CN102369838B - The production method of the liquid strain bacterium of mushroom - Google Patents
The production method of the liquid strain bacterium of mushroom Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The present invention provide the large-scale commercial applications of a kind of mushroom fruitbody to produce in cheap and effective liquid strain bacterium production method and use the mushroom fruitbody production method of this liquid strain bacterium。The present invention relates to the production method of the liquid strain bacterium of a kind of mushroom, it is the production method of the mushroom liquid kind bacterium for producing mushroom fruitbody, it is characterized in that, in the fluid medium containing vegetable oil, by using bubble agitation culture medium to carry out the cultivation of liquid deep。It addition, the invention still further relates to the production method of a kind of mushroom fruitbody, it is characterised in that in bottle is planted, use the bacterium bed cultivation culture medium that bacterium seat further increases。According to the present invention it is possible to significantly shorten kind of a bacterium production time, furthermore it is possible to the risk of production inhomogeneities when avoiding the mushroom fruitbody using solid kind bacterium to produce, and can stably produce excellent mushroom fruitbody。
Description
Technical field
The present invention relates to the production method of the mushroom liquid kind bacterium for producing mushroom fruitbody and the method using this liquid strain bacterium to produce mushroom fruitbody。
Background technology
In recent years, have been developed for the artificial cultivation technique of mushroom, thus providing various mushroom fruitbody (taking up an area mushroom, Folium Nelumbinis mushroom, Hypsizygus marmoreus, Pleurotus eryngii, Lentinus Edodes, Flammulina velutiper (Fr.) Sing etc.)。
Generally, so mushroom is carried out artificial culture: by being inoculated in by the kind bacterium of mushroom in fragment wood or solid medium, via operations such as cultivation, growths, gather in the crops sporophore。The bacterium of planting of mushroom includes using the solid kind bacterium of the culture medium such as sawdust and uses the liquid strain bacterium of fluid medium。In recent years, just at propelling machinery in the artificial culture of mushroom, for instance, enforcement etc. in the factory system of main equipment。But, produce mushroom fruitbody in a large number and stably in order to commercial, it is necessary to the kind bacterium that mass propgation uses。At present, have good keeping qualities from the kind bacterium of mushroom and can easily the reason such as transport consider, it is possible to use solid kind bacterium；But from the viewpoint of the risk of the production difference (it is due to the batch wise differences of solid kind bacterium) being prone to when avoiding mushroom fruitbody to produce or harmful bacteria insect to problems such as the severe contaminations (pollution planting the bottle caused by bacterium or container overflowed when it is due to inoculation solid kind bacterium) of equipment, can significantly shorten the production time of kind of bacterium and be easily enlarged scale etc., liquid strain bacterium is considered to be advantageous for, and its use increases。As the method for mass propgation liquid strain bacterium, liquid deep culture method (it is the mass propgation method for obtaining useful component from mycelium) is studied, in order to strictly control condition of culture etc., general is utilize carry out mechanical agitation, ventilation, temperature treatment small-sized fermentation tank carry out the method for air agitation (such as, non-patent literature 1)。But, in order to commercial mass production mushroom fruitbody, it is necessary to arrange the jumbo high price comprising small-sized fermentation tank and large-scale culture device。
[non-patent literature 1] ferment meeting, the 24th volume, the 7th phase, 293-304 page, 1966 years
Summary of the invention
The problem that invention to solve
In view of above-mentioned present situation, a kind of method that it is an object of the invention to provide during large-scale commercial applications at mushroom fruitbody produces inexpensively and effectively produce liquid strain bacterium and the method using this liquid strain bacterium to produce mushroom fruitbody。
The method of a large amount of mushroom liquid kind bacterium produced for large-scale commercial applications production has been concentrated on studies by the present inventor, found that, it is surprising that it is unexpectedly also useful to the production of the liquid strain bacterium for producing mushroom fruitbody to use the liquid deep of bubble to cultivate (unpowered agitation: air-blowing is steeped) in stirring。In addition, it is found that in this cultivation, by liquid medium within contains the vegetable oil of low concentration, such that it is able to effectively and stably produce mycelium in a large number when having mushroom fruitbody Forming ability。And then, by improving the bacterium seat of the bacterium bed cultivation culture medium used in solid kind bacterium up to now further, and make bacterium seating face planarize equably, such that it is able to carry out more stable bacterium circulation (bacterium り), can be effectively taking place and induce caused by mycelium stimulation (bacterium か I), compared with the situation using solid kind bacterium, excellent mushroom fruitbody can be produced, this mushroom fruitbody is uniform and commodity value is high, and this production method to comprise kind of the time of bacterium production time short, this completes the present invention。
The present invention is summarized as follows:
[1] production method of the liquid strain bacterium of a kind of mushroom, it is the production method of the mushroom liquid kind bacterium for producing mushroom fruitbody, it is characterized in that, in the fluid medium containing vegetable oil, by using bubble agitation culture medium that the mycelium of mushroom is carried out the cultivation of liquid deep；
[2] production method as described in [1], wherein, substantially only uses bubble to carry out stir culture base；
[3] production method as described in [1] or [2], wherein, described vegetable oil is one or more in salad oil, Testa oryzae oil, Oleum Brassicae campestris, safflower oil, Semen Maydis oil, olive oil, Oleum sesami, soybean oil and Oleum Gossypii semen；
[4] production method as according to any one of [1]～[3], wherein, the vegetable oil concentration in described culture medium is 0.001～0.1 volume %；
[5] production method of a kind of mushroom fruitbody, it is characterised in that use the mushroom liquid kind bacterium obtained by the production method according to any one of [1]～[4]；
[6] production method of the mushroom fruitbody as described in [5], it is bottle hydroponics；
[7] production method of the mushroom fruitbody as described in [5] or [6], it is characterised in that use the bacterium bed cultivation culture medium that bacterium seat improves。
[8] production method of the mushroom fruitbody as described in [7], it is characterised in that use the bacterium bed cultivation culture medium of bacterium seating face uniform planar。
Invention effect
According to the present invention it is possible to provide a kind of method that large-scale commercial applications for mushroom fruitbody produces, that produce mushroom liquid kind bacterium at short notice at a low price in a large number。Alternatively, it is also possible to provide a kind of method using this liquid strain bacterium to produce mushroom fruitbody。
Accompanying drawing explanation
[Fig. 1] is the figure that the bacterium seat of the bacterium bed cultivation culture medium illustrating that in the culture bottle for production method of the present invention, bacterium seat improves is put。
Detailed description of the invention
For can be used in " mushroom " of the present invention, there is no particular limitation, it is possible to enumerates Folium Nelumbinis mushroom, take up an area the edible mushrooms such as mushroom, Hypsizygus marmoreus, Pleurotus ostreatus, Lentinus Edodes, Flammulina velutiper (Fr.) Sing, Pholiota nameko, Pleurotus ostreatus, dance young pilose antler, Pleurotus eryngii, Agaricus blazei Murrill, Pleurotus abalonus, black ear Pleurotus ostreatus。Bacterial strain as these mushrooms, as long as can serve as liquid strain bacterium to carry out cultivating and carpogenic bacterial strain can being given birth to, it can be commercially available bacterial strain, it can also be the separate tissue strain from wild sporophore, it is also possible to the bacterial strain obtained for carrying out breeding by methods such as screening, hybridization, cell fusion, gene recombinaton。A preferred embodiment as the present invention, it is possible to enumerate Folium Nelumbinis mushroom (Lyophyllumdecastes)。As Folium Nelumbinis mushroom, known bacterial strain can be illustrated, such as, LyophyllumdecastesK-3303 strain (FERMBP-4347), LyophyllumdecastesK-3304 strain (FERMBP-4348), LyophyllumdecastesK-3305 strain (FERMBP-4349), LyophyllumdecastesF-623 strain (FERMP-13165), LyophyllumdecastesF-1154 strain (FERMP-13166), LyophyllumdecastesF-1488 strain (FERMP-13167) and be suitable to the mutant etc. of raw these bacterial strains carpogenic。
Additionally, example as the bacterial strain of this occupation of land mushroom (Lyophyllumshimeji) preferred in the present invention, it is possible to illustrate: LyophyllumshimejiLa01-27 (FERMBP-10960), LyophyllumshimejiLa01-20 (FERMBP-10959), LyophyllumshimejiLa01-37 (FERMP-17456), LyophyllumshimejiLa01-45 (FERMP-17457), LyophyllumshimejiLa01-46 (FERMP-17458) and be suitable to the mutant etc. of raw these bacterial strains carpogenic。
In addition, bacterial strain as Hypsizygus marmoreus preferred in the present invention, have and be expressed as the raw bacterial strain from pleat umbrella (Lyophyllumulmarium) of elm, it is expressed as the bacterial strain etc. of Hypsizygusmarmoreus, such as can illustrate: LyophyllumulmariumM-8171 (FERMBP-1415), LyophyllumulmariumK-0259 (FERMP-12981), LyophyllumulmariumLu1-172 strain (FERMBP-8354), LyophyllumulmariumLu1-173 strain (FERMBP-8355), LyophyllumulmariumLu1-174 strain (FERMBP-8356), LyophyllumulmariumLu1-181 strain (FERMBP-8357), HypsizygusmarmoreusK-4975 (FERMBP-11321), HypsizygusmarmoreusK-4979 (FERMBP-11322), HypsizygusmarmoreusK-4980 (FERMBP-11323), HypsizygusmarmoreusK-4981 (FERMBP-11324) and be suitable to the mutant etc. of raw these bacterial strains carpogenic。
In addition, bacterial strain as black ear Pleurotus ostreatus preferred in the present invention, it is possible to illustrate: Pleurotuscystidiosussubsp.abalonusK-4986 (FERMP-22064), Pleurotuscystidiosussubsp.abalonusK-4987 (FERMP-22065), Pleurotuscystidiosussubsp.abalonusK-4988 (FERMP-22066) and be suitable to the mutant etc. of raw these bacterial strains carpogenic。
Above-mentioned bacterial strains be there is no any restriction, as long as being can tame bacterial strain and the bacterial strain for the applicable present invention。
In present specification, the mycelium that so-called " mycelium " is above-mentioned mushroom, to it, there is no particular limitation, as long as the liquid strain bacterium that can cultivate and can be used as producing mushroom fruitbody in liquid medium within。
In present specification, so-called " the liquid deep using bubble is cultivated " refers to and makes culture fluid motion (flowing) by the bubble produced in culture tank, thus culture medium is implemented the cultural method of stirring。Preferably substantially only use bubble cultural method that culture medium is stirred, it is also possible to comprise the mechanical agitation undertaken by the device of agitator, the aerator etc of airfoil shape, as long as the degree not causing mycelia to cut off。It is particularly preferably the cultural method only using bubble that culture medium is stirred without mechanicalness stirring。For the culture tank for described cultural method, it is not necessary to for the use mechanokinetic device stirring or vibrating, as long as possessing the device producing bubble。In the present invention, the bubble-column-type culture apparatus etc. recorded in (such as) document " Japan I こ learns, the 8th volume, the 1st phase; 1-11 page (2000) " can be used, it is possible to use with producing the culture tank of device as aseptic bubble from bottom surface and/or side。Alternatively, it is also possible to the pipe etc. that can produce bubble is added directly in culture tank。Ventilation as the bubble in culture tank, preferably 0.2～0.8vvm (volumepervolumeperminute: the gas ventilation amount of per unit volume), it is preferably 0.2～0.6vvm, or can also be 10～50L/min (represent, in 1 minute, 10～50L filtrated air is vented to the amount in 25～83L fluid medium), it is preferred to 20～40L/min。
As long as the culture tank used in the present invention has the capacity that can keep desired amount culture fluid and is prevented from the container that microorganism is mixed into from outside。Preferably, it is possible to use there is the culture tank of the structure that the culture medium kept can be heated pressure sterilization, but if keeping the culture tank of culture fluid itself can be sterilized by autoclave etc., then need not be used for the function of pressure sterilization。Additionally, it is preferred that there is the function importing in container by filtrated air, the culture tank of the device that maybe can be additionally implemented for this function。Common small-sized fermentation tank can also be used, but in this case, mechanical agitation function during cultivation, need not be used。The size of the culture tank used in the present invention is suitable for adjusting according to the amount of the liquid strain bacterium produced。In the liquid strain bacterium of the present invention produces, it is possible to any one in cultivating for cultivation continuously or single, it is preferred to single is cultivated, i.e. preferably cultivation carries out the liquid strain bacterium that the per unit of mushroom fruitbody production uses every time。
It addition, when carrying out mass propgation (also referred to as " this cultivation " in present specification) in the production of liquid strain bacterium, it is possible to the mycelium in middle growths such as agar plates is directly used in this cultivation。As preferred embodiment, first pass through use flask etc. and be stirred or vibrate carrying out small-scale preculture to mycelium, then use this pre-culture to carry out this cultivation。In this case, by adopting the liquid strain bacterium production method of the present invention when this cultivation, it is possible to effectively and at a low price produce liquid strain bacterium in a large number。
The inventors discovered that, even if above by the small-scale preculture using the vibration of flask etc. to carry out, by vegetable oil containing low concentration in the fluid medium that uses, compared with the plant oil condition not containing low concentration, it is also possible to more obtain mycelium。Therefore, vegetable oil containing low concentration in the fluid medium used time by making preculture and during this cultivation, it is possible to more effective and produce liquid strain bacterium at a low price in a large number。
The fluid medium used in present specification uses those materials of the liquid culture nutrient source being typically used as mushroom as nutrient source, it is possible to use (such as) fluid medium of the nutrient source that can assimilate containing carbon source, nitrogenous source, inorganic salt etc.。Such as, the carbon sources such as carbohydrate such as glucose, maltose, molasses, dextrin, glycerol, starch can be used, and the nitrogenous source such as peptone, meat extract, cotton seed meal, Semen sojae atricolor powder, yeast extract, casein, Semen Maydis pulp, NZ-amine, ammonium sulfate, ammonium nitrate, ammonium chloride, the fluid medium of above-mentioned mushroom mycelium the inorganic salts such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium phosphate, Sal, calcium carbonate, magnesium sulfate, manganese chloride can also be contained, as long as can be cultivated。
To so-called in present specification " vegetable oil ", there is no particular limitation, as long as being the oil from plant, from the viewpoint of safety and obtain difficulty or ease, it is preferred to edible oil。As edible oil, it is possible to enumerate (such as) Testa oryzae oil, Oleum Brassicae campestris, safflower oil, Semen Maydis oil, olive oil, Oleum sesami, soybean oil, Semen Gossypii wet goods。It addition, with these oil be raw material salad wet goods processing oil can also be used for the present invention。These vegetable oil can be prepared by known method, it is possible to use commercially available product。In the present invention, it is also possible to the one kind or two or more mixing in these vegetable oil is used in the medium。
In the present invention, in culture medium, the content of vegetable oil can be low concentration relative to fluid medium total amount, it is suitably 0.001～0.1 volume % (v/v), being preferably 0.005～0.1 volume % (v/v), more preferably 0.01 to less than 0.05 volume % (v/v)。Utilize the vegetable oil of this low concentration, it is possible to use when not Covering Liguid media surface in liquid culture。
The liquid strain bacterium of the present invention is cultivated and can be undertaken by known method, and cultivation temperature and incubation time can determine aptly in the fertile scope of mushroom mycelium。As an example rather than being particularly limited to the present invention, for instance, it is possible to carry out according to following operation。At 200mLPGY fluid medium [composition glucose 1.49% (w/v), peptone 0.15% (w/v), yeast extract 0.15% (w/v), KH2PO40.0373% (w/v) and MgSO4·7H2O0.0373% (w/v), pH6.0] the middle mycelium inoculating Folium Nelumbinis mushroom, cultivate 14 days at 25 DEG C, prepare pre-culture。Then, in the 430LPGY fluid medium prepared separately be 0.001～0.04 volume % relative to culture medium amount add vegetable oil (such as, salad oil [day clear salad oil, day clear オ イ リ オ グ Le one プ Co., Ltd.'s system]), prepare the fluid medium of this cultivation。This fluid medium is filled in culture tank, at 118 DEG C, carries out 90 minutes autoclavings。Natural cooling utilizes filtrated air to ventilate immediately after starting, by furnishing malleation in culture tank。By this culture medium cool down after, inoculate above-mentioned whole pre-culture, cultivation temperature 25 DEG C, ventilation 0.26vvm, intrinsic pressure 0.075MPa, unpowered agitation (bubbling) condition of culture under, carry out 6～7 days cultivate, produce liquid strain bacterium。
As above the liquid strain bacterium produced is transferred aseptically in suitable container (such as, the round bottle etc. of polypropylene), until picked-up to bacterium bed culture medium this during, it is possible to be maintained at control be 3～-1 DEG C of scope environment under with preserve。
Then, aforesaid liquid kind bacterium is used, it is possible to planted as the bottle of bacterium bed cultivation method by (such as), bag cultivation, case cultivation etc. produce mushroom fruitbody。As an example, the production method of the mushroom fruitbody using liquid strain bacterium of the present invention utilizing bottle to plant is described, the method by the preparation of bacterium bed culture medium, bottling, sterilizing, the inoculation of liquid strain bacterium, bacterium bed cultivate, (the need to, mycelium stimulation), germinate, (if it is required, insert the separation of bud, insert transplanting of bud), growth and breeding, each operation such as results composition。For example, it is possible to be used as with bacterium of sowing by the liquid strain bacterium produced by the method for the present invention: if for Folium Nelumbinis mushroom, then it is used as the kind bacterium during bacterium bed cultivation carried out described in Japanese Unexamined Patent Publication 04-211308 publication；If being Lentinus Edodes, then it is used as the kind bacterium during bacterium bed cultivation carried out described in Japanese Unexamined Patent Publication 04-075538 publication；If being Hypsizygus marmoreus, then it is used as the kind bacterium during bacterium bed cultivation carried out described in Japanese Unexamined Patent Publication 05-268942 publication；Or, if for this occupation of land mushroom, then it is used as the kind bacterium during bacterium bed cultivation carried out described in Japanese Unexamined Patent Publication 2000-106752 publication。If it addition, for carrying out inserting the situation of the bacterium bed cultivation of the transplanting of the separation of bud and slotting bud, then the liquid strain bacterium produced by the method for the present invention can also be used as the kind bacterium during bacterium bed cultivation carried out described in Japanese Unexamined Patent Publication 2009-017872 publication。
But, with use solid kind bacterium time bacterium bed cultivation culture medium or normally used bacterium bed cultivation culture medium compared with, the bacterium bed cultivation culture medium used in the production method of the mushroom fruitbody of the present invention preferably makes bacterium seat improve, it is appropriate that make bacterium seat be positioned at the lower section of 2～10mm lower than bottleneck。Additionally, it is preferred that bacterium seating face is planarized equably。And then, the cave (also referred to as hole or inoculation hole) of bottle central part is not especially desirable, the bacterium bed cultivation culture medium that (that is, the periphery of bottle central part) near the bottle deep pool of each bottle opens the vesicle (also referred to as hole or inoculation hole) of 2～6 places, preferably 4 place bore about 3～10mm, preferred about 5mm, the degree of depth about 100～200mm, preferably about 110～150mm can be used in。
At this, in present specification, so-called " bacterium seat " refers to the bacterium inoculation position of cultivation culture medium upper face。Such as, in bottle cultivation culture medium, the media surface exposed of bottle peristome is called bacterium seat。In the present invention, preferably, with use solid kind bacterium time bacterium bed cultivation by culture medium (also referred to as bacterium bed cultivation solid medium, bacterium bed cultivation culture medium) compare, bacterium seat is made to improve (namely distance bottleneck is near), such as, in the situation (using the situation of solid kind bacterium as shown in Figure 1) that bottle is planted, it is recommended that, bottleneck (bottle deep pool) the 15mm place, lower section that common bottle is planted commercially available 850mL bottle or the 1100mL bottle used is set to bacterium seat, but in the present invention, preferably, compared with the bacterium seat of bacterium bed cultivation culture medium during with use solid kind bacterium, bacterium seat is made to improve, preferably will be located in 2～10mm place below bottleneck, it is preferred that 5～9mm place is set to bacterium seat below bottleneck。
When being seeded in bacterium bed cultivation culture medium by the liquid strain bacterium that the method by inventing produces, for the wide-mouth culture bottle of every bottle of 850mL, sterilely transplant (such as) about 10～20mL。Method as inoculation aforesaid liquid kind bacterium, it is not necessary to use special machinery to inoculate equably on bacterium seat and inoculation hole with spray form, only directly the liquid strain bacterium of necessary amounts is sterilely added on bacterium seat。Position on the bacterium seat of adding liquid kind bacterium is preferably placed near the central part of bacterium seat。
Compared with the solid kind bacterium of the commodity production for extensive mushroom fruitbody, in accordance with the invention it is possible to produce the liquid strain bacterium of the commodity production for mushroom fruitbody in very short time。Such as, when producing above-mentioned solid kind bacterium, by the liquid mycelia after preculture or solid mycelium inoculation in solid medium。Then, mycelia is made to spread about 30～60 days in solid medium。The solid medium having mycelia is spread as solid kind bacterium, for the commodity production of mushroom fruitbody using what obtain。On the other hand, when obtaining liquid strain bacterium by the inventive method, by the liquid mycelium inoculation after preculture in the fluid medium of the present invention。Then, by carrying out the cultivation of about 3～7 days, preferably 6～7 days, such that it is able to produce liquid strain bacterium, and may be used for the commodity production of mushroom fruitbody。
Additionally, the liquid strain bacterium produced by the inventive method is used for the commodity production of large-scale mushroom fruitbody, production difference when the mushroom fruitbody caused by batch difference of solid kind bacterium thus can be avoided to produce, and can stably produce excellent mushroom fruitbody。
Bottle cultivating method is illustrated by example listed above, but the liquid strain bacterium produced by the method for the present invention is not limited to the use in the bacterium bed cultivation utilizing above-mentioned bottle to plant。
[embodiment]
By the examples below the present invention is more particularly described, but the present invention is not limited to the scope of following example。
Embodiment 1
At 200mLPGY fluid medium [composition: glucose 1.49% (w/v), peptone 0.15% (w/v), yeast extract 0.15% (w/v), KH2PO40.0373% (w/v) and MgSO4·7H2O0.0373% (w/v), pH6.0] the middle mycelium inoculating LyophyllumdecastesK-3304 (FERMBP-4348), at 25 DEG C, carry out 14 days shaken cultivation (100rpm), prepare pre-culture。
Then, in the 430LPGY fluid medium prepared separately, using be about 0.02% (v/v) relative to culture medium ratio add salad oil (the day clear salad oil as vegetable oil, day is オ イ リ オ グ Le one プ Co., Ltd. system clearly), prepare the fluid medium of this cultivation。Culture tank uses the non-first kind pressure vessel not having stirring capacity。The culture tank being added with this cultivation fluid medium carries out high steam sterilization in 90 minutes at 118 DEG C, and natural cooling carries out utilizing the ventilation of filtrated air immediately after starting, by furnishing malleation in culture tank。By this culture medium cool down after, inoculate above-mentioned whole pre-culture, cultivation temperature 25 DEG C, ventilation 0.26vvm, intrinsic pressure 0.075MPa, unpowered agitation (bubbling) condition of culture under, carry out 6～7 days cultivate, prepare liquid strain bacterium。
Meanwhile, carry out unpowered agitation (bubbling) merely with PGY fluid medium and cultivate, as comparative control。Other condition of culture is identical with above-mentioned condition, compares the harvest yield of thalline。The results are shown in table 1。
[table 1]
| Condition | Dry thalline weight (g/L) |
| There is salad oil | 4.1 |
| Without salad oil | 3.0 |
Table 1 shows, use the present invention the culture medium being added with salad oil, use in the cultivation that carries out without churned mechanically bubble, compared with situation when not adding, it is possible to obtain the thalline of about many 1.36 times。
Embodiment 2
The Ability to form fruitbody of the liquid strain bacterium that the method in order to be identified through the present invention produces, carries out following sporophore and produces。
Liquid strain bacterium is prepared by method similarly to Example 1。As comparison other, solid kind bacterium is carried out as follows preparation。First, the wide-mouth culture bottle (850mL, Nong Cai Co., Ltd. of SHIN-ETSU HANTOTAI system) of polypropylene adds 100g sawdust (China fir material), 86g Testa oryzae, 2g Magnesiumaluminumsilicate (Fuji Chemical Industry Co., Ltd's system, trade name NeusilinFH1), 5g calcium carbonate (NacalaiTesque Co., Ltd. system, extra pure reagent), 3g citric acid monohydrate (NacalaiTesque Co., Ltd. system, extra pure reagent), and moisture is set as 63～65 weight %, being sufficiently mixed, compacting becomes the material of moisture state。Central authorities on compact surface open the inoculation hole portion of diameter about 1cm, after cover lid, carry out high steam sterilization in 90 minutes, naturally cool to 20 DEG C at 118 DEG C。To pre-culture described in the embodiment 1 of the solid medium inoculation about 10～20mL so obtained。Then, in the dark, when temperature 25 DEG C, humidity 55%, cultured mycelia about 60 days, makes mycelia spread to solid medium entirety, prepares solid kind bacterium。
Then, the wide-mouth culture bottle (1100mL, hillside plot Industry Co., Ltd system) of polypropylene adds 134g sawdust (China fir material), 130g Testa oryzae, 2.6g Magnesiumaluminumsilicate [Fuji Chemical Industry Co., Ltd's system, trade name NeusilinFH1], 6.5g calcium carbonate (NacalaiTesque Co., Ltd. system, extra pure reagent), 3.9g citric acid monohydrate (NacalaiTesque Co., Ltd. system, extra pure reagent), moisture is set as 63%～65 weight %, it is sufficiently mixed, bacterium seat is set in the lower section being about 15mm from bottleneck, and compacting becomes the material of moisture state。Bottle central authorities on compact surface open the inoculation hole portion of diameter about 2cm, after cover lid, carry out the sterilization of 90 minutes high steams at 118 DEG C, using the culture medium that naturally cools to 20 DEG C as bacterium bed cultivation culture medium。This bacterium bed cultivation culture medium is inoculated the liquid strain bacterium of the above-mentioned preparation of about 20mL。As comparison other, prepare to be inoculated with the culture medium of the solid kind bacterium of the above-mentioned preparation of about 35g。Each making 16 bottles is inoculated with the bacterium bed cultivation culture medium of liquid strain bacterium and solid kind bacterium respectively。By them in the dark, cultured mycelia about 60 days under when temperature 25 DEG C, humidity 55%, make mycelia spread to bacterium bed cultivation culture medium overall。Then, taking off lid, carry out the mycelium stimulation on bacterium bed surface top, then, add tap water to bottleneck, draining immediately, for germination operation。
Germination operation carries out under the following conditions in growing floor: illumination 50 lux, temperature 16 DEG C, humid control in 115%～120% scope of the indicating value as ヒ ユ mono-ミ ア イ 100 (aigret makes made), gas concentration lwevel control the scope at 1000～1500ppm。It addition, in order to avoid condensation, bottle is inverted, continues to cultivate 11 days, form fruit body primordium。Then, bottle reversion is just put, is transitioned into early growth operation。In early growth operation, cultivate under the following conditions in growth room 6 days: illumination 500 lux, temperature 16 DEG C, humid control in 115%～120% scope of the indicating value as ヒ ユ mono-ミ ア イ 100, gas concentration lwevel control the scope at 1000～2000ppm。Then, late growing stage operation it is transitioned into。In late growing stage operation, in growth room under the following conditions continue cultivate 7 days: illumination 500 lux, temperature 16 DEG C, humid control in 95～105% scopes of the indicating value as ヒ ユ mono-ア イ 100, gas concentration lwevel control the scope at 1000～2000ppm。After the mature sporophore that results so obtain, compare the average harvest yield (g/ bottle) of sporophore。The results are shown in table 2。
[table 2]
| Kind | Average harvest yield (g) |
| Solid kind bacterium | 226 |
| Liquid strain bacterium | 220 |
Table 2 shows, the liquid strain bacterium produced by the method for the present invention can be obtained and the harvest yield using the sporophore harvest yield during solid kind bacterium being used as generally to plant bacterium almost identical in the sporophore production of Folium Nelumbinis mushroom, therefore, the liquid strain bacterium produced by the method for the present invention has Ability to form fruitbody, furthermore it is possible to produce suitable mushroom fruitbody。
Embodiment 3
Just use the mushroom fruitbody production of the liquid strain bacterium produced by the inventive method, bacterium bed cultivation culture medium has been studied。
First, liquid strain bacterium is prepared by method similarly to Example 1。Prepare the bacterium bed cultivation culture medium (usual bacterium seat bacterium bed cultivation culture medium) described in 16 bottles of embodiments 2；And prepare 16 bottles of such bacterium bed cultivation culture medium (high bacterium seat bacterium bed cultivation culture medium): its ratio bacterium seat height of the bacterium bed cultivation culture medium described in embodiment 2, bacterium seat is set in about about 5～9mm place below bottleneck, compacting, and then make bacterium bed surface smooth。Use each bacterium bed cultivation culture medium, in addition, produce mushroom fruitbody by method similarly to Example 2。The generation rate (% is figured out by the bottle producing sporophore) of relatively more obtained sporophore and average harvest yield (g/ bottle)。It addition, also the situation occurred of obtained sporophore is observed simultaneously。The results are shown in table 3。
[table 3]
| Condition | Generation rate (%) | Average harvest yield (g) | Situation occurred |
| Usual bacterium seat bacterium bed cultivation culture medium | 81 | 220 | Slightly worse 7--> |
| High bacterium seat bacterium bed cultivation culture medium | 100 | 265 | Well |
Table 3 shows, in the production of the mushroom fruitbody of the liquid strain bacterium that use is produced by the inventive method, use high bacterium seat bacterium bed cultivation culture medium, thus, compared with the situation using usual bacterium seat bacterium bed cultivation culture medium, it is possible to effectively produce better mushroom fruitbody。
Embodiment 4
Just use solid kind bacterium and the production of the mushroom fruitbody of liquid strain bacterium produced by the inventive method, carry out the comparison of production inhomogeneities。
First, prepare solid kind bacterium by method similarly to Example 2, prepare liquid strain bacterium by method similarly to Example 1。Then, prepare respectively 10,000 bottles as solid kind bacterium inoculation embodiment 2 described in bacterium bed cultivation culture medium and as liquid strain bacterium inoculation embodiment 3 described in high bacterium seat bacterium bed cultivation culture medium。Then, solid kind bacterium or liquid strain bacterium are inoculated in respective bacterium bed cultivation culture medium, carry out the production of mushroom fruitbody according to method similarly to Example 2。In units of bottle, the defective work in when calculating the mycelium stimulation in this production process, germination and grow, calculate disqualification rate (%)。The results are shown in table 4。It addition, defective work during mycelium stimulation is bacterium circulates incomplete product。Defective work during germination is the product not germinateed, the product germinateing uneven product or bud number rareness。Defective work during growth confirms afterwards in early growth operation (that is, germinating growth operation) on the 17th day, confirms product that the growth of mushroom fruitbody stops, the product of shape defect or does not grow into the product of uniform strain shape。It addition, underproof product abandonment will be become in each operation, follow-up operation all uses in units of bottle certified products。
[table 4]
By table 4 it can be shown that compared with the production using solid kind bacterium, the production inhomogeneities that the mushroom fruitbody of the liquid strain bacterium that use is produced by the inventive method produces is little, it is useful in large-scale business is cultivated。
Embodiment 5
At 200mLPGY fluid medium [composition: glucose 2.0% (w/v), peptone 0.2% (w/v), yeast extract 0.2% (w/v), KH2PO40.05% (w/v), MgSO4·7H2O0.05% (w/v)] the middle mycelium inoculating Pleurotuscystidiosus.abalonusK-4988 (FERMP-22066), at 25 DEG C, carry out 14 days shaken cultivation (90rpm), prepare pre-culture。
Then, in the 60LPGY fluid medium prepared separately, with be about 0.1% (v/v) relative to culture medium ratio add Testa oryzae oil (trade name: シ ヤ リ this こ め oil, ジ ヤ パ Application ラ イ ス Co., Ltd. system), prepare the fluid medium of this cultivation。Culture tank uses the non-first kind pressure vessel not having stirring capacity。The culture tank being added with the fluid medium of this cultivation carries out high steam sterilization in 30 minutes at 118 DEG C, and natural cooling carries out utilizing the ventilation of filtrated air immediately after starting, by furnishing malleation in culture tank。By this culture medium cool down after, inoculate above-mentioned whole pre-culture, cultivation temperature 27 DEG C, ventilation 0.35vvm, unpowered agitation (bubbling) condition of culture under, carry out 8 days cultivate, prepare liquid strain bacterium。
Meanwhile, carry out unpowered agitation (bubbling) merely with PGY fluid medium and cultivate, as comparative control。From cultivating the 5th day, sampling every day, compares the harvest yield of thalline。Result is shown in table 5。
[table 5]
By table 5 it can be shown that in the cultivation using the use of fluid medium being added with Testa oryzae oil to carry out without churned mechanically bubble, compared with not adding Testa oryzae oil condition, it is possible to obtain more thalline。Especially show, cultivating the 8th day, compared with not adding Testa oryzae oil condition, with the addition of the fluid medium of Testa oryzae oil and can obtain the thalline of about 2.28 times。
Embodiment 6
In the PGY fluid medium that 4L is identical with embodiment 5, with ratio interpolation Semen Maydis oil (the AJINOMOTO plumule favour body U one Application oil being about 0.1% (v/v) relative to culture medium, J-オ イ Le ミ Le ズ Co., Ltd. system), prepare the fluid medium of this cultivation。Culture tank employs microbial cultivation device (BMS-05PI, エ イ ブル Co., Ltd. system)。The culture tank being added with the fluid medium of this cultivation is carried out at 121 DEG C high steam sterilization in 15 minutes, after being cooled to room temperature, the mycelium of inoculation Pleurotuscystidiosus.abalonusK-4988 (FERMP-22066), cultivation temperature 27 DEG C, ventilation 0.35vvm, unpowered agitation (bubbling) condition of culture under, carry out 8 days cultivating, prepare liquid strain bacterium。
Meanwhile, carry out unpowered agitation (bubbling) merely with PGY fluid medium and cultivate, as comparative control。From cultivating the 5th day, sampling every day, compares the harvest yield of thalline。Result is shown in table 6。
[table 6]
By table 6 it can be shown that in the cultivation using the use of fluid medium being added with Semen Maydis oil to carry out without churned mechanically bubble, compared with being not added with Semen Maydis oil condition, it is possible to obtain more thalline。Especially show, cultivating the 8th day, compared with not adding Semen Maydis oil condition, with the addition of the fluid medium of Semen Maydis oil and can obtain the thalline of about 15.4 times。
Embodiment 7
In the PGY fluid medium that 200mL is identical with embodiment 5, respectively be about 0.01% (v/v) relative to culture medium, the ratio of about 0.025% (v/v), about 0.05% (v/v), about 0.075% (v/v), about 0.1% (v/v) add Testa oryzae oil (trade name: シ ヤ リ this こ め oil, ジ ヤ パ Application ラ イ ス Co., Ltd. system) or Semen Maydis oil (AJINOMOTO plumule favour body U one Application oil, J-オ イ Le ミ Le ズ Co., Ltd. system), prepare fluid medium。At the mycelium of this inoculation of medium Pleurotuscystidiosus.abalonusK-4988 (FERMP-22066), at 25 DEG C, shaken cultivation (90rpm) 14 days, prepare liquid strain bacterium。By Testa oryzae oil or Semen Maydis oil, the result of the thalline harvest yield under the different content relative to fluid medium is shown in table 7。
[table 7]
By table 7 it can be shown that be added with in the shaken cultivation of a small amount of fluid medium of Testa oryzae oil or Semen Maydis oil in use, compared with not adding Testa oryzae oil or Semen Maydis oil condition, it is possible to the thalline obtained is more。
Embodiment 8
In the PGY fluid medium that 200mL is identical with embodiment 5, respectively be about 0.01% (v/v) relative to culture medium, the ratio of about 0.025% (v/v), about 0.05% (v/v), about 0.075% (v/v) add Testa oryzae oil (trade name: シ ヤ リ this こ め oil, ジ ヤ パ Application ラ イ ス Co., Ltd. system), prepare fluid medium。At this inoculation of medium by fine and soft (Grifolafrondosa (Fr.) S.F.Gray of commercially available dance, snow state ま い け Co., Ltd. system) according to the obtained mycelium of known method, at 25 DEG C, shaken cultivation (90rpm) 14 days, prepare liquid strain bacterium。The result of the thalline harvest yield under the Testa oryzae oil different content relative to fluid medium is shown in table 8。
[table 8]
| Testa oryzae oil concentration | Dry thalline weight (g/L) |
| 0% | 1.60 |
| 0.01% | 1.65 |
| 0.025% | 1.80 |
| 0.05% | 2.20 |
| 0.075% | 2.30 |
By table 8 it can be shown that be added with in the shaken cultivation of a small amount of fluid medium of Testa oryzae oil in use, compared with not adding Testa oryzae oil condition, it is possible to the thalline obtained is more。
Embodiment 9
At 200mLSGY fluid medium [composition: glucose 2.0% (w/v), soy peptone 0.2% (w/v), yeast extract 0.2% (w/v), KH2PO40.05% (w/v), MgSO4·7H2O0.05% (w/v)] in, respectively with relative to culture medium for about 0.01% (v/v), about 0.025% (v/v), the ratio of about 0.05% (v/v) adds salad oil (day clear salad oil, day is オ イ リ オ グ Le one プ Co., Ltd. system clearly), prepare fluid medium, and respectively with relative to culture medium for about 0.01% (v/v), about 0.025% (v/v), about 0.05% (v/v), about 0.075% (v/v), the ratio of about 0.1% (v/v) adds Testa oryzae oil (trade name: シ ヤ リ this こ め oil, ジ ヤ パ Application ラ イ ス Co., Ltd. system), prepare fluid medium。At the mycelium of this inoculation of medium LyophyllumulmariumLu1-181 (FERMBP-8357), at 25 DEG C, shaken cultivation (90rpm) 10 days, prepare liquid strain bacterium。The result of the thalline harvest yield under the salad oil different content relative to fluid medium is shown in table 9。
[table 9]
By table 9 it can be shown that be added with in the shaken cultivation of a small amount of fluid medium of salad oil or Testa oryzae oil in use, compared with not adding salad oil or Testa oryzae oil condition, it is possible to the thalline obtained is more。
Being described in detail by the present invention with reference to specific embodiment, but can increase various change and amendment without departing from the spirit and scope of the present invention, this will be apparent to those skilled in the art。
The Japanese patent application (Japanese Patent Application 2010-179999) that the application submitted to based on August 11st, 2010, its content is introduced into as reference herein。
Industrial applicibility
According to the present invention it is possible to provide the production method of a kind of liquid strain bacterium inexpensively and effectively with mushroom fruitbody Forming ability。And then by using this liquid strain bacterium, the situation planting the bacterium production time needing 30～60 days during relative to generally use solid kind bacterium, it is possible to significantly shorten kind of a bacterium production time；Furthermore it is possible to the risk of production inhomogeneities when avoiding the mushroom fruitbody using solid kind bacterium to produce, and can stably produce excellent mushroom fruitbody, therefore, it is useful in the large-scale commercial applications cultivation of mushroom fruitbody。
Claims (3)
1. the production method of a mushroom fruitbody, it is in the fluid medium of vegetable oil containing concentration being 0.001～0.1 volume %, by using bubble agitation culture medium that the mycelium of mushroom is carried out the cultivation of liquid deep, and use the production method of the liquid strain bacterium of obtained mushroom, it is characterized in that, bacterium seat improved and bacterium seating face is planarized equably, and not needing the inoculation hole of bottle central part and described liquid strain bacterium is sterilely implanted on bacterium seating face。
2. production method as claimed in claim 1, wherein, substantially only uses bubble that culture medium is stirred。
3. production method as claimed in claim 1 or 2, wherein, described vegetable oil is one or more in salad oil, Testa oryzae oil, Oleum Brassicae campestris, safflower oil, Semen Maydis oil, olive oil, Oleum sesami, soybean oil and Oleum Gossypii semen。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
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| JP2010179999 | 2010-08-11 | ||
| JP2010-179999 | 2010-08-11 |
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| KR (1) | KR20120024421A (en) |
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| CN103952367B (en) * | 2013-04-16 | 2016-06-01 | 中国人民解放军军事医学科学院基础医学研究所 | A kind of fermention medium and utilize the method for its production melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA |
| KR101616887B1 (en) * | 2014-06-05 | 2016-04-29 | 전라북도 | A artificial culture method for mass producing mycelium of morchella esculenta |
| CN104350948A (en) * | 2014-11-14 | 2015-02-18 | 张家港市大新利群农民专业合作社 | Method for culturing mushroom cultivated species |
| CN105272542A (en) * | 2015-09-30 | 2016-01-27 | 山东晨阳菌业有限公司 | Preparation method of medium for liquid culture of Hypsizygus marmoreus |
| CN108676729B (en) * | 2018-05-28 | 2022-05-03 | 杭州雪域生物技术有限公司 | Culture medium composition containing fermentation waste liquid, and preparation method and fermentation method thereof |
| CN112772295A (en) * | 2021-01-29 | 2021-05-11 | 贵州省生物研究所 | Preparation method of morchella liquid strain and improved cultivar |
| CN115413530A (en) * | 2022-10-08 | 2022-12-02 | 福建农林大学 | Liquid strain culture medium for velvet antler mushroom and method for cultivating velvet antler mushroom by using culture medium |
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| CN1338200A (en) * | 2000-08-11 | 2002-03-06 | 株式会社世界 | Preparation of liquid fungus culture and culturing apparatus |
| CN101194569A (en) * | 2007-10-15 | 2008-06-11 | 冯伟 | Method and device for culturing liquid cultivation seed of mushroom for eating and medicine |
| CN101690453A (en) * | 2007-11-15 | 2010-04-07 | 宝生物工程株式会社 | Hon-shimeji mushroom-fungal bed culture |
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| JPS55118389A (en) * | 1978-12-27 | 1980-09-11 | Takeda Chem Ind Ltd | Incubation of mycorrhiza-forming fungi |
| JPS606127A (en) * | 1983-06-22 | 1985-01-12 | 株式会社幸茸園 | Auxiliary tool for opening part of mushroom culture bag |
| JPS62171618A (en) * | 1986-01-22 | 1987-07-28 | バブコツク日立株式会社 | Artificial culture method for mushroom |
| JP4341759B2 (en) * | 1999-05-27 | 2009-10-07 | 協全商事株式会社 | Mushroom cultivation method |
| JP2002159218A (en) * | 2000-11-28 | 2002-06-04 | Nagano Kida Kogyo Kk | Cap for mushroom cultivation and method for cultivating mushroom using the same |
| JP4054584B2 (en) * | 2002-02-15 | 2008-02-27 | スン、ジャ−モ | Liquid inoculum culture device for mass production of Cordyceps fruit body |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1338200A (en) * | 2000-08-11 | 2002-03-06 | 株式会社世界 | Preparation of liquid fungus culture and culturing apparatus |
| CN101194569A (en) * | 2007-10-15 | 2008-06-11 | 冯伟 | Method and device for culturing liquid cultivation seed of mushroom for eating and medicine |
| CN101690453A (en) * | 2007-11-15 | 2010-04-07 | 宝生物工程株式会社 | Hon-shimeji mushroom-fungal bed culture |
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