-11226BP), Lactobacillus genus nhs221 strain (Accession number: KCTC-11749BP), Saccharomyces genus CCRG321 yeast strain (Accession number: KCTC-13300BP) ) And Saccharomyces nhs321 yeast strain (Accession No .: KCTC-11227BP).

After drying the bakery product and pulverizing it, 300-500 ml of purified water is added to 100 g of the obtained bakery powder, and 2 g of whey powder, sugar, and / or fructose are added to adjust the sugar content to a concentration of 10-15 ° Brix. Lactobacillus genus nhs221 strain (Accession No. KCTC-11749BP) culture solution 1.0ml (1.0 ~ 8.0 × 10 ⁸ CFU) was added to the obtained mixture for fermentation at room temperature for 50 days to ferment and ferment the lactic acid bacteria having the virus disease prevention effect of the present invention To obtain

(Production Comparative Example 1) Preparation of lactic acid bacteria fermentation broth (whey powder not added)

The dough is prepared by using 100 g of raw material dough. To the prepared dough, 0.5 g of a mixed probiotic mixture of two yeast strains of Lactococcus and Lactobacillus and Saccharomyces is added to fermentation and aging to prepare a bakery product. At this time, the mixed probiotic preparations include Leukonostock genus nhs210 strain (Accession number: KCTC-11226BP), Lactobacillus genus nhs221 strain (Accession number: KCTC-11749BP), Saccharomyces genus CCRG321 yeast strain (Accession number: KCTC-13300BP) ) And Saccharomyces nhs321 yeast strain (Accession No .: KCTC-11227BP). After drying the bakery product and grinding it, 300-500 ml of purified water is added to 100 g of the obtained bakery powder, and sugar and / or fructose are added to adjust the sugar content to a concentration of 10-15 ° Brix. Lactobacilli nhs221 strain (Accession No. KCTC-11749BP) culture solution 1.0ml (1.0 ~ 8.0 × 10 ⁸ CFU) was added to the obtained mixture to fermentation and aging at room temperature for 50 days to obtain a lactic acid fermentation broth.

(Production Comparative Example 2) Preparation of lactic acid bacteria fermentation broth (bread made with a chemical expanding agent)

2 g of whey powder is added to 100 g of the raw material dough to prepare the dough by intermediate method. After aging by adding a chemical expanding agent to the prepared dough, bake in an oven to prepare a bakery product. After drying the bakery product and pulverizing it, 300-500 ml of purified water is added to 100 g of the obtained bakery powder, and 2 g of whey powder, sugar, and / or fructose are added to adjust the sugar content to a concentration of 10-15 ° Brix. Lactobacilli nhs221 strain (Accession No. KCTC-11749BP) culture solution 1.0ml (1.0 ~ 8.0 × 10 ⁸ CFU) was added to the obtained mixture to fermentation and aging at room temperature for 50 days to obtain a lactic acid fermentation broth.

(Production Comparative Example 3) Preparation of lactic acid bacteria fermentation broth (difference of lactic acid bacteria)

2 g of whey powder is added to 100 g of the raw material dough to prepare the dough by intermediate method. To the prepared dough, 0.5 g of a mixed probiotic of lactic acid bacteria of genus Leuconostock and two yeast strains of genus Saccharomyces is added and fermented and aged and baked in an oven to prepare a bakery product. At this time, the mixed probiotic preparations include Leukonostock genus nhs210 strain (Accession No .: KCTC-11226BP), Saccharomyces genus CCRG321 yeast strain (Accession No .: KCTC-13300BP) and Saccharomyces genus nhs321 strain (Accession No .: KCTC-11227BP).

After drying the bakery product and pulverizing it, 300-500 ml of purified water is added to 100 g of the obtained bakery powder, and 2 g of whey powder, sugar, and / or fructose are added to adjust the sugar content to a concentration of 10-15 ° Brix. To the obtained mixture, 1.0 ml (1.0 to 8.0 × 10 ⁸ CFU) of commercially available lactic acid bacteria Lactobacillus rhamnosus GG culture used for the production of fermented milk was added and fermented and aged at room temperature for 50 days to obtain a lactic acid bacteria fermentation broth.

(Example 1) Antiviral test

(Test group) After mixing 1 ml of lactic acid bacteria fermentation broth prepared in the production example of the present invention and 1 ml of physiological saline (PBS), 0.1 ml of mixed lactic acid bacteria fermentation broth was dispensed into mouse macrophage cell line RAW 264.7 and inoculated with virus.

(Comparative test group) After mixing 1 ml of lactic acid bacteria fermentation broth prepared in Comparative Example 2 and 1 ml of physiological saline (PBS), 0.1 ml of mixed lactic acid bacteria fermentation broth was dispensed into mouse macrophage cell line RAW 264.7 and inoculated with virus.

(Negative control) Only 0.1 ml of physiological saline (PBS) was dispensed into the mouse macrophage cell line RAW 264.7, and the virus was not inoculated.

(Positive control) 0.1 ml of physiological saline (PBS) was dispensed into RAW 264.7, a mouse macrophage cell line, and the virus was inoculated.

Antiviral tests of the lactic acid bacteria fermentation broth of the present invention against influenza virus and Herpes simplex virus were performed as follows.

After culturing the mouse macrophage cell line RAW 264.7 in a 6-well TC plate at a concentration of 1 × 10 ⁶ cells / well, the lactic acid bacteria fermentation broth of the present invention was compared to the test group in DMEM added with 10% (v / v) of FBS. The lactic acid bacteria fermentation broth prepared in Comparative Example 2 was dispensed into the test group, and physiological saline was dispensed into the negative control group and the positive control group. The concentration of the test group was treated to be 50 μg / ml. 12 hours after treatment with the test group, influenza virus PR8-GFP (MOI: 1) and herpes simplex virus HSV-GFP (MOI: 1) were inoculated into RAW 264.7 cells of each test group, comparative test group, and positive control group. After 2 hours of inoculation, the inoculum was removed, and RAW 264.7 cells were washed 3 times with PBS, replaced with fresh DMEM containing 10% FBS, and after 12-24 hours, the degree of virus infection was measured using an ultraviolet (345 nm) filter. Observed and measured.

In addition, in order to more accurately compare the GFP fluorescence expression in each condition, it was confirmed through a conventional GFP expression level confirmation system. FIG. 1B shows data measuring GFP expression for influenza virus (PR8), and FIG. 2B shows data measuring GFP expression for herpes simplex virus (HSV).

At this time, in the negative control group, only 0.1 ml of physiological saline (PBS) was dispensed into the mouse macrophage cell line RAW 264.7 and the virus was not inoculated. In addition, the positive control group was inoculated with virus after dispensing 0.1 ml of physiological saline (PBS) into mouse macrophage cell line RAW 264.7.

1B and 2B, the PR8-GFP and HSV-GFP relative units of the test group of the present invention were significantly reduced than the PR8-GFP and HSV-GFP relative units of the comparative test group.

The viability of RAW 264.7 cells in the virus infection state was confirmed by MTT assay. To perform the MTT assay, RAW 264.7 was transplanted into a 96-well plate, treated with GFP expressing virus, and after 48 hours, 100 μl of 1 mg / ml MTT solution was added to the cells and reacted for 4 hours. Thereafter, 150 μl of DMSO was added to dissolve the formazanitis produced in the cells, and the amount of the formazan was measured at 540 nm using a microplate reader.

1A shows the cell viability of RAW 264.7 against influenza virus (PR8), and in the case of the test group in which the lactic acid bacteria fermentation broth of the present invention was dispensed, it showed a cell viability of about 85% of the mouse macrophage cell line RAW 264.7 and the comparative test group. In the case of mouse macrophage cell line RAW 264.7 showed about 70% cell viability.

On the other hand, in the positive control group (PR8-GFP) inoculated only with PR8 virus, the macrophage cell line RAW 264.7 showed a cell viability of about 60%. Through this, the test group of the present invention increased the viability of RAW 264.7, a macrophage cell line of about 25%, compared to the positive control group inoculated with only PR8 virus. Increased. This confirms the PR8 antiviral activity of the lactic acid bacteria fermentation broth of the present invention.

Figure 2A shows the cell viability of RAW 264.7 against herpes simplex virus (HSV), in the case of the test group in which the lactic acid bacteria fermentation broth of the present invention was dispensed, it showed a cell viability of about 80% of the mouse macrophage cell line RAW 264.7 and a comparative test The group showed cell viability of about 70% of the mouse macrophage cell line RAW 264.7.

On the other hand, in the positive control group (HSV-GFP) inoculated only with the HSV virus, the macrophage cell line RAW 264.7 showed a cell viability of about 50%. Through this, the test group of the present invention increased the viability of RAW 264.7, a macrophage cell line of about 35%, compared to the positive control group inoculated with HSV virus alone. Increased. This confirms the HSV antiviral activity of the lactic acid bacteria fermentation broth of the present invention.

(Example 2) palatability sensory test

1. Preparation of beverage 1

After adding 5% by weight of oligosaccharides and 3% by weight of fructose to 100ml of lactic acid bacteria fermentation broth prepared in the production example, purified water is further added to prepare a beverage 1 so that the total capacity is 200ml.

2. Preparation of Comparative Drink 1

After adding 5% by weight of oligosaccharides and 3% by weight of fructose to 100ml of lactic acid bacteria fermentation broth prepared in Comparative Example 1, a beverage 2 was prepared by further adding purified water so that the total capacity was 200ml.

3. Preparation of Comparative Drink 2

Comparative beverage 1 was prepared by adding 5% by weight of oligosaccharides and 3% by weight of fructose to 100ml of lactic acid bacteria fermentation broth prepared in Comparative Example 2, and further adding purified water so that the total capacity was 200ml.

4. Preparation of Comparative Drink 3

Comparative beverage 2 was prepared by adding 5% by weight of oligosaccharides and 3% by weight of fructose to 100ml of lactic acid bacteria fermentation broth prepared in Comparative Example 3, and further adding purified water so that the total capacity was 200ml.

The palatability of beverage 1 and comparative beverages 1, 2, and 3 was performed by selecting 10 sensory inspectors. Items of palatability were measured by dividing them into taste, smell, and drinking, and were measured by a 5-point grading scale to show 5 (very good), 4 (good), 3 (normal), 2 (bad), and 1 (very bad). . Table 3 shows the results.

flavor smell Drinking grade Drink 1 4.8 4.9 4.7 4.80 Comparative Drink 1 4.5 4.4 4.5 4.47 Comparative Drink 2 4.4 4.5 4.5 4.47 Comparative Drink 3 4.4 4.4 4.3 4.37

The highest palatability sensory test results were measured in beverage 1 using the lactic acid bacteria fermentation broth prepared in Preparation Example as a raw material.

In addition, the comparative beverage 2 using the lactic acid bacteria fermentation liquid of the manufacturing comparative example 2 using a chemical expanding agent as a raw material and the comparative beverage 3 using the lactic acid bacteria fermentation liquid of the manufacturing comparative example 3 fermented using a conventional lactic acid bacteria as a raw material also compared with the beverage 1 of the present invention It showed a low taste, smell, and drinking.

Therefore, the beverage 1 prepared by using the lactic acid bacteria fermentation broth of the present invention has a high sensory palatability along with health functionalities, thereby increasing the possibility of commercialization.

Korea Research Institute of Bioscience and Biotechnology KCTC11226BP 20071025 Korea Research Institute of Bioscience and Biotechnology KCTC11227BP 20071025 Korea Research Institute of Bioscience and Biotechnology KCTC11749BP 20100824 Korea Research Institute of Bioscience and Biotechnology KCTC13300BP 20170718

Claims (4)

Iii) adding 1.0 to 10 parts by weight of whey powder to 100 parts by weight of the bakery raw material dough to prepare a dough by a medium method;
Ii) adding 0.01 to 1.0 parts by weight of a mixed probiotic mixture of two yeast strains of the genus Lactococcus and Lactobacillus and Saccharomyces, followed by fermentation and aging to prepare a bakery product;
Iv) 300 to 500 parts by weight of purified water is added to 100 parts by weight of the bakery powder obtained by drying and then pulverizing the bakery product, and 0.1 to 10 parts by weight of whey powder, sugar or fructose is added to increase the sugar content to 10 to 15 ° Brix. Adjusting; And
Iv) 0.5 to 2.0 parts by weight (1.0 to 8.0 × 10 ⁸ CFU) of Lactobacillus nhs221 strain (Accession No. KCTC-11749BP) culture solution was added and fermented for 40 to 70 days at room temperature to obtain a lactic acid bacteria fermentation broth;
In the method for producing a lactic acid bacteria fermentation broth having a virus disease prevention effect consisting of,
The mixed probiotic preparations include Leukonostock genus nhs210 strain (Accession number: KCTC-11226BP), Lactobacillus genus nhs221 strain (Accession number: KCTC-11749BP), Saccharomyces genus CCRG321 yeast strain (Accession number: KCTC-13300BP) And Saccharomyces genus nhs321 yeast strain (Accession No .: KCTC-11227BP), characterized in that the influenza virus or herpes simplex virus disease prevention method of producing a fermentation broth fermentation broth.
delete The method of claim 1, wherein the Saccharomyces genus CCRG321 yeast strain (Accession No .: KCTC-13300BP) is passaged through a plate plate with PDA (Potato dextrose agar) separation medium to which 3% by weight of whey powder is added. , A method for producing a lactic acid bacteria fermentation broth having a virus disease preventing effect, characterized in that the strain obtained by inoculating and cultivating wild-type baker's yeast and separating and culturing the yeast strain colonies having excellent growth 3 times.
An animal feed having a viral disease preventing effect, comprising 1 part by weight of the fermentation broth of claim 1 and 100 to 10,000 parts by weight of feed.

Images