201210465 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種用於菇類子實體之製造之菇類液體種 菌之製造方法、使用該液體種菌之菇類子實體之製造方 法。 【先前技術】 近年來，業界正開發菇類之人工栽培技術，提供各種菇 類子實體（真姬離褶傘（Lyophyllum shimeji)、荷葉離褶傘 (Lyophyllum decastes)、缚喜兹（Hypsizygus marmoreus)、 杏鲍菇（Pleurotus eryngii)、香菇（lentinus edodes)、金針菇 (flammulina velutipes)等）。 通常’菇類之人工栽培係藉由將菇類之種菌接種於搰林 或固體培養基，經過培養、生長等步驟，並收穫子實體而 進行。於菇類之種菌中存在使用鋸屬等培養基之固體種菌 與使用液體培養基之液體種菌。近年來，業界正推進以大 型設備之工廠系統實施菇類之人工栽培等機械化。然而， 為了商業性地大量且穩定地製造菇類子實體，必需大量地 培養所使用之種菌。先前，於菇類之種菌中就保存性良 好’可容易地輸送等原因而言使用固體種菌，但就如下方 面而言’認為液體種菌有利，而增加其使用：可避免由固 體種菌之批量差引起之菇類子實體製造時之製造不均之風 險，或因接種固體種菌時溢出之種菌而產生之瓶或容器之 污染而引起之嚴重之設備之害菌.害蟲污染等，及可大幅 度縮短種菌之製造時間，且規模擴大較容易等。作為用以 157547.doc 201210465 $量地培養液體種菌之方法，討論有作為心自菌絲體獲 得有用成分之大量培養方法的液體深部培養法，為了嚴密 地控制培養條件等，通常使用藉由進行機械攪拌;;通氣、 溫度管理之缸式礙酵槽Gar fermenter)之通氣攪拌方法（例 如非專利文獻υ。然而，為了以商業規模製造兹類子實 體，必需包括大容量之缸式醱酵槽之高價且大規模之培養 設備之設置。 [先前技術文獻] [非專利文獻] 非專利文獻1 :醱酵協會志，第24卷，第7號，293_3〇4 頁，1966年 【發明内容】 [發明所欲解決之問題] 鑒於上述現狀，本發明之目的在於提供一種菇類子實體 之大規模之商業製造中之廉價且高效之液體種菌之製造方 法、使用該液體種菌之菇類子實體之製造方法。 [解決問題之技術手段] 本發明者等人對用於大規模之商業製造的菇類之液體種 菌之大量製造方法進行努力研究，結果驚奇地發現，於攪 拌中使用氣泡之液體深部培養（無動力攪拌：氣泡（Air bubbling))意外地對用以製造菇類之子實體之液體種菌製 造有用。又’發現於該培養中，藉由使液體培養基中含有 低濃度之植物油’可於具有菇類之子實體形成能力之狀態 下尚效且穩定地大量製造菌絲體。進而，藉由使迄今為止 157547.doc 201210465 用於固體種菌之菌床栽培用培養基之菌座升高，並且使菌 座表面均勻地平面化，可實現更為穩定之發菌，並高效地 進行利用搔菌之產生誘導，可與使用固體種菌之情形相 比，製造包括種菌製造時間更短時間且均勻、商品價值更 高之優良之菇類子實體’從而完成本發明。 若概述本發明，本發明係關於： Π]種益類之液體種菌之製造方法，其特徵在於：其係用 以製造菇類子實體者，並且於含有植物油之液體培養基 中，藉由使用氣泡攪拌培養基而進行菇類之菌絲體之液體 深部培養； [2]如[1]之製造方法，其實質上僅使用氣泡而進行培養基 之攪拌； [3] 如[1]或[2]之製造方法’其中植物油為選自沙拉油、米 糠油、菜籽油、紅花油、玉米油、撖欖油、芝麻油、大豆 油及棉籽油中之一種以上； [4] 如[1]至[3]中任一項之製造方法，其中培養基中之植物 油濃度為0.001〜0.1體積％ ; [5] —種菇類子實體之製造方法，其特徵在於：其使用藉由 U]至[4]中任一項之製造方法而獲得之菇類之液體種菌； [6] 如[5]之菇類子實體之製造方法，其為瓶栽培； [7] 如[5]或[6]之菇類子實體之製造方法，其使用升高菌座 之菌床栽培用培養基； [8] 如[7]之菇類子實體之製造方法’其使用使菌座表面均 勻地平面化之菌床栽培用培養基。 157547.doc 201210465 [發明之效果] 根據本發明，提供一種用於菇類子實體之大規模之商業 製造，短時間且廉價之菇類之液體種菌之大量製造方法。 又，亦提供一種使用該液體種菌之菇類子實體之製造方法。 【實施方式】 可用於本發明之「菇類」並無特別限定，可列舉：荷葉 離褶傘、真姬離稍傘、轉喜益、平益（Pleurotus ostreatus))、香兹、金針益、滑兹（Pholiota nameko)、平 蒜、舞兹（Grifola frondosa)、杏鮑兹、巴西磨兹（Agaricus blazei Murill)、大平兹、鲍魚益（Pleurotus cystidiosus)等 食用菇類。作為該等菇類之菌株，可為市售之菌株，或可 為自野生之子實體之組織分離株，亦可為藉由選拔、交 配、細胞融合、基因重組等方法而育種之株，只要為可作 為液體種菌培養且可製造子實體之菌株即可。作為本發明 之較佳之一態樣，可列舉荷葉離梢傘（Lyophyllum decastes)。作為荷葉離褶伞，例示有公知之菌株，例如： 荷葉離褶傘K-3303株（FERM BP-4347)、荷葉離褶傘K-3304 株（FERM BP-4348)、荷葉離褶傘 K-3305 株（FERM BP-4349) 、 荷葉 離褶傘F-623 株 （FERM P-13165) 、 荷葉 離褶傘 F-1154 株（FERM P-13166)、荷葉離褶傘F-1488 株（FERM P-13167)及適於子實體之製造之該等之變異株等。 又’作為本發明較佳之真姬離指傘（Lyophyllum shimeji) 之菌株之例，例示有真姬離褶傘La 01-27(FERM BP-10960)、 真姬離褶傘 La 01-20(FERM BP-10959)、 真姬離褶 157547.doc 201210465 傘 La 01-37(FERM P-17456)、真姬離褶伞 La 01-45(FERM P-17457)、真姬離褶傘 La 01-46(FERM P-17458)及適於子 實體之製造之該等之變異株等》 進而，作為本發明較佳之鴻喜菇之菌株，有作為榆生離 糟伞（Lyophyllum ulmarium)而表示之菌株、作為鴻喜益 (Hypsizygus marmoreus)而表示之菌株等，例如例示有： 榆生離褶傘M-8171(FERM BP-1415)、榆生離褶傘K-0259(FERM P-12981)、榆生離褶傘Lul-172 株（FERM BP-8354) 、 榆生 離褶傘Lul-173 株（FERM BP-8355) 、 榆生離褶 傘 Lul-174 株（FERM BP-8356)、榆生離褶傘 Lul-181 株 (FERM BP-8357)、鴻喜菇 K-4975(FERM BP-11321)、鴻喜 蒜 K-4979(FERM BP-11322)、鴻喜菇 K-4980(FERM BP-11323)、 鴻喜菇 K-4981(FERM BP-11324) 及適合製造子實 體之該等之變異株等。 進而，作為本發明較佳之鲍魚菇之菌株，例示有：鮑魚 兹臺灣亞種（Pleurotus cystidiosus subsp. abalones)K-4986(FERM P-22064)、鮑魚菇臺灣亞種K-4987(FERM Ρ· 22065)、鮑魚菇臺灣亞種K-4988(FERM P-22066)及適於子 實體之製造之該等之變異株等。 只要為可人工栽培之菌株且可應用本發明之菌株，則上 述菌株並無任何限制。 於本案說明書中，所謂「菌絲體」，係指上述菇類之菌 絲體，只要可於液體培養基中培養，且可用作用以製造菇 類之子實體之液體種菌，則並無特別限定。 157547.doc 201210465 於本案說明書中，所謂「使用氣泡之液體深部培養」， 係指藉由於培養槽内產生之氣泡而產生培養液之運動（流 動），並實施培養基之㈣的培養方法。較佳為僅實質地 使用氣泡而進行培養基之㈣之培養方法，只要為不產生 菌絲之切斷之程度，則亦可包括利用攪拌器、如螺旋槳形 狀之扇般之裝置之機械授拌。尤佳為不伴隨機械搜拌而僅 使用氣泡來進行培養基之_之培養方法。上述培養方法 所使用之培養槽中無需用以㈣或振動之使用機械動力之 裝置’只要具備使氣泡產生之裝置即可。於本發明中，例 如可使用日本應用軸學會誌，第8卷，第丨號，^七頁 (膽)所記載之氣泡塔型培養裝置等，並可使用附有如可 自底面及/或側面無菌地產生氣泡般之裝置的培養槽。 又’亦可將可產生氣泡之管等直接放入培養槽中進行。作 為培養槽中之氣泡之通氣量，較佳為G.2〜0.8 vvm(volume per vohnne Per minute:每單位體積之氣體通氣量），適宜 為0.2〜0.6 vvm，或亦可為1()〜5〇升/min(表示於卜分鐘内將 10 50升之無菌空氣向25〜83 L之液體培養基中通氣之 量），適宜為20〜40升/min。 本發明所使用之培養槽只要為具有可保持所期望之量之 培養液之谷量’且可防止自外部之微生物之混人的容器即 可。較佳使用有可對所保持之培養基加熱加壓殺菌之結構 之培養槽，但只要可將保持培養液之培養槽本身利用高壓 釜等殺菌，則不需要用以加壓殺菌之功能。又，較佳為具 有將製成無菌之空氣導入容器内之功能，或可附加用於此 157547. doc 201210465 之褒置者。亦可使用通常之缸式醋酵槽，但於該情形時， 則無需於培養時使用機械攪拌功能。本發明所使用之培養 槽之大小只要根據所製造之液體種菌之量而適當調整即 可。於本發明之液體種菌之製造中，進行連續或單次培養 之任一者均可，適宜為單次培養，即較佳為每次培養每個 進行菇類子貫體製造之單元所使用之液體種菌。 再者，於液體種菌之製造中進行大量培養（於本說明書 中，亦稱為主培養）之情形時，亦可將於瓊脂平板等生長 之菌絲體直接用於主培養。作為較佳之態樣，首先，進行 使用燒瓶等之利用g絲體之髮或振動之小規模下之預培 養，使用該預培養物進行主培養。於該情形時，藉由於主 培養時使用本發明之液體種菌之製造方法’可高效、廉價 且大量地製造液體種菌。 本發明者等人發現：於上述使用燒瓶等之利用震動之小 規模下之預培養中’藉由使所使用之液體培養基中含有低 濃度之植物油，與不含有低濃度之植物油情形相比亦可 獲付較多g絲體。因此’藉由使預培養時及主培養時所使 用之液體培養基中含有低濃度之植物油，可進而高效、廉 價且大量地製造液體種菌。 於本案說明書中，所使用之液體培養基可使用通常用作 兹類之液體培養之營養源者作為營養源，例如含有碳源、 氣源、無機鹽等可同化之營養源之液體培養基。例如可使 用：葡萄糖、麥芽糖、糖漿、糊精、甘油、澱粉等碳水化 合物等碳源，及蛋白臊、肉彳、棉籽粉、大豆粉、酵母萃 157547.doc 201210465 取物、路蛋白、玉米浸液（CC)rn steep叫㈣、胺硫 酸錄、硝酸錄、氣化錄等氮源，進而含有鱗酸二氣卸、^ 酸氩二鉀、碌酸鈉、食鹽、碳_、硫酸鎮、氣化般等無 機鹽’只要為可培養上㈣類之g絲體之液體培#基即可。 於本案說明書中’所謂「植物油」，只要為來源於植物 之油則並無特別限定，就安全性及獲取容易性之觀點而言 較佳為食用油。作為食用油，例如可列舉米糠油、菜籽 油、紅花油、玉米油、橄欖油、芝麻油、大豆油、棉籽油 等。又，以該等油作為原料之沙拉油等加工油亦可用於本 發明》該等植物油可利用公知之方法製備，亦可使用市售 品。於本發明中，亦可將該等植物油之一種或兩種以上於 培養基中混合而使用。 本發明中之培養基中之植物油之含有量可相對於液體培 養基總量為低濃度，適宜為0·001〜0 i體積％(v/v)、較佳為 未達0.005〜0· 1體積％(v/v)、進而較佳為未達〇 〇i〜〇 〇5體積 /°(v/v)。藉由該低濃度之植物油，可於液體培養中不覆蓋 液體培養基表面而使用。 本發明之液體種菌之培養可藉由公知之方法而進行，培 養溫度或培養時間可於菇類菌絲體可增殖之範圍適當決 定。並非對本發明加以特別限定，作為一例，例如可以下 述步驟進行。於PGY(Pe〇t〇ne Glucose Yeast，蛋白脒、葡 萄糖、酵母萃取物）液體培養基[組成：葡萄糖149% (w/v)、蛋白腺 〇.15%(w/v)、酵母萃取物 〇 i5%(w/v)、201210465 VI. [Technical Field] The present invention relates to a method for producing a mushroom liquid seed fungus for producing a mushroom fruit body, and a method for producing a mushroom fruit body using the liquid seed fungus. [Prior Art] In recent years, the industry is developing artificial cultivation techniques for mushrooms, providing various mushroom fruit bodies (Lyophyllum shimeji, Lyophyllum decastes, Hypsizygus marmoreus). , Pleurotus eryngii, lentinus edodes, flammulina velutipes, etc.). Usually, artificial cultivation of mushrooms is carried out by inoculating the mushroom inoculum in a eucalyptus forest or a solid medium, culturing, growing, and the like, and harvesting the fruiting bodies. Among the inoculums of the mushroom, there are a solid inoculum using a medium such as Saw, and a liquid inoculum using a liquid medium. In recent years, the industry is promoting the mechanization of artificial cultivation of mushrooms by the factory system of large equipment. However, in order to commercially and efficiently produce mushroom fruiting bodies, it is necessary to cultivate the inoculum used in a large amount. Previously, in the case of mushroom inoculum, solid inoculum was used for reasons such as good preservation, and it was easy to transport, but in terms of the following, 'the liquid inoculum was considered to be advantageous, and its use was increased: the batch difference from the solid inoculum could be avoided. The risk of uneven manufacturing during the manufacture of the mushroom fruiting body, or the serious damage to the equipment caused by the contamination of the bottle or container caused by the inoculating the inoculum when inoculated with the solid inoculum, and the Shorten the manufacturing time of the inoculum, and it is easier to expand the scale. As a method for cultivating liquid inoculum by 157547.doc 201210465, a liquid deep culture method in which a large number of culture methods for obtaining useful components from mycelium is discussed is used, and in order to strictly control culture conditions and the like, it is usually used Mechanical agitation; aeration and agitation method for ventilation and temperature management (eg, fermenter) (for example, non-patent literature). However, in order to manufacture a class of fruit bodies on a commercial scale, it is necessary to include a large-capacity tank-type fermentation tank. The setting of the high-priced and large-scale culture equipment. [Prior Art Document] [Non-Patent Document] Non-Patent Document 1: Fermentation Association, Vol. 24, No. 7, 293_3〇4, 1966 [Invention] [Problems to be Solved by the Invention] In view of the above circumstances, an object of the present invention is to provide a method for producing an inexpensive and highly efficient liquid inoculum in a large-scale commercial manufacture of a mushroom fruiting body, and a mushroom fruiting body using the liquid inoculum [Technical method for solving the problem] The inventors of the present invention have mass-produced a liquid inoculum for use in large-scale commercial manufacture of mushrooms. Efforts were made to study the results, and it was surprisingly found that liquid deep culture (air bubbling) using bubbles during agitation was unexpectedly useful for the manufacture of liquid inoculum for the production of fruiting bodies of mushrooms. In this culture, the mycelium can be produced in a large amount in a state in which the liquid culture medium contains a low concentration of the vegetable oil, and the fruit body can be produced in a state in which the fruiting body has the ability to form a mushroom. Further, by making 157547.doc 201210465 The bacteria base for the culture bed for the cultivation of the solid inoculum is raised, and the surface of the fungus is uniformly planarized, so that a more stable bacterium can be obtained, and the use of the bacterium can be efficiently induced, and the solid can be used. Compared with the case of the inoculum, the present invention is completed by producing an excellent mushroom fruiting body including a shorter time and uniform production time and higher commercial value of the inoculum. The present invention relates to: A method for producing a liquid inoculant, which is characterized in that it is used for the manufacture of mushroom fruit bodies, and in a liquid medium containing vegetable oil The liquid deep culture of the mycelium of the mushroom is carried out by stirring the culture medium using a bubble; [2] The production method according to [1], wherein the medium is substantially agitated using only bubbles; [3] as [1] Or the manufacturing method of [2] wherein the vegetable oil is one or more selected from the group consisting of salad oil, rice bran oil, rapeseed oil, safflower oil, corn oil, eucalyptus oil, sesame oil, soybean oil, and cottonseed oil; [4] as [ The production method according to any one of [1], wherein the vegetable oil concentration in the medium is 0.001 to 0.1% by volume; [5] a method for producing a mushroom-like fruit body, characterized in that it is used by U] a liquid inoculum of a mushroom obtained by the method of any one of [4]; [6] a method for producing a mushroom fruiting body according to [5], which is bottle cultivation; [7] such as [5] or [ 6] a method for producing a mushroom fruit body, which uses a culture substrate for raising a mushroom bed; [8] a method for producing a mushroom fruit body according to [7], which uses the surface of the mushroom seat to be uniformly planarized The culture medium for the cultivation of the bacteria bed. 157547.doc 201210465 [Effect of the Invention] According to the present invention, there is provided a mass production method for a large-scale commercial manufacture of a mushroom fruiting body, a liquid inoculum of a short-time and inexpensive mushroom. Further, a method for producing a mushroom fruit body using the liquid inoculum is also provided. [Embodiment] The "mushroom" which can be used in the present invention is not particularly limited, and examples thereof include a lotus leaf pleated umbrella, a true yam, a snail, a syllabus, a Pleurotus ostreatus, a fragrant scent, and a golden needle. Pholiota nameko, Grifola frondosa, Apricot Bautz, Agaricus blazei Murill, Dapingz, Pleurotus cystidiosus and other edible mushrooms. The strains of the mushrooms may be commercially available strains, or may be tissue isolates derived from wild fruiting bodies, or may be strains selected by methods such as selection, mating, cell fusion, genetic recombination, etc., as long as A strain which can be cultured as a liquid inoculum and which can produce a fruit body can be used. As a preferred aspect of the present invention, a lotus leaf leave umbrella (Lyophyllum decastes) can be cited. As the lotus leaf pleated umbrella, well-known strains are exemplified, for example, lotus leaf pleated umbrella K-3303 strain (FERM BP-4347), lotus leaf pleated umbrella K-3304 strain (FERM BP-4348), lotus leaf pleated umbrella K- 3305 strain (FERM BP-4349), lotus leaf pleated umbrella F-623 strain (FERM P-13165), lotus leaf pleated umbrella F-1154 strain (FERM P-13166), lotus leaf pleated umbrella F-1488 strain (FERM P -13167) and such variants suitable for the manufacture of the fruiting body. Further, as an example of the strain of the preferred Lyophyllum shimeji of the present invention, there are exemplified by the true pleated umbrella La 01-27 (FERM BP-10960), and the true detached umbrella La 01-20 (FERM) BP-10959), Zhen Ji pleats 157547.doc 201210465 Umbrella La 01-37 (FERM P-17456), Zhen Ji pleated umbrella La 01-45 (FERM P-17457), Zhen Ji pleated umbrella La 01-46 (FERM P-17458) and such strains suitable for the production of the fruiting body, etc. Further, as a strain of the sage mushroom of the present invention, there is a strain represented by the genus Lyophyllum ulmarium, Examples of strains and the like represented by Hypsizygus marmoreus include, for example, a detached umbrella M-8171 (FERM BP-1415), a detached umbrella K-0259 (FERM P-12981), and a twin. Pleated umbrella Lul-172 strain (FERM BP-8354), twin detached umbrella Lul-173 strain (FERM BP-8355), twin detached umbrella Lul-174 strain (FERM BP-8356), twin detachment Umbrella Lul-181 strain (FERM BP-8357), Hongxi mushroom K-4975 (FERM BP-11321), Hongxi garlic K-4979 (FERM BP-11322), Hongxi mushroom K-4980 (FERM BP-11323) , Hongxi Mushroom K-4981 (FERM BP-11324) and suitable for manufacturing Variants of those of peers. Further, as a preferred strain of abalone mushroom of the present invention, there are exemplified: Pleurotus cystidiosus subsp. abalones K-4986 (FERM P-22064), abalone mushroom Taiwan subspecies K-4987 (FERM Ρ·22065) ), abalone mushroom Taiwan subspecies K-4988 (FERM P-22066) and such variants suitable for the manufacture of fruiting bodies. The above strain is not limited as long as it is an artificially cultivated strain and the strain of the present invention can be applied. In the present specification, the "mycelium" refers to the mycelium of the above-mentioned mushroom, and is not particularly limited as long as it can be cultured in a liquid medium and can be used as a liquid inoculum for producing a fruit body of the mushroom. 157547.doc 201210465 In the present specification, the term "deep culture of liquid using a bubble" refers to a culture method in which the movement (flow) of the culture liquid is generated by the bubbles generated in the culture tank, and the medium (4) is applied. It is preferable to carry out the culture method of the medium (4) by using only the bubbles substantially, and it is also possible to include mechanical agitation using a stirrer or a device such as a propeller-shaped fan as long as the hyphae are not cut. It is particularly preferable to use a culture method in which a medium is not used only with mechanical mixing. The culture tank used in the above culture method does not require a device for mechanical power using (4) or vibration, as long as it has a device for generating bubbles. In the present invention, for example, a bubble column type culture device described in Japanese Society of Applied Axis, Vol. 8, No. ,, ^7 (Bile) can be used, and can be attached to the bottom surface and/or the side surface. A culture tank that produces a bubble-like device aseptically. Further, a tube which can generate bubbles can be directly placed in the culture tank. The aeration amount of the bubble in the culture tank is preferably G.2 to 0.8 vvm (volume per vohnne Per minute), suitably 0.2 to 0.6 vvm, or 1 ()~ 5 liters/min (indicating that 10 50 liters of sterile air is ventilated in a liquid medium of 25 to 83 L in a minute), suitably 20 to 40 liters/min. The culture tank used in the present invention may be any container having a volume of a culture liquid which can maintain a desired amount and which prevents mixing of microorganisms from the outside. It is preferable to use a culture tank having a structure capable of heating and pressurizing the culture medium to be held. However, as long as the culture tank holding the culture liquid itself can be sterilized by an autoclave or the like, a function for pressure sterilization is not required. Further, it is preferred to have a function of introducing sterile air into the container, or may be attached to the apparatus of 157547.doc 201210465. It is also possible to use a conventional tank type vinegar tank, but in this case, it is not necessary to use a mechanical stirring function for the culture. The size of the culture tank used in the present invention may be appropriately adjusted depending on the amount of the liquid inoculum to be produced. In the manufacture of the liquid inoculum of the present invention, any one of continuous or single culture may be carried out, and it is preferably a single culture, that is, preferably used for each unit for manufacturing the mushroom per body. Liquid inoculum. Further, when a large amount of culture is carried out in the production of a liquid inoculum (also referred to as a main culture in the present specification), the mycelium grown on an agar plate or the like may be directly used for the main culture. As a preferred aspect, first, a pre-culture using a small scale of hair or vibration using a g-body or the like is carried out, and the preculture is used for main culture. In this case, the liquid inoculum can be produced efficiently, inexpensively, and in a large amount by the use of the method for producing a liquid inoculum of the present invention in the main culture. The inventors of the present invention have found that in the above-mentioned pre-culture using a small scale using vibrations such as a flask, 'by using a low concentration of vegetable oil in the liquid medium to be used, compared with the case of not containing a low concentration of vegetable oil. More g filaments can be paid. Therefore, by allowing the liquid medium used in the pre-culture and the main culture to contain a low concentration of vegetable oil, the liquid inoculum can be produced efficiently, inexpensively, and in a large amount. In the present specification, the liquid medium used can be used as a nutrient source for a nutrient source generally used for liquid culture, for example, a liquid medium containing an assimilable nutrient source such as a carbon source, a gas source, or an inorganic salt. For example, carbon sources such as glucose, maltose, syrup, dextrin, glycerin, starch, etc., and peptone, meat emulsion, cottonseed meal, soy flour, and yeast extract 157547.doc 201210465 can be used, path protein, corn dip Liquid (CC) rn steep called (four), amine sulfuric acid record, nitric acid record, gasification record and other nitrogen sources, and then contain sulphuric acid two gas discharge, argon dipotassium, sodium sulphate, salt, carbon _, sulfuric acid town, gas The inorganic salt such as the like may be a liquid culture base which can culture the g of the upper (four) type. In the present specification, the term "vegetable oil" is not particularly limited as long as it is a plant-derived oil, and is preferably an edible oil from the viewpoint of safety and ease of availability. Examples of the edible oil include rice bran oil, rapeseed oil, safflower oil, corn oil, olive oil, sesame oil, soybean oil, cottonseed oil, and the like. Further, a processing oil such as salad oil using these oils as a raw material can also be used in the present invention. These vegetable oils can be prepared by a known method, and commercially available products can also be used. In the present invention, one or two or more kinds of these vegetable oils may be used in combination in a medium. The content of the vegetable oil in the medium of the present invention may be a low concentration with respect to the total amount of the liquid medium, and is suitably 0.001 to 0% by volume (v/v), preferably less than 0.005 to 0.1% by volume. (v/v), further preferably less than 〇〇i~〇〇5 volume/° (v/v). By using the low concentration vegetable oil, it can be used without covering the surface of the liquid medium in liquid culture. The culture of the liquid inoculum of the present invention can be carried out by a known method, and the culture temperature or the culture time can be appropriately determined in the range in which the mycelium of the mushroom can be proliferated. The present invention is not particularly limited, and as an example, for example, the following steps can be performed. Liquid medium for PGY (Pe〇t〇ne Glucose Yeast, peptone, glucose, yeast extract) [composition: glucose 149% (w/v), protein adenine. 15% (w/v), yeast extract 〇 I5%(w/v),
KH2P04 0.0373%(W/V)、及 MgS04.7H20 0.0373%(w/v)、pH 157547.doc •10· 201210465 值6.0] 200 mL中接種荷葉離褶傘之菌絲體，於25°C下培養 I4天，製備預培養物。繼而，於另外製備之PGY液體培養 基430 L中’以相對於培養基成為o.ooi〜〇_〇4體積％之方式 添加植物油、例如沙拉油[曰清沙拉油、日清奥利友集團 公司製造]’製備主培養用之液體培養基。將該液體培養 基填充至培養槽中，於118。〇下進行90分鐘高壓蒸氣殺 菌。放置冷卻開始後立刻進行利用無菌空氣之通氣，使培 養槽内成為正壓。冷卻該培養基後，接種上述預培養物之 總量，於培養溫度為25t、通氣量為〇.26 vvm、内壓為 〇.〇75 MPa、無動力攪拌（Air BubMing)之培養條件下，進 行6〜7天培養，製造液體種菌。 a利用如上所述之方式製造<液體種@亦可無菌地移至適 當之容器、例如聚丙烯製之圓型瓶等，於直至攝取至菌床 培養基為止之間，於控制為3〜·以之範圍之環境下保持並 1之用上述液體種菌 屬rrn 例如可利用作為菌床栽培方 ，之瓶栽培、袋栽培、箱栽培等而製造菇類子實體。作為 體2制由瓶裁培之本發明之使用液體種菌之蒜類子實 穿觀^㈣行描料，該方法包含g床料基製備、 菌、液體種菌之接種、菌床培養、(視需要搔 菌）發穿、（視需要插芽之分離、㈣之# 穫等各步驟。例如，若A # )生長收 特開平队2113_ ^ 7#_㈣作為騎日本專利 為香兹則作之菌床裁培時之種菌，若 本專利㈣平队〇75538號公報所記 157547.doc 201210465 載之菌床栽培時之種菌，若為鴻喜兹則作為進行曰本專利 特開平05-268942號公報所記載之菌床栽培時之種菌，或 若為真姬離褶傘則作為進行日本專利特開2〇〇〇1〇6752號 公報所記載之菌床栽培時之種菌，可使用藉由本發明之方 法所製造之液體種菌。X ’若為進行插芽之分離及插芽之 移植之g床栽培之情形，則作為進行日本專㈣開 017872號公報記載之菌床栽培時之種菌，亦可使用藉由本 發明之方法所製造之液體種菌。 其中’本發明之益類子實體之製造方法所使用之菌床栽 培用培養基較佳為使菌座高於使用固體種菌之情形之菌床 栽培用培養基或通常所使用之菌床栽培用培養基，適宜為 使菌座低於瓶口2〜1() mm即可。又，菌座表面較佳為均勾 地平面化。進而，瓶中央部之孔穴（亦稱為孔或接種孔）並 非特別需要，亦可使用對於每丨瓶於瓶頸附近、即瓶中央 部之周邊開有2〜6處、較佳為4處口徑為3〜1〇 mm左右較 佳為5 mm左右，深度為1〇〇〜2〇〇 mm左右，較佳為ιι〇~ΐ5〇 mm左右之小孔穴（亦稱為孔或接種孔）之菌床栽培用培養 基。 此處，於本案說明書中，所謂「菌座」，係指栽培用培 養基上部之表面之菌接種部位。例如於瓶栽培用培養基 中，瓶開口部露出之培養基表面稱為菌座。於本發明中， 較佳為使菌座高於使用固體種菌之情形之菌床栽培用培養 基（亦稱為菌床栽培用固體培養基、菌床栽培用培養基）（即 更接近瓶口），例如，於瓶栽培之情形時，於如圖丨所示， 157547.doc 12 201210465 使用固體種菌之情形時，推薦將低於通常之瓶栽培所使用 之市售之850 mL瓶或11〇〇 mL之瓶口（瓶頸）15 mm設為菌 座’但於本發明中，較佳為高於使用固體種菌之情形之菌 床栽培用培養基之菌座，適宜為將低於瓶口 2〜10 mm、進 而較佳為將低於5〜9 mm設為菌座。 將利用本發明之方法而製造之液體種菌接種於菌床栽培 用培養基時’對於每一瓶850 mL之廣口培養瓶，例如無菌 地接種約10〜20 mL即可。作為接種上述液體種菌之方法， 無需使用特殊之機械而噴霧狀地無遺漏地接種於菌座及接 種孔中’只要將液體種菌直接無菌地添加必要量於菌座上 即可。添加液體種菌之菌座上之位置較佳為菌座之中央部 附近。 根據本發明，與大規模之菇類子實體之商業製造用之固 體種菌相比，可飛躍地於短時間製造菇類子實體之商業製 造用之液體種菌。例如，於製造上述固體種菌之情形時， 將預培養後之液體菌絲或固體菌絲接種於固體培養基。其 後約30〜60天，使菌絲蔓延於固體培養基上。將所獲得之 蔓延有菌絲之固體培養基作為固體種菌而用於菇類子實體 之商業製造。相對於此，於利用本發明之方法而獲得之液 體種菌之情形時’將預培養後之液體菌絲接種於本發明之 液體培養基。其後於約3〜7天，宜於6〜7天進行培養，藉此 可製造液體種菌，且可用於菇類子實體之商業製造。 又’藉由將利用本發明之方法而製造之液體種菌用於大 規模之菇類子實體之商業製造，可避免因固體種菌之批量 157547.doc •13- 201210465 差所引起之菇類子實體製造時之製造不均’且可穩定地製 造優良之益類子實體。 以上，對瓶栽培方法舉例進行說明’但利用本發明之方 法而製造之液體種菌並不限定於上述利用瓶栽培之菌床栽 培中之用途。 [實施例] , 以下，藉由實施例對本發明進而具體地進行說明，但本 發明並不僅限定於以下實施例之範圍。 實施例1 於PGY液體培養基[組成：葡萄糖1.49%(w/v)、蛋白腺 0.15%(w/v)、酵母萃取物 〇.15%(w/v)、KH2P〇4 0.0373% (w/v)、及 MgS〇4.7H20 0.0373%(w/v)，pH值 6·〇] 200 mL 中 接種荷葉離褶傘K-33 04(FERM BP-4348)之菌絲體，於25°c 下進行14天振盪培養（1〇〇 rpm)，製備預培養物。 繼而，於另外製備之PGY液體培養基430 L中以相對於 培養基成為約0·02%(ν/ν)之方式添加作為植物油之沙拉油 (曰清沙拉油’曰清奥利友集團公司製造），製備主培養用 之液體培養基。培養槽使用無攪拌能力之非第一種壓力容 器。將放入有主培養用之液體培養基之培養槽於118。〇下 進行90分鐘高壓蒸氣殺菌’放置冷卻開始後立刻進行利用 無菌空氣之通氣，使培養槽内成為正壓。冷卻該培養基 後，接種上述預培養物之總量，於培養溫度為25它、通氣 量為0.26 vvm、内壓為0 075 Mpa、無動力攪拌（Air Bubbling)之培養條件下，進行6〜7天培養，製備液體種 157547.doc •14- 201210465 菌。 同時，進行僅利用PGY液體培養基之無動力檀拌（Air BubMing)培養，設為比較對照。將其他培養條件設為與上 述相同，進行菌體之產量比較。將其結果示於表1。 [表1] 條件 乾燥菌體重量(g/L) 有沙拉油 4.1 無沙拉油 r 3.0 根據表1可知，於本發明之使用添加有沙拉油之培養基 之不伴隨機械攪拌而使用氣泡之培養中，可與不添加沙拉 油之情形相比獲得多約1.36倍之菌體。 實施例2 為了進行利用本發明之方法而製造之液體種菌之子實體 形成能力之確認，以如下所述之方式進行子實體之製造。 液體種菌係利用與實施例1相同之方法而製備。固體種 菌係以如下之方式製備作為比較對象。首先，將設定為鑛 屑（杉材）100 g、米糠86 g、偏矽酸鋁酸鎂（富士化學工業公 司製造，商品名Neusilin FH丨）2 g、碳酸鈣（Nacalai Tesque 公司製造，試劑一級）5 g、檸檬酸一水合物（Nacalai Tesque公司製造、試劑一級）3 g、水分含量63〜65重量〇/〇 , 並良好地混合而成為濕潤狀態者填壓於聚丙烯製之廣口培 養瓶（850 mL，信越農材公司製造）中。於填壓物表面之中 央打開直徑為1 cm左右之接種孔部，打栓後，於丨丨8它下 進行90分鐘高壓蒸氣殺菌，放置冷卻至2(rc。對於以如上 所述之方式獲得之固體培養基，接種實施例1所記載之預 157547.doc -15· 201210465 培養物約10〜20 mL。其後，於暗處溫度25°C、濕度55%之 條件下培養菌絲體約60天，使菌絲蔓延至固體培養基整 體，製備固體種菌。 繼而，將設定為鋸屑（杉材）134 g、米糠13〇 g、偏石夕酸 鋁酸鎂[富士化學工業股份有限公司製造，商品名Neusilin FH丨]2.6 g、碳酸舜（Nacalai Tesque公司製造，試劑一 級）6.5 g、檸檬酸一水合物（Nacalai Tesque公司製造，試劑 一級）3.9 g、水分含量63〜65重量％ ’並良好地混合而成為 濕潤狀態者設定為菌座低於瓶口約1 5 mm，並填壓至聚丙 稀製之廣口培養瓶中（1100 mL，Yamada產業公司製造）。 於填壓物表面之瓶中央打開直徑為2 cm左右之接種孔部， 打栓後，於118°C下進行90分鐘高壓蒸氣殺菌，將放置冷 卻至20 C者作為菌床栽培用培養基。於該菌床栽培用培養 基接種上述所製備之液體種菌約2〇 mL。準備接種上述所 製備之固體種菌約35 g者作為比較對象。製備分別接種液 體種菌及固體種菌之菌床栽培用培養基各16個。將該等於 暗處溫度25°C、濕度55。/。之條件下培養菌絲體約6〇天，使 菌絲蔓延至菌床栽培用培養基整體。其後，打開封蓋，進 行菌床面上之搔菌，繼而添加自來水直至瓶口後，立刻 排水’供至發芽步驟。 發芽步驟係、於照度5G勒克司、溫度阶、加濕控制為作 ^Humid Eyel00(鷺宮製作所製造）之指示值為 範圍中、二氧化碳濃度控制為1000〜1500 ppm之範圍中之 出芽至進行。X ’為了避免冷凝水，倒置瓶，繼續培養^ 157547.doc • 16 - 201210465 天’使子f it U形成。其後，反轉正置瓶，轉移向前期 生長步驟。於刖期生長步驟中，於照度500勒克司、溫度 16C、加濕控制為作為Humid EyelOO之指示值為 115〜120%之範圍中、二氧化碳濃度控制為1〇〇〇〜2〇〇〇叩瓜 之範圍中之生長室中，進行培養6天。繼而，轉移向後期 生長步驟。於後期生長步驟中，於照度500勒克司、溫度 WC、加濕控制為作為Humid Eyel〇〇之指示值為95〜1〇5〇/〇 之範圍中、二氧化碳濃度控制為1000~2000 ppm之範圍中 之生長至中，繼續培養7天。收穫以如上所述之方式獲得 之成熟子實體後’進行子實體之平均產量（g/瓶）之比較。 將其結果示於表2» [表2] 種類 平均產量(g) 固體種菌 226 液體種菌 220 根據表2可知，利用本發明之方法而製造之液體種菌可 獲知·與使用荷葉離褶傘之子實體製造中用作通常種菌之固 體種菌之情形的子實體之產量大致相同之產量，因而利用 本發明之方法而製造之液體種菌具有子實體形成能力， 又’可製造適宜之菇類之子實體。 實施例3 進行使用藉由本發明之方法而製造之液體種菌的菇類子 實體之製造中之菌床栽培用培養基之研究。 首先’利用與實施例丨相同之方法製備液體種菌。製備 實施例2所記載之菌床栽培用培養基（通常菌座菌床栽培用 157547.doc -17· 201210465 培養基）’及設定為高於實施例2所記載之菌床栽培用培養 基之菌座，且菌座低於瓶口約5~9 mm左右，並填壓，進 而使菌床面平整之菌床栽培用培養基（高菌座菌床栽培用 培養基）各16個。除使用各菌床栽培用培養基以外，利用 與實施例2相同之方法製造菇類子實體。進行獲得之子實 體之產生率（％’根據產生子實體之瓶之數算出）及平均產 量（g/瓶）之比較。又，同時亦對獲得之子實體之產生狀況 進行觀察。將其結果示於表3。 [表3] 條件 產生率(％) 平均產量(g) 產生狀況 通常菌座菌床栽培用培養基 81 220 稍有不良 南菌座g床栽培用培養基 100 265 良好 根據表3可知，使用藉由本發明之方法而製造之液體種 菌的菇類子實體之製造藉由使用高菌座菌床栽培用培養 基’可與使用通常菌座菌床栽培用培養基之情形相比，更 高效地製造進而優良之菇類子實體。 實施例4 進行使用固體種菌及藉由本發明之方法而製造之液體種 菌的菇類子實體之製造中之製造不均之比較。 首先’固體種菌係利用與實施例2相同之方法製備，液 體種菌係利用與實施例1相同之方法製備。繼而，製備實 施例2所記載之菌床栽培用培養基作為固體種菌接種用、 及實施例3所記載之高菌座菌床栽培用培養基作為液體種 菌接種用各1萬個。其後’將固體種菌或液體種菌接種至 各自之菌床栽培用培養基上，利用與實施例2相同之方法 157547.doc •18· 201210465 進行菇類子實體之製造。 ^ 將該製造步驟中之搔菌、發芽及 生長時之不良產品以瓶罩 發穿及 * 。十數，算出不良率（〇/。）。將其 結果不於表I再者，搔 將其 ^ ^ ΐέ B± 夺之不良產品係設為發菌不完 全者。發芽時之不良產0 ^70 老…— 设為未出芽者、出芽不均句 :芽稀疏且較少者。生長時之不良產品係於前期生 步驟後’即發芽、生長步驟第17天進行4認，係設為 類子實體之生長停止者、形狀不良者、或未生長為均；之 株狀者。X ’對各步财成為不良者進行廢棄，於其次之 步驟中以瓶單位均使用良品而進行。 [表4] 種類 不良率一 搔菌時 發芽時1 生長時 固體種菌 1.8 1.8 2.1 液體種菌 Π 〇 0.03 0.3 根據表4可知，使用藉由本發明之方法而製造之液體種 菌的菇類子實體之製造與使用固體種菌之製造相比，製造 不均較少’可用於大規模之商業栽培。 實施例5 於PGY液體培養基[組成：葡萄糖2 〇%(w/v)、蛋白脒 0_2°/〇(w/v)、酵母萃取物 〇.2%(w/v)、kh2P04 0.05%(w/v)、 MgS04.7H2〇 0.05%(w/v)]200 mL 中接種Pleurotus cystidiosus. abalones K-4988(FERM P-22066)之菌絲體，於 25°C 下振盪 培養（90 rpm) 14天，製備預培養物。 繼而，於另外製備之PGY液體培養基60 L中，以相對於 培養基成為約0.1%(ν/ν)之方式添加米糠油（商品名： 157547.doc •19- 201210465 ginsyarihonpo米糠油，Japan Rice公司製造），製備主培養 用之液體培養基。培養槽係使用無攪拌能力之非第一種壓 力容器。將放入有主培養用之液體培養基之培養槽於 118 C下進行30分鐘高壓蒸氣殺菌，放置冷卻開始後立刻 進行利用無菌空氣之通氣，使培養槽内成為正壓。冷卻該 培養基後，接種上述預培養物之總量，於培養溫度27=、 通氣量0.35 vvm、無動力攪拌（Air Bubbiing)之培養條件 下，進行8天培養’製備液體種菌。 同時，進行僅利用PGY液體培養基之無動力攪拌（八卜 Bubbling)培養，作為比較對照。培養第5天起每天進行取 樣’進行菌體之產量比較。將其結果示於表5。 [表5] 培養天數 乾燥菌葡 ^量(g/L)— 0% 0.1% 第5天 0.39 1.62 第6天 0.62 2.35 第7天 0.82 3.19 ' 第8天 1.69 3.86 根據表5可知，藉由使用添加米糠油之液體培養基之不 伴隨機械攪拌而使用氣泡之培養，可與不添加米糠油之情 形相比獲得更多之菌體。尤其是可知於培養第8天中，可 與不添加米糠油之情形相比獲得約2 28倍之菌體。 實施例6 於與實施例5相同之PGY液體培養基4 L中以相對於培養 基成為約0.1%(v/v)之方式添加玉米油（AJIN〇M〇T〇胚芽之 惠玉米油，j_〇il Mills公司製造），製備主培養用之液體捭 157547.doc •20· 201210465 養基。培養槽係使用微生物培養裝置（BMS-05PI，Able公 司製造）。將放入有主培養用之液體培養基之培養槽於 12 1°C下進行15分鐘高壓蒸氣殺菌，於室溫下冷卻後，接 種 Pleurotus cystidiosus. abalones K.-4988(FERM P-22066) 之菌絲體，於培養溫度27°C、通氣量0.35 vvm、無動力授 拌（Air Bubbling)之培養條件下，進行8天培養，製備液體 種菌。 同時，進行僅利用PGY液體培養基之無動力攪拌（Air Bubbling)培養作為比較對照。自培養第5天起每天進行取 樣，進行菌體之產量比較。將其結果示於表6。 [表6] 培養曰數 乾燥菌H t重量(g/L) 0% 0.1% 第5天 0.13 1.13 第6天 0.20 2.19 第7天 0.25 3.38 第8天 0.23 3.55 根據表6可知，藉由使用添加有玉米油之液體培養基之 不伴隨機械攪拌而使用氣泡之培養，可與不添加玉米油之 情形相比獲得更多之菌體。尤其是可知於培養第8天中， 可與不添加玉米油之情形相比獲得約15.4倍之菌體。 實施例7 製作於與實施例5相同之PGY液體培養基200 mL中，分 別以相對於培養基成為約0.01%(v/v)、約0·025%(ν/ν)、約 0·05%(ν/ν)、約 0.075%(ν/ν)、約 0.1°/〇(ν/ν)之方式添加米糠 油（商品名：ginsyarihonpo米糠油，Japan Rice公司製造）或 157547.doc -21 - 201210465 玉米油（AJINOMOTO胚芽之惠玉米油，j_〇ii Mills公司製 造）而成之液體培養基。於該培養基中接種Pleurotus cystidiosus. abalones K-4988(FERM P-22066)之菌絲:體，於 25°C下進行14天振盪培養（90rpm)，製備液體種菌。將按 米糠油或玉米油對於液體培養基之含量分組之菌體產量之 結果示於表7。 [表7] 油濃度 乾燥菌體重量(g/L) 米糠油 玉米油 0% 3.42 3.42 0.01% 4.35 3.65 0.025% 4.91 3.86 0.05% 5.19 3.76 0.075% 5.39 4.59 0.1% 6.11 5.74 根據表7可知’即便於使用添加有米糠油或玉米油之少 量之液體培養基之振盪培養中’亦可與不添加米糠油或玉 米油之情形相比獲得更多之菌體。 實施例8 製作於與實施例5相同之PGY液體培養基2〇〇 mL中，分 別以相對於培養基成為約〇_〇1%(ν/ν)、約〇.〇25%(v/v)、約 0.〇5%(v/v)、約〇·〇75%(ν/ν)之方式添加米糠油（商品名： ginsyarihonpo米糠油，Japan Rice公司製造）而成之液體培 養基。於該培養基中接種自市售之舞菇（Grif〇la fr〇nd〇sa (Fr.) S. F. Gray ’ Yukiguni Maitake公司製造）利用公知之方 法而獲得之菌絲體’於25°C下進行14天振盤培養（9〇 rpm) ’製備液體種菌。將按米糠油相對於液體培養基之含 157547.doc -22- 201210465 量分組之菌體產量之結果示於表8。 [表8] 米糠油濃度 乾燥菌體重量(g/L) 0% 1.60 0.01% 1.65 0.025% 1.80 0.05% 2.20 0.075% 2.30 根據表8可知，即便於使用添加米糠油之少量之液體培 養基之振盪培養中，亦可與不添加米糠油之情形相比獲得 更多之菌體。 實施例9 製作於SGY液體培養基[組成：葡萄糖2.0%(w/v)、大豆 蛋白腺 0.2%(w/v)、酵母萃取物 0.2%(w/v)、KH2P〇4 0.05%(w/v)、MgS04.7H20 0.05%(w/v)]200 mL 中分別以相 對於培養基成為約〇.〇1%(ν/ν)、約0.025%(v/v)、約 0.05%(v/v)之方式添加沙拉油（曰清沙拉油、曰清奥利友集 團公司製造）而成之液體培養基、及分別以相對於培養基 成為約 0·01%(ν/ν)、約 0.025%(v/v)、約 0.05%(v/v)、約 0.075°/〇(v/v)、約0.1%(v/v)之方式添加米糠油（商品名： ginsyarihonpo米糠油，Japan Rice公司製造）而成之液體培 養基。於該培養基中接種榆生離褶傘Lul-181(FERM BP-8357)之菌 絲體 ，於25°C 下進行 10天振盪培養（90 rpm) ， 製 備液體種菌。將按沙拉油相對於液體培養基之含量分組之 菌體產量之結果示於表9。 [表9] 157547.doc •23· 201210465 油濃度 乾燥菌體重 沙拉油 米糠油 0% 2.27 7.90 0.01% 4.30 10.20 0.025% 4.60 9.90 0.05% 5.17 8.30 0.075% - 10.05 0.1% - 10.65 根據表9可知，即便於使用添加有沙拉油或米糖油之少 量之液體培養基之振盈培養中’亦可與不添加沙拉油或米 糠油之情形相比獲得更多之菌體。 — 對本發明詳細地且參照特定之實施態樣進行說明，但業 者應知曉，可不脫離本發明之精神與範圍而加以各種變更 或修正。 本申凊案係基於2010年8月11曰申請之曰本專利申請（曰 本專利特願2010-179999號）者，其内容作為參照而併入本 文中。 [產業上之可利用性] 根據本發明，可提供一種廉價、高效之具有菇類子實體 形成能力之液體種菌之製造方法。進而，藉由使用該液體 種菌’可大幅度縮短通常於固體種菌之情形時需要3〇〜6〇 曰天之種菌製造時間，又，可避免使用固體種菌製造菇類 子實體時之製造不均之風險，並且可穩定地製造優良之菇 類子實體，因而可用於菇類子實體之大規模之商業栽培。 【圖式簡單說明】 圖1係表示使本發明之製造方法所使用之栽培瓶中之升 高菌座之菌床栽培用培養基之菌座位置之圖。 157547.doc -24·KH2P04 0.0373% (W/V), and MgS04.7H20 0.0373% (w/v), pH 157547.doc •10· 201210465 Value 6.0] 200 mL of mycelium inoculated with lotus leaf pleated umbrella at 25 ° C The culture was prepared for 4 days to prepare a preculture. Then, in a separately prepared PGY liquid medium 430 L, vegetable oil, such as salad oil, is added in the form of o.ooi~〇_〇4% by volume relative to the medium [Muqing salad oil, manufactured by Nisshin Olympus Group Co., Ltd. ]'Preparation of liquid medium for main culture. The liquid medium was filled into a culture tank at 118. The sputum was sterilized by high pressure steam for 90 minutes. Immediately after the start of cooling, ventilation with sterile air was performed to make the inside of the culture tank positive. After cooling the medium, the total amount of the preculture was inoculated, and the culture condition was carried out under the culture conditions of a culture temperature of 25 t, aeration volume of 〇.26 vvm, internal pressure of 〇.〇75 MPa, and no power agitation (Air BubMing). 6 to 7 days of cultivation to produce liquid inoculum. a manufactured by the method described above, <liquid species@ can also be aseptically transferred to a suitable container, for example, a round bottle made of polypropylene, etc., until the ingestion to the bed culture medium, the control is 3~· In the environment of the above-mentioned range, the above-mentioned liquid genus rrn can be used for the production of mushroom fruit bodies, for example, bottle culture, bottle cultivation, box cultivation, and the like. The invention discloses a method for preparing a garlic seed using a liquid inoculum according to the invention, which comprises a g bed base preparation, a bacteria, a liquid inoculum inoculation, a mushroom bed culture, and (see) Need to carry out the sputum), (depending on the need to insert the buds, (4) #, etc.. For example, if A # ) growth, the special Kaiping team 2113_ ^ 7#_ (four) as the Japanese patent for the Ziz If the inoculum of the bed is cultivated, if the inoculum is cultivated in the mushroom bed of 157547.doc 201210465, which is recorded in the Gazette No. 75538 of this patent, if it is Hongxiz, the patent is issued as a special patent No. 05-268942. In the case of the inoculum of the present invention, the inoculum of the present invention can be used as the inoculum in the case of the cultivation of the mushroom bed described in Japanese Laid-Open Patent Publication No. Hei. The liquid inoculum produced by the method. In the case of the g bed cultivation in which the insertion of the buds and the transplantation of the buds are carried out, the inoculum in the case of the mushroom bed culture described in Japanese Patent Publication No. 017872 can also be produced by the method of the present invention. Liquid inoculum. The culture medium for the cultivation of the mushroom bed used in the method for producing the beneficial fruit body of the present invention is preferably a culture medium for bacterial bed cultivation in which the bacterial seat is higher than the case where the solid inoculum is used, or a culture medium for mushroom bed which is generally used. In order to make the bacteria seat 2~1() mm below the bottle mouth. Further, the surface of the fungus is preferably flattened. Further, the hole in the central portion of the bottle (also referred to as a hole or an inoculating hole) is not particularly required, and it is also possible to use 2 to 6, preferably 4 cavities for each bottle near the neck of the bottle, that is, the periphery of the bottle. It is about 3~1〇mm, preferably about 5 mm, and the depth is about 1〇〇~2〇〇mm, preferably a small hole (also called a hole or an inoculation hole) of about ιι〇~ΐ5〇mm. Bed cultivation medium. Here, in the present specification, the term "bacteria" refers to a bacterial inoculation site on the surface of the upper portion of the cultivation medium. For example, in the medium for bottle cultivation, the surface of the medium in which the opening of the bottle is exposed is called a fungus. In the present invention, it is preferred that the culture base for bacteria bed cultivation (also referred to as solid medium for mushroom bed cultivation, culture medium for mushroom bed cultivation) in which the bacterial seat is higher than the case of using the solid inoculum (i.e., closer to the bottle mouth), for example, In the case of bottle cultivation, as shown in Figure 157547.doc 12 201210465 When using solid inoculum, it is recommended to use a commercially available 850 mL bottle or 11 mL bottle than the usual bottle cultivation. The mouth (bottleneck) 15 mm is set as the bacterium 'but in the present invention, it is preferably higher than the bacterium for the bed culture medium in the case of using the solid inoculum, and is preferably 2 to 10 mm below the mouth of the bottle, and further Preferably, less than 5 to 9 mm is set as a microbial holder. When the liquid inoculum produced by the method of the present invention is inoculated into a culture medium for mushroom bed cultivation, for each bottle of 850 mL of a wide-mouth culture bottle, for example, about 10 to 20 mL of the inoculum can be inoculated aseptically. As a method of inoculating the above-mentioned liquid inoculum, it is possible to inoculate the cells and the seeding holes in a spray-like manner without using a special machine. The liquid inoculum can be directly and aseptically added to the fungus base. The position on the colony to which the liquid inoculum is added is preferably near the central portion of the fungus. According to the present invention, liquid inoculum for commercial manufacture of mushroom fruit bodies can be produced in a short period of time as compared with solid inoculum for commercial manufacture of large-scale mushroom fruit bodies. For example, in the case of producing the above solid inoculum, the precultured liquid hyphae or solid hyphae are inoculated to a solid medium. Thereafter, about 30 to 60 days, the hyphae spread on the solid medium. The obtained solid medium in which hyphae was spread was used as a solid inoculum for commercial manufacture of mushroom fruiting bodies. On the other hand, in the case of the liquid inoculum obtained by the method of the present invention, the liquid mycelium after the preculture is inoculated to the liquid medium of the present invention. Thereafter, the culture is carried out for about 6 to 7 days, preferably for 6 to 7 days, whereby liquid inoculum can be produced and used for commercial manufacture of mushroom fruiting bodies. In addition, by using the liquid inoculum manufactured by the method of the present invention for the commercial manufacture of large-scale mushroom fruiting bodies, the mushroom fruit bodies caused by the difference of the solid inoculum 157547.doc •13-201210465 can be avoided. Manufacturing is uneven at the time of manufacture' and it is possible to stably manufacture excellent beneficial fruit bodies. As described above, the bottle cultivation method will be described as an example. However, the liquid inoculum produced by the method of the present invention is not limited to the use in the above-mentioned mushroom cultivation by bottle cultivation. [Examples] Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to the scope of the following examples. Example 1 In PGY liquid medium [composition: glucose 1.49% (w/v), protein gland 0.15% (w/v), yeast extract 〇.15% (w/v), KH2P〇4 0.0373% (w/ v), and MgS〇4.7H20 0.0373% (w/v), pH 6·〇] 200 mL of mycelium inoculated with lotus leaf pleated umbrella K-33 04 (FERM BP-4348) at 25 ° c A preculture was prepared by shaking culture (1 rpm) for 14 days. Then, in a separately prepared PGY liquid medium 430 L, a salad oil as a vegetable oil was added in a manner of about 0. 02% (v/v) with respect to the medium (Muqing Salad Oil's manufactured by Minqing Aoliyou Group Co., Ltd.) , preparing a liquid medium for main culture. The culture tank uses a non-first type of pressure vessel without agitation. The culture tank in which the liquid medium for main culture is placed is placed at 118. Under the squat, the high-pressure steam sterilization was carried out for 90 minutes. Immediately after the start of the cooling, the aeration was performed using sterile air to make the inside of the culture tank positive. After cooling the medium, the total amount of the preculture was inoculated, and the culture temperature was 25, the aeration was 0.26 vvm, the internal pressure was 0 075 Mpa, and the air bubbling was carried out under the conditions of 6 to 7 Day culture, preparation of liquid species 157547.doc •14- 201210465 bacteria. At the same time, air bubming culture using only PGY liquid medium was carried out, and comparison was made. The other culture conditions were set to be the same as described above, and the yield of the cells was compared. The results are shown in Table 1. [Table 1] Condition-dried cell weight (g/L) Salad oil 4.1 No salad oil r 3.0 According to Table 1, it is known that the culture medium to which the salad oil is added is cultured without using mechanical agitation in the culture of the present invention. It can obtain about 1.36 times more bacteria than if no salad oil is added. Example 2 In order to confirm the formation ability of the liquid inoculum produced by the method of the present invention, the production of the fruit body was carried out in the following manner. The liquid inoculum was prepared by the same method as in Example 1. Solid strains were prepared as follows in the following manner. First, it will be set to 100 g of mineral chips (cedar), 86 g of rice bran, magnesium bismuth aluminate (manufactured by Fuji Chemical Industry Co., Ltd., trade name Neusilin FH) 2 g, calcium carbonate (manufactured by Nacalai Tesque, reagent level) 5 g, citric acid monohydrate (manufactured by Nacalai Tesque Co., Ltd., reagent first grade) 3 g, moisture content 63 to 65 weight 〇 / 〇, and well mixed to become wet state, filled with polypropylene in wide mouth culture Bottle (850 mL, manufactured by Shin-Etsu Chemical Co., Ltd.). Open the inoculation hole with a diameter of about 1 cm at the center of the surface of the filler. After the plug, perform high-pressure steam sterilization for 90 minutes under the crucible 8 and place it to cool to 2 (rc. For the above method) The solid medium was inoculated with about 10 to 20 mL of the pre-157547.doc -15·201210465 culture described in Example 1. Thereafter, the mycelium was cultured under the conditions of a dark temperature of 25 ° C and a humidity of 55%. In the day, the mycelium is spread to the whole of the solid medium to prepare a solid inoculum. Then, it is set to 134 g of sawdust (cedar), 13 〇g of rice bran, and magnesium bismuth aluminate [manufactured by Fuji Chemical Industry Co., Ltd., Neusilin FH丨]2.6 g, cesium carbonate (manufactured by Nacalai Tesque, reagent grade) 6.5 g, citric acid monohydrate (manufactured by Nacalai Tesque, reagent grade) 3.9 g, moisture content 63 to 65 wt% 'and well Mix and become wet. Set to about 15 mm below the mouth of the bottle, and pressurize it into a polypropylene wide-mouth culture bottle (1100 mL, manufactured by Yamada Industries Co., Ltd.). Open diameter is 2 After inoculation, the inoculation hole portion of about cm is subjected to high-pressure steam sterilization at 118 ° C for 90 minutes, and is placed in a bed culture medium for cooling to 20 C. The liquid prepared in the above-mentioned bed cultivation medium is inoculated with the above prepared liquid. The inoculum was about 2 mL, and about 35 g of the solid inoculum prepared as described above was prepared for comparison. Sixteen culture mediums for inoculating the liquid inoculum and the solid inoculum were prepared, respectively, which was equal to a dark temperature of 25 ° C, The mycelium is cultured for about 6 days under the condition of a humidity of 55., so that the hyphae spread to the whole culture medium for the mushroom bed. Thereafter, the cover is opened, the bacteria on the surface of the bacteria bed are carried out, and then tap water is added to the bottle. Immediately after the mouth, the drainage is supplied to the germination step. The germination step is in the range of 5G lux, temperature step, humidification control for the Humid Eyel00 (made by Heron Palace), and the carbon dioxide concentration is controlled to 1000~ In the range of 1500 ppm, the sprouting is carried out. X 'To avoid condensation, invert the bottle and continue to culture ^ 157547.doc • 16 - 201210465 days 'Makoto f it U formation. Thereafter, reverse the positive The bottle is transferred to the forward growth step. In the growth stage of the flood season, the illumination is 500 lux, the temperature is 16C, the humidification is controlled as the indicator value of Humid EyelOO is 115 to 120%, and the carbon dioxide concentration is controlled to 1〇〇. In the growth chamber in the range of 〇~2 〇〇〇叩 melon, culture is carried out for 6 days. Then, the transfer is carried out to the later growth step. In the later growth step, the illuminance is 500 lux, the temperature is WC, and the humidification is controlled as Humid. The indication value of Eyel〇〇 is in the range of 95~1〇5〇/〇, and the growth of carbon dioxide is controlled in the range of 1000~2000 ppm, and the cultivation is continued for 7 days. A comparison of the average yield (g/bottle) of the fruiting bodies after harvesting the mature fruiting bodies obtained as described above was carried out. The results are shown in Table 2» [Table 2] Type average yield (g) Solid inoculum 226 Liquid inoculum 220 According to Table 2, liquid inoculum produced by the method of the present invention can be known and used as a fruiting body using a lotus leaf pleated umbrella The production of the fruiting body in the case of use as a solid inoculum of the usual inoculum is substantially the same as the yield, and thus the liquid inoculum produced by the method of the present invention has a fruiting body forming ability and can produce a fruit body of a suitable mushroom. Example 3 A culture medium for mushroom bed cultivation in the production of a mushroom fruit body using a liquid inoculum produced by the method of the present invention was carried out. First, liquid inoculum was prepared by the same method as in Example 。. The culture medium for mushroom bed cultivation (usually 157547.doc -17·201210465 medium for cultivation of the bacteria bed) described in Example 2, and the mushroom seat set to be higher than the culture medium for mushroom bed culture described in Example 2, The fungus base is about 5 to 9 mm lower than the mouth of the bottle, and is filled with pressure, and the culture bed culture medium (the culture medium for the cultivation of the high-bacteria bacteria bed) of the bacterial bed surface is 16 each. A mushroom fruit body was produced in the same manner as in Example 2 except that the culture medium for each mushroom bed was used. The yield of the obtained sub-subjects (%' is calculated based on the number of bottles from which the sub-entities are produced) and the average yield (g/bottle). At the same time, the state of production of the obtained fruiting entities is also observed. The results are shown in Table 3. [Table 3] Conditional production rate (%) Average yield (g) Production condition Normal bacteria bed culture medium 81 220 Slightly defective South base g bed cultivation medium 100 265 Good According to Table 3, use of the present invention The production of the mushroom fruit body of the liquid inoculum produced by the method can be more efficiently produced and the mushroom is better than the case of using the culture medium for the cultivation of the common bacteria bed. Class child entity. Example 4 A comparison of manufacturing unevenness in the manufacture of mushroom fruiting bodies using solid inoculum and liquid inoculum produced by the method of the present invention was carried out. First, the solid strain was prepared in the same manner as in Example 2, and the liquid strain was prepared in the same manner as in Example 1. Then, the culture medium for the cultivation of the bacteria bed described in the second embodiment was prepared as a medium for inoculating the solid inoculum and the medium for the cultivation of the high-bacteria bacteria bed described in the third embodiment as each of the 10,000 inoculations for liquid seeding. Thereafter, solid inoculum or liquid inoculum was inoculated to the respective culture medium for bed culture, and the production of the mushroom fruit body was carried out by the same method as in Example 2, 157547.doc • 18·201210465. ^ The defective product of the bacteria, germination and growth in the manufacturing step is pierced with a bottle cover and *. Ten, calculate the bad rate (〇 /.). If the result is not in Table I, then the defective product line of ^ ^ ΐέ B± is not completely sterilized. Bad production at the time of germination 0 ^ 70 Old... - Set as a non-germination, uneven sprouting sentence: buds sparse and less. The defective product at the time of growth is subjected to the pre-production step, i.e., on the 17th day of the germination and growth step, and is determined to be a growth stopr of the fruit-like entity, a shape-defective person, or a non-growth; X ’ is discarded for each person who has become a bad person, and in the next step, the bottle unit is used as a good product. [Table 4] Type of defective rate at the time of germination at the time of germination 1 Solid inoculum at the time of growth 1.8 1.8 2.1 Liquid bacterium Π 0.03 0.3 According to Table 4, production of a mushroom fruit body using the liquid inoculum produced by the method of the present invention Compared with the manufacture of solid inoculum, the manufacturing is less uneven 'can be used for large-scale commercial cultivation. Example 5 In PGY liquid medium [composition: glucose 2% (w/v), peptone 0 2 ° / 〇 (w / v), yeast extract 〇 2% (w / v), kh2P04 0.05% (w /v), MgS04.7H2〇0.05% (w/v)] 200 mL of mycelium inoculated with Pleurotus cystidiosus. abalones K-4988 (FERM P-22066), shake culture at 25 ° C (90 rpm) 14 Days, pre-cultures were prepared. Then, rice bran oil (trade name: 157547.doc •19-201210465 ginsyarihonpo rice bran oil, manufactured by Japan Rice Co., Ltd.) was added to 60 L of the separately prepared PGY liquid medium in a manner of about 0.1% (v/v) with respect to the medium. ), preparing a liquid medium for main culture. The culture tank uses a non-first pressure vessel without agitation. The culture tank in which the liquid medium for main culture was placed was subjected to high-pressure steam sterilization at 118 C for 30 minutes, and immediately after the start of cooling, aeration air was used to make the inside of the culture tank positive. After cooling the medium, the total amount of the above preculture was inoculated, and the liquid inoculum was prepared by performing culture for 8 days under the culture conditions of the culture temperature of 27 =, the aeration amount of 0.35 vvm, and the air bubbiing. At the same time, a non-powered agitation (eight Bubbling) culture using only PGY liquid medium was carried out as a comparative control. The sample was taken every day from the 5th day of culture to compare the yield of the cells. The results are shown in Table 5. [Table 5] Culture days Dry bacteria amount (g/L) - 0% 0.1% Day 5 0.39 1.62 Day 6 0.62 2.35 Day 7 0.82 3.19 ' Day 8 1.69 3.86 According to Table 5, by using The culture of the liquid medium to which rice bran oil is added without using mechanical agitation can obtain more bacterial cells than in the case where no rice bran oil is added. In particular, it was found that on the 8th day of culture, about 2 to 28 times of the cells were obtained as compared with the case where no rice bran oil was added. Example 6 Corn oil (AJIN〇M〇T〇 germ benefit corn oil, j_〇) was added in 4 L of the same PGY liquid medium as in Example 5 in a manner of about 0.1% (v/v) relative to the medium. Il Mills company), preparation of liquid for main culture 捭157547.doc •20· 201210465 Nutrient. The culture tank was a microbial culture apparatus (BMS-05PI, manufactured by Able Corporation). The culture medium in which the liquid medium for main culture is placed is subjected to autoclaving at 12 1 ° C for 15 minutes, and after cooling at room temperature, the bacteria of Pleurotus cystidiosus. abalones K.-4988 (FERM P-22066) are inoculated. The silk body was cultured for 8 days under the culture conditions of a culture temperature of 27 ° C, aeration of 0.35 vvm, and air bubbling to prepare a liquid inoculum. At the same time, air bubbling culture using only PGY liquid medium was performed as a comparative control. Samples were taken every day from the fifth day of culture, and the yield of the cells was compared. The results are shown in Table 6. [Table 6] Cultured dry bacteria H t weight (g/L) 0% 0.1% Day 5 0.13 1.13 Day 6 0.20 2.19 Day 7 0.25 3.38 Day 8 0.23 3.55 According to Table 6, it can be seen that by using The culture of a liquid medium containing corn oil without using mechanical agitation can obtain more cells than in the case where corn oil is not added. In particular, it was found that in the eighth day of culture, about 15.4 times of the cells were obtained as compared with the case where corn oil was not added. Example 7 was prepared in 200 mL of the same PGY liquid medium as in Example 5, and was about 0.01% (v/v), about 0.025% (ν/ν), and about 0.05% with respect to the medium, respectively. Rice bran oil (trade name: ginsyarihonpo rice bran oil, manufactured by Japan Rice Co., Ltd.) or 157547.doc -21 - 201210465, ν/ν), about 0.075% (ν/ν), about 0.1°/〇 (ν/ν) A liquid medium made from corn oil (AJINOMOTO Germ Corn Oil, manufactured by J_〇ii Mills). The culture medium was inoculated with mycelium of Pleurotus cystidiosus. abalones K-4988 (FERM P-22066), and shake culture (90 rpm) was carried out at 25 ° C for 14 days to prepare a liquid inoculum. The results of the cell yields classified by the content of rice bran oil or corn oil for the liquid medium are shown in Table 7. [Table 7] Oil concentration dry cell weight (g/L) Rice bran oil corn oil 0% 3.42 3.42 0.01% 4.35 3.65 0.025% 4.91 3.86 0.05% 5.19 3.76 0.075% 5.39 4.59 0.1% 6.11 5.74 According to Table 7, it is known that In the shaking culture using a small amount of liquid medium supplemented with rice bran oil or corn oil, it is also possible to obtain more bacteria than in the case where rice bran oil or corn oil is not added. Example 8 It was prepared in 2 mL of the same PGY liquid medium as in Example 5, and was about % 〇 1% (ν/ν), about 〇. 〇 25% (v/v), respectively, with respect to the medium. A liquid medium obtained by adding rice bran oil (trade name: ginsyarihonpo rice bran oil, manufactured by Japan Rice Co., Ltd.) in a manner of about 5% (v/v) and about 5% (v/v). The culture medium was inoculated with a commercially available mushroom (Grif〇la fr〇nd〇sa (Fr.) SF Gray 'Yukiguni Maitake Co., Ltd.) and the mycelium obtained by a known method was carried out at 25 ° C. Day vibration plate culture (9 rpm) 'Preparation of liquid inoculum. The results of the cell yields of rice bran oil in relation to the liquid medium containing 157547.doc -22-201210465 are shown in Table 8. [Table 8] Rice bran oil concentration Dry cell weight (g/L) 0% 1.60 0.01% 1.65 0.025% 1.80 0.05% 2.20 0.075% 2.30 According to Table 8, even in the shaking culture using a small amount of liquid medium supplemented with rice bran oil In addition, more bacteria can be obtained than if rice bran oil is not added. Example 9 Manufactured in SGY liquid medium [composition: glucose 2.0% (w/v), soybean protein gland 0.2% (w/v), yeast extract 0.2% (w/v), KH2P〇4 0.05% (w/ v), MgS04.7H20 0.05% (w/v)] 200 mL, respectively, relative to the medium, about 1% (v / v), about 0.025% (v / v), about 0.05% (v / v) The liquid medium obtained by adding salad oil (Muqing Salad Oil, manufactured by Minqing Orly Group), and about 0. 01% (v/v), about 0.025%, respectively (relative to the medium) Add rice bran oil (v/v), about 0.05% (v/v), about 0.075°/〇 (v/v), about 0.1% (v/v) (trade name: ginsyarihonpo rice bran oil, manufactured by Japan Rice Co., Ltd. ) a liquid medium. The culture medium was inoculated with the mycelium of the detached umbrella Lul-181 (FERM BP-8357), and subjected to shaking culture (90 rpm) at 25 ° C for 10 days to prepare a liquid inoculum. The results of the bacterial yields grouped according to the contents of the salad oil relative to the liquid medium are shown in Table 9. [Table 9] 157547.doc •23· 201210465 Oil concentration dry bacteria weight salad oil rice bran oil 0% 2.27 7.90 0.01% 4.30 10.20 0.025% 4.60 9.90 0.05% 5.17 8.30 0.075% - 10.05 0.1% - 10.65 According to Table 9, even In the vibrating culture using a small amount of liquid medium supplemented with salad oil or rice sugar oil, it is also possible to obtain more bacteria than in the case where no salad oil or rice bran oil is added. The present invention has been described in detail with reference to the specific embodiments thereof, and it is understood that various modifications and changes may be made without departing from the spirit and scope of the invention. The present application is based on a patent application filed on Jan. 11, 2010, the entire disclosure of which is hereby incorporated by reference. [Industrial Applicability] According to the present invention, it is possible to provide an inexpensive and highly efficient method for producing a liquid inoculum having a mushroom fruiting body forming ability. Further, by using the liquid inoculum, the production time of the inoculum which usually takes 3 to 6 days in the case of solid inoculum can be greatly shortened, and the manufacturing unevenness when the mushroom seed body is produced by using the solid inoculum can be avoided. The risk, and the stable mushroom fruit body can be stably produced, and thus can be used for large-scale commercial cultivation of mushroom fruiting bodies. [Brief Description of the Drawings] Fig. 1 is a view showing the position of the locus of the culture substrate for the mushroom bed for raising the bacteria in the cultivation bottle used in the production method of the present invention. 157547.doc -24·