JP2012136436A - Lactic acid bacterium fermentation product using polyphenol component and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a lactic acid bacterium fermentation product using a polyphenol component, such as a water extract of pine bark, astringent skin of peanuts, grape seeds or azuki beans, and a method for producing the same.SOLUTION: The lactic acid bacterium fermentation product is obtained by lactic acid-fermenting, for example, a water extract of azuki beans with Lactobacillus piantarum. As the water extract of azuki beans, use can be made of, for example, a broth that is obtained by cooking azuki beans in water in the course of producing a sweat azuki bean paste. The lactic acid bacterium fermentation product has an anti-SARS (Sub-Acute Respiratory Syndrome) corona virus activity thrice or more as high as the activity of the unfermented material. This lactic acid bacterium fermentation product is widely usable as a starting material to be added to drinks, foods such as ice creams, baked products, candies, chewing gums and desserts, tablets, injections, nasal drop compositions for preventive and therapeutic uses, drops, gargle solutions and mouth washers.

Description

  The present invention relates to the fact that lactic acid bacteria fermented products of water extracts such as polyphenol components such as pine bark, peanut astringent skin, grape seeds, and red beans have strong antiviral activity, and a method for producing the same, and in particular, lactobacilli as lactic acid bacteria -It is characterized in that lactic acid fermentation is performed using plantarum (Lactobacillus plantarum).

  As countermeasures against viral diseases, prevention by vaccine is the greatest measure, but there are many viral diseases for which vaccine development is difficult, and antiviral agents effective after onset are very limited. In recent years, there is an urgent need for prevention and treatment measures against emerging and reviving viral diseases, such as the emergence of a virus resistant to Tamiflu, a therapeutic drug for the new avian influenza virus, which has become a social problem.

  Conventionally, an anti-SARS virus agent containing proanthocyanidins as an active ingredient has been proposed (see Patent Documents 1 and 2). Moreover, as a plant body for extracting such proanthocyanidins, grapes, cranberries, cacao, apples, red beans, straw, cabbage, barley, malt, walnuts, almonds, cedar, straw, pine, tochi, straw (Citrus), bonito (myoga), leaf ginger (ginger) and the like are disclosed (see paragraph 0013 of Patent Document 1).

  Among the above plant bodies, red beans are a crop that has been found in Japan since the beginning of the Jomon period to the early Kofun period, and charcoal seeds of red beans have been found in the early Nara period. Azuki beans have been closely associated with people's lives since ancient times, and in Japan, China and Korea, it is believed that the redness of red beans has a mysterious power such as amulet and has been used for events and ceremonies. In addition, it seems to have been used for medicinal purposes, and the effects and prescriptions of red beans are written in Chinese books of the Ming era.

  The effects of red beans have been known for a long time, such as the elimination of hangover and diuretic action. Among them, anthocyan, which is a pigment of red beans, and proanthocyanidin, which is a precursor thereof, have recently attracted attention as red bean polyphenols (antioxidants). It is a material. Moreover, although this red bean polyphenol is contained in the product koji, it is known that it is contained in a large amount in the broth discarded during the koji making process.

  Therefore, the present inventors aimed to develop a food material with added value from red bean broth discarded as industrial waste.

  Here, as a technique for using the boiled legume soup as a food material, a technique is disclosed in which soybean soup is mixed with fruit juice, vegetable juice, or the like, and the mixture is lactic acid-fermented with lactic acid bacteria and used as a food or drink. (See Patent Document 3). Patent Document 3 describes that, when soybean broth is used as a substrate, various lactic acid bacteria have fermentability and that Lactococcus lactis has been selected in terms of imparting an appropriate acidity to the fermentation broth. ing.

JP 2005-314316 A JP 2008-143840 A JP 2005-304322 A

  However, according to the study by the present inventors, it has been clarified that lactic acid bacteria having fermentative ability with respect to red bean broth are extremely limited.

  The present invention has been made in view of the above circumstances, and the main purpose thereof is a fermented lactic acid bacterium using a polyphenol component, for example, an aqueous extract of pine bark, peanut astringent skin, grape seeds, red beans, and the like, and a method for producing the same. Is to provide.

  The present inventors have found that, among many lactic acid bacteria, only Lactobacillus plantarum has the ability to ferment with red bean broth and the like, thereby completing the present invention.

That is, the gist of the present invention is as follows.
[1] A fermented lactic acid bacterium with improved antiviral activity, obtained by lactic acid fermentation of a polyphenol component with Lactobacillus plantarum,
[2] The polyphenol component is an aqueous extract of one or more raw materials selected from pine bark, peanut astringent skin, grape seeds, grape skins, red beans, red rice, cowpeas, and gold bean [1] Lactic acid bacteria fermented product,
[3] The fermented lactic acid bacterium according to the above [1], wherein the polyphenol component is an aqueous extract of red beans.
[4] The fermented lactic acid bacterium according to [3], wherein the red bean water extract is red bean broth.
[5] A food, pharmaceutical or cosmetic containing the lactic acid bacteria fermented product according to any one of [1] to [4],
[6] A method for producing a fermented lactic acid bacterium having improved antiviral activity, characterized by subjecting a water extract of red beans to lactic acid fermentation with Lactobacillus plantarum,
[7] The method for producing a fermented lactic acid bacterium according to [6], wherein the water extract of red beans is a soup of red beans.
[8] A method for improving the antiviral activity of an aqueous extract of red beans, characterized by subjecting the aqueous extract of red beans to lactic acid fermentation with Lactobacillus plantarum,
[9] Human immunodeficiency virus (HIV), papilloma virus, infectious molluscumoma virus, wart virus, herpes virus, influenza virus, parainfluenza virus, adenovirus, rhinovirus, coronavirus, norwalk virus, rotavirus An aqueous extract of red beans according to [8] above, which exhibits antiviral activity against any of echovirus, enterovirus, norovirus, koi herpesvirus, iridovirus, rhabdovirus group, or white spot virus For improving the antiviral activity of.

  According to the present invention, there is provided a fermented lactic acid bacterium using a polyphenol component, for example, a water extract such as pine bark, peanut astringent skin, grape seed, grape skin, red beans, red rice, cowpea, and gold bean, and a method for producing the same. Is done. The fermented product of lactic acid bacteria according to the present invention has an antiviral activity that is improved by 3 times or more compared to the unfermented product.

(1) shows the TLC of the red bean broth fermentation concentrate and the red bean broth concentrate, and (2) shows the antiviral activity (IC ₅₀ value) for each fraction of (1). It is the figure (left side: CBB dyeing | staining, right side: immunoblot) which showed the influence of the red bean juice concentrate on the proliferation, survival, and metabolism of Vero cell. It is the figure (left side: CBB dyeing | staining, right side: immunoblot) which showed the influence of the red bean broth fermentation concentrate on the proliferation, survival, and metabolism of Vero cell. TLC of fermentation concentrate of various raw material broths.

  As described above, the fermented lactic acid bacteria of the present invention is characterized in that it is obtained by lactic acid fermentation of a polyphenol component with Lactobacillus plantarum.

  The polyphenol component that can be used in the present invention is capable of lactic acid fermentation by Lactobacillus plantarum, among the polyphenol component contained in the water extract of the raw material plant or the water extract of the raw material plant itself, And the thing provided with the property which the antiviral activity of the said lactic-acid-bacteria fermented material improves. The form of the polyphenol component may be either liquid or powder, and the polyphenol component may be prepared from a raw plant, or a commercially available product may be used. Examples of the raw material plant that can be used in the present invention include red beans, pine bark, peanut astringent skin, grape seeds, grape skins, red rice, cowpeas, and gold bean. As a polyphenol component, 1 type of water extracts among the said plants may be used, and 2 or more types of water extracts may be mixed and used.

  The water extract of red beans among polyphenol components means a component extracted from red beans soaked in water, and the temperature during extraction may be room temperature or higher. For example, in the koji making process, boiled soup (also referred to as “astringent soup” or “steamed soup”) obtained when boiled red beans with water can be mentioned. The above-mentioned red bean broth is discarded in the process of making koji and contains red bean proanthocyanidins as a component. The sugar content of the red bean broth is not particularly limited, but usually about 1.5 ± 0.5 ° is used. Moreover, the variety of red beans is not particularly limited, and examples thereof include Erimoshozu.

  Lactic acid bacteria that can be used in the present invention are limited to bacterial species having fermentative ability for the water extract of the above-mentioned raw material plant. As the lactic acid bacteria, the present inventors have selected as Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii ss delbrueckii, (Streptococcus thermophilus), Lactobacillus casei (Lactobacillus casei), Pediococcus pentosaceus (Pediococcus pentosaceus), Lactobacillus acidophilus (Lactobacillus acidophilus), Bifidobacteria bifisum (Bifidobacterium bru actus) (Lactobacillus delbrecky subspices bulgaricus), Lactococcus lactis (Lactococcus lactis) and inoculated into the water extract of red beans, Lactobacillus plantarum (Lactobacillus plantarum) was confirmed to have only fermentability.

  In producing a lactic acid bacteria fermented product, for example, an aqueous extract of red beans is sterilized by heating and then cooled, and then inoculated with Lactobacillus plantarum. Specifically, 1-10 parts by weight of Lactobacillus plantarum per 100 parts by weight of the red bean water extract at 85-140 ° C. and cooled to 25-45 ° C. Inoculate. After inoculating lactic acid bacteria, it is preferable to ferment at 25-45 degreeC for 10 to 40 hours.

  The fermented lactic acid bacterium according to the present invention has improved antiviral activity compared to the unfermented product. According to the study by the present inventors, the content of the polyphenol component having a reduced molecular weight in the extracted component of the fermented lactic acid bacteria is higher than that of the unfermented product, which improves the antiviral activity. Presumed to have contributed. Therefore, the present invention also discloses a method for improving the antiviral activity of the water extract of the above-mentioned raw material plant.

  The antiviral activity of the fermented lactic acid bacterium is non-specific to the virus species, such as human immunodeficiency virus (HIV), papilloma virus, infectious molluscumoma virus, wart virus, herpes virus, influenza virus Antiviral against any of parainfluenza virus, adenovirus, rhinovirus, coronavirus, norwalk virus, rotavirus, echovirus, enterovirus, norovirus, koi herpes virus, iridovirus, rhabdovirus group, or white spot virus Shows activity.

  The fermented lactic acid bacteria obtained above include, for example, food additives such as beverages, ice, baked confectionery, candy, gum, dessert, tablets, injections, nasal solution compositions for prevention and treatment, gargle, mouthwash It can be used as an additive material for liquids, drops and the like. The form of use is not particularly limited. In addition to the fermented product itself (hereinafter sometimes referred to as “liquid product”), for example, a concentrated product obtained by concentrating the liquid product, and a mixture obtained by mixing skim milk powder or soy milk with the liquid product. The liquid product can be used in various forms such as powdered powder, and a sugar-added product obtained by adding sugar to the liquid product. For example, when it provides as a concentrated product, what concentrated the liquid product under reduced pressure so that it may become 1/5-1/20 volume can be illustrated. In the case of serving as a powder, a liquid product added with an excipient such as dextrin or lactose and powdered until the water content is less than 10% can be exemplified. The fermented lactic acid bacteria can be filtered and sterilized by a known method after the end of fermentation.

  Hereinafter, the present invention will be described in more detail with reference to test examples and the like, but the present invention is not limited thereto.

1. Example of production of fermented lactic acid bacteria of red bean broth 1-1. Example of production of red bean broth Add 60 liters of water to 60 kg of red bean soup, heat at 85 ° C. for 30 minutes, transfer the broth to another container, add 180 L of water to the red beans again, and heat at 85 ° C. for 30 minutes. Then, add 180 L of water to the red beans, heat at 100 ° C. for 2 hours and 30 minutes, transfer the broth to the above containers, sterilize by holding at 90 ° C. for 5 minutes, cool to 30 ° C. and red beans Boiled juice was produced.

1-2. Examination of Lactic Acid Bacteria Having Fermentation Capability for Azuki Soup Add 5 parts by weight of one of the lactic acid bacteria shown in Table 1 with respect to 95 parts by weight of Azuki soup obtained in “1-1. It culture | cultivated at the temperature shown in Table 1 for 20 hours, and examined whether a fermented liquid was obtained. As a result, the fermentation broth was obtained only from the red bean broth added with Lactobacillus plantarum ATCC14917. From the above results, it was found that the lactic acid bacteria having the ability to ferment with red bean broth were only Lactobacillus plantarum among the many lactic acid bacteria shown in Table 1.

  As a starter for various strains of lactic acid bacteria shown in Table 1, a medium consisting of 1 part by weight of glucose, 1 part by weight of yeast extract and 97 parts by weight of ion-exchanged water was autoclaved, and 1 part by weight of various lactic acid strains were inoculated. What was cultured at 0 degreeC was used.

1-3. Preparation example of test substance (preparation example of fermented red bean juice concentrate)
In Table 1, the fermentation broth fermented with red bean broth with Lactobacillus plantarum was centrifuged to remove solids, and the supernatant was concentrated under reduced pressure to a sugar content of 20 ° and stored frozen. The test substance (hereinafter sometimes referred to as “red bean broth fermentation concentrate”) was subjected to the following test.

(Examples of preparation of various polyphenol component fractions in the red bean broth fermentation concentrate)
About 300 mg of the red bean broth fermentation concentrate obtained above is dissolved in 20 ml of Milli-Q water, and this solution is adsorbed on a column (20 ml) using Lipophilic Sephadex (Pharmacia) as a carrier, with 3 column volumes of water. After washing, elution was successively carried out with 3 column volumes of 30% ethanol, 70% ethanol and 70% acetone. The obtained 30% ethanol fraction, 70% ethanol fraction, and 70% acetone fraction were concentrated under reduced pressure to 1/10 volume and then stored frozen, and these were used as the test substances in the following tests.

(Examples of preparation of red bean broth concentrate and various polyphenol component fractions)
Centrifugation of the red bean broth obtained in “1-1. Production example of red bean broth” above removes solid matter, concentrates the supernatant under reduced pressure to 1/10 volume, and stores it in a frozen state. And then subjected to the following test (hereinafter sometimes referred to as “red bean broth concentrate”). Furthermore, 30% ethanol fraction, 70% ethanol fraction and 70% acetone fraction were prepared from this red bean broth concentrate by the same operation as above (preparation example of various polyphenol component fractions of red bean broth fermented concentrate). did. Then, each of the obtained fractions was centrifuged to remove solids, and the supernatant was concentrated to 1/10 volume under reduced pressure and stored frozen, and these were used as the test substances for the following tests.

2. Component analysis of test substance The polyphenol component of the test substance obtained in "1-3. Preparation example of test substance" was analyzed using silica gel thin layer chromatography. Specifically, a silica gel thin layer plate (Merck, 60F254) was developed with a solvent (isopropanol: acetic acid: water = 60: 20: 10), and 1% iron chloride: 1% potassium ferrocyanide = 1: 1 (v / v) The phenolic hydroxyl group was colored with the solution. The results are shown in FIG.

3. 3. Measurement of antiviral effect of test substance 3-1. Measurement of anti-SARS (Sub-Acute Respiratory Syndrome) coronavirus activity of test substance SARS coronavirus (hereinafter also referred to as “SARS-CoV”) is FFM-1 strain (Dr. HW Doerr, Frankfrut University of Medicine, Germany). More). As the cultured cells sensitive to the FFM-1 strain, Vero (African mizorisal kidney cells, E-64) is used, and a medium in which 10% fetal bovine serum is added to Dulbecco's minimum essential medium (DMEM) is used. The cells were cultured at 37 ° C. in the presence of CO ₂ . The Vero cells were similarly distributed by Dr. HW Doerr.
SARS-CoV virus stock solution was used to infect cultured Vero cells with 90% monolayers under the conditions that the virus amount per cell was 0.1 (MOI = 0.1, Multiplicity of infection) (Tuker PC et al., J. Virol. 71: 6106, 1997), 24 hours later, the culture supernatant was collected, and the virus titer was measured by the plaque method described below.

The supernatant of the collected culture broth was added 10% with PBA (−) (0.05M phosphate buffer, 0.15M NaCl, pH 7.0 not containing Mg ²⁺ and Ca ²⁺ ) supplemented with 1% bovine serum albumin (BSA). The serial dilution was performed, and 0.2 ml of the diluted solution at each stage was inoculated into each well (6-well plate). After infecting at 25 ° C. for 60 minutes, the cells were cultured in DMEM (containing 5% fetal calf serum) supplemented with 1.0% methylcellulose for 4-7 days. After culturing, methylcellulose was removed, and the cells were stained in 2.5% crystal violet (30% ethyl alcohol, 1% ammonium oxalate), washed / decolored three times with PBS (−), and the average number of plaques ( The amount of virus in 1 ml was calculated from 3 wells) as PFU (Plaque Forming Unit) / ml (Tuker, PC et al., J. Virol. 71: 6106, 1997).

The antiviral activity of the red bean broth fermentation concentrate by the plaque method was determined by infecting 60-200 PFU / 0.2 ml of SARS-CoV per well of a 6-well plate at 22 ° C. for 60 minutes, and then diluting the test solution DMEM added with 1.0% methylcellulose containing the above was layered, and cultured for 4-7 days. The number of plaques was measured, and the test substance concentration IC ₅₀ value (the concentration that inhibited growth of SARS-CoV by 50%) (Inhibitory concentration, (μg / ml)) As shown in Fig. 1 (2), the red bean broth fermentation concentrate showed an IC ₅₀ value of about 1/7 compared to the red bean broth concentrate. In other words, the anti-SARS coronavirus activity of the red bean broth fermented concentrate increased by about 7 times compared to the red bean broth concentrate, and the plaques formed on the cultured monolayer cells were denatured by virus infection. It means cysts, but at the same time, the toxicity of the test solution to normal cells can be measured from the staining degree of non-infected cells.The used red bean broth fermentation concentrate is significantly more stained than the culture without the test solution. There was no difference and no cytotoxicity was observed.

3-2. Measurement of anti-influenza virus activity of red bean broth fermentation concentrate In order to show that the anti-viral action of red bean broth fermentation concentrate is non-specific to the virus species, the effect on influenza virus was examined. For the influenza virus A (Flu-A), the PR8 strain (distributed by the National Institute of Infectious Diseases) was used. Sensitive cells MDCK (canine kidney cells, Dainippon Pharmaceutical KK) were cultured in the same manner as the above-mentioned Vero cells, and 90% monolayered cells were infected with 0.1% BSA-DMEM. After washing twice and Flu-A infection, the same medium was used in the presence of 2.5 μg / ml trypsin.

The anti-Flu-A effect of the test solution was measured by measuring the Tissue Culture Infective Dose (TCID) of cytopathic effect. As with SARS-CoV, 0.1 TCID ₅₀ /0.2 ml of MDCK cells 90% monolayered on a 6-well plate (the virus concentration at which ₅₀ % of cells undergo cytopathy is the reciprocal of the dilution of the virus solution) Flu-A for 60 minutes at 22 ° C. and 0.1-6% BSA-DMEM (2.5 μg / ml trypsin added) containing each diluted test solution for 4-6 days After culturing, the cytopathic effect by Flu-A was measured. The antiviral action was compared based on the properties of cultured cells with and without the test solution. Results The anti-Flu-A activity of the red bean broth fermentation concentrate was improved by about 3 times compared to the red bean broth concentrate. Table 2 shows the results.

4). Characteristics of the polyphenol component of the red bean broth fermentation concentrate It is widely known that grape seeds containing red beans and pine bark proanthocyanidins exhibit antiviral activity. The feature of the patent is that the lactic acid fermentation product of red bean broth enhances the antiviral effect. The nature of the polyphenol component on TLC was examined for the basis of this enhancing action. As shown in FIG. 1 (1), when comparing the molecular weight of the polyphenol component of the red bean broth fermentation concentrate (lane: 0-B) and the red bean broth concentrate (lane: 0-A), A decrease in the polymer region component and an increase in the oligo region component are observed. Since it is known that the components of the oligo region have strong antiviral activity, it is suggested that the increase in the components of this region is involved in the antiviral activity.

Furthermore, the properties of the polyphenol component on the Lipophilic Sephadex chromatography of the red bean broth concentrate and the red bean broth fermentation concentrate were examined. Glycosides containing saccharides and saponins are collected in the non-adsorbed fraction (lane: 1) or 30% ethanol fraction (lane: 3). Polyphenols having antiviral activity are eluted in the latter half of 70% ethanol or 70% acetone fraction (lanes: 5, 6). Many of the polyphenols eluted in the latter half of 70% ethanol are polymeric, and the 70% acetone fraction contains oligopolyphenol and organic acid as the main components. The red bean broth concentrate contained a lot of polyphenols eluted with 70% ethanol (lane: 4), and the red bean broth fermentation concentrate contained a large amount of polyphenols eluted with 70% acetone (lanes: 5, 6). The results of measuring the antiviral activity (IC ₅₀ value) after drying each fraction under reduced pressure are shown in FIG. 1 (2). The red bean broth concentrate is in the 70% ethanol fraction, and the red bean broth fermentation concentrate is 70%. Antiacetic activity was high in the acetone fraction, and low molecular weight of polyphenol was observed by fermentation. These results suggest that by fermenting red bean broth, polyphenols in the broth are reduced in molecular weight and antiviral activity is enhanced. In addition, the presence of antiviral substances other than polyphenols in red bean broth has not been reported.

5. Effect of Azuki Soup Fermented Concentrate on Cultured Cells As described above, it was shown that the red bean broth fermented concentrate has stronger antiviral action and relatively less cytotoxic effect than the red bean soup concentrate. In order to examine the mechanism by which this antiviral action is induced, after adding a test substance to Vero cells and culturing, the total protein is analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). They were isolated and examined for cell proliferation, effects on cellular protein synthesis, presence or absence of apoptosis, induction of intracellular antioxidant factors, and the like. For each, tubulin, phosphorylated ribosome S6 protein), PARP (polyADP ribosyl polymerase), and Mn-SOD (Manganese superoxide dismutase) were stained by immunoblotting, and the influence of the red bean broth fermentation concentrate on the cultured cells was examined. .

5-1. Measurement of total cellular protein Vero cells (2.5 ml medium per well, 2 × 10 ⁶ cells) cultured in a 6-well plate were mixed with red bean broth concentrate or red bean broth fermented concentrate (see “1-3. Tests” above). 0,1,3,10,30,100 μg / ml), cultured at 37 ° C., and after 48 hours, all cells were recovered in 0.4 ml of sample buffer for SDS-PAGE. Total protein was separated by SDS-PAGE. Next, the gel after electrophoresis was stained with Coomassie Brilliant Blue R250 (CBB) and then decolorized with 7% acetic acid. Images were taken with a CCD camera, and the staining concentration of the actin band was measured with an image analysis software (The Analytical Imaging System AIS. Amersham / Pharmacia) and used as an index of cell growth inhibition (Nakatsue, M. et al., Biochem. Biophys. Res.Commun.253: 59,1998). FIG. 2 (unfermented content) and FIG. 3 (fermented content) show the results.

5-2. Measurement of apoptosis-related protein and Mn-SOD After separating all proteins of Vero cells cultured in 6-well plate by SDS-PAGE, the protein was transferred to PVDF membrane (Polyvinylidene fluolide, Millipore), and tubulin (cell Indicator of increase / decrease in total protein, Santa Cruz), PARP (Poly ADP ribosyl polymerase, indicator of apoptosis induction, Santa Cruz), phosphorylated ribosome S6 protein (promoting cellular protein synthesis, indicator of inhibition, Cell Signaling) and An Mn-SOD antibody (intracellular antioxidant factor, Stressgen), and an anti-rabbit-goat antibody (Santa Cruz) labeled with alkaline phosphatase as the secondary antibody, and coloring with NBT (nitroblue tetrazolium salt, Boehringer) Detect with immunity Tsu theft was carried out (Yoshinaka, Y et al., Biochem Biophy.Res.Commun.261:.. 139,1999). The relative expression level was measured from image analysis. The results are shown in FIGS.

  First, looking at the effects on cell proliferation and cell protein of the red bean broth concentrate and the red bean broth fermentation concentrate, the concentration of total cell protein by CBB staining is suppressed when the concentration is high. The amount of tubulin protein showed a similar tendency. Cell protein synthesis using S6 protein phosphorylation as an indicator was strongly suppressed depending on the concentration, and these phenomena were found to be stronger in the red bean broth fermentation concentrate. Furthermore, it has been found that these inhibitory effects are restored to normal by removing them from the medium.

  On the other hand, PARP, which is an indicator of cell death, does not detect cleavage products and induces cell death even when the concentration of the red bean juice concentrate and the red bean juice concentrate is high. Although cell growth was suppressed, apoptosis was not induced, and the red bean broth concentrate and the red bean broth fermentation concentrate showed an effect of inducing the cell survival state to a quiescent state.

  Viral infection induces oxidative stress in cells and proliferates, so suppressing oxidative stress induction is thought to be one way to control viral growth. Therefore, it was examined whether the red bean broth fermentation concentrate induces antioxidant factors. The amount of total cellular protein and amount of tubulin observed by CBB staining and phosphorylation of S6 protein, which is an index of protein synthesis, are high depending on the concentration of red bean broth concentrate and red bean broth fermentation concentrate. It is suppressed, and the red bean broth fermentation concentrate is strongly suppressed. On the other hand, the expression of Mn-SOD is increased conversely, and the action is strong in the red bean broth fermentation concentrate, and it is considered that the antiviral action is exhibited. By the way, in normal virus infection, virus protein synthesis proceeds even though cell protein synthesis is suppressed by virus infection.

  The above results indicate that the red bean broth concentrate and the red bean broth ferment concentrate inhibit cell growth and metabolism that do not induce cell death, and increase the expression of cell survival factors (cell dormancy state). Induction). It is considered that the induction of the cell state and the induction of antioxidant factors by the red bean broth concentrate and the red bean broth ferment concentrate act on protection against cell damage caused by various external factors.

6). Examination of lactic acid fermentability and antiviral effect when using raw materials other than red beans Red beans (prepared in-house), pine bark (Pycnogenol, Nippon Sebel), peanut astringent skin (peanut astringent skin extract, Kishimoto Sangyo), grape seeds (gravinol, Prepare a diluted solution of Kikkoman), red rice (prepared in-house), cowpea (prepared in-house), gold bean (prepared in-house) and concentrated grape juice (concentrated red grape juice, welch). The fermentation ability of Lactobacillus plantarum ATCC14917 was examined in accordance with the method described in “Investigation of Lactic Acid Bacteria Having Fermentation Ability for Lactobacillus”.
Next, among the above-mentioned test substances that have been observed to be fermented, according to the method described in “3-1. Measurement of Anti-SARS (Sub-Acute Respiratory Syndrome) Coronavirus Activity of Test Substances” above. Antiviral action was examined. The results are shown in Table 3, Table 4 and FIG.

7). Various production examples The red bean broth fermented concentrate prepared in the above "1-3. Preparation examples of test substances" was pulverized, and the following various products were produced using this as a blending raw material.
7-1. Example of tablet production Tablets having a composition of 1 tablet as shown in Table 5 were produced by a conventional method.

7-2. Example of production of injection An injection having the composition shown in Table 6 below was produced.

  The parabens, sodium metabisulfite and sodium chloride are dissolved in about half of the distilled water at 80 ° C. with stirring. The resulting solution was cooled to 40 ° C., and the red bean fermented product (powder), vitamin C, and then polyethylene glycol and polyoxyethylene sorbitan monooleate were dissolved in the solution. Next, distilled water for injection was added to the solution to prepare the final volume, and the solution was sterilized by sterilizing filtration using an appropriate filter paper, and dispensed in 1 ml aliquots to obtain the intended injection. .

7-3. Preparation of nasal spray for prevention and treatment Nasal spray containing Azuki proanthocyanidin and Vitamin C, fermented red beans (powder) 5ml per 1ml of physiological saline (0.9% (w / v) NaCl aqueous solution) It was prepared by adding 3 mg of vitamin C.

7-4. Example of Producing and Preventing Nasal Solution Composition A nasal spray containing the components shown in Table 7 was prepared.

7-5. Example of production of gargle liquid A gargle liquid containing the components shown in Table 8 was prepared.

7-6. Manufacturing Example of Mouthwash Liquid Mouthwashes containing the components shown in Table 9 were prepared.

7-7. Beverage production example 1
A fermented red bean (powder) and 1000 ml of vitamin C-containing beverage (10 bottles) were produced according to the formulation shown in Table 10.

7-8. Beverage production example 2
Fermented red beans (powder) and 1000 ml of vitamin C-containing beverage (for 10 bottles) were produced by the formulation shown in Table 11.

7-9. Production Example of Chewing Gum A fermented red bean (powder) and vitamin C-containing chewing gum (30 pieces) were produced by a conventional method with the formulation shown in Table 12.

7-10. Production Example of Drops Fermented red beans (powder) and vitamin C-containing drops were produced with the formulation shown in Table 13.

Sugar was dissolved in 5.5 liters of water and glucose containing corn syrup was added and mixed well. At this point, any desired dye may be added to give the desired color. Use a dye that dissolves sufficiently.

The mixture was placed in a steam jacket kettle heated to 125 ° C. From there, the mixture was pumped into a storage container and fed to a continuous reading cooker.
A temperature of 125-150 ° C. was reached as the syrup passed through the coil in the cooker. The drug was then fed by a steam vacuum ejector to a receiving kettle maintained at 28-29 inches Hg vacuum for 6-7 minutes. During this time, water was removed until the water content was reduced to about 1% or less, and a molten candy base was formed. The candy base was then slowly cooled.

  Fermented red beans (powder), vitamin C and wild cherry mimetic flavor (powder) were added to polyethylene glycol and the resulting mixture was fluidized by heating at 70 ° C.

The resulting hot fluid mixture was quickly added with proper blending into the steam candy base (temperature reduced to 100 ° C. or slightly below). Next, after the whole lump was sufficiently kneaded, it was transferred to a spinning machine and extruded onto a lozenge forming die. Alternatively, the molten candy mass to which Azuki Proanthocyanidin and Vitamin C are added is poured onto a cooling table, solidified to a semi-solid mass on the cooling table, and then used to dispense a unit dose of the drug. Alternatively, it may be formed into a desired shape.

  The fermented lactic acid bacteria of the present invention include, for example, food additives such as beverages, ice, baked confectionery, candy, gum, dessert, tablets, injections, nasal solution compositions for prevention / treatment, gargle, mouthwash, It can be widely used as an additive material such as drops.

Claims (9)

  A fermented lactic acid bacterium with improved antiviral activity, which is obtained by subjecting a polyphenol component to lactic acid fermentation with Lactobacillus plantarum.   The lactic acid bacteria fermentation according to claim 1, wherein the polyphenol component is an aqueous extract of one or more raw materials selected from pine bark, peanut astringent bark, grape seeds, grape skins, red beans, red rice, cowpeas, and gold beans. object.   The fermented lactic acid bacterium according to claim 1, wherein the polyphenol component is an aqueous extract of red beans.   The fermented lactic acid bacterium according to claim 3, wherein the water extract of red beans is boiled red beans.   A food, medicine or cosmetic containing the fermented lactic acid bacterium according to any one of claims 1 to 4.   A method for producing a fermented lactic acid bacterium with improved antiviral activity, characterized by subjecting an aqueous extract of red beans to lactic acid fermentation with Lactobacillus plantarum.   The method for producing a fermented lactic acid bacterium according to claim 6, wherein the water extract of red beans is boiled red beans.   A method for improving the antiviral activity of an aqueous extract of red beans, characterized by subjecting the aqueous extract of red beans to lactic acid fermentation with Lactobacillus plantarum.   Human immunodeficiency virus (HIV), papilloma virus, infectious molluscumoma virus, wart virus, herpes virus, influenza virus, parainfluenza virus, adenovirus, rhinovirus, coronavirus, norwalk virus, rotavirus, echovirus The antiviral activity of the water extract of red bean according to claim 8, which exhibits antiviral activity against any one of enterovirus, norovirus, koi herpes virus, iridovirus, rhabdovirus group, or white spot virus How to improve.