JPS6237015B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing an antiviral agent by treating cane molasses with basidiomycetes. It is well known that viruses are filterable pathogens, that they are smaller than Rickettsia (20 to 60 millimicrons), that they parasitize organisms other than viruses, and that they multiply only within living cells. In animals, there are Japanese encephalitis, influenza, hepatitis, etc., and in plants, tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), etc. are typical. By the way, the present inventors first inoculated a solid medium such as bagasse or a liquid medium with basidiomycetes such as Vertebrae,
A mixture of these mycelia and a medium (mycelium culture)
We have developed an antiviral agent whose active ingredients are polysaccharides and cytokinin-based active substances obtained from . In addition, the present inventors focused on metabolites of mycelium and partial decomposition products of culture medium components, and as a result of further research and experiments, they found that plant cell wall components such as bagasse themselves also have antiviral effects. We have developed an antiviral agent (for example, see JP-A-57-106624). That is, it is thought that heteroglycans such as xyloglucan and arabinoxylan, which constitute hemicellulose of plant cell walls, have antiviral activity. In order to develop an antiviral agent that is more easily available and cheaper, the present inventors conducted extensive research and surprisingly discovered that cane molasses has excellent antiviral activity. A separate patent application was filed at the same time as this application for a method for suppressing viral diseases in agricultural and horticultural crops. The present invention has been completed as a result of further pursuing the above invention. That is, the present invention is a method for producing an antiviral agent, in which a medium containing cane molasses as a main component is inoculated with mycelium of edible mushrooms belonging to Basidiomycetes and cultured in a deep culture. Cane molasses is the dark black, viscous molasses that remains at the end of the process of producing sugar from sugar cane, which cannot be recovered for economic reasons. This cane molasses can be obtained at extremely low cost and has been commonly used as a medium component for alcohol fermentation, glutamic acid fermentation, etc. However, in such conventional techniques, cane molasses is merely used as a nutritional source for microorganisms, and there is no example of using cane molasses by focusing on the special activity of the cane molasses itself. As mentioned above, heteroglycans such as xyloglucan and arabinoxylan, which are constituent components of hemicellulose in plant cell walls such as bagasse, have been confirmed to have antiviral activity. By the way, when sugar cane is pressed to obtain squeezed juice in the sugar manufacturing process, these heteroglycans are mixed into the squeezed juice and remain in cane molasses. Therefore,
Cane molasses contains a large amount of heteroglycans, and these components are thought to contribute to the expression of antiviral activity. The present invention further enhances the antiviral activity of cane molasses by treating it with basidiomycetes. That is,
When the mycelium of edible mushrooms belonging to Basidiomycetes is inoculated into a medium containing cane molasses as its main component and cultured deeply, many carbohydrases such as cellulase, α-glucidase, β-galactosidase, etc. are released from the mycelium as extracellular enzymes. is produced, and the heteroglycans contained in cane molasses are partially degraded by these extracellular enzymes. As a result, more active groups of the heteroglycan are exposed, and the antiviral activity is thought to be further enhanced. The medium used in the present invention is preferably a liquid medium prepared by diluting cane molasses with water or the like. Other components such as peptone and yeast may be supplementarily added to this liquid medium in order to support the growth of mycelium. Furthermore, when using cane molasses, it is preferable to carry out pretreatment such as steam injection to precipitate and remove colloidal substances and metals. The adjusted liquid medium is placed in an autoclave or the like and sterilized. The edible mushrooms belonging to Basidiomycetes used in the present invention include, for example, shiitake mushrooms, oyster mushrooms, enokitake mushrooms, nameko mushrooms, and shimeji mushrooms, but it is most preferable to use shiitake mushrooms in terms of ease of culturing and antiviral activity. Next, the mycelia of these edible mushrooms are inoculated into the liquid medium and cultured in depth. In this case, the mycelium may be precultured before inoculation. Culture at 15-30°C by shaking culture or aerated culture.
It is appropriate to carry out the test for about 10 days under the temperature conditions of . As a result, the mycelium sufficiently proliferates, produces various types of extrahyphal enzymes, and utilizes the medium components. That is, heteroglycans contained in cane molasses are partially decomposed by various types of carbohydrases to expose active groups. After culturing, the culture is filtered or centrifuged to separate the mycelium and the culture solution. The culture solution from which the mycelium has been removed can be concentrated to an appropriate concentration using an evaporator, a filtration membrane, etc., and then passed through a sterilization filter or the like to form a liquid antiviral agent. or,
A powdered antiviral agent can also be produced by concentrating the culture solution from which the mycelium has been removed to an appropriate concentration and freeze-drying it. In the present invention, the culture solution from which the mycelium has been removed may be subjected to Sephadex, DEAE cellulose, etc. to further separate and purify effective components. In addition, although the present invention was made with a focus on the antiviral activity of cane molasses, it is also expected that the mycelium of edible mushrooms itself has antiviral activity, and in some cases, the mycelium may be homogenized. Alternatively, an antiviral agent that also contains mycelium components may be produced by autolysis. Examples of the present invention will be described below. Example 1 Cane molasses subjected to steam injection was diluted with water to prepare a culture solution. This culture solution was placed in an autoclave and sterilized according to a conventional method, inoculated with shiitake mushroom mycelium, and aerated culture was performed in a thermostatic chamber at 25°C.
After culturing for 10 days, the culture was filtered to remove mycelium, and the culture filtrate was concentrated using an evaporator.
Lyophilization gave a brown powder. The LD ₅₀ of this brown powder when administered orally to rats was 18.5 g/Kg. Next, antiviral activity was tested using this brown powder. (1) Tobacco mosaic virus (TMV) Nicotiana glutinosa (N.glutinosa) was used as the test plant. The above brown powder was diluted to various concentrations, and TMV was added to this diluted solution.
A 2000-fold dilution was mixed and inoculated into half leaves. In addition, control half leaves were inoculated with only a diluted solution of TMV. Then, the number of local lesions in the test plot and the control plot was measured, and the inhibition rate of local lesion formation in the test plot relative to the control plot was determined. The results are shown in Table 1.

【table】

[Table] (*Number of local lesions)
As is clear from the table above, the brown powder has excellent antiviral activity at low concentrations. (2) Influenza virus Influenza A type ₂ grown using pig kidney cells as host cells was inoculated into a medium supplemented with various concentrations of brown powder, and after culturing the cells at 37℃ for 2 days, the chicken hemagglutination value was determined. I looked into it. The results are shown in Table 2.

[Table] As is clear from the above table, the brown powder has excellent antiviral activity against influenza viruses even at low concentrations. (3) Hepatitis B virus A 34-year-old man diagnosed with active chronic hepatitis B as a result of liver biopsy was orally administered 6 g of brown powder per day. As a result, before administration
The GOT value was 779 and the GPT value was 838, but it suddenly decreased from the second week after starting administration, and by the first month, the GOT value was 7 and the GPT value was 22, which were normal. In addition, the HBe antigen and HBe antibodies were 5.6 and 6.0 before administration, but seroconversion occurred in the first month of administration, and in the second month,
The HBe antigen was 0.4 and the HBe antibody was 73. Example 2 A brown powder was produced in the same manner as in Example 1 using enokitake mycelium. The LD ₅₀ of this brown powder when administered orally to rats was 17.4 g/Kg. This brown powder was used to test for antiviral activity. (1) Tobacco mosaic virus (TMV) Nicotiana glutinosa (N.glutinosa) was used as the test plant. The above brown powder was diluted to various concentrations, and TMV was added to this diluted solution.
A 2000-fold dilution was mixed and inoculated into half leaves. In addition, control half leaves were inoculated with only a diluted solution of TMV. Then, the number of local lesions in the test plot and the control plot was measured, and the inhibition rate of local lesion formation in the test plot relative to the control plot was determined. The results are shown in Table 3.

[Table] The antiviral activity is clear from the above table.
(2) Influenza virus Influenza A type ₂ grown using pig kidney cells as host cells was inoculated into a medium supplemented with various concentrations of brown powder, and after culturing the cells at 37℃ for 2 days, the chicken hemagglutination value was determined. I looked into it. The results are shown in Table 4.

[Table] The antiviral activity is clear from the above table.
As explained above, according to the present invention, an antiviral agent having high activity can be produced using cane molasses, an extremely easily available and inexpensive raw material. In addition, since a liquid medium can be used, the mycelium can be removed after culturing to easily obtain a culture solution with antiviral activity.
Operations such as extraction of the active ingredient are greatly simplified, making it suitable for production on an industrial scale. Furthermore, cane molasses is an extremely safe substance, and the edible mushroom mycelium that acts on this cane molasses is also non-toxic, so the obtained antiviral agent has no toxicity or side effects. There is no problem at all.

Claims (1)

[Claims] 1. A method for producing an antiviral agent, which comprises inoculating a medium containing cane molasses as a main component with mycelium of edible mushrooms belonging to Basidiomycetes and culturing them in deep culture.