JP5934896B2 - Basidiomycetous culture method - Google Patents

Description

  The present invention relates to a basidiomycete culture method for culturing basidiomycetous mushrooms in a woody culture medium or the like.

  Conventionally, fungus bed cultivation of edible mushrooms has been performed by the process of medium adjustment, sterilization, cooling, inoculation, culture, and generation. Each process is an indispensable process, but the process that requires the most expensive equipment and requires specialized knowledge and skills is sterilization and inoculation. In edible mushroom cultivation, contamination of germs and poor sterilization in the medium have serious consequences to be described later and must be avoided. For example, if the sterilization pot malfunctions or misoperates, the medium becomes poorly sterilized. If mold or bacteria are generated, the entire medium sterilized together is contaminated, and the contamination further spreads throughout the production facility. When various bacteria are mixed into the medium in the inoculation step, the adjacent medium is infected with air. Furthermore, if the sterilization / inoculation process is not successful, production may be stopped. For this reason, a sterilization pot that is surely sterilized is placed, and in the process after the medium is taken out from the sterilization pot, the process is allowed to cool, inoculate, and the initial stage of the culture is handled in a non-sterile room, from sterilization to inoculation. In this process, careful attention must be paid from work clothes to operation. In addition, sterilization pots and sterilization chambers are expensive equipment, and expertise and techniques relating to sterilization operations are required to reliably perform inoculation.

  In the cultivation of logs such as shiitake mushrooms and sea cucumbers, no sterilization / sterilization room facilities are required, and the inoculation work is completed by drilling the logs with a drill and inoculating seed pieces there. Log cultivation can be easily done by ordinary farmers, but fungal bed cultivation is extremely difficult for the reasons described above.

  In order for farmers to incorporate into the fungus bed cultivation, a large amount of capital investment and expertise and techniques regarding flame sterilization (flame sterilization, alcohol disinfection, clean bench operation) are required, which are major obstacles. . If conventional sterilization operations, inoculation work under aseptic conditions, and initial culture in an aseptic room can be omitted, mushroom fungus bed cultivation will be easy for farmers.

  On the other hand, enokitake, oyster mushroom, and himematsutake, which are basidiomycetes and edible mushrooms, have both a substrate-degrading enzyme and an alcohol dehydrogenase. These can decompose lignocellulosic biomass by substrate decomposition and saccharify, and monosaccharides can be ethanol-fermented. In recent years, there has been a demand for producing bioethanol at low cost from non-cereal biomass, that is, lignocellulosic biomass such as rice straw and wood, in order to avoid an increase in grain prices. As an inexpensive ethanol production method, a linked bioprocess (CBP) that performs all steps of enzyme production, substrate decomposition (saccharification), and fermentation in a lump attracts attention. Based on these backgrounds, a project project commissioned by the Secretariat of the Agriculture, Forestry and Fisheries Technology Council of the Ministry of Agriculture, Forestry and Fisheries on the assumption of using the enokitake mushroom bed to perform a connected bioprocess with lignocellulosic biomass, “Unused crops such as rice straw, etc. Development of technology to efficiently convert parts, resource crops, and woody biomass into ethanol. " The waste fungus bed is one of the woody biomass discharged in large quantities, but the area where fresh waste fungus beds can be obtained is limited to the vicinity of the edible mushroom production facility. In order to obtain a wider range of fresh waste beds, it is possible to easily cultivate fungi beds such as enokitake mushrooms in general farmers, but this involves expensive capital investment and aseptic operation such as sterilization facilities and aseptic rooms Technology has become a major obstacle. Simple farming in a general farmer is required both in the field of producing bioethanol and in the field of harvesting cultivated fruiting bodies.

  Here, miscellaneous germs that prevail in mushroom cultivation are molds such as Trichodem and Penicillium. Streptomyces spp. Are known to secrete antibiotics against mold at the culture stage, and several methods for preventing the propagation of miscellaneous bacteria using this fact have been considered.

  For example, a method for increasing the production of matsutake or hon-shimeji using a mixed microorganism disclosed in Patent Document 1 is described as follows. Or, it can be applied to white shimeji mushrooms to increase the amount of fruiting bodies.

  In addition, the method for producing an extract of Agaricus blazei mycelium and / or fruiting body disclosed in Patent Document 2 is based on conditions in which the medium raw material is at a temperature of 55 ° C. to 65 ° C. and a water content of 60% by mass to 75% by mass. Step (1) of producing a medium by fermenting for 7 to 20 days, and then propagating the fungus on the mycelium formed using the obtained medium, and the mycelium and / or fruiting body of Agaricus blazei Step (2) of culturing, and then using any extraction solvent selected from water, ethanol, a mixed solution of water and ethanol, from the obtained mycelium and / or fruiting body of Agaricus blazei Step (3) of extracting the extract. Thermophilic actinomycetes and / or thermophilic bacteria are added at any stage until step (2) is performed. According to this, molds and the like that adversely affect the cultivation of Agaricus blazei can be eliminated, and the extract of mycelium and / or fruiting body of Agaricus blazei can be stably supplied at low cost.

  Moreover, the auxiliary material for cultivation of mushrooms and the cultivation method of mushrooms disclosed in Patent Document 3 were made by mixing a base material for cultivation of mushrooms and an auxiliary material for cultivation of mushrooms in a container for cultivation of mushrooms. A medium is accommodated, and CN chitin is mixed in the lower part of the medium to prevent the propagation of filamentous fungi harmful to the growth of mushrooms in the container. CN chitin constitutes the cell membrane of filamentous fungi, but the filamentous fungi have the property that actinomycetes prefer and prey on them, and by mixing CN chitin, the actinomycetes are allowed to grow, Prevent the breeding of. When mixing CN chitin, a particularly effective actinomycete is mixed in a small amount, and when this actinomycete is grown, the effect is particularly high. Thereby, even if preliminary work, such as sterilization of the mushroom cultivation base material and the container, is omitted, the growth environment of the mushrooms can be favorably maintained.

JP-A-5-153854 JP 2001-178404 A Japanese Patent Laid-Open No. 2-177820

  The method for increasing production in Patent Document 1 of the background art described above is applied to white matsutake and white shimeji mushrooms, and the mycorrhizal fungi that form mycorrhiza with basidiomycetes having a symbiotic relationship with trees such as matsutake and honshimeji, Points to the area where the child entity is generated. It was intended to increase the yield of fruiting bodies by manipulating mycorrhizal fungi on an outdoor white paper, and was not used in the cultivation method of wood-rotting basidiomycetes. In addition, the basidiomycetes to be targeted are limited to matsutake and hon-shimeji mushrooms, and the place for cultivation is also limited to the outdoors. Therefore, it is not used for indoor cultivation such as enokitake.

  The production method of Patent Document 2 is limited to Agaricus blazei and is not used for cultivation of enokitake. Moreover, it sterilizes at the high temperature of 55-65 degreeC, and an installation and a technique are required and it is difficult to carry out easily with a general farmer.

  The cultivation method disclosed in Patent Document 3 requires the addition of actinomycetes and mixing of CN chitin, which takes time. Moreover, the species of actinomycetes was not specified, and it was not efficient to select a suitable one from various actinomycetes.

  The present invention has been made in view of the problems of the background art described above, and can eliminate the sterilization process in mushroom cultivation and the inoculation / initial culture process in an aseptic room, and can reliably propagate various germs in a simple process. An object of the present invention is to provide a basidiomycete culturing method capable of culturing basidiomycetes while suppressing the above.

This invention inoculates the mixture obtained by mixing the medium base material and nutrients and adjusting the water content, in a normal room, with the actinomycetes Streptomyces genus and basidiomycetes that secrete antibiotics , at 3-12 ° C. A basidiomycete culture method for culturing basidiomycetes to obtain fruiting bodies.

  The actinomycete Streptomyces genus is Streptomyces rimosus.

The Streptomyces rimosus and the basidiomycete are mixed and cultured together with the medium base material containing rice bran .

  The basidiomycete culture method of the present invention is a culture method that can suppress the propagation of germs with a simple process by omitting the sterilization process in the fungus bed cultivation and the inoculation / initial culture process in the sterile room. By eliminating the sterilization process and the inoculation / initial culture process in the sterile room, there is no need for expensive capital investment in sterilization facilities, sterile rooms, etc. Simple and safe mushroom bed cultivation.

In the culture method of this invention, it is the graph which measured two types of substrate degrading enzyme activities as a parameter | index of the prevalence of the enokitake mushroom mycelium by the difference in the inoculation method of a microbe. In the culture method of this invention, it is the graph which measured three other types of substrate-degrading enzyme activities as a parameter | index of the prevalence of Enokitake mycelium by the difference in the inoculation method of a microbe. In the culture method of this invention, in order to examine the presence or absence of the suitable period which harvests fruit bodies in a culture process, it is the graph which measured two types of substrate-degrading enzyme activities. In the culture method of this invention, in order to examine the presence or absence of the suitable period which harvests a fruit body in a culture | cultivation process, it is the graph which measured three other types of substrate-degrading enzyme activities.

  Hereinafter, an embodiment of the present invention will be described. In this embodiment, a basic medium prepared by mixing normal materials such as sawdust and nutrients prepared by normal adjustment to adjust the water content, and in a normal room that is not a sterile room or clean room, Oga inoculum belonging to the genus Streptomyces The inoculum of basidiomycete is mixed and inoculated, and cultured at 3 to 12 ° C, preferably at 5 to 10 ° C. The basidiomycetes are cultured for a certain period of time to form mushroom fruit bodies and harvested. The species of the genus Streptomyces is preferably Streptomyces rimosus and secretes antibiotics against various bacteria during the culturing process. The basidiomycete is enokitake.

  The optimum temperature for mycelium growth of basidiomycetes is 22-27 ° C, but grows even at 10 ° C or less. At the culture temperature of 3 to 12 ° C., which is the culture temperature of this embodiment, the growth rate of basidiomycetes is about the same as or higher than molds and bacteria in the air. Better than mold and bacteria. Furthermore, by secreting antibiotics from basidiomycetes, as a result, basidiomycetes dominate and occupy bacteria and molds in the medium. Therefore, the sterilization process is omitted, and the culture method does not require a sterilization facility or a sterile room. Below 3 ° C., the mycelium of basidiomycetes do not extend but become dormant and almost stagnant. When the temperature exceeds 12 ° C., mycelium of basidiomycetes grows, but mold and bacteria grow faster than that, resulting in occupation of the medium and hindering good growth of basidiomycetes. Therefore, selection of actinomycetes that grow sufficiently at 3 to 12 ° C. is a point.

  According to the basidiomycete culture method of this embodiment, the sterilization process in the fungus bed cultivation and the inoculation / initial culture process in the aseptic room can be omitted, and the propagation of germs can be reliably suppressed by a simple process. By eliminating the sterilization process, inoculation in the aseptic room, and initial culture process, there is no need for expensive capital investment in sterilization facilities and sterilization rooms, no cost, no specialized knowledge and aseptic operation techniques However, it is possible to perform simple and safe microbial bed cultivation that suppresses the propagation of various bacteria. Basidiomycetes can be easily cultured, and mycelium components, metabolites, etc. of basidiomycetes can be obtained at low cost in addition to fruit bodies.

  As a result, fungus bed cultivation of mushrooms will be practical and easy for general farmers and the like and can be expected to spread. Moreover, fungus bed cultivation of mushrooms becomes simple, and simple edible mushroom cultivation is possible at the site where forest maintenance is carried out, which can contribute to promotion of mountainous areas without sterilization facilities and promotion of forest maintenance. The amount of waste bacterial bed used for fungus bed cultivation increases, and it becomes easy to obtain materials for bioethanol production using lignocellulosic biomass.

  In the basidiomycete culturing method of the present invention, the medium base material, the nutrient material, the mixing ratio, the size of the container in which the medium is placed, and the like can be freely set. The mixing ratio of the culture medium, basidiomycetes, and actinomycetes can be set as appropriate. In culture, conditions such as setting of temperature and humidity, and culture period can be appropriately set. Actinomycetes to be mixed may not be Oga species. The kind of basidiomycete can be used for various things.

The first embodiment of the basidiomycete culture method of the present invention will be described with specific numerical values. 1st Example shows the process by which the actinomycete seed | species is selected to Streptomyces rimosus. The germs that are prevalent in the mushroom basidiomycete culture stage are molds such as Trichoderma and Penicillium. Therefore, 100 μl (10 ⁵ to 10 ⁶ cfu / ml, 0.3% (w / v) peptone, 0.3% (w / v) yeast extract, 0. 10 μl of Trichoderma reesei, which is one of various bacteria. 1% (w / v) NaH ₂ PO ₄ , 0.01% (w / v) MgSO ₄ .7H ₂ O) is added with 10 μl of Streptomyces culture filtrate, and cultured at 25 ° C. for 60 to 84 hours. Absorbance was measured. When the conidia of Trichoderma reesei germinate and mycelium growth occurs, the culture becomes dark green, and the state of germination and mycelium growth are determined by the degree of dark green. Those having antibacterial activity do not increase the absorbance.

  As the actinomyces Streptomyces sp. fradiae NBRC13147 (S-1), S. griseosporeus NBRC13458 (S-2), S. grieseus subsp. griseus NBRC12875 (S-3), S. cattleya NBRC14057 (S-4), S. cattleya. rimosus subsp. rimousus NBRC12907 (S-5), S. Venezuelae NBRC13096 (S-6), S. tendae NBRC12822 (S-7), S. griseochromogenes subsp. suitaensis NBRC13776 (S-8), S. et al. Culture and measurement were performed using 9 species of Spectabilis NBRC1544 (S-9).

  As a result, S.E. griseochromogenes subsp. Suitaensis NBRC13776 (S-8) and S. rimosus subsp. rimosus NBRC12907 (S-5) had antibacterial activity. S. griseochromogenes subsp. Situaensis NBRC13776 (S-8) rimosus subsp. It showed stronger antibacterial activity than rimosus NBRC12907 (S-5).

  Therefore, S. cerevisiae having antibacterial activity against Trichoderma reesei. griseochromogenes subsp. Suitaensis NBRC13776 (S-8) and S. rimosus subsp. rimosus NBRC12907 (S-5) was screened for these two species having antibacterial activity, and S. rimosus having no antibacterial activity for comparison. The sterilization-free culture test was conducted using a total of three kinds of fradiae NBRC13147 (S-1). Corncob / rice bran medium (dry weight 3: 2, water content 65%) is packed in 100 g of biopot (PP, diameter 80 × height 100 mm) and inoculated with the above three actinomycetes previously cultured in beech / rice bran medium. 10 g was inoculated as a source, and further 10 g of Enokitake Fv-1 inoculum was inoculated and cultured at 5, 10, 15, 20, and 25 ° C. (each has a ± 2 ° C. temperature range) for 60 days. Table 1 shows the situation in which three actinomycetes and enokitake were cultured for 60 days.

この結果、Ｓ．ｒｉｍｏｓｕｓ ｓｕｂｓｐ．ｒｉｍｏｓｕｓ ＮＢＲＣ１２９０７（Ｓ‐５）において、１０℃以下ではＴｒｉｃｈｏｄｅｒｍａ ｒｅｅｓｅｉ等雑菌が繁殖することなくエノキタケが占有して培養された。１５℃では雑菌の混入が認められたが、エノキタケが占有して培養された。１５℃では雑菌の混入が認められたが、エノキタケが優勢であった。２０℃、２５℃では雑菌が占有した。Ｓ．ｆｒａｄｉａｅ ＮＢＲＣ１３１４７（Ｓ‐１）では１０℃以下で、Ｓ．ｇｒｉｓｅｏｃｈｒｏｍｏｇｅｎｅｓ ｓｕｂｓｐ．ｓｕｉｔａｅｎｓｉｓ ＮＢＲＣ１３７７６（Ｓ‐８）では５℃で雑菌の混入が認められ、エノキタケが優勢であった。それら以外の温度条件では雑菌が占有した。このことから、Ｓ．ｒｉｍｏｓｕｓ ｓｕｂｓｐ．ｒｉｍｏｓｕｓ ＮＢＲＣ１２９０７（Ｓ‐５）とエノキタケを接種し、３〜１２℃で培養すれば、無殺菌でエノキタケを占有して培養できることが明らかになった。表１で△も含めると１５℃以下となる。培養器が±２℃の制度なので、５℃−２℃＝３℃、１０℃＋２℃＝１２℃となる。表を遵守すると、５〜１０℃あるいは５〜１５となる。また、試供した３種の放線菌のいずれを用いても３〜７℃で培養すれば、エノキタケを優先的に培養できることが明らかになった。なお、３℃以下では、エノキタケ菌糸体は伸長せず、休眠状態となり、ほぼ停滞する。１２℃以上では、エノキタケ菌子体は生長するが、それ以上にＴｒｉｃｈｏｄｅｒｍａ ｒｅｅｓｅｉの生長がはやく、結果としてＴｒｉｃｈｏｄｅｒｍａ ｒｅｅｓｅｉが占有する。

As a result, S.E. rimosus subsp. In Rimosus NBRC12907 (S-5), at 10 ° C. or lower, Trichoderma reesei and other germs occupy without being propagated and cultured. Although contamination with germs was observed at 15 ° C., it was cultured with enokitake occupying it. At 15 ° C., contamination with germs was observed, but enokitake was predominant. At 20 ° C and 25 ° C, miscellaneous bacteria occupied. S. fradiae NBRC13147 (S-1) at 10 ° C. or lower. griseochromogenes subsp. In Situensis NBRC13776 (S-8), contamination with germs was observed at 5 ° C., and enokitake was dominant. Other temperature conditions occupied the various bacteria. From this, S.I. rimosus subsp. It was clarified that rimosus NBRC12907 (S-5) and enokitake were inoculated and cultured at 3-12 ° C., and occupying enokitake without sterilization. When Δ is included in Table 1, it is 15 ° C. or lower. Since the incubator is a system of ± 2 ° C., 5 ° C.−2 ° C. = 3 ° C., 10 ° C. + 2 ° C. = 12 ° C. If it adheres to a table | surface, it will be 5-10 degreeC or 5-15. Moreover, it became clear that enokitake can be preferentially cultured if it is cultured at 3-7 ° C. using any of the three actinomycetes tested. At 3 ° C. or lower, the enokitake mushroom mycelium does not elongate, becomes dormant and almost stagnates. At 12 ° C. or higher, enokitake mushrooms grow, but Trichoderma reesei grows faster than that, and as a result, Trichoderma reesei occupies.

  It should be noted that S. aureus having the most antibacterial activity against Trichoderma reesei. griseochromogenes subsp. Suitaensis NBRC13776 (S-8) is an S. aureus that has no antibacterial activity. fradiae NBRC13147 (S-1), the result is inferior. rimosus subsp. Rimosus NBRC12907 (S-5) gave the best results. This is because S.A. griseochromogenes subsp. In Suitaensis NBRC13776 (S-8), the secreted antibacterial substance also damages the enokitake mushroom. rimosus subsp. Since rimosus NBRC12907 (S-5) exhibits antibacterial activity only against mold, it is presumed that such a result was obtained.

  Next, a second embodiment of the basidiomycete culture method of the present invention will be described with specific numerical values. The second example shows the process of examining the inoculation method, and 100 g of corn cob / rice bran medium (dry weight 3: 2, moisture content 65%) in a biopot (PP, diameter 80 × height 100 mm). Stuffed, Actinomycetes rimosus subsp. Rimosus NBRC12907 (S-5) and Enokitake Fv-1 inoculum were inoculated by A, B, and C methods. Inoculation method A is a method in which 10 g of actinomycetes is first inoculated on the test medium, and 10 g of enokitake is superimposed on the actinomycetes. Inoculation method B is a method in which 10 g of enokitake is first inoculated on the test medium, and 10 g of actinomycetes are stacked on the enokitake. Inoculation method C is a method in which a test medium, 10% by mass of actinomycetes of the test medium, and 10% by mass of enokitake mushroom inoculum are mixed and 120 g is packed in a biopot.

  The culture medium prepared by the inoculation methods A, B, and C was cultured at 10 ° C. for 12 weeks, and the substrate degrading enzyme activity was measured. By measuring the substrate-degrading enzyme activity, the prevalence of enokitake mycelium can be known indirectly. It is difficult to measure only the enokitake mycelium in the solid medium, and it can be confirmed by the naked eye that the enokitake mycelium is spreading in the incubator, but it is not known any more. . Therefore, the polysaccharide degrading enzyme activity secreted from the enokitake mushroom mycelium is measured, and the production of various enzymes related to the degradation of the polysaccharide is not considered other than enokitake. Therefore, the enzyme activity is regarded as an indicator of the spread of enokitake mycelium. did. Substrate-degrading enzymes were measured for β-D-Xylopyranosidase, β-D-Glucosidase, β-D-Cellobiosidase, CMCase, and Xylanase. The measurement results of enzyme activity are shown in FIGS.

  As a result, enokitake was cultivated exclusively by inoculation method A, B, or C. In any of the enzymes, the highest enzyme activity was obtained by inoculation method C. In inoculation method A and inoculation method B, β-D-Xylopyranosidase and β-D-Cellobiosidase shown in FIG. 1 are higher in inoculation method A, and in β-D-Glucosidase and Xylanase shown in FIG. In the CMCase shown in FIG. That is, no significant difference was observed between the inoculation methods A and B. When non-sterile culture of actinomycetes and basidiomycetes is performed, the inoculation method C in which each inoculum and the substrate of the medium are mixed is most effective.

  Next, a third embodiment of the basidiomycete culture method of the present invention will be described with specific numerical values. The third example shows a process for examining the presence or absence of an appropriate period for harvesting fruit bodies in the culturing process. Corn cob / rice bran medium (dry weight 3: 2, moisture content 65%) and test medium 10% by weight of actinomycetes rimosus subsp. Rimosus NBRC12907 (S-5) and enokitake inoculum were mixed, and 100 g was packed in a biopot (PP, diameter 80 × height 100 mm). Thereafter, the cells were cultured at 10 ° C. for 4, 8, and 12 weeks. The culture status was determined using the substrate degrading enzyme activity (β-D-Xylopyranosidase, β-D-Glucosidase, β-D-Cellobiosidase, CMCase, Xylanase) as an index. The measurement results of enzyme activity are shown in FIGS. In addition, it was confirmed by the naked eye that the enokitake mycelium invaded the biopot in 4 weeks of culture, formed fruit bodies in 8 weeks of culture, and self-dissolved in 12 weeks of culture.

  As a result, all enzyme activities were highest in 8 weeks of culture, and slightly lower in 12 weeks than in 8 weeks. All of the enzymes are energetic as enokitake mushrooms spread and form fruiting bodies, indicating that there is an appropriate period in the culture process. That is, fruit body formation can be expected with this medium in an appropriate culture period. It is expected that fruit bodies will be generated by manipulating so as to promote fruit body formation at the peak of culture. In the biopot (PP made, diameter 80 × height 100 mm) used in this example, a suitable culture period is around 8 weeks, but the optimal culture period varies depending on the size of the medium. .

As shown in Examples 1 to 3 above, the basidiomycete culture method of the present invention is a non-sterile culture of actinomycetes and basidiomycetes, as long as the basidiomycetes are firmly cultured and covered with mycelium. Even when cultivated at a low temperature at which the propagation of potato is suppressed, basidiomycetous fruit bodies are formed and harvesting is expected.

Claims (2)

A mixture obtained by mixing the medium base material and nutrients and adjusting the water content is inoculated with Streptomyces rimosus that is an actinomycete Streptomyces genus and secretes antibiotics , and basidiomycetes, in a normal room, 3-12 ° C Basidiomycete culture method cultivated in The basidiomycete culture method according to claim 1, wherein the Streptomyces rimosus and the basidiomycete are mixed and cultured together with the medium substrate containing rice bran .

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