TWI510615B - Solid medium for culturing ganoderma strains and application thereof - Google Patents
Solid medium for culturing ganoderma strains and application thereof Download PDFInfo
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Description
本發明是關於一種靈芝固態培養基，特別是關於一種添加小麥胚芽之靈芝固態培養基。The invention relates to a solid medium of Ganoderma lucidum, in particular to a solid medium of Ganoderma lucidum added with wheat germ.
隨著社會變遷，生活步調日趨緊湊，壓力伴隨而生，使得人類罹患憂鬱症及失眠症狀之比例逐漸提高，影響人類生活品質及健康甚鉅。With the changes of society, the pace of life is becoming more and more compact, and the pressure is accompanied. The proportion of people suffering from depression and insomnia is gradually increasing, which affects the quality of life and health of human beings.
γ-胺基丁酸(gamma-aminobutyric acid，簡稱GABA)為哺乳類動物的抑制性神經傳導物質，具有抗憂鬱、改善失眠、改善自律神經系統功能失調問題等多種生理活性。已知許多微生物中都含有γ-胺基丁酸，因此，可藉由食用或飲用發酵食品，攝取足量的γ-胺基丁酸，達到抗憂鬱症及改善失眠症狀的效果。Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in mammals. It has many physiological activities such as anti-depression, improving insomnia, and improving autonomic nervous system dysfunction. Many microorganisms are known to contain γ-aminobutyric acid, and therefore, a sufficient amount of γ-aminobutyric acid can be ingested by eating or drinking a fermented food to achieve anti-depression and an effect of improving symptoms of insomnia.
雖然現今可透過化學方法合成γ-胺基丁酸，然而化學方法製成的γ-胺基丁酸有食用安全上之疑慮。另一方面，雖然也有利用微生物發酵方式產生天然的γ-胺基丁酸，唯目前之微生物發酵方式仍無法產生高濃度之γ-胺基丁酸，使得發酵食品、飲品之抗憂鬱、抗失眠效果有限，亟待開發一種能產生高γ-胺基丁酸含量之微生物培養基及其發酵方法。Although gamma-aminobutyric acid can be chemically synthesized today, gamma-aminobutyric acid produced by chemical methods has safety concerns. On the other hand, although natural γ-aminobutyric acid is produced by microbial fermentation, the current microbial fermentation method still cannot produce high concentration of γ-aminobutyric acid, which makes anti-depression and anti-insomnia of fermented foods and drinks. The effect is limited, and it is urgent to develop a microbial culture medium capable of producing high γ-aminobutyric acid content and a fermentation method thereof.
鑑於習知技術的缺陷，本發明提供一種靈芝固態培養基，經靈芝發酵代謝後可產生大量的γ-胺基丁酸。In view of the defects of the prior art, the present invention provides a solid medium of Ganoderma lucidum which can be fermented by Ganoderma lucidum to produce a large amount of γ-aminobutyric acid.
本發明又提供一種靈芝固態培養基之用途，係用於發酵產生大量的γ-胺基丁酸。The invention further provides a use of a solid medium of Ganoderma lucidum for fermentation to produce a large amount of γ-aminobutyric acid.
本發明再提供一種靈芝固態培養基之用途，係用於產生富含γ-胺基丁酸之食品。The invention further provides a use of a solid medium of Ganoderma lucidum for producing a food rich in γ-aminobutyric acid.
本發明再提供一種生產大量高濃度γ-胺基丁酸之方法，使每公克靈芝固態培養基至少包括20毫克γ -胺基丁酸。The present invention further provides a method for producing a large amount of high-concentration γ-aminobutyric acid, which comprises at least 20 mg of γ -aminobutyric acid per gram of Ganoderma lucidum solid medium.
於一較佳實施例中，本發明提供一種靈芝固態培養基，係以複數製備步驟所製成之靈芝固態培養基，該複數製備步驟包括：(a)將複數種培養基成分混合，得到一培養基成分混合物，該培養基成分混合物中包括：小麥胚芽，重量百分濃度為10%~60%；以及水，重量百分濃度為20%~50%；以及(b)經高溫及滅菌處理，製得該靈芝固態培養基，其中，該高溫係指100℃~150℃。In a preferred embodiment, the present invention provides a solid medium of Ganoderma lucidum, which is a solid medium of Ganoderma lucidum prepared by a plurality of preparation steps, wherein the plurality of preparation steps comprises: (a) mixing a plurality of medium components to obtain a mixture of medium components. The medium component mixture includes: wheat germ, 10% to 60% by weight; and water, 20% to 50% by weight; and (b) high temperature and sterilization treatment to obtain the ganoderma lucidum A solid medium, wherein the high temperature means 100 ° C to 150 ° C.
於一較佳實施例中，該培養基成分混合物中，小麥胚芽之重量百分濃度為30%~40%。In a preferred embodiment, the concentration of the wheat germ in the medium component mixture is 30% to 40% by weight.
於一較佳實施例中，該培養基成分混合物中更包含胚芽米，且該胚芽米之重量百分濃度為10%~40%。In a preferred embodiment, the medium component mixture further comprises germ rice, and the concentration of the germ rice is 10% to 40% by weight.
於一較佳實施例中，該培養基成分混合物中，小麥胚芽之重量百分濃度為30%~40%，胚芽米之重量百分濃度為10%~20%，且水之重量百分濃度為50%。In a preferred embodiment, the concentration of the wheat germ is 30% to 40%, the weight percentage of the germ rice is 10% to 20%, and the weight concentration of the water is 50%.
於一較佳實施例中，該培養基成分混合物中更包含酪蛋白(casein)、大豆蛋白、或大豆。In a preferred embodiment, the medium component mixture further comprises casein, soy protein, or soybean.
於再一較佳實施例中，本發明提供一種靈芝固態培養基，係以複數製備步驟所製成之靈芝固態培養基，該複數製備步驟包括：(a)將複數種培養基成分混合，得到一培養基成分混合物，該培養基成分混合物中包括：小麥胚芽；以及水；以及(b)經高溫及滅菌處理，製得該靈芝固態培養基，其中，該高溫係指100℃~150℃；步驟(a)中，小麥胚芽之成分包括麥膠蛋白，且該培養基成分混合物中之麥膠蛋白之重量百分濃度為0.4%~2.6%。In a further preferred embodiment, the present invention provides a solid medium of Ganoderma lucidum, which is a solid medium prepared from a plurality of preparation steps, wherein the plurality of preparation steps comprises: (a) mixing a plurality of medium components to obtain a medium component. a mixture, the medium component mixture includes: wheat germ; and water; and (b) the high temperature and sterilization treatment to obtain the ganoderma lucidum solid medium, wherein the high temperature means 100 ° C ~ 150 ° C; in step (a), The composition of the wheat germ includes gliadin, and the concentration of the gliadin in the mixture of the components of the medium is 0.4% to 2.6%.
於另一較佳實施例中，本發明提供一種靈芝固態培養基，係以複數製備步驟所製成之靈芝固態培養基，該複數製備步驟包括：(a)將複數種培養基成分混合，得到一培養基成分混合物，該培養基成分混合物中包括：小麥胚芽；以及水；以及(b)經高溫及滅菌處理，製得該靈芝固態培養基，其中，該高溫 係指100℃~150℃；步驟(a)中，小麥胚芽之成分包括小麥榖蛋白，且該培養基成分混合物中之小麥榖蛋白之重量百分濃度為0.007%~0.07%。In another preferred embodiment, the present invention provides a solid medium of Ganoderma lucidum, which is a solid medium prepared from a plurality of preparation steps, wherein the plurality of preparation steps comprises: (a) mixing a plurality of medium components to obtain a medium component. a mixture, the medium component mixture includes: wheat germ; and water; and (b) high temperature and sterilization treatment to obtain the ganoderma lucidum solid medium, wherein the high temperature Refers to 100 ° C ~ 150 ° C; in step (a), the components of wheat germ include wheat glutinous protein, and the concentration of wheat gluten in the mixture of the components of the medium is 0.007% to 0.07% by weight.
於一較佳實施例中，該步驟(b)中，係在121℃作用至少15分鐘，以同時達到高溫蒸煮及滅菌效果。In a preferred embodiment, in step (b), it is applied at 121 ° C for at least 15 minutes to simultaneously achieve high temperature cooking and sterilization.
於一較佳實施例中，其係用於培養靈芝以產生高濃度之γ -胺基丁酸；其中，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少產生20毫克γ -胺基丁酸。In a preferred embodiment, it is used to culture Ganoderma lucidum to produce a high concentration of γ -aminobutyric acid; wherein, the high concentration of γ -aminobutyric acid means at least 20 mg of γ -amine per gram of Ganoderma lucidum solid medium. Butyric acid.
於一較佳實施例中，其係用於培養靈芝以生產含高濃度γ -胺基丁酸之食品；其中，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少包含20毫克γ -胺基丁酸。In a preferred embodiment, it is used for cultivating Ganoderma lucidum to produce a food containing a high concentration of γ -aminobutyric acid; wherein a high concentration of γ -aminobutyric acid means at least 20 mg γ per gram of Ganoderma lucidum solid medium. - Aminobutyric acid.
於又一較佳實施例中，本發明提供一種生產γ -胺基丁酸的方法，步驟包括：In still another preferred embodiment, the present invention provides a method of producing γ -aminobutyric acid, the steps comprising:
(a)將複數種培養基成分混合，得到一培養基成分混合物，該培養基成分混合物中包括：小麥胚芽，重量百分濃度為10%~60%；以及水，重量百分濃度為20%~50%；以及(a) mixing a plurality of medium components to obtain a medium component mixture comprising: wheat germ, 10% to 60% by weight; and water, 20% to 50% by weight ;as well as
(b)經高溫及滅菌處理，製得一靈芝固態培養基，其中，該高溫係指100℃~150℃。(b) A high temperature medium and a sterilization process are used to prepare a solid medium of Ganoderma lucidum, wherein the high temperature means 100 ° C to 150 ° C.
(c)加入靈芝菌株，發酵生產γ -胺基丁酸。(c) Adding a Ganoderma lucidum strain to produce γ -aminobutyric acid by fermentation.
於又一較佳實施例中，經步驟(c)發酵後，每公克該靈芝固態培養基至少包括20毫克γ -胺基丁酸。In still another preferred embodiment, after the fermentation in step (c), the ganoderma lucidum solid medium comprises at least 20 mg of γ -aminobutyric acid per gram.
圖1A：於30℃條件下，以不同米飯固態培養基底培養A靈芝菌株7天所得之菌絲量。Fig. 1A: The amount of hyphae obtained by culturing A strain of Ganoderma lucidum for 7 days under different conditions of solid rice medium at 30 °C.
圖1B：於30℃條件下，以不同米飯固態培養基底培養C靈芝菌株7天所得之菌絲量。Fig. 1B: The amount of hyphae obtained by culturing C ganoderma lucidum strains for 7 days under different rice solid medium cultures at 30 °C.
圖2A：胚芽米飯固態培養基底添加不同濃度氮源後，於30℃條件下培養A靈芝菌株7天所得之菌絲量。Fig. 2A: The amount of mycelium obtained by culturing A strain of Ganoderma lucidum for 7 days at 30 ° C after adding different concentrations of nitrogen source to the bottom of the solid medium of the germ rice.
圖2B：胚芽米飯固態培養基底添加不同濃度氮源後，於30℃條件下培養C靈芝菌株7天所得之菌絲量。Fig. 2B: The amount of hyphae obtained by culturing C ganoderma lucidum strain at 7 ° C for 7 days after adding different concentrations of nitrogen source to the bottom of the solid medium of the germ rice.
圖3A：胚芽米飯固態培養基底添加1%麩胺酸鈉後，於30℃條件下培養靈芝菌株7天所得之菌絲量。Fig. 3A: The amount of mycelium obtained by culturing Ganoderma lucidum strain at 7 ° C for 7 days after adding 1% glutamate at the bottom of the solid medium of the germ rice.
圖3B：胚芽米飯固態培養基底添加1%麩胺酸鈉後，於30℃條件下培養靈芝菌株7天所得之γ-胺基丁酸產量。Fig. 3B: γ-aminobutyric acid yield obtained by culturing Ganoderma lucidum strain for 7 days at 30 ° C after adding 1% glutamate at the bottom of the solid medium of the germ rice.
圖4：將不同比例之胚芽米及小麥胚芽混合後，所製成的靈芝固態培養基之γ-胺基丁酸含量。Figure 4: γ-aminobutyric acid content of the manufactured Ganoderma lucidum solid medium after mixing different proportions of germ rice and wheat germ.
圖5A：於30℃條件下，以不同比例之胚芽米及小麥胚芽培養A靈芝菌株7天所得之菌絲量。Fig. 5A: The amount of hyphae obtained by culturing A ganoderma lucidum strains in different proportions of germ rice and wheat germ at 7 ° C for 7 days.
圖5B：於30℃條件下，以不同比例之胚芽米及小麥胚芽培養A靈芝菌株7天所得之γ-胺基丁酸量。Fig. 5B: The amount of γ-aminobutyric acid obtained by culturing A ganoderma lucidum strains at different ratios of germ rice and wheat germ for 7 days at 30 °C.
圖6A：於30℃條件下，以不同比例之胚芽米及小麥胚芽培養C靈芝菌株7天所得之菌絲量。Fig. 6A: The amount of hyphae obtained by culturing C. ganoderma strains in different proportions of germ rice and wheat germ at 7 °C for 7 days.
圖6B：於30℃條件下，以不同比例之胚芽米及小麥胚芽培 養C靈芝菌株7天所得之γ-胺基丁酸量。Figure 6B: Different ratios of germ rice and wheat germ culture at 30 °C The amount of γ-aminobutyric acid obtained by raising C strain of Ganoderma lucidum for 7 days.
圖7：於32℃條件下，以第2靈芝固態培養基(含20%之小麥 胚芽、50%之水、以及30%之胚芽米)培養A靈芝菌株21天所得之菌絲量以及γ-胺基丁酸量。Figure 7: 2nd Ganoderma lucidum solid medium (containing 20% wheat) at 32 °C Germ, 50% water, and 30% germ rice) The amount of mycelium obtained by culturing A strain of Ganoderma lucidum for 21 days and the amount of γ-aminobutyric acid.
圖8A：於第2靈芝固態培養基(含20%之小麥胚芽、50%之 水、以及30%之胚芽米)中添加0.1%不同氮源後，在30℃條件下培養A靈芝菌株7天所得之菌絲量。Figure 8A: In the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% After adding 0.1% of different nitrogen sources to water and 30% of germ rice, the amount of mycelium obtained by culturing A strain of Ganoderma lucidum for 7 days at 30 ° C was obtained.
圖8B：於第2靈芝固態培養基(含20%之小麥胚芽、50%之 水、以及30%之胚芽米)中添加0.1%不同氮源後，在30℃條件下培養A靈芝菌株7天所得之γ-胺基丁酸量。Figure 8B: In the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% The amount of γ-aminobutyric acid obtained by culturing A ganoderma lucidum strain for 7 days at 30 ° C after adding 0.1% of different nitrogen sources to water and 30% of germ rice.
圖9A：於第2靈芝固態培養基(含20%之小麥胚芽、50%之 水、以及30%之胚芽米)中添加0.1%不同氮源後，在30℃條件下培養C靈芝菌株7天所得之菌絲量。Figure 9A: In the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% After adding 0.1% of different nitrogen sources to water and 30% of germ rice, the amount of mycelium obtained by culturing C ganoderma lucidum strain at 7 ° C for 7 days was obtained.
圖9B：於第2靈芝固態培養基(含20%之小麥胚芽、50%之 水、以及30%之胚芽米)中添加0.1%不同氮源後，在30℃條件下培養C靈芝菌株7天所得之γ-胺基丁酸量。Figure 9B: In the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% The amount of γ-aminobutyric acid obtained by culturing C ganoderma lucidum strain for 7 days at 30 ° C after adding 0.1% of different nitrogen sources to water and 30% of germ rice.
鑒於習知技術的缺陷，發明人依據多年的研究成果，認為應能發展出一種靈芝固態培養基，經靈芝發酵產生高濃度之γ -胺基丁酸。In view of the shortcomings of the prior art, the inventors believe that based on the research results of many years, it is believed that a solid medium of Ganoderma lucidum can be developed, and a high concentration of γ -aminobutyric acid is produced by fermentation of Ganoderma lucidum.
以下係利用本發明之實施例之詳細說明書，以及本發明之技術、特點。然本實施例並非用以限定本發明，任何熟悉此技術者，在不脫 離本發明之精神和範圍內所作之各種更動、潤飾，均應包含在本發明之申請專利範圍內。The following is a detailed description of embodiments of the invention, as well as the techniques and features of the invention. However, the present embodiment is not intended to limit the present invention, and any person familiar with the technology does not take off. Various changes and modifications made within the spirit and scope of the present invention are intended to be included in the scope of the present invention.
靈芝(Ganoderma spp.)為靈芝屬的生物，係一年生白腐型真 菌，主要分佈於熱帶、亞熱帶及溫帶。目前全世界約有150~200種天然靈芝被鑑定發表，台灣有17種，包括G.australe ，G.tropicum ，G.weberianum ，G.boninense ，G.lucidum (赤芝)，G.japonicum (紫芝)，與G.formosana 。市面上所販售之靈芝，以G.lucidum (赤芝)為最大宗。 Ganoderma spp. is a genus of Ganoderma lucidum, an annual white rot fungus mainly distributed in the tropics, subtropics and temperate zones. At present, there are about 150 to 200 kinds of natural ganoderma lucidum in the world, and there are 17 species in Taiwan, including G.australe , G.tropicum , G.weberianum , G.boninense , G.lucidum (Ganoderma lucidum), G.japonicum ( Zizhizhi ). With G.formosana . Ganoderma lucidum sold in the market is dominated by G. lucidum (Ganoderma lucidum).
靈芝子實體內或菌絲體內的成分具有降血糖、降血壓、抗氧化、抗腫瘤、抗凝血、免疫調節、護肝等生理活性，菌絲體內的生理活性物質包括：多醣(polysaccharide)、三萜類(triterpenoids)、超氧化歧化酶(superoxide dismutase,SOD)、蛋白質(protein)、鍺金屬(germanium)、腺苷(adenosine)、以及類固醇(steroid)。The components in the fruit body of the ganoderma lucidum or the mycelium have physiological activities such as blood sugar lowering, blood pressure lowering, anti-oxidation, anti-tumor, anti-coagulation, immune regulation, liver protection, etc. The physiological active substances in the mycelium include: polysaccharide (polysaccharide), Triterpenoids, superoxide dismutase (SOD), protein, germanium, adenosine, and steroid.
本發明所使用的二株靈芝屬菌株A、C在分類上均為赤芝(Ganoderma lucidum )，且均購自新竹食品工業發展研究所生物資源保存及研究中心。依專利法之相關規定，不需寄存(A菌株產品編號BCRC36111；C菌株產品編號BCRC36674)。亦可使用其他靈芝菌株，例如食品工業發展研究所生物資源保存及研究中心中的其他靈芝菌株，但不以此為限。The two strains of Ganoderma lucidum strains A and C used in the present invention are all classified as Ganoderma lucidum , and are purchased from the Bioresource Conservation and Research Center of Hsinchu Food Industry Development Research Institute. According to the relevant provisions of the Patent Law, no deposit is required (A strain product number BCRC36111; C strain product number BCRC36674). Other Ganoderma lucidum strains, such as other Ganoderma lucidum strains in the Center for Bioresource Conservation and Research of the Food Industry Development Institute, may also be used, but are not limited thereto.
稻屬(Oryza )有24個物種(species)，包括2個栽培種和22個野生種，分布於全球的熱帶與亞熱帶地區。栽培種包括水稻(Oryza sativa )及西非栽培稻(Oryza glaberrima )。水稻及西非栽培稻的種子(seed)，即一般所稱的稻穀(rice)。其中，水稻又包括秈稻(Oryza sativa indica )以及粳稻 (Oryza sativa japonica )等不同亞種。 Oryza has 24 species, including 2 cultivars and 22 wild species, distributed in tropical and subtropical regions of the world. The cultivars include rice ( Oryza sativa ) and West African cultivated rice ( Oryza glaberrima ). Seeds of rice and West African cultivated rice, commonly known as rice. Among them, rice includes different subspecies such as indica ( Oryza sativa indica ) and indica ( Oryza sativa japonica ).
稻穀經加工脫去穀殼後即得糙米(brown rice)，本發明之糙米購自愛買(產品名稱：大地有機糙米；廠牌：池上，台灣)，但不以此為限。糙米碾去米糠層且保留住胚芽者，即為胚芽米(embryo rice)，本發明之胚芽米購自愛買(產品名稱：胚芽米；廠牌：中興米，台灣)，但不以此為限。The rice is processed to remove the chaff and then the brown rice is obtained. The brown rice of the present invention is purchased from Aibu (product name: organic organic brown rice; brand: Chihshang, Taiwan), but not limited thereto. The brown rice is ground to remove the rice bran layer and retain the germ, which is the embryo rice. The germ rice of the present invention is purchased from Aibu (product name: germ rice; label: Zhongxing Rice, Taiwan), but not limited thereto. .
日本粳稻在台灣改良後得一粳稻新品系，其稻穀經加工脫去穀殼、米糠層、胚芽後所得之白米，稱為蓬萊米(Pon Lai rice，也稱為Japonica rice)。本發明之蓬萊米愛買(產品名稱：三好米；廠牌：三好米，台灣)，但不以此為限。Japanese japonica rice is improved in Taiwan and has a new line of japonica rice. The white rice obtained by processing the rice, the rice bran layer and the germ after processing is called Pon Lai rice (also known as Japonica rice). The Penglai Mi Aibu of the invention (product name: three good rice; label: three good rice, Taiwan), but not limited to this.
秈稻之稻穀經加工脫去穀殼、米糠層、胚芽後所得之白米，稱為秈米(indica rice)。本發明之秈米購自東里碾米工廠(產品名稱：御皇長秈米；廠牌：御皇米，台灣)，但不以此為限。The white rice obtained by processing the rice in the indica rice and removing the chaff, rice bran layer and germ is called indica rice. The glutinous rice of the invention is purchased from the Dongli rice milling factory (product name: Yuhuangchang glutinous rice; brand: Yuhuangmi, Taiwan), but not limited thereto.
本發明使用之酪蛋白(casein)係購自Sigma-Aldrich公司(Sigma-Aldrich,Saint Louis,MO,U.S.A.)，大豆蛋白(soy protein)係購自中美聯合實業股份有限公司(台灣)，大豆(soybean)係購自愛買(台灣)，麩胺酸鈉(monosodium glutamate，簡稱MSG)係購自Sigma-Aldrich公司(Sigma-Aldrich,Saint Louis,MO,U.S.A.)，但不以此為限。The casein used in the present invention was purchased from Sigma-Aldrich (Sigma-Aldrich, Saint Louis, MO, USA), and the soy protein was purchased from Sino-US Union Industrial Co., Ltd. (Taiwan), soybean. (Soybean) was purchased from Aibu (Taiwan), and monosodium glutamate (MSG) was purchased from Sigma-Aldrich (Sigma-Aldrich, Saint Louis, MO, USA), but not limited thereto.
實驗一：各種米飯固態培養基底對靈芝生長的影響Experiment 1: Effect of the solid medium of various rice on the growth of Ganoderma lucidum
(一)製備米飯固態培養基底(1) Preparation of rice solid medium bottom
將40g蓬萊米、40g秈米、40g糙米、或40g胚芽米分別與40mL之一次水混合，使該蓬萊米、秈米、糙米、或胚芽米之重量百分濃度為 50%，放入醬菜瓶(指含金屬蓋的玻璃瓶)中，經高溫及滅菌處理後，分別製得蓬萊米飯固態培養基底、秈米飯固態培養基底、糙米飯固態培養基底、或胚芽米飯固態培養基底。Mix 40g of porphyria, 40g of glutinous rice, 40g of brown rice, or 40g of germ rice with 40mL of primary water, respectively, so that the weight percentage of the plenum, glutinous rice, brown rice, or germ rice is 50%, placed in a pickles bottle (referring to a glass bottle containing a metal lid), after high temperature and sterilization treatment, respectively, to obtain a solid medium bottom of Penglai rice, a solid medium bottom of glutinous rice, a solid medium bottom of brown rice, or a solid of germ rice The substrate is cultured.
其中，該高溫係指100℃~150℃。較佳者，該高溫係指 121℃。較佳者，在121℃條件下作用至少15分鐘，以同時達到高溫蒸煮及滅菌效果。於本案最佳實施例中，係於121℃作用20分鐘，以同時達到高溫蒸煮及滅菌效果。The high temperature refers to 100 ° C to 150 ° C. Preferably, the high temperature means 121 ° C. Preferably, it is allowed to act at 121 ° C for at least 15 minutes to simultaneously achieve high temperature cooking and sterilization. In the preferred embodiment of the present invention, it is applied at 121 ° C for 20 minutes to simultaneously achieve high temperature cooking and sterilization effects.
(二)接種靈芝菌株，並觀測菌絲生長情形(2) Inoculation of Ganoderma lucidum strains and observation of mycelial growth
分別在蓬萊米飯固態培養基底、秈米飯固態培養基底、糙米 飯固態培養基底、或胚芽米飯固態培養基底中接種A靈芝菌株或C靈芝菌株，於30℃避光靜置培養7天，得一蓬萊米飯暨菌絲混合物、一秈米飯暨菌絲混合物、一糙米飯暨菌絲混合物、或一胚芽米飯暨菌絲混合物(以下統稱為米飯暨菌絲混合物)。測量各種米飯暨菌絲混合物中的靈芝菌絲量。In the bottom of the solid medium of Penglai rice, the bottom of the solid medium of glutinous rice, brown rice The rice solid medium bottom or the germ rice solid medium bottom is inoculated with A ganoderma lucidum strain or C ganoderma lucidum strain, and cultured at 30 ° C for 7 days in the dark, to obtain a Penglai rice and hyphae mixture, a glutinous rice and hyphae mixture, Brown rice and hyphae mixture, or a mixture of germ rice and hyphae (hereinafter collectively referred to as rice and hyphae mixture). The amount of Ganoderma lucidum mycelium in various rice and hyphae mixtures was measured.
發明人係偵測米飯暨菌絲混合物中的麥角固醇(egosterol)含量，以間接獲得靈芝菌絲量，步驟包括：The inventor detects the content of ergosterol in the mixture of rice and hyphae to indirectly obtain the amount of ganoderma lucidum, and the steps include:
A、製作靈芝菌絲體標準檢量線A. Making Ganoderma lucidum mycelium standard calibration curve
在液體培養基中接種A靈芝菌株或C靈芝菌株，以30℃、110rpm條件震盪培養7天，離心收集菌絲體，再清洗、凍乾。其中，該液體培養基可以是例如馬鈴薯葡萄糖培養基(產品名稱，Potato Dextrose Broth，PDB；廠牌BD)，但不以此為限。接下來，以無水酒精將凍乾菌絲體粉末配成10、25、50、及100mg/L菌絲粉末液，於室溫萃取1小時。離心(8000×g、10min)，收集上清液。以無水酒精定量至5mL，經0.22μm濾膜過濾，再 利用高效能液相層析法(HPLC)分析麥角固醇含量。最後，利用菌絲粉末液之菌絲濃度與麥角固醇含量關係，製作出靈芝菌絲體標準檢量線。A strain of Ganoderma lucidum or C. ganoderma lucidum strain was inoculated in a liquid medium, cultured at 30 ° C, 110 rpm for 7 days, and the mycelium was collected by centrifugation, washed, and lyophilized. The liquid medium may be, for example, potato dextrose medium (product name, Potato Dextrose Broth, PDB; brand BD), but is not limited thereto. Next, the freeze-dried mycelium powder was formulated into 10, 25, 50, and 100 mg/L mycelium powders with absolute alcohol, and extracted at room temperature for 1 hour. The supernatant was collected by centrifugation (8000 x g, 10 min). Quantify to 5mL with absolute alcohol, filter through 0.22μm filter, and then The ergosterol content was analyzed by high performance liquid chromatography (HPLC). Finally, using the relationship between the mycelial concentration of mycelial powder and the ergosterol content, a standard calibration curve for Ganoderma lucidum mycelium was prepared.
B、測量米飯暨菌絲混合物中的靈芝菌絲量B. Measuring the amount of Ganoderma lucidum mycelium in the mixture of rice and hyphae
將米飯暨菌絲混合物凍乾後，添加1g凍乾樣品至2mL無水酒精中，於室溫萃取1小時。離心(8000×g、10min)，收集上清液。以無水酒精定量至5mL，經0.22μm濾膜過濾，再利用高效能液相層析法分析麥角固醇含量。利用靈芝菌絲體標準檢量線將麥角固醇量換算為菌絲體量。After the rice and hyphae mixture was lyophilized, 1 g of the lyophilized sample was added to 2 mL of absolute alcohol, and extracted at room temperature for 1 hour. The supernatant was collected by centrifugation (8000 x g, 10 min). The residue was diluted to 5 mL with absolute alcohol, filtered through a 0.22 μm filter, and the ergosterol content was analyzed by high performance liquid chromatography. The amount of ergosterol is converted into the amount of mycelium by using the Ganoderma lucidum mycelium standard calibration curve.
請參見圖1A與表一。圖1A係於30℃條件下，以不同米飯固態培養基底培養A靈芝菌株7天所得之菌絲量。橫座標由左至右分別為胚芽米飯固態培養基底、糙米飯固態培養基底、蓬萊米飯固態培養基底及秈米飯固態培養基底。縱座標則為菌絲量。若兩組間之字母相同時，表示兩組間不具有顯著差異；反之則具有顯著差異。圖1A中，A靈芝菌株培養於每克胚芽米飯固態培養基底中所得之菌絲量為0.242±0.060克、培養於每克糙米飯固態培養基底中所得之菌絲量為0.143±0.006克、培養於每克蓬萊米飯固態培養基底中所得之菌絲量為0.099±0.001克、培養於每克秈米飯固態培養基底中所得之菌絲量為0.139±0.003克。其中，A靈芝菌株培養於胚芽米飯固態培養基底可獲得最高之菌絲量。See Figure 1A and Table 1. Fig. 1A shows the amount of hyphae obtained by culturing A strain of Ganoderma lucidum for 7 days under different conditions of solid rice medium at 30 °C. The horizontal coordinates from left to right are the solid medium bottom of the germ rice, the solid medium bottom of the brown rice, the bottom of the solid medium of the Penglai rice, and the bottom of the solid medium of the glutinous rice. The ordinate is the amount of hyphae. If the letters between the two groups are the same, it means that there is no significant difference between the two groups; otherwise, there is a significant difference. In Fig. 1A, the amount of hyphae obtained by culturing A strain of Ganoderma lucidum in the bottom of solid medium of gram of germ rice is 0.242±0.060 g, and the amount of hyphae obtained by culturing in the bottom of solid medium per gram of brown rice is 0.143±0.006 g, and culture is carried out. The amount of hyphae obtained in the bottom of the solid medium per gram of lentil rice was 0.099±0.001 g, and the amount of hyphae obtained in the bottom of the solid medium per gram of glutinous rice was 0.139±0.003 g. Among them, the A ganoderma lucidum strain is cultured on the bottom of the solid medium of the germ rice to obtain the highest amount of mycelium.
請參見圖1B與表一。圖1B係於30℃條件下，以不同米飯固態培養基底培養C靈芝菌株7天所得之菌絲量。橫座標由左至右分別為胚芽米飯固態培養基底、糙米飯固態培養基底、蓬萊米飯固態培養基底及秈米飯固態培養基底。縱座標則為菌絲量。若兩組間之字母相同時，表示兩組間不具有顯著差異；反之則具有顯著差異。圖1B中，將C靈芝菌株培養於每 克胚芽米飯固態培養基底中所得之菌絲量為0.181±0.007克、培養於每克糙米飯固態培養基底中所得之菌絲量為0.170±0.006克、培養於每克蓬萊米飯固態培養基底中所得之菌絲量為0.022±0.003克、培養於每克秈米飯固態培養基底中所得之菌絲量為0.064±0.004克。其中，C靈芝菌株培養於胚芽米飯固態培養基底可獲得最高之菌絲量。See Figure 1B and Table 1. Fig. 1B shows the amount of hyphae obtained by culturing C ganoderma lucidum strains for 7 days under different rice solid medium cultures at 30 °C. The horizontal coordinates from left to right are the solid medium bottom of the germ rice, the solid medium bottom of the brown rice, the bottom of the solid medium of the Penglai rice, and the bottom of the solid medium of the glutinous rice. The ordinate is the amount of hyphae. If the letters between the two groups are the same, it means that there is no significant difference between the two groups; otherwise, there is a significant difference. In Figure 1B, C strain of Ganoderma lucidum is cultured in each The amount of hyphae obtained in the bottom of the solid medium of the gram germ rice is 0.181±0.007 g, and the amount of the hyphae obtained in the bottom of the solid medium per gram of brown rice is 0.170±0.006 g, and is cultivated in the bottom of the solid medium per gram of porphyria rice. The amount of hyphae was 0.022±0.003 g, and the amount of hyphae cultured in the bottom of the solid medium per gram of glutinous rice was 0.064±0.004 g. Among them, the C ganoderma lucidum strain is cultured on the bottom of the solid medium of the germ rice to obtain the highest amount of mycelium.
圖1A、1B與表一之實驗結果顯示，A或C靈芝菌株培養於胚芽米飯固態培養基底時，均可獲得最高之菌絲量，因此將以胚芽米飯固態培養基底為基礎，進行後續實驗。The experimental results of Figs. 1A, 1B and Table 1 show that the highest mycelial amount can be obtained when the A or C Ganoderma lucidum strain is cultured at the bottom of the solid medium of the germ rice, so the subsequent experiment will be carried out based on the solid medium of the germ rice.
上述各個實驗均進行三重覆，數據表示方式為平均值±標準差。Each of the above experiments was triple-covered, and the data representation was mean ± standard deviation.
實驗二：胚芽米飯固態培養基底添加不同氮源對靈芝菌株生長的影響Experiment 2: Effect of different nitrogen sources on the growth of Ganoderma lucidum strains in the solid medium of germ rice
(一)製備內含不同氮源之胚芽米飯固態培養基(1) Preparing a solid medium for germ rice containing different nitrogen sources
將40g胚芽米與40mL之一次水混合，並添加重量百分濃度為0.1%或1%之不同氮源，放入醬菜瓶中，經高溫及滅菌處理後，製得內含 不同氮源之胚芽米飯固態培養基。Mix 40g of germ rice with 40mL of primary water, add 0.1% or 1% by weight of different nitrogen sources, put in the pickles bottle, and after high temperature and sterilization, make the inclusion Germ rice solid medium with different nitrogen sources.
於本案最佳實施例中，係於121℃作用20分鐘，以同時達到高溫蒸煮及滅菌效果。In the preferred embodiment of the present invention, it is applied at 121 ° C for 20 minutes to simultaneously achieve high temperature cooking and sterilization effects.
其中，不同氮源係指酪蛋白(casein)、大豆蛋白(soy protein)或大豆(soybean)。Among them, different nitrogen sources refer to casein, soy protein or soybean.
(二)接種靈芝菌株A或C，步驟、方法均與實驗一相同。(2) Inoculation of Ganoderma lucidum strain A or C, the steps and methods are the same as in Experiment 1.
請參見圖2A，係胚芽米飯固態培養基底添加不同濃度氮源後，於30℃條件下培養A靈芝菌株7天所得之菌絲量。橫座標由左至右分別為控制組(無添加任何蛋白質)、酪蛋白組(添加0.1%或1%之酪蛋白)、大豆蛋白組(添加0.1%或1%之大豆蛋白)及大豆組(添加0.1%或1%之大豆)。縱座標則為菌絲量。圖2A顯示，將A靈芝菌株培養於內含不同蛋白質之胚芽米飯固態培養基7天後，A靈芝菌株之生長情形並未顯著增加。Please refer to FIG. 2A , which is a mycelial amount obtained by culturing A strain of Ganoderma lucidum for 7 days at 30 ° C after adding different nitrogen sources at the bottom of the solid medium of the germ rice. The horizontal coordinates from left to right are control group (no protein added), casein group (addition of 0.1% or 1% casein), soy protein group (addition of 0.1% or 1% soy protein) and soybean group ( Add 0.1% or 1% soy). The ordinate is the amount of hyphae. 2A shows that the growth condition of the A ganoderma lucidum strain was not significantly increased after the A-Ganoderma lucidum strain was cultured for 7 days in the solid medium containing the different protein.
請參見圖2B，係胚芽米飯固態培養基底添加不同濃度氮源後，於30℃條件下培養C靈芝菌株7天所得之菌絲量。橫座標及縱座標均與圖2A相同。圖2B顯示，C靈芝菌株培養於內含0.1%不同蛋白質之胚芽米飯固態培養基7天後，每公克米飯暨菌絲體混合物內含之菌絲量皆顯著高於控制組，其中又以培養於添加0.1%酪蛋白之培養基所得之菌絲量最高，為0.323±0.004克(控制組之1.76倍)。此外，添加1%不會使C靈芝菌株之菌絲量顯著增加。Please refer to FIG. 2B , which is a mycelial amount obtained by culturing C ganoderma lucidum strain at 7 ° C for 7 days after adding different nitrogen sources at the bottom of the solid medium of the germ rice. Both the abscissa and the ordinate are the same as in Fig. 2A. 2B shows that after the G. lucidum strain cultured in the solid medium of the germ rice containing 0.1% different proteins for 7 days, the amount of mycelium contained per gram of rice and mycelium mixture was significantly higher than that of the control group, and The amount of hyphae obtained from the medium supplemented with 0.1% casein was the highest, 0.323 ± 0.004 g (1.76 times the control group). In addition, the addition of 1% did not significantly increase the amount of hyphae of the C. ganoderma strain.
實驗三：胚芽米飯固態培養基底添加麸氨酸納對靈芝菌株生長及γ -胺基丁酸產量的影響Experiment 3: Effect of adding glutamic acid sodium on the growth of Ganoderma lucidum strain and the yield of γ -aminobutyric acid in the solid medium of germ rice
麸氨酸(glutamate)為良好的胺基酸氮源，故探討麸氨酸鈉(monosodium glutamate,MSG)是否可提升菌絲體及γ -胺基丁酸之產量。Glutamate is a good source of amino acid nitrogen, so whether the monosodium glutamate (MSG) can increase the production of mycelium and γ -aminobutyric acid.
(一)製備內含1%麸氨酸鈉之胚芽米飯固態培養基(1) Preparing a solid medium for germ rice containing 1% sodium glutamate
將40g胚芽米與40mL之一次水混合，並添加重量百分濃度為1%之麸氨酸鈉，放入醬菜瓶中，經高溫及滅菌處理後，製得內含1%麸氨酸鈉之胚芽米飯固態培養基。Mix 40g of germ rice with 40mL of primary water, add 1% by weight of sodium glutamate, put it into the pickle bottle, and after high temperature and sterilization, obtain 1% sodium glutamate. Germ rice solid medium.
(二)接種靈芝菌株A或C，步驟、方法均與實驗一相同。(2) Inoculation of Ganoderma lucidum strain A or C, the steps and methods are the same as in Experiment 1.
(三)γ -胺基丁酸含量分析(III) Analysis of γ -aminobutyric acid content
將米飯暨菌絲混合物與100mM鹽酸混合，於室溫振盪45分鐘。以孔徑為0.45mm之濾膜過濾，濾液再與o-phthaladehyde反應，以製備γ -胺基丁酸衍生物。以高效能液相層析法搭配UV 345nm波長，分析γ -胺基丁酸衍生物之含量。The rice and hyphae mixture was mixed with 100 mM hydrochloric acid and shaken at room temperature for 45 minutes. The membrane was filtered through a membrane having a pore size of 0.45 mm, and the filtrate was further reacted with o-phthaladehyde to prepare a γ -aminobutyric acid derivative. The content of γ -aminobutyric acid derivatives was analyzed by high performance liquid chromatography with UV 345 nm wavelength.
請參見圖3A，係胚芽米飯固態培養基底添加1%麩胺酸鈉後，於30℃條件下培養靈芝菌株7天所得之菌絲量。圖3A由左至右為A及C靈芝菌株；縱座標則為菌絲量。若兩組間之字母相同時，表示兩組間不具有顯著差異；反之則具有顯著差異。圖3A顯示，將A靈芝菌株培養於內含1%麸氨酸鈉之胚芽米飯固態培養基中或控制組中，兩者所得之菌絲量無顯著差異。將C靈芝菌株培養於內含1%麸氨酸鈉之胚芽米飯固態培養基中或控制組中，兩者所得之菌絲量也無顯著差異。Referring to FIG. 3A, the amount of mycelium obtained by culturing Ganoderma lucidum strain for 7 days at 30 ° C after adding 1% glutamate at the bottom of the solid medium of the germ rice is used. Figure 3A shows A and C Ganoderma lucidum strains from left to right; the ordinate is the amount of hyphae. If the letters between the two groups are the same, it means that there is no significant difference between the two groups; otherwise, there is a significant difference. Fig. 3A shows that the A ganoderma lucidum strain was cultured in a solid medium containing 1% glutamate or in a control group, and the amount of hyphae obtained by the two was not significantly different. The C. lucidum strain was cultured in a solid medium containing 1% glutamate or in a control group, and the amount of hyphae obtained by the two was also not significantly different.
請參見圖3B，係胚芽米飯固態培養基底添加1%麩胺酸鈉後，於30℃條件下 培養靈芝菌株7天所得之γ -胺基丁酸產量。圖3B由左至右為A及C靈芝菌株；縱座標則為γ -胺基丁酸含量。圖3B顯示，將A靈芝菌株培養於內含1%麸氨酸鈉之胚芽米飯固態培養基中，所得之γ -胺基丁酸產量顯著高於控制組(p<0.01)。將C靈芝菌株培養於內含1%麸氨酸鈉之胚芽米飯固態培養基中，所得之γ -胺基丁酸產量也顯著高於控制組(p<0.01)。Referring to FIG. 3B, the γ -aminobutyric acid yield of the Ganoderma lucidum strain was cultured at 30 ° C for 7 days after adding 1% glutamate at the bottom of the solid medium of the germ rice. Figure 3B shows A and C Ganoderma lucidum strains from left to right; the ordinate is γ -aminobutyric acid content. Fig. 3B shows that the A-Ganoderma lucidum strain was cultured in a solid medium containing 1% glutamate in a germ rice, and the yield of γ -aminobutyric acid was significantly higher than that of the control group (p<0.01). The G. ganoderma strain was cultured in a solid medium containing 1% glutamate in the germ rice, and the yield of γ -aminobutyric acid was also significantly higher than that of the control group (p<0.01).
實驗四：小麥胚芽之一般組成分析Experiment 4: Analysis of the general composition of wheat germ
小麥(wheat)係小麥屬(Triticum spp.)植物之穀粒(grain)。小麥胚芽為小麥穀粒中的胚芽(germ)，約佔整個小麥穀粒(whole grain of wheat)的2.5%。Wheat is the grain of the Triticum spp. plant. The wheat germ is the germ in the wheat grain, accounting for about 2.5% of the whole grain of wheat.
小麥胚芽中的蛋白質含量極高，蛋白質含量約為29%~30%。小麥胚芽的蛋白質成分中，球蛋白(globulin)約佔18.9%，麥膠蛋白(Gliadin)約佔14.0%，小麥榖蛋白(Glutenin)約佔0.3%~0.37%。The protein content in wheat germ is extremely high, and the protein content is about 29% to 30%. Among the protein components of wheat germ, globulin accounts for about 18.9%, Gliadin accounts for about 14.0%, and wheat glutenin (Glutenin) accounts for about 0.3% to 0.37%.
本發明使用之小麥胚芽係購自生命海生物科技有限公司(中國山東)。The wheat germ system used in the present invention was purchased from Life Sea Biotechnology Co., Ltd. (Shandong, China).
其中，本發明使用之小麥胚芽是從小麥穀粒切割出胚芽，經磨成粉末、製成片狀、或提煉乾燥而得，然而，並不以此為限。The wheat germ used in the present invention is obtained by cutting a germ from a wheat grain, grinding it into a powder, forming a sheet, or refining and drying. However, it is not limited thereto.
為了瞭解小麥胚芽的一般組成分，以一般常用之A.O.A.C.(1980)方法分析之。In order to understand the general composition of wheat germ, it is analyzed by the commonly used A.O.A.C. (1980) method.
請參閱表二，為本發明所使用之小麥胚芽之一般組成份。表二中，小麥胚芽粉中的水分含量為1.65%、粗蛋白質為29.26%、粗脂肪為7.50%、灰分為5.10%、碳水化合物為56.38%。Please refer to Table 2 for the general composition of the wheat germ used in the present invention. In Table 2, the water content in the wheat germ powder was 1.65%, the crude protein was 29.26%, the crude fat was 7.50%, the ash was 5.10%, and the carbohydrate was 56.38%.
上述各個實驗均進行三重複，數據表示方式為平均值±標準差。Each of the above experiments was performed in triplicate, and the data representation was mean ± standard deviation.
實驗五：製備靈芝固態培養基及其γ -胺基丁酸濃度分析Experiment 5: Preparation of Ganoderma lucidum solid medium and its concentration of γ -aminobutyric acid
(一)製備靈芝固態培養基(1) Preparation of Ganoderma lucidum solid medium
步驟1：將複數種培養基成分混合，得到一第0培養基成分混合物、第1培養基成分混合物、第2培養基成分混合物、第3培養基成分混合物、第4培養基成分混合物或第5培養基成分混合物；以及步驟2：經高溫及滅菌處理，分別製得第0靈芝固態培養基、第1靈芝固態培養基、第2靈芝固態培養基、第3靈芝固態培養基、第4靈芝固態培養基、或第5靈芝固態培養基。其中，該高溫係指100℃~150℃。Step 1: mixing a plurality of medium components to obtain a 0th medium component mixture, a first medium component mixture, a second medium component mixture, a third medium component mixture, a fourth medium component mixture, or a fifth medium component mixture; and a step 2: After the high temperature and sterilization treatment, the 0th Ganoderma lucidum solid medium, the 1st Ganoderma lucidum solid medium, the 2nd Ganoderma lucidum solid medium, the 3rd Ganoderma lucidum solid medium, the 4th Ganoderma lucidum solid medium, or the 5th Ganoderma lucidum solid medium were respectively prepared. The high temperature refers to 100 ° C to 150 ° C.
步驟1中，該第0培養基成分混合物中包括0%之小麥胚芽、50%之水、以及50%之胚芽米。該第1培養基成分混合物中包括10%之小麥胚芽、50%之水、以及40%之胚芽米。該第2培養基成分混合物中包括20% 之小麥胚芽、50%之水、以及30%之胚芽米。該第3培養基成分混合物中包括30%之小麥胚芽、50%之水、以及20%之胚芽米。該第4培養基成分混合物中包括40%之小麥胚芽、50%之水、以及10%之胚芽米。該第5培養基成分混合物中包括50%之小麥胚芽、50%之水、以及50%之胚芽米。上述第0~5培養基成分混合物中各成分之濃度百分比，均為重量百分濃度。In step 1, the 0th medium component mixture includes 0% wheat germ, 50% water, and 50% germ rice. The first medium component mixture includes 10% wheat germ, 50% water, and 40% germ rice. 20% of the second medium ingredient mixture Wheat germ, 50% water, and 30% germ rice. The third medium component mixture includes 30% wheat germ, 50% water, and 20% germ rice. The fourth medium component mixture includes 40% wheat germ, 50% water, and 10% germ rice. The fifth medium component mixture includes 50% wheat germ, 50% water, and 50% germ rice. The concentration percentage of each component in the above 0 to 5 medium component mixture is a weight percent concentration.
步驟2中，係在121℃作用至少15分鐘，以同時達到高溫蒸煮及滅菌效果。In step 2, it is applied at 121 ° C for at least 15 minutes to simultaneously achieve high temperature cooking and sterilization.
再者，步驟2中之第0靈芝固態培養基係第0培養基混合物經高溫及滅菌處理所製得；第1靈芝固態培養基係第1培養基混合物經高溫及滅菌處理所製得；第2靈芝固態培養基係第2培養基混合物經高溫及滅菌處理所製得；第3靈芝固態培養基係第3培養基混合物經高溫及滅菌處理所製得；第4靈芝固態培養基係第4培養基混合物經高溫及滅菌處理所製得；第5靈芝固態培養基係第5混合物經高溫及滅菌處理所製得。Furthermore, the 0th medium of the Ganoderma lucidum solid medium in the step 2 is obtained by high temperature sterilization treatment; the first medium of the Lingzhi solid medium is prepared by high temperature and sterilization; the second solid medium of Ganoderma lucidum The second medium mixture is prepared by high temperature sterilization; the third medium of the Lingzhi solid medium is prepared by high temperature sterilization; the fourth medium of the Lingzhi solid medium is prepared by high temperature and sterilization. The fifth mixture of the 5th Ganoderma lucidum solid medium is prepared by high temperature and sterilization treatment.
(二)γ -胺基丁酸含量分析(II) Analysis of γ -aminobutyric acid content
將靈芝固態培養基與100mM鹽酸混合，於室溫振盪45分鐘。以孔徑為0.45mm之濾膜過濾，濾液再與o-phthaladehyde反應，以製備γ -胺基丁酸衍生物。以高效能液相層析法搭配UV 345nm波長，分析γ -胺基丁酸衍生物之含量。The Ganoderma lucidum solid medium was mixed with 100 mM hydrochloric acid and shaken at room temperature for 45 minutes. The membrane was filtered through a membrane having a pore size of 0.45 mm, and the filtrate was further reacted with o-phthaladehyde to prepare a γ -aminobutyric acid derivative. The content of γ -aminobutyric acid derivatives was analyzed by high performance liquid chromatography with UV 345 nm wavelength.
請參閱圖4，係將不同比例之胚芽米及小麥胚芽混合後，所製成的靈芝固態培養基之γ -胺基丁酸含量。橫座標由左至右依序為：第0靈芝固態培養基、第1靈芝固態培養基、第2靈芝固態培養基、第3靈芝固態培養基、第4靈芝固態培養基、以及第5靈芝固態培養基。縱座標則為該靈 芝固態培養基所含之γ -胺基丁酸濃度。圖4顯示，小麥胚芽之比例越高，該靈芝固態培養基中之γ -胺基丁酸含量越高。其中，未包含小麥胚芽之第0靈芝固態培養基中，每克培養基的γ -胺基丁酸濃度為7.567±0.376毫克；包含50%小麥胚芽之第5靈芝固態培養基中，每克培養基的γ -胺基丁酸濃度為11.379±0.550毫克。Please refer to FIG. 4, which is a γ -aminobutyric acid content of the solid medium of Ganoderma lucidum prepared by mixing different proportions of germ rice and wheat germ. The horizontal coordinates are from left to right: 0th Ganoderma lucidum solid medium, 1st Ganoderma lucidum solid medium, 2nd Ganoderma lucidum solid medium, 3rd Ganoderma lucidum solid medium, 4th Ganoderma lucidum solid medium, and 5th Ganoderma lucidum solid medium. The ordinate is the concentration of γ -aminobutyric acid contained in the Ganoderma lucidum solid medium. Figure 4 shows that the higher the proportion of wheat germ, the higher the content of γ -aminobutyric acid in the solid medium of Ganoderma lucidum. Among them, in the 0th Ganoderma lucidum solid medium containing no wheat germ, the concentration of γ -aminobutyric acid per gram of the medium is 7.567±0.376 mg; in the 5th Ganoderma lucidum solid medium containing 50% wheat germ, γ per gram of the medium The aminobutyric acid concentration was 11.379 ± 0.550 mg.
實驗六：各種靈芝固態培養基對A靈芝菌株菌絲量及γ -胺基丁酸產量之影響Experiment 6: Effect of various solid mediums of Ganoderma lucidum on the amount of mycelial strain and the yield of γ -aminobutyric acid
將A靈芝菌株接種於上述第0~5靈芝固態培養基中，在30℃條件下培養7天，測量菌絲量及γ-胺基丁酸含量。A strain of Ganoderma lucidum was inoculated into the above-mentioned 0~5 Ganoderma lucidum solid medium, and cultured at 30 ° C for 7 days, and the amount of mycelium and the content of γ-aminobutyric acid were measured.
將培養後所得之米飯暨菌絲混合物與100mM鹽酸混合，於室溫振盪45分鐘。以孔徑為0.45mm之濾膜過濾，濾液再與o-phthaladehyde反應，以製備γ -胺基丁酸衍生物。以高效能液相層析法搭配UV 345nm波長，分析γ -胺基丁酸衍生物之含量。The rice and hyphae mixture obtained after the cultivation was mixed with 100 mM hydrochloric acid, and shaken at room temperature for 45 minutes. The membrane was filtered through a membrane having a pore size of 0.45 mm, and the filtrate was further reacted with o-phthaladehyde to prepare a γ -aminobutyric acid derivative. The content of γ -aminobutyric acid derivatives was analyzed by high performance liquid chromatography with UV 345 nm wavelength.
請參閱圖5A及表三。圖5A係於30℃條件下，以不同比例之胚芽米及小麥胚芽培養A靈芝菌株7天所得之菌絲量。橫座標由左至右分別為第0靈芝固態培養基、第1靈芝固態培養基、第2靈芝固態培養基、第3靈芝固態培養基、第4靈芝固態培養基及第5靈芝固態培養基。縱座標則為培養7天後每公克米飯暨菌絲混合物內含之菌絲量。圖5A及表三顯示，將A靈芝菌株培養於第0~5靈芝固態培養基後，每克米飯暨菌絲混合物所含之菌絲量依序為0.292±0.060克、0.579±0.018克、0.745±0.032克、0.665±0.002克、0.573±0.004克、以及0.335±0.020克。Please refer to Figure 5A and Table 3. Fig. 5A shows the amount of hyphae obtained by culturing A ganoderma lucidum strains in different proportions of germ rice and wheat germ at 7 °C for 7 days. From the left to the right, the horizontal coordinate is 0th Ganoderma lucidum solid medium, the first Ganoderma lucidum solid medium, the 2nd Ganoderma lucidum solid medium, the 3rd Ganoderma lucidum solid medium, the 4th Ganoderma lucidum solid medium, and the 5th Ganoderma lucidum solid medium. The ordinate is the amount of hyphae contained per gram of rice and hyphae mixture after 7 days of culture. Fig. 5A and Table 3 show that after the A-Ganoderma lucidum strain is cultured in the 0~5 Ganoderma lucidum solid medium, the amount of hyphae per gram of rice and hyphae mixture is 0.292±0.060 g, 0.579±0.018 g, 0.745±. 0.032 g, 0.665 ± 0.002 g, 0.573 ± 0.004 g, and 0.335 ± 0.020 g.
實驗結果顯示，第1至第4靈芝固態培養基培養所得之菌絲量 較多。因此，靈芝固態培養基中以同時包含胚芽米及小麥胚芽為較佳。較佳者，小麥胚芽之濃度為10%~40%，且胚芽米之濃度為10%~40%。The experimental results show that the amount of mycelium obtained by the first to fourth Ganoderma lucidum solid medium culture More. Therefore, it is preferred that the Ganoderma lucidum solid medium contains both germ rice and wheat germ. Preferably, the concentration of the wheat germ is 10% to 40%, and the concentration of the germ rice is 10% to 40%.
請參閱圖5B及表三。圖5B係於30℃條件下，以不同比例之 胚芽米及小麥胚芽培養A靈芝菌株7天所得之γ-胺基丁酸量。橫座標由左至右分別為第0靈芝固態培養基、第1靈芝固態培養基、第2靈芝固態培養基、第3靈芝固態培養基、第4靈芝固態培養基及第5靈芝固態培養基。縱座標則為γ -胺基丁酸濃度，其中，原始培養基中的γ -胺基丁酸含量以網底表示；培養7天後每公克米飯暨菌絲混合物內含之γ -胺基丁酸總量則以網底及斜紋疊加之方式呈現。圖5B及表三顯示，將A靈芝菌株培養於第0~5靈芝固態培養基後，每克米飯暨菌絲體混合物所含之γ -胺基丁酸濃度依序為11.187±0.134克、14.607±0.271克、20.373±0.044克、25.514±0.129克、30.616±0.089克、以及25.151±0.214毫克。Please refer to Figure 5B and Table 3. Fig. 5B is the amount of γ-aminobutyric acid obtained by culturing A ganoderma lucidum strains in different proportions of germ rice and wheat germ at 7 ° C for 7 days. From the left to the right, the horizontal coordinate is 0th Ganoderma lucidum solid medium, the first Ganoderma lucidum solid medium, the 2nd Ganoderma lucidum solid medium, the 3rd Ganoderma lucidum solid medium, the 4th Ganoderma lucidum solid medium, and the 5th Ganoderma lucidum solid medium. The ordinate is the concentration of γ -aminobutyric acid, wherein the content of γ -aminobutyric acid in the original medium is represented by the bottom of the mesh; the γ -aminobutyric acid contained in the mixture of the rice and the hyphae after 7 days of culture is cultured. The total amount is presented in the form of a mesh bottom and a twill overlay. Fig. 5B and Table 3 show that the concentration of γ -aminobutyric acid per gram of rice and mycelium mixture was 11.187±0.134 g and 14.607± after the A-Ganoderma lucidum strain was cultured in the 0~5 Ganoderma lucidum solid medium. 0.271 g, 20.373 ± 0.044 g, 25.514 ± 0.129 g, 30.616 ± 0.089 g, and 25.151 ± 0.214 mg.
實驗結果顯示，第1至第5靈芝固態培養基培養所得之γ -胺基丁酸量較多。因此，靈芝固態培養基中以包含10%~50%小麥胚芽為較佳。較佳者，小麥胚芽之濃度為30~40%。較佳者，胚芽米之濃度為0%~40%。較佳者，胚芽米之濃度為10%~40%。較佳者，胚芽米之濃度為10%~20%。The experimental results showed that the amount of γ -aminobutyric acid obtained by the culture of the first to fifth Ganoderma lucidum solid medium was large. Therefore, it is preferred that the Ganoderma lucidum solid medium contains 10% to 50% wheat germ. Preferably, the concentration of wheat germ is 30-40%. Preferably, the concentration of the germ rice is from 0% to 40%. Preferably, the concentration of the germ rice is 10% to 40%. Preferably, the concentration of the germ rice is 10% to 20%.
實驗七：各種靈芝固態培養基對C靈芝菌株菌絲量及γ -胺基丁酸產量之影響Experiment 7: Effect of various solid mediums of Ganoderma lucidum on the amount of mycelial strain of C and Ganoderma lucidum and the yield of γ -aminobutyric acid
將C靈芝菌株接種於上述第0~5靈芝固態培養基中，在30℃條件下培養7天，測量菌絲量及γ-胺基丁酸含量。The G. lucidum strain was inoculated into the above-mentioned 0~5 Ganoderma lucidum solid medium, and cultured at 30 ° C for 7 days, and the amount of mycelium and the content of γ-aminobutyric acid were measured.
請參閱圖6A及表三。圖6A係於30℃條件下，以不同比例之 胚芽米及小麥胚芽培養C靈芝菌株7天所得之菌絲量。橫座標由左至右分別為第0靈芝固態培養基、第1靈芝固態培養基、第2靈芝固態培養基、第3靈芝固態培養基、第4靈芝固態培養基及第5靈芝固態培養基。縱座標則為培養7天後每公克米飯暨菌絲體混合物內含之菌絲量。圖6A顯示，將C靈芝菌株培養於第0~5靈芝固態培養基後，每克米飯暨菌絲體混合物所含之菌絲量依序為0.185±0.007克、0.479±0.014克、0.635±0.002克、0.608±0.007克、0.635±0.014克、以及0.451±0.009克。Please refer to Figure 6A and Table 3. Figure 6A is at 30 ° C, in different proportions The amount of hyphae obtained from germ rice and wheat germ culture C ganoderma strain for 7 days. From the left to the right, the horizontal coordinate is 0th Ganoderma lucidum solid medium, the first Ganoderma lucidum solid medium, the 2nd Ganoderma lucidum solid medium, the 3rd Ganoderma lucidum solid medium, the 4th Ganoderma lucidum solid medium, and the 5th Ganoderma lucidum solid medium. The ordinate is the amount of hyphae contained per gram of rice and mycelium mixture after 7 days of culture. 6A shows that the amount of hyphae per gram of rice and mycelium mixture is 0.185±0.007 g, 0.479±0.014 g, 0.635±0.002 g after the C. lucidum strain is cultured in the 0~5 Ganoderma lucidum solid medium. , 0.608 ± 0.007 g, 0.635 ± 0.014 g, and 0.451 ± 0.009 g.
實驗結果顯示，第1至第5靈芝固態培養基所得之菌絲量較 多。The experimental results show that the amount of hyphae obtained from the first to the fifth Ganoderma lucidum solid medium is higher. many.
請參閱圖6B及表三。圖6B係於30℃條件下，以不同比例之 胚芽米及小麥胚芽培養C靈芝菌株7天所得之γ-胺基丁酸量。橫座標由左至右分別為第0靈芝固態培養基、第1靈芝固態培養基、第2靈芝固態培養基、第3靈芝固態培養基、第4靈芝固態培養基及第5靈芝固態培養基。縱座標則為γ -胺基丁酸濃度，其中，原始培養基中的γ -胺基丁酸含量以網底表示；培養7天後每公克米飯暨菌絲混合物內含之γ -胺基丁酸總量則以網底及斜紋疊加之方式呈現。圖6B及表三顯示，將C靈芝菌株培養於第0~5靈芝固態培養基後，每克米飯暨菌絲體混合物所含之γ -胺基丁酸濃度依序為9.108±0.243毫克、20.629±0.099毫克、23.775±0.167毫克、29.351±0.120毫克、22.901±0.111毫克、以及22.533±0.068毫克。Please refer to Figure 6B and Table 3. Fig. 6B is the amount of γ-aminobutyric acid obtained by culturing C. ganoderma strains in different proportions of germ rice and wheat germ at 7 °C for 7 days. From the left to the right, the horizontal coordinate is 0th Ganoderma lucidum solid medium, the first Ganoderma lucidum solid medium, the 2nd Ganoderma lucidum solid medium, the 3rd Ganoderma lucidum solid medium, the 4th Ganoderma lucidum solid medium, and the 5th Ganoderma lucidum solid medium. The ordinate is the concentration of γ -aminobutyric acid, wherein the content of γ -aminobutyric acid in the original medium is represented by the bottom of the mesh; the γ -aminobutyric acid contained in the mixture of the rice and the hyphae after 7 days of culture is cultured. The total amount is presented in the form of a mesh bottom and a twill overlay. Figure 6B and Table 3 show that the concentration of γ -aminobutyric acid per gram of rice and mycelium mixture is 9.108±0.243 mg, 20.629± after the C. lucidum strain is cultured in the 0~5 Ganoderma lucidum solid medium. 0.099 mg, 23.775 ± 0.167 mg, 29.351 ± 0.120 mg, 22.901 ± 0.111 mg, and 22.533 ± 0.068 mg.
實驗結果顯示，第1至第5靈芝固態培養基所得之γ -胺基丁 酸量較多。因此，靈芝固態培養基中以包含10%~50%小麥胚芽為較佳。較 佳者，小麥胚芽之濃度為20~40%。較佳者，胚芽米之濃度為0%~40%。較佳者，胚芽米之濃度為10%~40%。較佳者，胚芽米之濃度為10%~30%。The experimental results showed that the amount of γ -aminobutyric acid obtained from the first to fifth Ganoderma lucidum solid medium was large. Therefore, it is preferred that the Ganoderma lucidum solid medium contains 10% to 50% wheat germ. Preferably, the concentration of wheat germ is 20-40%. Preferably, the concentration of the germ rice is from 0% to 40%. Preferably, the concentration of the germ rice is 10% to 40%. Preferably, the concentration of the germ rice is 10% to 30%.
上述各個實驗均進行三重複，數據表示方式為平均值±標準差。Each of the above experiments was performed in triplicate, and the data representation was mean ± standard deviation.
實驗八：培養時間對γ -胺基丁酸產量之影響Experiment 8: Effect of culture time on the yield of γ -aminobutyric acid
將重量百分濃度為20%之小麥胚芽、5o%之水、以及30%之胚芽米混合，放入醬菜瓶中，經高溫及滅菌處理後，製得內含不同氮源之第2靈芝固態培養基。Mixing 20% by weight of wheat germ, 5% by weight of water, and 30% of germ rice into a pickle bottle, and after high temperature sterilization, the second solid of Ganoderma lucidum containing different nitrogen sources is obtained. Medium.
於本案最佳實施例中，係於121℃作用20分鐘，以同時達到高溫蒸煮及滅菌效果。In the preferred embodiment of the present invention, it is applied at 121 ° C for 20 minutes to simultaneously achieve high temperature cooking and sterilization effects.
在32℃條件下，培養A靈芝菌株21天，於第4、7、10、14、 18及21天測量菌絲量及γ-胺基丁酸含量。Culture A strain of Ganoderma lucidum for 21 days at 32 ° C, on the 4th, 7th, 10th, 14th, The amount of hyphae and the content of γ-aminobutyric acid were measured on days 18 and 21.
請參閱圖7，係於32℃條件下，以第2靈芝固態培養基(含20%之小麥胚芽、50%之水、以及30%之胚芽米)培養A靈芝菌株21天所得之菌絲量以及γ-胺基丁酸量。圖7顯示，以靈芝固態培養基培養靈芝菌株至第21天，可使每克米飯暨菌絲體混合物所含之γ -胺基丁酸濃度提升至119毫克，顯著高於僅培養7天所得之γ -胺基丁酸量(請參見表三，以第2靈芝固態培養基培養A靈芝菌株7天，每克米飯暨菌絲體混合物中含有20.373毫克之γ -胺基丁酸)。Referring to Figure 7, the amount of mycelium obtained by culturing A ganoderma lucidum strain for 21 days in the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% water, and 30% germ rice) at 32 ° C and The amount of γ-aminobutyric acid. Figure 7 shows that the Ganoderma lucidum strain was cultured in Ganoderma lucidum solid medium until the 21st day, and the γ -aminobutyric acid concentration per gram of rice and mycelium mixture was increased to 119 mg, which was significantly higher than that obtained only for 7 days. The amount of γ -aminobutyric acid (see Table 3, culturing A ganoderma lucidum strain for 7 days in the second Ganoderma lucidum solid medium, containing 20.373 mg of γ -aminobutyric acid per gram of rice and mycelium mixture).
實驗九：添加氮源對γ -胺基丁酸產量之影響Experiment 9: Effect of nitrogen source on the yield of γ -aminobutyric acid
將重量百分濃度為20%之小麥胚芽、50%之水、以及30%之胚芽米混合，並添加重量百分濃度為0.1%或1%之不同氮源，放入醬菜瓶中，經高溫及滅菌處理後，製得內含不同氮源之第2靈芝固態培養基。Mix 20% by weight of wheat germ, 50% water, and 30% of germ rice, and add 0.1% or 1% by weight of different nitrogen sources into the pickles bottle. After the sterilization treatment, the second solid medium of Ganoderma lucidum containing different nitrogen sources is prepared.
於本案最佳實施例中，係於121℃作用20分鐘，以同時達到高溫蒸煮及滅菌效果。In the preferred embodiment of the present invention, it is applied at 121 ° C for 20 minutes to simultaneously achieve high temperature cooking and sterilization effects.
其中，不同氮源係指酪蛋白(casein)、大豆蛋白(soy protein)或大豆(soybean)。Among them, different nitrogen sources refer to casein, soy protein or soybean.
在30℃條件下，培養A或C靈芝菌株7天，測量γ-胺基丁酸含量。The A or C Ganoderma lucidum strain was cultured for 7 days at 30 ° C, and the γ-aminobutyric acid content was measured.
請參閱圖8A、8B、9A、9B。其中，圖8B係於第2靈芝固態培養基(含20%之小麥胚芽、50%之水、以及30%之胚芽米)中添加0.1%不同氮源後，在30℃條件下培養A靈芝菌株7天所得之γ-胺基丁酸量。圖9B則係 於第2靈芝固態培養基(含20%之小麥胚芽、50%之水、以及30%之胚芽米)中添加0.1%不同氮源後，在30℃條件下培養C靈芝菌株7天所得之γ-胺基丁酸量。Please refer to Figures 8A, 8B, 9A, 9B. 8B is a strain of the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% water, and 30% germ rice), and then culturing A ganoderma lucidum strain 7 at 30 ° C. The amount of γ-aminobutyric acid obtained in days. Figure 9B is After adding 0.1% different nitrogen sources to the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% water, and 30% germ rice), the γ- obtained by culturing C ganoderma strain for 7 days at 30 ° C Amount of aminobutyric acid.
圖8B以及圖9B中，控制組係本案之第2靈芝固態培養基，其他組別則為本案第2靈芝固態培養基再添加不同氮源所得之培養基。由圖8B以及圖9B可知，添加0.1%酪蛋白(casein)、0.1%大豆蛋白(soy protein)、或0.1%大豆(soybean)，都能使利用本案靈芝固態培養基培養靈芝菌株所得之γ-胺基丁酸產量顯著提升(p<0.05)。In Fig. 8B and Fig. 9B, the control group is the second Ganoderma lucidum solid medium in the present case, and the other group is the medium obtained by adding the different nitrogen sources to the second Ganoderma lucidum solid medium of the present case. As can be seen from FIG. 8B and FIG. 9B, the addition of 0.1% casein, 0.1% soy protein, or 0.1% soybean can make the γ-amine obtained by culturing the Ganoderma lucidum strain using the Ganoderma lucidum solid medium in the present case. The yield of butylbutyrate was significantly increased (p<0.05).
上述圖5B、圖6B及表三顯示，將靈芝菌株培養於本案靈芝固態培養基中，可產生高濃度之γ -胺基丁酸。據此，本案靈芝固態培養基可用於培養靈芝，以生產含高濃度γ -胺基丁酸之食品。其中，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少包含20毫克γ -胺基丁酸。上述圖7顯示，較佳者，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少包含30毫克γ -胺基丁酸。較佳者，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少包含50毫克γ -胺基丁酸。較佳者，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少包含60毫克γ -胺基丁酸。較佳者，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少包含100毫克γ -胺基丁酸。較佳者，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少包含200毫克γ -胺基丁酸。The above-mentioned FIG. 5B, FIG. 6B and Table 3 show that the Ganoderma lucidum strain is cultured in the solid medium of the Ganoderma lucidum in the present case, and a high concentration of γ -aminobutyric acid can be produced. Accordingly, the solid medium of Ganoderma lucidum can be used to culture Ganoderma lucidum to produce a food containing a high concentration of γ -aminobutyric acid. Among them, the high concentration of γ -aminobutyric acid means that at least 20 mg of γ -aminobutyric acid is contained per gram of the Ganoderma lucidum solid medium. Figure 7 above shows that, preferably, a high concentration of gamma -aminobutyric acid means at least 30 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, the high concentration of gamma -aminobutyric acid means at least 50 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, the high concentration of gamma -aminobutyric acid means at least 60 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, the high concentration of gamma -aminobutyric acid means at least 100 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, the high concentration of gamma -aminobutyric acid means at least 200 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium.
其中，較佳者，製備本案靈芝固態培養基之步驟包括：(a)將複數種培養基成分混合，得到一培養基成分混合物，該培養基成分混合物中包括： 小麥胚芽(wheat germ)，重量百分濃度為10%~60%；以及水，重量百分濃度為20%~50%；以及(b)經高溫及滅菌處理，製得該靈芝固態培養基，其中，該高溫係指100℃~150℃。Preferably, the step of preparing the solid medium of the ganoderma lucidum in the present case comprises: (a) mixing a plurality of medium components to obtain a mixture of medium components, the medium component mixture comprising: Wheat germ (wheat germ, weight percent concentration of 10% to 60%; and water, weight percent concentration of 20% to 50%; and (b) high temperature and sterilization treatment, the ganoderma lucidum solid medium is prepared, wherein The high temperature means 100 ° C ~ 150 ° C.
培養基成分混合物中包含10%~60%小麥胚芽時，可以例如是相當於培養基成分混合物中包含0.4%~2.6%麥膠蛋白，抑或是，可以例如是相當於培養基成分混合物中包含0.007%~0.07%小麥榖蛋白。When the medium component mixture contains 10% to 60% wheat germ, it may, for example, correspond to 0.4% to 2.6% of gliadin in the mixture of the medium components, or may be, for example, equivalent to 0.007% to 0.07 in the mixture of the medium components. % wheat prion protein.
較佳者，該培養基成分混合物中，小麥胚芽之重量百分濃度為30%~40%。Preferably, the concentration of the wheat germ in the medium component mixture is 30% to 40% by weight.
較佳者，該培養基成分混合物中更包含胚芽米(embryo rice)，且該胚芽米之重量百分濃度為10%~40%。Preferably, the medium component mixture further comprises embryo rice, and the concentration of the germ rice is 10% to 40% by weight.
於本案之另一較佳實施例中，該培養基成分混合物中，小麥胚芽之重量百分濃度為30%~40%，胚芽米之重量百分濃度為10%~20%，且水之重量百分濃度為50%。In another preferred embodiment of the present invention, the concentration of the wheat germ is 30% to 40%, the weight percentage of the germ rice is 10% to 20%, and the weight of the water is 100% by weight. The concentration is 50%.
培養基成分混合物中包含30%~40%小麥胚芽時，可以例如是相當於培養基成分混合物中包含1.0%~2.0%麥膠蛋白，抑或是，可以例如是相當於培養基成分混合物中包含0.02%~0.05%小麥榖蛋白。When the medium component mixture contains 30% to 40% wheat germ, it may be, for example, 1.0% to 2.0% of gliadin in the mixture of the medium components, or may be, for example, 0.02% to 0.05 in the mixture of the medium components. % wheat prion protein.
較佳者，該培養基成分混合物中更包含酪蛋白(casein)、大豆蛋白(soy protein)、大豆(soybean)。Preferably, the medium component mixture further comprises casein, soy protein, and soybean.
於本案另一較佳實施例中，該靈芝固態培養基之製備步驟包括：(a)將複數種培養基成分混合，得到一培養基成分混合物，該培 養基成分混合物中包括：小麥胚芽(wheat germ)；以及水；以及(b)經高溫及滅菌處理，製得該靈芝固態培養基，其中，該高溫係指100℃~150℃；且步驟(a)中，小麥胚芽之成分包括麥膠蛋白(Gliadin)，且該培養基成分混合物中之麥膠蛋白之重量百分濃度為0.4%~2.6%。In another preferred embodiment of the present invention, the preparation step of the Ganoderma lucidum solid medium comprises: (a) mixing a plurality of medium components to obtain a medium component mixture, the culture The nutrient component mixture includes: wheat germ (wheat germ; and water; and (b) high temperature and sterilization treatment to obtain the ganoderma lucidum solid medium, wherein the high temperature means 100 ° C ~ 150 ° C; and the step (a In the wheat germ component, Gliadin is included, and the concentration of the gliadin in the mixture of the components of the medium is 0.4% to 2.6%.
較佳者，該培養基混合物中之麥膠蛋白之重量百分濃度為1.0%~2.0%。Preferably, the concentration of the gliadin in the medium mixture is from 1.0% to 2.0% by weight.
於本案之再一較佳實施例中，該靈芝固態培養基之製備步驟包括：(a)將複數種培養基成分混合，得到一培養基成分混合物，該培養基成分混合物中包括：小麥胚芽(wheat germ)；以及水；以及(b)經高溫及滅菌處理，製得該靈芝固態培養基，其中，該高溫係指100℃~150℃；且步驟(a)中，小麥胚芽之成分包括小麥榖蛋白(Glutenin)，且該培養基成分混合物中之小麥榖蛋白之重量百分濃度為0.007%~0.07%。In a further preferred embodiment of the present invention, the preparation step of the Ganoderma lucidum solid medium comprises: (a) mixing a plurality of medium components to obtain a medium component mixture, the medium component mixture comprising: wheat germ (wheat germ); And (b) obtaining the solid medium of the ganoderma lucidum by high temperature and sterilization, wherein the high temperature means 100 ° C ~ 150 ° C; and in the step (a), the components of the wheat germ include wheat gluten (Glutenin) And the concentration of the wheat gluten protein in the mixture of the medium components is 0.007% to 0.07% by weight.
較佳者，該培養基成分混合物中之小麥榖蛋白之重量百分濃度為0.02%~0.05%。Preferably, the concentration of the wheat gluten protein in the mixture of the medium components is 0.02% to 0.05% by weight.
較佳者，係在121℃作用至少15分鐘，以同時達到高溫蒸煮 及滅菌效果。Preferably, it is applied at 121 ° C for at least 15 minutes to simultaneously achieve high temperature cooking. And sterilization effect.
較佳者，係用於培養靈芝以產生高濃度之γ -胺基丁酸；其中，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少產生20毫克γ -胺基丁酸。較佳者，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少產生30毫克γ -胺基丁酸。較佳者，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少產生50毫克γ -胺基丁酸。較佳者，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少產生60毫克γ -胺基丁酸。較佳者，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少產生100毫克γ -胺基丁酸。較佳者，高濃度之γ -胺基丁酸指每公克靈芝固態培養基至少產生200毫克γ -胺基丁酸。Preferably, it is used to culture Ganoderma lucidum to produce a high concentration of γ -aminobutyric acid; wherein, a high concentration of γ -aminobutyric acid means at least 20 mg of γ -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, a high concentration of gamma -aminobutyric acid means at least 30 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, the high concentration of gamma -aminobutyric acid means that at least 50 mg of gamma -aminobutyric acid is produced per gram of Ganoderma lucidum solid medium. Preferably, a high concentration of gamma -aminobutyric acid means at least 60 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, a high concentration of gamma -aminobutyric acid means at least 100 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, a high concentration of gamma -aminobutyric acid means at least 200 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium.
本案更提供一種利用微生物發酵方式生產高濃度γ -胺基丁酸的方法，以解決現今只能利用化學方法製備γ -胺基丁酸之產業問題。其中，利用該微生物發酵方式生產γ -胺基丁酸之步驟包括：The present invention further provides a method for producing high concentration γ -aminobutyric acid by microbial fermentation to solve the industrial problem that only γ -aminobutyric acid can be prepared by chemical methods. The step of producing the γ -aminobutyric acid by the microbial fermentation method comprises:
(a)將複數種培養基成分混合，得到一培養基成分混合物，該培養基成分混合物中包括：小麥胚芽(wheat germ)，重量百分濃度為10%~60%；以及水，重量百分濃度為20%~50%；以及(a) mixing a plurality of medium components to obtain a mixture of medium components comprising: wheat germ (by weight, concentration of 10% to 60%; and water, weight percent concentration of 20) %~50%; and
(b)經高溫及滅菌處理，製得一靈芝固態培養基，其中，該高溫係指100℃~150℃。(b) A high temperature medium and a sterilization process are used to prepare a solid medium of Ganoderma lucidum, wherein the high temperature means 100 ° C to 150 ° C.
(c)加入靈芝菌株，發酵生產γ -胺基丁酸。(c) Adding a Ganoderma lucidum strain to produce γ -aminobutyric acid by fermentation.
其中，高濃度之γ -胺基丁酸係指每公克該靈芝固態培養基至少包括20毫克γ -胺基丁酸。較佳者，高濃度之γ -胺基丁酸係指每公克該靈芝固態培養基至少包括30毫克γ -胺基丁酸。較佳者，高濃度之γ -胺基丁 酸係指每公克該靈芝固態培養基至少包括100毫克γ -胺基丁酸。較佳者，高濃度之γ -胺基丁酸係指每公克該靈芝固態培養基至少包括200毫克γ -胺基丁酸。Wherein, the high concentration of γ -aminobutyric acid means that the ganoderma lucidum solid medium contains at least 20 mg of γ -aminobutyric acid per gram. Preferably, the high concentration of γ -aminobutyric acid means that the ganoderma lucidum solid medium contains at least 30 mg of γ -aminobutyric acid per gram. Preferably, the high concentration of γ -aminobutyric acid means that the ganoderma lucidum solid medium contains at least 100 mg of γ -aminobutyric acid per gram. Preferably, the high concentration of γ -aminobutyric acid means that the ganoderma lucidum solid medium contains at least 200 mg of γ -aminobutyric acid per gram.
本案之各個實施例中，第0~5培養基成分混合物之各成分之濃度百分比，均為重量百分濃度。本發明中之各個濃度百分比，均為重量百分濃度。In each of the examples of the present invention, the concentration percentages of the components of the 0 to 5 medium component mixture are all weight percent concentrations. Each concentration percentage in the present invention is a weight percent concentration.
以上所述僅為本發明之較佳實施例，並非用以限定本發明之申請專利範圍，因此凡其它未脫離本發明所揭示之精神下所完成之各種更動或潤飾等，均應包含於本案之申請專利範圍內。The above is only the preferred embodiment of the present invention, and is not intended to limit the scope of the claims of the present invention. Therefore, various other modifications or retouchings, etc., which are not departing from the spirit of the present invention, should be included in the present invention. Within the scope of the patent application.
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| CN1759760A (en) * | 2005-10-13 | 2006-04-19 | 南京农业大学 | Method for producing fruit juice of germinant unpolished rice and ganoderma lucidum, and production |
| CN102154392A (en) * | 2010-12-20 | 2011-08-17 | 安徽丰原发酵技术工程研究有限公司 | Method for improving yield of fermentation of epsilon-polylysine |
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| CN1759760A (en) * | 2005-10-13 | 2006-04-19 | 南京农业大学 | Method for producing fruit juice of germinant unpolished rice and ganoderma lucidum, and production |
| CN102154392A (en) * | 2010-12-20 | 2011-08-17 | 安徽丰原发酵技术工程研究有限公司 | Method for improving yield of fermentation of epsilon-polylysine |
| CN103146599A (en) * | 2013-02-01 | 2013-06-12 | 浙江五养堂药业有限公司 | Lactobacillus plantarum producing high yield gamma-aminobutyric acid and application of lactobacillus plantarum |
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