TWI550084B - Liquid culture method for improving the content of ergot sulfate in mycelia of - Google Patents
Liquid culture method for improving the content of ergot sulfate in mycelia of Download PDFInfo
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本發明係關於提高珊瑚菇菌絲體麥角硫因含量之液態培養方法。更特別地，本發明係關於利用反應曲面法，決定胺基酸前驅物之最佳添加濃度與比例，生產得到高麥角硫因含量的珊瑚菇菌絲體，並用於以最佳液體培養條件，製備得含有高麥角硫因含量的培養液。 The present invention relates to a liquid culture method for increasing the content of ergot sulfur in mycelium of coral mushroom. More particularly, the present invention relates to the use of a reaction surface method to determine the optimum concentration and ratio of amino acid precursors to produce a high ergot sulfur content of the coral mushroom mycelium, and to be used for optimal liquid culture conditions. A culture solution containing a high ergothione content is prepared.
麥角硫因是一種Tanret於1909年研究麥角菌(Claviceps purpurea)時所分離出的一種低分子量硫醇化合物，麥角硫因為真菌之代謝產物，存在於植物及動物組織中，但人體與脊椎動物無法合成麥角硫因，因此僅能藉由攝取食物來吸收。Genghof(Journal of Bacteriology. 103(2)：475-478,1970)研究發現麥角硫因及其前驅物組胺酸三甲基內鹽(hercynine)在真菌及放線菌中可生成，另外，黏菌(Physarum polycephalum)也具有合成麥角硫因之能力。Askari(Journal of Biological Chemistry. 237：1615-1618,1962)指出麥角硫因可以由真菌合成，透過胺基酸來合成：組織胺(histidine)、甲硫胺酸(methionine)及半胱胺酸(cysteine)。麥角硫因是唯一可以發生2-硫-咪唑的胺基酸，在自然界以硫酮型和硫醇型存在，水溶液下以硫酮型為優勢(Franzoni et al,Biomedicine & Pharmacotherapy. 60：453-457,2006)。 Ergosine is a low molecular weight thiol compound isolated by Tanret in 1909 when it studied Claviceps purpurea . The ergot sulfur is present in plant and animal tissues because of the metabolites of fungi, but the human body Vertebrates cannot synthesize ergot sulfur, so they can only be absorbed by ingesting food. Genghof ( Journal of Bacteriology. 103 (2): 475-478, 1970) found that ergothione and its precursor, hiscynine, can be produced in fungi and actinomycetes. Physarum polycephalum also has the ability to synthesize ergot sulfur. Askari ( Journal of Biological Chemistry. 237 :1615-1618, 1962) pointed out that ergot can be synthesized by fungi and synthesized by amino acids: histidine, methionine and cysteine. (cysteine). Ergosine is the only amino acid that can occur 2-thio-imidazole, which exists in the natural form as a thioketone and a thiol type, and in an aqueous solution with a thioketone type (Franzoni et al, Biomedicine & Pharmacotherapy. 60 :453 -457, 2006).
黃齡誼等人曾利用深層培養提高杏鮑菇菌絲體之麥角硫因含量，於溫度25℃、10%接菌量、2%葡萄糖為碳源、0.5%酵母萃取物為氮源，以125rpm搖瓶培養，並在培養7天後加入4mM組胺酸，所得菌絲體麥角硫因含量為5.05mg/g dw，所以調整培養基組成或添加胺基酸前驅物，可 提高菇類菌絲體麥角硫因含量(毛正倫、梁志弘、黃齡誼。2013。中華民國發明專利第I-408226號)。 Huang Lingyi et al. used deep culture to increase the ergot sulfur content of Pleurotus eryngii mycelium at a temperature of 25 ° C, 10% inoculation, 2% glucose as carbon source, and 0.5% yeast extract as nitrogen source. The flask was cultured at 125 rpm, and 4 mM histidine was added after 7 days of culture, and the obtained mycelium ergothione content was 5.05 mg/g dw, so the medium composition was adjusted or the amino acid precursor was added. Increasing the content of ergot sulfur in mushroom mycelium (Mao Zhenglun, Liang Zhihong, Huang Lingyi. 2013. Republic of China invention patent No. I-408226).
珊瑚菇(Pleurotus citrinopileatus Sing.)分類上隸屬於擔子菌綱(Basidiomycetes)，傘菌目(Agaricales)，側耳科(Pleurotaceae)，側耳屬(Pleurotus)。其子實體外型像珊瑚因而得名，在中國大陸又名金頂蘑、榆黃蘑、玉皇蘑，在台灣則以金頂側耳、黃金菇、玉米菇或玉皇菇命名(黃，1998)。珊瑚菇食味鮮美，香甜可口，富含蛋白質、胺基酸和維生素等多種營養成分，是美味食用菌之一，且因色澤金黃，豔麗美觀，受大眾歡迎。除了其食用性外，根據福建省三明真菌研究所所長黃年來編撰的中國大型真菌原色圖鑑(1998)中記載，珊瑚菇有入藥及滋補強壯的功效，對腎虛、陽萎和痢疾等狀都有一定的幫助。『中華本草』中記載，珊瑚菇性味甘，具有滋補強壯、止痢。主虛弱萎症、肺氣腫、痢疾。 Pleurotus citrinopileatus Sing. is classified into Basidiomycetes, Agaricales, Pleurotaceae, and Pleurotus. Its fruit body is named after coral, and it is also known as the golden mushroom, the yellow mushroom and the jade mushroom in mainland China. In Taiwan, it is named after the golden ear, golden mushroom, corn mushroom or jade mushroom (Huang, 1998). ). Coral mushroom tastes delicious, sweet and delicious, rich in protein, amino acid and vitamins. It is one of the delicious edible fungi, and it is popular because of its golden color, beautiful appearance. In addition to its edible properties, according to the Chinese large-scale fungal primary color map (1998) compiled by Huang Nianlai, director of the Sanming Fungal Research Institute of Fujian Province, coral mushrooms have the effect of being used as medicine and nourishing and strengthening, and have kidney deficiency, impotence and dysentery. Certain help. According to "Chinese Materia Medica", the coral mushroom is sweet and tonic, and it is nourishing and strong. Main weakness, emphysema, dysentery.
人工培養的珊瑚菇其營養要求，一般以木屑、棉籽殼、甘蔗渣及廢棉等作為原料均可充分提供。pH 4以下，8以上出菇有困難，出菇最適pH為6.0~6.5。珊瑚菇是一種分解纖維素、木質素能力較強的食用菌，栽培時需要豐富的碳源和氮源，特別是氮源豐富時，菌絲生長速度快，子實體產量高(黃仕政、楊梅春、陳勁初。金頂側耳。鄉間小路。11：45,1999)。 The nutrient requirements of artificially cultivated coral mushrooms are generally provided by using wood chips, cottonseed hulls, bagasse and waste cotton as raw materials. Below pH 4, it is difficult to produce mushrooms above 8 or higher, and the optimum pH for mushrooming is 6.0 to 6.5. Coral mushroom is an edible fungus with strong ability to decompose cellulose and lignin. It needs abundant carbon and nitrogen sources when planting. Especially when the nitrogen source is rich, the mycelium grows fast and the fruit body yield is high (Huang Shizheng, Yang Meichun) Chen Jinchu. Jinding Pleurotus. Country Road. 11:45, 1999).
近年來國內外利用深層發酵來培養菌絲體已十分普遍。此法可大量培養菌絲體，且所需時間甚短，又有幾項優點：1.菌絲體生長快速且生長週期短：由於液態培養可將通氣量、溫度、酸鹼度等設備，控制在最佳培養條件，於短時間內增殖大量菌絲體和具有生理活性的代謝產物。2.可工廠化生產、無季節性：食用菌液態培養於發酵設備中，不受季節性的限制。利用液體發酵培養，雖然可以縮短食用真菌類的培養時間，但是如何控制菌絲體的生產、如何有效控制生產出有用的二次代謝物(如抗腫瘤物質或降血糖物質)、如何 分離出有效的成分，以及有效成分的產品差異如何，技術與品質的控制都是必須克服的問題，這樣才能提升食用菇菌類產品品質而有助於人體健康(王培銘。食品工業。34：32-35,2002；蔡淑瑤，國立中興大學食品暨應用生物科技學系博士論文，2006)。 In recent years, it has been common to culture mycelium by deep fermentation at home and abroad. This method can culture mycelium in a large amount, and the time required is very short, and has several advantages: 1. The mycelium grows fast and the growth cycle is short: the liquid culture can control the ventilation, temperature, pH and other equipment. The optimal culture conditions proliferate a large amount of mycelium and physiologically active metabolites in a short time. 2. It can be factory-produced and has no seasonality: the edible fungus is liquid cultured in the fermentation equipment, and is not subject to seasonal restrictions. Using liquid fermentation culture, although the cultivation time of edible fungi can be shortened, how to control the production of mycelium, how to effectively control the production of useful secondary metabolites (such as anti-tumor substances or hypoglycemic substances), how to Separation of effective ingredients, as well as product differences in active ingredients, technology and quality control are issues that must be overcome, in order to improve the quality of edible mushroom products and contribute to human health (Wang Peiming. Food Industry. 34:32- 35, 2002; Cai Shuyao, Ph.D., Department of Food and Applied Biotechnology, National Chung Hsing University, 2006).
而深層培養之培養基是微生物生長的基本食物，也是合成代謝產物的反應物來源，適當的選配營養源成分與比例，才能充分利用微生物來生產所需的代謝物，若無法搭配出適當的營養源，則可能會使得單位時間內的發酵成果無法符合經濟效益，造成後續的分離、純化難度提高並增加操作成本，因此有效率地選出營養成分與搭配培養基的比例是非常重要的。本研究首先以一次因子方式針對珊瑚菇菌絲體液態培養之生長條件加以探討，並在培養過程中分析菌絲體乾重、麥角硫因含量、發酵液殘糖及pH值變化之關係。 The medium for deep culture is the basic food for microbial growth and the source of reactants for anabolic products. Appropriate selection of nutrient source components and ratios can make full use of microorganisms to produce the desired metabolites. The source may make the fermentation result in unit time unable to meet the economic benefits, which will make the subsequent separation and purification difficult and increase the operation cost. Therefore, it is very important to efficiently select the ratio of nutrients to the medium. In this study, the growth conditions of mycelium liquid culture of Pleurotus ostreatus were firstly studied in a single factor method, and the relationship between the dry weight of mycelium, the content of ergot sulfur, the residual sugar of fermentation broth and the pH value were analyzed during the cultivation.
本發明基於以上之目的發現，利用一次一因子及反應曲面法，針對珊瑚菇菌絲體液態培養之生長條件加以探討，找出添加麥角硫因的三種胺基酸前驅物最佳濃度比例，可獲得高麥角硫因含量之珊瑚菇菌絲體。 Based on the above object, the present invention finds out the growth conditions of the liquid culture of the mycelium of the coral mushroom by using the one-factor and the reaction surface method, and finds the optimal concentration ratio of the three amino acid precursors added with ergothione. Coral mushroom mycelium with high ergot sulphur content can be obtained.
於是，本發明之一方面係關於提高珊瑚菇菌絲體麥角硫因含量之液態培養方法。 Accordingly, one aspect of the present invention relates to a liquid culture method for increasing the content of ergot sulfur in mycelium of coral mushroom.
於本發明之一些具體實施態樣，所述之高含量麥角硫因之珊瑚菇菌絲體製備方法包含：將珊瑚菇菌絲體於25ºC下振盪培養7天進行活化；將菌絲體培養液接種原以5%接種量，於初始pH值為10.0或不調整培養基初始pH值且包含2%碳源及0.5%氮源之培養基中，於溫度20-25℃下進行振盪培養10-20天，並於培養第7天時添加綜合胺基酸液(包含半胱胺酸8mM、組胺酸4mM及甲硫胺酸0.5mM)；及收取高含量麥角硫因之珊瑚菇菌絲體。 In some embodiments of the present invention, the method for preparing a high content of ergot sulfur mycelium mycelium comprises: vibrating the mycelium of the coral mushroom at 25 ° C for 7 days for activation; and culturing the mycelium The liquid was inoculated with 5% inoculum, and the culture was carried out at a temperature of 20-25 ° C at a temperature of 20-25 ° C at an initial pH of 10.0 or a medium containing no initial pH of the medium and containing 2% carbon source and 0.5% nitrogen source. Day, and on the 7th day of culture, add a comprehensive amino acid solution (containing 8 mM cysteine, 4 mM histidine and 0.5 mM methionine); and a high content of ergot sulfur mycelium mycelium .
於本發明之一項具體實施態樣，所述之碳源為葡萄糖。於本發明之另一項具體實施態樣，所述之碳源為胰蛋白腖。 In one embodiment of the invention, the carbon source is glucose. In another embodiment of the invention, the carbon source is tryptone.
於本發明之一項具體實施態樣，所述之氮源為酵母抽出物。於本發明之另一項具體實施態樣，所述之氮源為胰蛋白腖。 In a specific embodiment of the invention, the nitrogen source is a yeast extract. In another embodiment of the invention, the nitrogen source is tryptone.
於本發明之一項具體實施態樣，所述之高含量麥角硫因之珊瑚菇菌絲體製備方法進一步包含將收取之菌絲體進行冷凍乾燥的步驟。 In a specific embodiment of the present invention, the method for preparing a high content of ergot sulfur mycelium mycelium further comprises the step of freeze-drying the collected mycelium.
本發明之另一方面，係關於一種經由本發明之液態培養方法製得之高含量麥角硫因珊瑚菇菌絲體。 Another aspect of the invention relates to a high content of ergoporphyrin mycelium mycelium produced by the liquid culture method of the present invention.
於本發明之一些具體實施態樣，所述之高含量麥角硫因珊瑚菇菌絲體含有之麥角硫因含量為5.0mg/g dw以上。於其他具體實施態樣，所述之高含量麥角硫因珊瑚菇菌絲體含有之麥角硫因含量為10-20mg/g dw。 In some embodiments of the present invention, the high content of ergot sulfur is contained in the mycelium of the coral mushroom, and the ergot sulfur content is 5.0 mg/g dw or more. In other specific embodiments, the high content of ergot sulfur due to the mycelium content of the coral mushroom mycelium is 10-20 mg/g dw.
本發明之又另一方面，係關於一種食品添加劑，其包含經由本發明之液態培養方法製得之高含量麥角硫因的珊瑚菇菌絲體。 Still another aspect of the present invention relates to a food additive comprising a high content of ergothione-derived mycelium mycelium produced by the liquid culture method of the present invention.
本發明之功效：本發明所使用之菌種原麥角硫因原本含量僅為1.81mg/g dw(控制組)，在經調整初始pH值為10後，可得麥角硫因含量為10.65mg/g dw，較控制組顯著提高了5.88倍。再者，本發明於培養第7天添加綜合胺基酸液，培養至第22天時麥角硫因含量最高可達14.57mg/g dw，較控制組提高了8.05倍。而添加綜合胺基酸液所得之珊瑚菇菌絲體於培養第10天時菌絲體中麥角硫因含量(2.54mg/g dw)，已達到相似於不添加前驅物之菌絲體培養22天(2.87mg/g dw)之麥角硫因含量，顯示本發明具體縮短了培養時間。 The effect of the invention: the original ergot sulfur content of the strain used in the invention is only 1.81 mg/g dw (control group), and after adjusting the initial pH value of 10, the ergot sulfur content is 10.65. Mg/g dw was significantly increased by 5.88 times compared with the control group. Furthermore, in the present invention, a comprehensive amino acid solution was added on the 7th day of culture, and the ergot sulfur content was up to 14.57 mg/g dw at the 22nd day, which was 8.05 times higher than that of the control group. The mycelium content of the mycelium mycelium obtained by adding the integrated amino acid solution on the 10th day of culture showed that the content of ergothione in the mycelium (2.54 mg/g dw) had reached a mycelial culture similar to that without the addition of the precursor. The ergothione content of 22 days (2.87 mg/g dw) showed that the present invention specifically shortened the culture time.
圖1為珊瑚菇液態發酵不同天數後調整pH值至10，對發酵22天之菌絲體乾重、最終pH值(A)及麥角硫因含量(B)之影響。 Figure 1 shows the effect of adjusting the pH value to 10 after different days of liquid fermentation of coral mushroom, the dry weight of mycelium, the final pH value (A) and the content of ergot sulfur (B).
圖2係顯示珊瑚菇液態發酵期間，分別添加濃度4mM之胺基酸(半胱胺酸、組胺酸及甲硫胺酸)對發酵22天之珊瑚菇菌絲體乾重(A)及麥角硫因含量(B)之影響。 Figure 2 shows the dry weight (A) and wheat of mycelium mycelium fermented for 22 days during the liquid fermentation of coral mushroom by adding amino acid (cysteine, histidine and methionine) at a concentration of 4 mM. The effect of the sulphur content (B).
圖3為固定組胺酸(X 2)濃度為4.00mM時，半胱胺酸(X 1)及甲硫胺酸(X 3)對深層培養珊瑚菇菌絲體麥角硫因含量(mg/g dw)之等高線圖(A)與曲面圖(B)。 3 is fixed histidine (X 2) concentration is 4.00mM, homocysteine (X 1) and methionine (X-3) of the submerged culture mycelium coral ergothioneine Quantity (mg / g dw) contour map (A) and surface map (B).
圖4係顯示以胰蛋白腖(Tryptone)為氮源時珊瑚菇液態發酵之菌絲體乾重、殘糖量、最終pH值(A)及麥角硫因含量(B)之變化。 Fig. 4 is a graph showing changes in the dry weight of the mycelium, the amount of residual sugar, the final pH (A), and the ergosulfur content (B) of the liquid fermentation of the coral mushroom with Tryptone as a nitrogen source.
圖5係顯示圖6-4、培養第7天添加RSM最適條件胺基酸時珊瑚菇菌絲體乾重、殘糖量、最終pH值(A)及麥角硫因含量(B)之變化。 Fig. 5 is a graph showing the changes of dry weight, residual sugar amount, final pH value (A) and ergothione content (B) of coral mushroom mycelium in Figure 6-4 and the optimum conditions for adding RSM on the 7th day of culture. .
圖6係顯示珊瑚菇子實體和菌絲體70%乙醇萃取物之抗氧化力。 Figure 6 shows the antioxidant power of 70% ethanol extract of coral mushroom fruit bodies and mycelia.
圖7係顯示珊瑚菇子實體和菌絲體70%乙醇萃取物之還原力。 Figure 7 shows the reducing power of the 70% ethanol extract of the coral mushroom fruiting body and mycelium.
本發明之其他特色及優點將於下列實施範例中被進一步舉例與說明，而該實施範例僅作為輔助說明，並非用於限制本發明之範圍。 The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.
本研究所使用之珊瑚菇(Pleurotus citrinopileatus)菌絲體，係由彰化縣大村鄉蕈優生物科技有限公司所購得之珊瑚菇子實體所繼代培養而得。珊瑚菇菌種保存於PDA(potato dextrose agar)斜面培養基並存放於4℃冰箱中，使用前已滅菌過之接種刀切取一小塊至PDA平板培養基上並置 於25℃培養箱中進行活化。接種時則取於平板培養基上活化完成之菌種，同樣已滅菌過之接種刀切取外圍活性較強之菌絲作為菌種，其大小均為5mm x 5mm，接入PDB(potato dextrose broth)液體培養基中並置於25℃培養箱靜置培養，觀察菌絲體生長型態，培養12天菌落直徑平均為70~80mm，菌絲呈現白色。收集菌絲並凍乾。 The mycelium of Pleurotus citrinopileatus used in this study was subcultured from the coral mushroom fruit body purchased by Changcun County Dicun Biotechnology Co., Ltd. The coral mushroom strains were stored in PDA (potato dextrose agar) slant medium and stored in a refrigerator at 4 ° C. A small piece of the sterilized sterilized knife was used to cut into a PDA plate medium and placed in a 25 ° C incubator for activation. When inoculation, the strain is activated on the plate medium, and the inoculated knife which has been sterilized is also used to cut the hyphae with strong peripheral activity as the strain, the size is 5mm x 5mm, and the PDB (potato dextrose broth) liquid is connected. The culture medium was placed in a 25 ° C incubator for static growth, and the mycelial growth pattern was observed. The average colony diameter was 70-80 mm for 12 days, and the hyphae showed white color. The hyphae were collected and lyophilized.
以一次一因子方式探討含高麥角硫因之珊瑚菇菌絲體培養條件Study on culture conditions of mycelium of coral oyster mushroom containing high ergot sulfur by one-factor method
本實施例主要係利用深層培養方式，探討各式培養條件對於珊瑚菇菌絲體生成麥角硫因之影響，並在培養過程中分析菌絲體乾重、麥角硫因含量、發酵液殘糖量及pH值變化之關係。珊瑚菇菌絲體保存於PDA(potato dextrose agar)培養基中並儲存於4℃。接種時，取活化完成之培養皿，以滅菌後之接種刀切取外圍活性較強之菌絲作為菌種，其大小約為5mm×5mm，並放於25ºC下培養活化。於無菌操作台中，將保存於PDA之菌種以滅菌解剖刀切取5塊5mm×5mm大小之菌酛，將菌酛接種於盛裝100mL液態培養基(每升含有：20g葡萄糖、5g酵母抽出物、2g(NH4)2SO4、0.5g KH2PO4,、0.5g K2HPO4及0.5g MgSO4．7 H2O)之250mL凹槽三角錐型瓶，以125rpm，25ºC下進行振盪培養7天做為種菌酛。將所得之菌絲體及培養基以Waring blender攪碎15秒，得一菌絲體均質液，然後取5mL均質液放入內裝95mL液態培養基之250mL三角錐型瓶中，作為接種原。本實驗則循環以培養於三角瓶振盪培養基中之菌絲體為接種原。 In this embodiment, the effects of various culture conditions on the formation of ergot sulfur in the mycelium of Pleurotus ostreatus were studied by means of deep culture, and the dry weight of the mycelium, the content of ergot sulfur, and the residue of the fermentation broth were analyzed during the cultivation. The relationship between the amount of sugar and the change in pH. Mycelium of coral mushroom was preserved in PDA (potato dextrose agar) medium and stored at 4 °C. At the time of inoculation, the culture dish which was activated was taken, and the hyphae having strong peripheral activity was cut out as a strain by the inoculation knife after sterilization, and the size was about 5 mm×5 mm, and cultured and activated at 25 °C. In the aseptic table, the bacteria stored in the PDA were cut into 5 pieces of 5 mm × 5 mm size bacteria by a sterilizing scalpel, and the bacteria were inoculated into 100 mL of liquid medium (containing 20 g of glucose, 5 g of yeast extract, 2 g per liter). 250 mL grooved triangular cone bottle of (NH 4 ) 2 SO 4 , 0.5 g KH 2 PO 4 , 0.5 g K 2 HPO 4 and 0.5 g MgSO 4 .7 H 2 O), shake culture at 125 rpm, 25 ° C 7 days as an inoculum. The obtained mycelium and medium were pulverized with a Waring blender for 15 seconds to obtain a mycelium homogenate, and then 5 mL of the homogenate was placed in a 250 mL triangular cone bottle containing 95 mL of liquid medium as an inoculum. In this experiment, the mycelium cultured in the shake flask shaking medium was circulated as an inoculum.
本實驗使用兩種培養基，分別為基礎培養基與礦物質培養基，兩種培養基組成成分如下表1所示。分別盛裝95mL之基礎培養基及礦物質培養基於250mL三角錐型瓶中，經由殺菌釜滅菌後，加入5mL製備好之接種原(5%)，於125rpm，25ºC下進行振盪培養。培養期間每2天分析菌絲乾重與麥角硫因含量、發酵液pH值及殘糖量，觀察珊瑚菇利 用基礎培養基和礦物質培養基液態發酵之生長曲線，以選擇較適合的培養基做為後續實驗培養基，並進一步探討其最適溫度(實驗溫度為20、25及30ºC)、接種量(1、5、10、15及20%接種原)、初始pH值(實驗組為以0.1N HCl或0.1N NaOH將培養基pH值調整為2.0、4.0、8.0、10.0、12.0及14.0，而對照組為不調pH值)、碳源(包括：果糖、乳糖、麥芽糖、蔗糖及葡萄糖(作為控制組)，濃度皆為2%)，以及氮源(包括：玉米浸出液、麥芽抽出物、蛋白腖、胰蛋白腖及酵母抽出物(作為控制組)，濃度皆為0.5%)等培養之因子，以找出液態發酵珊瑚菇產生麥角硫因之最適培養基組成及最佳生長採收天數，以三重覆進行各項試驗。 In this experiment, two kinds of mediums were used, which were basic medium and mineral medium. The composition of the two mediums is shown in Table 1 below. 95 mL of the basic medium and mineral medium were respectively placed in a 250 mL triangular cone type bottle, and after sterilization through a sterilization pot, 5 mL of the prepared inoculum (5%) was added, and shaking culture was carried out at 125 rpm at 25 °C. Analyze the dry weight of mycelium and the content of ergot sulfur, the pH value of the fermentation broth and the amount of residual sugar every 2 days during the culture period. The growth curve of the liquid fermentation of the basic medium and the mineral medium was used to select the suitable medium as the subsequent experimental medium, and further explored the optimum temperature (experimental temperature of 20, 25 and 30oC) and the inoculum amount (1, 5, 10). , 15 and 20% vaccination), initial pH (experimental group adjusted the pH of the medium to 2.0, 4.0, 8.0, 10.0, 12.0 and 14.0 with 0.1N HCl or 0.1N NaOH, while the control group did not adjust the pH value) Carbon sources (including: fructose, lactose, maltose, sucrose, and glucose (as control groups) at 2%), and nitrogen sources (including: corn extract, malt extract, peptone, tryptone, and yeast extract) (as a control group), the concentration of 0.5%) and other culture factors, in order to find the optimum medium composition and the best growth and harvest days for the liquid fermented coral mushroom to produce ergot sulfur, and carry out various tests in triple overlap.
總合所有以一次一因子方式，利用不同培養基成分及培養條件進行之實驗結果可知，在5%接種量，使用葡萄糖為碳源和酵母抽出物為氮源，並調酸鹼值pH至10，於溫度25℃下經22天的時間培養，可得到最高量菌絲體(8.28g/L)及麥角硫因(10.65mg/g dw)產量。培養初期，基礎培養基及 礦物質培養基兩種培養基中，珊瑚菇菌絲體乾重皆隨著培養時間增加而增加，當使用基礎培養基時，其菌絲體乾重於第14天達最高為8.70g/L；使用礦物質培養基時，菌絲體乾重則於第6天可達最高為11.22g/L。 The total results of the experiments were carried out in a one-factor, one-factor manner using different medium components and culture conditions. In the 5% inoculation amount, glucose was used as the carbon source and the yeast extract was used as the nitrogen source, and the pH value was adjusted to 10, The highest amount of mycelium (8.28 g/L) and ergothione (10.65 mg/g dw) were obtained by culturing at a temperature of 25 ° C for 22 days. In the early stage of culture, the basic medium and In the two culture media, the dry weight of mycelium of coral mushroom increased with the increase of culture time. When using the basic medium, the dry weight of mycelium reached the highest at 8.70g/L on the 14th day; In the case of the medium, the dry weight of the mycelium reached a maximum of 11.22 g/L on the sixth day.
以二階段刺激探討含高麥角硫因之珊瑚菇菌絲體最適培養條件Optimum culture conditions of mycelium of coral oyster mushroom containing high ergot sulfur by two-stage stimulation
接著，進一步探討調整液態培養基pH值及添加前驅物之時機，觀察不同時間點調整pH值或添加前驅物對於菌絲體之乾重及麥角硫因含量之影響。本實驗設計於培養期間第0、7及14天時，以0.1N NaOH，將培養基pH值調整為10，以二階段刺激方式進行菌絲體麥角硫因含量之探討。此外，也設計於培養第0、7及14天，分別添加麥角硫因之三種胺基酸前驅物：半胱胺酸(cysteine)、組胺酸(histidine)及甲硫胺酸(methionine)。培養22天後，分析珊瑚菇菌絲乾重與麥角硫因含量、發酵液pH值。 Next, further explore the timing of adjusting the pH value of the liquid medium and adding the precursor, and observe the effects of adjusting the pH value or adding the precursor at different time points on the dry weight of the mycelium and the content of ergot sulfur. The experiment was designed to adjust the pH of the culture medium to 10 in 0.1N NaOH on the 0th, 7th and 14th day of the culture period, and to discuss the content of mycelium ergot in the second stage stimulation mode. In addition, it was also designed to add the three amino acid precursors of ergothione on the 0th, 7th and 14th days of culture: cysteine, histidine and methionine. . After 22 days of culture, the dry weight of the mycelium of the coral mushroom and the content of ergot sulfur and the pH of the fermentation broth were analyzed.
已知珊瑚菇菌絲最適生長pH為5.0至6.5，但由前述以一次一因子探討珊瑚菇菌絲體生成麥角硫因之結果發現，調整培養基pH值至10，能有效提升菌絲體之麥角硫因含量。 It is known that the optimum growth pH of mycelium of coral mushroom is 5.0 to 6.5. However, it is found that the ergot sulfur production of coral mushroom mycelium by one factor is as follows. Adjusting the pH value of the medium to 10 can effectively improve the mycelium. Ergot sulfur content.
結果由圖1之結果顯示，於培養第0天即調整培養基pH值所得到的珊瑚菇菌絲體乾重最高，依序為第0天(8.28g/L)>控制組(7.59g/L)>第14天(6.20g/L)>第7天(4.41g/L)。而菌絲體麥角硫因含量方面，也同樣是培養第0天含量最高(10.65mg/g dw)，其他依序為控制組(1.81mg/g dw)>第14天(1.43mg/g dw)>第7天(0.55mg/g dw)。考慮每公升所得菌絲產量經換算，可得每公升發酵液中菌絲體之麥角硫因含量為第0天(88.19mg/L)>控制組(13.75mg/L)>第14天(8.46mg/L)>第7天(2.40mg/L)。由以上結果可知，於培養第0天即調整培養基pH值所得到的珊瑚菇菌絲體乾重(8.28g/L)及麥角硫因含量最高(10.65mg/g dw)，每公 升發酵液中菌絲體之麥角硫因含量為88.19mg/L，比控制組提高了6.4倍。 The results shown in Fig. 1 showed that the mycelium of the coral mushroom mycelium obtained by adjusting the pH value of the culture medium on the 0th day of culture was the highest, which was on the 0th day (8.28g/L)> control group (7.59g/L). > Day 14 (6.20 g/L) > Day 7 (4.41 g/L). The content of mycelium ergot sulfur was also the highest on the 0th day of culture (10.65mg/g dw), and the other order was control group (1.81mg/g dw)> 14th day (1.43mg/g) Dw)> Day 7 (0.55 mg/g dw). Considering the conversion of the hyphae yield per liter, the ergot sulfur content of the mycelium per liter of fermentation broth is 0 day (88.19 mg/L)> control group (13.75 mg/L) > day 14 ( 8.46 mg/L) > Day 7 (2.40 mg/L). From the above results, it can be seen that the dry weight of the mycelium of the coral mushroom (8.28 g / L) and the highest content of ergot sulfur (10.65 mg / g dw) obtained by adjusting the pH value of the medium on the 0th day of culture, per liter The content of ergot sulfur in the mycelium of the fermentation broth was 88.19 mg/L, which was 6.4 times higher than that of the control group.
而於不同生長階段添加胺基酸前驅物，皆對珊瑚菇菌絲體生長造成影響(圖2A)，但都能有效提高菌絲體麥角硫因之含量，其中又以培養第7天時添加4mM半胱胺酸所得菌絲體麥角硫因含量最高為16.31mg/g dw，比控制組提升了將近9倍(圖2B)。 The addition of amino acid precursors at different growth stages affected the growth of mycelium of coral mushrooms (Fig. 2A), but all of them could effectively increase the content of mycelium ergot sulfur, which was also cultivated on the 7th day. The mycelium ergosulfide content of the mycelium obtained by adding 4 mM cysteine was 16.31 mg/g dw, which was nearly 9 times higher than that of the control group (Fig. 2B).
以反應曲面法探討含高麥角硫因之珊瑚菇菌絲體最適培養條件Study on optimum culture conditions of mycelium of coral mushroom containing high ergot sulfur by reaction surface methodology
反應曲面法經常用來探討最佳培養基組成與改善食品生產製程等，透過實驗設計可有效降低實驗次數及了解各因子間的交互關係。於是，本實驗進一步利用反應曲面法探討三種前驅物之間交互作用，以找出前驅物之最佳濃度比例。 The reaction surface method is often used to explore the optimal medium composition and improve the food production process. The experimental design can effectively reduce the number of experiments and understand the interaction between various factors. Therefore, the experiment further uses the reaction surface method to explore the interaction between the three precursors to find the optimal concentration ratio of the precursor.
由前述液態培養一次一因子之試驗所得結果，選出對珊瑚菇液態發酵生長時具有顯著差異之培養條件。本實驗首先以半胱胺酸(X 1)、組胺酸(X 2)及甲硫胺酸(X 3)三因子為獨立變數，所選取的濃度範圍為0.64~7.36mM進行第一批實驗，經三因子之中心混成設計(central composite design,CCD)，可得到20組不同添加濃度之組合。以半胱胺酸(cysteine)、組胺酸(histidine)及甲硫胺酸(methionine)為三獨立變數，進行三因子五階層中心混成設計，總共需進行20組試驗；進行試驗時，則依表所設計的各因子濃度或條件配製於液態培養基中，並於培養箱中培養22天。所得珊瑚菇菌絲體凍乾後分析菌體乾重及麥角硫因含量，發酵液pH值，實驗結果以電腦軟體SAS(8.0版,SAS Institute Inc.,Cary,NC)及SigmaPlot(12.0版,SPSS,Inc.,Chicago,IL)計算並繪圖，可分別獲得菌絲體乾重及麥角硫因含量之二次方程式，再繪製成反應曲面圖及等高線。 From the results of the above-mentioned liquid culture one-factor test, the culture conditions which have significant differences in the liquid fermentation growth of the coral mushroom are selected. In this experiment, the three factors of cysteine ( X 1 ), histidine ( X 2 ) and methionine ( X 3 ) were used as independent variables, and the selected concentrations ranged from 0.64 to 7.36 mM for the first batch of experiments. Through a three-factor central composite design (CCD), a combination of 20 different concentrations can be obtained. With three independent variables of cysteine, histidine and methionine, a three-factor and five-level central mixing design was carried out, and a total of 20 sets of tests were required. The concentration or conditions of each factor designed in the table were prepared in a liquid medium and cultured in an incubator for 22 days. After the freeze-dried mycelium of the coral mushroom was analyzed, the dry weight of the fungus and the content of ergot sulfur and the pH of the fermentation broth were analyzed. The experimental results were obtained by computer software SAS (version 8.0, SAS Institute Inc., Cary, NC) and SigmaPlot (version 12.0). , SPSS, Inc., Chicago, IL) Calculate and plot, respectively, can obtain the quadratic equation of mycelial dry weight and ergot sulfur content, and then draw into the reaction surface map and contour.
由第二批菌絲體麥角硫因含量之變異數分析可 知，半胱胺酸(X 1)、組胺酸(X 2)及甲硫胺酸(X 3)，三種獨立變數對於珊瑚菇菌絲體麥角硫因含量而言，在0.1%下皆具有顯著性。而且，由下表2顯示，描述三種獨立變數半胱胺酸(X 1)、組胺酸(X 2)及甲硫胺酸(X 3)對於麥角硫因含量之三元二次數學模式的P值小於0.0001，統計上具有顯著性差異，也就是說，即此反應曲面模式之信賴度高達99.99%；Lack of fit之P值為0.6633，所以此描述生物質之三元二次數學模式是成立的。變異係數為10.12%，表示描述生物質之數學模式具有高準確性及可靠性。而檢定係數為0.9880，也代表其相關係數為0.9940。 From the analysis of the variation of the second batch of mycelium ergot sulfur content, cysteine ( X 1 ), histidine ( X 2 ) and methionine ( X 3 ), three independent variables for coral mushrooms The mycelium ergot sulfur content was significant at 0.1%. Moreover, as shown in Table 2 below, the ternary quadratic mathematical model for the content of ergot sulfur in three independent variables, cysteine ( X 1 ), histidine ( X 2 ) and methionine ( X 3 ), is described. The P value is less than 0.0001, and there is a statistically significant difference, that is, the reliability of the reaction surface mode is as high as 99.99%; the P value of the Lack of fit is 0.6633, so this describes the ternary quadratic mathematical model of biomass. It was established. The coefficient of variation is 10.12%, indicating that the mathematical model describing biomass has high accuracy and reliability. The verification coefficient is 0.9880, which also means that the correlation coefficient is 0.9940.
經ANOVA(analysis of variance)分析後所得經由三變數深層培養發酵珊瑚菇菌絲體麥角硫因含量之三元二次方程式：
Y 麥角硫因 ：深層培養珊瑚菇菌絲體之麥角硫因含量(mg/g)。 Y ergothione : the ergot sulfur content (mg/g) of the mycelium of the coral mushroom in the deep culture.
表3係列示利用反應曲面法所得之液態發酵珊瑚菇麥角硫因其不同變數分析結果，顯示針對獨立變數組胺酸(X 2)之P值皆有顯著差異，而獨立變數半胱胺酸(X 1)、及甲硫胺酸(X 3)及X 1 X 2、X 1 X 3、X 2 X 3之交互效應皆不具有顯著性差異；在二次效應中，和皆具有顯著差異。因此，固定組胺酸(X 2)之濃度為4mM，探討半胱胺酸(X 1)及甲硫胺酸(X 3)之間的交互作用，並繪製出菌絲體麥角硫因含量 之等高線圖及反應曲面圖，如圖3所示，兩圖中皆可清楚看到最適點的產生。 Table 3 series shows the results of different variables of the liquid fermented coral mushroom ergot sulfur obtained by the reaction surface method, which shows that the P value of the independent variable array of amine acid ( X 2 ) is significantly different, while the independent variable cysteine ( X 1 ), and methionine ( X 3 ) and X 1 X 2 , X 1 X 3 , X 2 X 3 have no significant difference in interaction; in the secondary effect, with Both have significant differences. Therefore, the concentration of immobilized histidine ( X 2 ) was 4 mM, and the interaction between cysteine ( X 1 ) and methionine ( X 3 ) was investigated, and the content of mycelium ergot sulfur was drawn. The contour map and the reaction surface map are shown in Fig. 3. The optimum points can be clearly seen in both figures.
綜合以上結果，此三元二次數學模式是成立的，而在等高線圖中與反應曲面圖中可清楚看到最適點的產生。從等高線圖中可預測當半胱胺酸、組胺酸及甲硫胺酸之正規值為0時菌絲體麥角硫因含量可達到最高，回推至實際濃度依序為半胱胺酸8mM、組胺酸4mM及甲硫胺酸0.5mM，帶正規值於方程式中計算可得麥角硫因預測值，其值為13.90mg/g dw，而實際添加前驅物所得珊瑚菇菌絲體麥角硫因含量之實驗值為14.57mg/g dw，由此可見實際實驗值與經方程式所得之預測值結果相近，其菌絲體麥角硫因含量甚至更高。 Based on the above results, the ternary quadratic mathematical model is established, and the optimal point can be clearly seen in the contour map and the reaction surface map. It can be predicted from the contour map that when the normal value of cysteine, histidine and methionine is 0, the content of mycelium ergot can be highest, and the actual concentration is cysteine. 8 mM, histidine 4 mM and methionine 0.5 mM, with the normal value calculated in the equation, the predicted value of ergothione can be obtained, the value is 13.90 mg / g dw, and the actual addition of the precursor obtained coral mushroom mycelium The experimental value of ergot sulfur content is 14.57 mg/g dw, which shows that the actual experimental value is similar to the predicted value obtained by the equation, and the mycelium ergot sulfur content is even higher.
可提高菌絲體麥角硫因含量之因子對珊瑚菇菌絲體生長曲線之影響Effect of factors on the growth curve of mycelium mycelium of mycelium
由先前一次一因子條件探討時發現，透過調整培養基pH值至10、以胰蛋白腖為氮源及適時添加前驅物胺基酸都可以有效提升珊瑚菇菌絲體麥角硫因之含量，然而先前實驗探討天數皆為22天，而改變培養因子或添加前驅物之濃度及比例，皆可能影響珊瑚菇菌絲體之生長曲線，因此，本實驗進一步探討因子對於菌絲體生長曲線的影響，以找出能有效提高麥角硫因含量同時縮短珊瑚菇菌絲體深層發酵培養時間的因子。 From the previous one-factor condition, it was found that by adjusting the pH of the medium to 10, using tryptone as a nitrogen source and adding the precursor amino acid in time, the content of ergot sulfur in the mycelium of coral mushroom can be effectively improved. The number of days in the experiment is 22 days, and changing the concentration and proportion of the culture factor or the added precursor may affect the growth curve of the mycelium of the coral mushroom. Therefore, this experiment further explores the influence of the factor on the growth curve of the mycelium. Find out the factors that can effectively increase the ergot sulfur content and shorten the culture time of the deep fermentation of the coral mushroom mycelium.
根據珊瑚菇菌絲體於培養基pH值為10時之生長曲線，於培養至第20天時菌絲體麥角硫因含量達最高7.48mg/g dw，但隨著培養時間增加培養基所產生類似氨之物質越多氣味濃厚。以胰蛋白腖為氮源時，珊瑚菇菌絲體之麥角硫因含量培養至第20天時，麥角硫因含量可達最高，其值為5.50mg/g dw，此時菌絲體乾重為7.12g/L，經換算後每公升可得之麥角硫因含量為39.17mg/L，相較於以酵母萃取物為氮源時之1.81mg/g dw及18.17mg/L高出許多，此外以胰蛋白腖作為珊瑚菇深層培養之氮源時，培養至12天菌絲體之麥角硫 因，即可達以酵母萃取物為氮源時22天之含量，確實能有效縮短發酵天數(圖4)。 According to the growth curve of mycelium of coral mushroom mycelium at pH 10, the mycelium content of mycelium reached the highest at 7.48 mg/g dw when cultured until the 20th day, but the medium produced similarly with the increase of culture time. The more the ammonia is, the more odor is. When tryptone was used as the nitrogen source, the content of ergot sulfur in the mycelium of the coral mushroom mycelium was up to the 20th day, and the content of ergot sulfur was the highest, and the value was 5.50 mg/g dw. The weight is 7.12g/L, and the converted ergot sulfur content per liter is 39.17mg/L, which is higher than 1.81mg/g dw and 18.17mg/L when the yeast extract is nitrogen source. Many, in addition to tryptone as a nitrogen source for deep culture of coral mushroom, ergot sulfur cultured to 12 days mycelium Because it can reach the content of 22 days when the yeast extract is used as a nitrogen source, it can effectively shorten the fermentation days (Fig. 4).
針對胺基酸前驅物之探討，於培養第7天添加綜合胺基酸液於培養基時，珊瑚菇菌絲體於第8天後隨著培養時間的增加菌絲乾重逐漸下降。菌絲體麥角硫因含量方面，與不添加任何胺基酸前驅物時珊瑚菇菌絲體之生長曲線比較，同樣培養第16天時，添加綜合胺基酸液所得之珊瑚菇菌絲體中麥角硫因含量較不添加前驅物之菌絲體麥角硫因含量提高了34倍；而添加綜合胺基酸液所得之珊瑚菇菌絲體於培養第10天時菌絲體中麥角硫因含量(2.54mg/g dw)，已達到相似於不添加前驅物之菌絲體培養22天(2.87mg/g dw)之麥角硫因含量(參見圖5)。與添加單一胺基酸前驅物(4mM半胱胺酸)時珊瑚菇菌絲體之生長曲線比較，同樣培養第16天時，僅添加單一種胺基酸濃度之菌絲體麥角硫因含量為8.04mg/g dw，可知，添加綜合胺基酸液所得菌絲體麥角硫因含量，較只添加單一種胺基酸時好。由以上結果可知，於深層培養珊瑚菇之培養基中添加綜合胺基酸液，可有效縮短珊瑚菇菌絲體生成麥角硫因之時間，且能顯著提升珊瑚菇菌絲體中麥角硫因之含量。 In view of the discussion of amino acid precursors, the hyalin mycelium gradually decreased with the increase of culture time after the 8th day when the comprehensive amino acid solution was added to the culture medium on the 7th day. Compared with the growth curve of mycelium mycelium in the mycelium, compared with the growth curve of the mycelium of the coral mushroom, without adding any amino acid precursor, the coral mushroom mycelium obtained by adding the integrated amino acid was also cultured on the 16th day of culture. The content of sulphur in the middle ergot was 34 times higher than that in the mycelium without the addition of precursors. The mycelium of the coral mushroom was added to the mycelium of the mycelium on the 10th day of culture. The sulphur content (2.54 mg/g dw) has reached an ergothione content similar to that of the mycelium culture without addition of the precursor for 22 days (2.87 mg/g dw) (see Figure 5). Compared with the growth curve of coral mushroom mycelium when adding a single amino acid precursor (4 mM cysteine), the mycelium ergothione content of only one single amino acid concentration was added on the 16th day of culture. When it was 8.04 mg/g dw, it was found that the mycelium ergosulfide content obtained by adding the integrated amino acid solution was better than when only a single amino acid was added. From the above results, it can be seen that the addition of a comprehensive amino acid solution to the culture medium of the deep-layered coral mushroom can effectively shorten the time of producing ergot sulfur in the mycelium of the coral mushroom, and can significantly increase the ergot sulfur in the mycelium of the coral mushroom. The content.
高麥角硫因之珊瑚菇菌絲體之活性物質及抗氧化分析Active substances and antioxidation analysis of mycelium of coral oyster mushroom
由先前反應曲面法之實驗結果可知，珊瑚菇液態培養期間，添加綜合胺基酸液於培養基中可有效提高菌絲體麥角硫因之含量，而所得之菌絲簡稱為含高麥角硫因之珊瑚菇菌絲體(high ergothioneine Pleurotus citrinopileatus mycelia,HEPM)。目前利用液態發酵所得之菇類菌絲體，已有許多使用在營養食品或機能性食品的配方上，作為食品原料，在產品化學組成和非揮發性呈味成分方面，與產品接受度有高度相關性，然而在文獻回顧過程中發現，針對珊瑚菇子實體與菌絲體非揮發性呈味成分部分較少相關之研究。因此，本研究 目的欲了解HEPM和低麥角硫因含量之菌絲體(low ergothioneine Pleurotus citrinopileatus mycelia,LEPM)的一般組成成分、非揮發性呈味成分包括可溶性糖和糖醇、游離胺基酸及5'-核苷酸等成分上之差異性，也探討兩種菌絲體中生物活性成分，如麥角固醇、腺苷及GABA含量上之差異，並與珊瑚菇子實體比較。 According to the experimental results of the previous reaction surface method, during the liquid culture of the coral mushroom, the addition of the comprehensive amino acid solution in the medium can effectively increase the content of the mycelium ergot sulfur, and the obtained mycelium is abbreviated as high ergot sulfur. High ergothioneine Pleurotus citrinopileatus mycelia (HEPM). At present, the mushroom mycelium obtained by liquid fermentation has been used in the formulation of nutritious foods or functional foods. As a food raw material, it has a high degree of product acceptance in terms of chemical composition and non-volatile taste components. Correlation, however, during the literature review, it was found that there was little correlation between the coral mushroom fruiting bodies and the non-volatile taste components of the mycelium. Therefore, the purpose of this study is to understand the general composition of HEPM and low ergothioneine Pleurotus citrinopileatus mycelia (LEPM), non-volatile taste components including soluble sugars and sugar alcohols, free amino acids And the difference in the composition of 5'-nucleotides, etc., also explored the difference between the bioactive components of the two mycelium, such as ergosterol, adenosine and GABA, and compared with the coral mushroom fruiting body.
菌絲體培養時，取0.250L珊瑚菇液態菌種接於裝有4.75L液態培養基之10L桌上型發酵槽中，所使用的液態培養基組成同液態菌種，攪拌轉速125rpm，通氣量1vvm於25ºC下培養，培養期間不添加胺基酸前驅物，培養第14天回收菌絲，利用100mesh篩網將菌絲體與發酵液分離，以去離子水清洗菌絲體，並將菌絲體凍乾，所得菌絲體為低麥角硫因之珊瑚菇菌絲體，以下即簡稱為LEPM菌絲體。取20mL液態菌種接裝有180液態培養基之500mL三角瓶中並於25ºC，125rpm培養，使用之培養基組成同液態菌種，於培養第7天時添加綜合胺基酸液，並於培養第22天回收菌絲，利用100mesh篩網將菌絲體與發酵液分離，菌絲體以去離子水清洗，隨即冷凍乾燥，所得菌絲體為含高麥角硫因之珊瑚菇菌絲體，以下簡稱為HEPM(high ergothioneine Pleurotus citrinopileatus mycelia)菌絲體。綜合胺基酸液為配製高濃度之胺基酸液，以0.22μl濾膜過濾後再添加，使其於培養基中含量為8mM半胱胺酸、4mM組胺酸及0.5mM甲硫胺酸。 When the mycelium is cultured, 0.250L of the coral mushroom liquid strain is taken in a 10L table type fermentation tank containing 4.75L of liquid medium. The liquid medium used is composed of the same liquid species, the stirring speed is 125 rpm, and the aeration rate is 1 vvm. Cultured at 25oC, no amino acid precursor was added during the culture, the hyphae were recovered on the 14th day of culture, the mycelium was separated from the fermentation broth by a 100mesh sieve, the mycelium was washed with deionized water, and the mycelium was frozen. Dry, the obtained mycelium is a mycelium of coral oyster mushroom with low ergot sulfur, and the following is simply referred to as LEPM mycelium. Take 20 mL of liquid bacteria in a 500 mL flask containing 180 liquid medium and incubate at 25 ° C, 125 rpm. The medium used is the same liquid species, and the combined amino acid solution is added on the 7th day of culture, and the culture is carried out. The hyphae were recovered in the day, and the mycelium was separated from the fermentation broth by a 100 mesh sieve. The mycelium was washed with deionized water and then freeze-dried. The obtained mycelium was a coral mushroom mycelium containing high ergot sulfur. Referred to as HEPM (high ergothioneine Pleurotus citrinopileatus mycelia) mycelium. The synthetic amino acid solution was prepared by preparing a high-concentration amino acid solution, filtering it through a 0.22 μl filter, and adding it to a medium content of 8 mM cysteine, 4 mM histidine, and 0.5 mM methionine.
珊瑚菇子實體和兩種麥角硫因含量不同之菌絲體經凍乾後其水分含量於統計下不具有顯著性差異，水分含量範圍在8.88~9.03%。菇類主要成分之一為碳水化合物之糖質，占菇類成分的40~70%。以乾重為基準來看，HEPM含有較高量的碳水化合物，其含量為53.47%。在粗蛋白質含量方面，菇類蛋白質含量約為乾物重之19%~35%，雖然蛋白質含量低於動物性食品，但能為良好之蛋白質來源，由分析結果顯示，珊瑚菇子實體與HEPM和LEPM兩種菌絲體之粗蛋白 質含量不具有顯著性差異，其蛋白質範圍為33.83~34.23%。 The water content of the mycelium of the coral mushroom fruit body and the two ergot sulphur content was not significantly different after lyophilization, and the moisture content ranged from 8.88 to 9.03%. One of the main ingredients of mushrooms is the carbohydrate of carbohydrates, which accounts for 40 to 70% of the components of mushrooms. Based on dry weight, HEPM contains a higher amount of carbohydrates, which is 53.47%. In terms of crude protein content, the protein content of the mushroom is about 19% to 35% of the dry weight. Although the protein content is lower than the animal food, it can be a good source of protein. The analysis results show that the coral mushroom fruit body and HEPM and LEPM two kinds of mycelium crude protein There is no significant difference in the quality content, and the protein range is 33.83~34.23%.
珊瑚菇子實體含有之可溶性糖及糖醇有***糖(arabinose)、乳糖(lactose)、甘露糖醇(mannitol)、核糖(ribose)和海藻糖(trehalose)，其中甘露糖醇含量最高為46.57mg/g，占總可溶性糖的50.5%，***糖及海藻糖次之，含量分別為16.28和15.27mg/g。在HEPM含有之可溶性糖及糖醇為甘露糖醇、核糖和海藻糖。HEPM中核糖含量最高，其值為18.58mg/g，占總可溶性糖的39.17%，甘露糖醇及海藻糖次之，分別為16.07和12.87mg/g。由以上結果可知，在珊瑚菇子實體及HEPM菌絲體中皆含有甘露糖醇、核糖和海藻糖。 The soluble sugar and sugar alcohols contained in the coral mushroom fruit body are arabinose, lactose, mannitol, ribose and trehalose, and the highest mannitol content is 46.57 mg. /g, which is 50.5% of the total soluble sugar, followed by arabinose and trehalose, and the contents are 16.28 and 15.27 mg/g, respectively. The soluble sugars and sugar alcohols contained in HEPM are mannitol, ribose and trehalose. The highest content of ribose in HEPM was 18.58 mg/g, which accounted for 39.17% of the total soluble sugar, followed by mannitol and trehalose, which were 16.07 and 12.87 mg/g, respectively. From the above results, it was found that mannitol, ribose and trehalose were contained in both the coral mushroom fruit body and the HEPM mycelium.
珊瑚菇子實體之游離胺基酸含量顯著高於HEPM及LEPM菌絲體；此外，在菌絲體游離胺基酸組成中與麥角硫因有關之胺基酸前驅物含量，甲硫胺酸皆未偵測到；由於培養HEPM菌絲體之培養基中添加了高濃度4mM的組胺酸，所以組胺酸含量方面，HEPM菌絲體較LEPM菌絲體高出許多。在核甘酸含量方面，HEPM菌絲體中總5'-核苷酸含量為200.44mg/g，顯著高於子實體和LEPM菌絲體。在生理活性物質方面，HEPM菌絲體之麥角固醇含量與GABA含量，皆顯著高於珊瑚菇子實體和LEPM菌絲體。腺苷含量則以子實體含量較高，其值為5.53mg/g，顯著高於HEPM和LEPM菌絲體。 The free amino acid content of the coral mushroom fruiting body is significantly higher than that of HEPM and LEPM mycelium; in addition, the content of amino acid precursor related to ergothione in the free amino acid composition of mycelium, methionine None of them were detected; since the high concentration of 4 mM histidine was added to the medium in which the HEPM mycelium was cultured, the HEPM mycelium was much higher than the LEPM mycelium in terms of histidine content. In terms of nucleotide content, the total 5'-nucleotide content in HEPM mycelia was 200.44 mg/g, which was significantly higher than that of fruiting bodies and LEPM mycelia. In terms of physiologically active substances, the ergosterol content and GABA content of HEPM mycelium were significantly higher than that of coral mushroom fruit body and LEPM mycelium. The adenosine content was higher in the fruiting body and its value was 5.53 mg/g, which was significantly higher than that of HEPM and LEPM mycelium.
針對珊瑚菇子實體、HEPM及LEPM兩種麥角硫因含量不同之菌絲體，探討其70%乙醇萃取物之抗氧化性質及所含抗氧化成分。以70%乙醇萃取珊瑚菇子實體、HEPM與LEPM菌絲體，萃取後經減壓濃縮至乾燥，再直接用以抗氧化成分或以70%乙醇稀釋成不同濃度(0.1~10.0mg/mL)作為抗氧化性質分析用。本研究所分析的抗氧化性質之項目包括：抗氧化力、還原力、清除DPPH自由基、螯合亞鐵離子及TEAC抗氧化能力，並測定70%乙醇萃取物中抗氧化物質的含量。更進一步將實驗所得的各種抗氧化性質的評估結果，利用線性迴歸之內、外差法加以換算後，以EC50(Effective concentration)值來表示，可更了解珊瑚菇子實體、HEPM與LEPM兩種菌絲體其70%乙醇萃取物之抗氧化性質；而再經70%乙醇萃取率的換算將更明白多少量的珊瑚菇子實體和菌絲體粉末可表現出EC50的效果。並利用相關係數(r)評估抗氧化成分與抗氧化能力之EC50值之間的相關性。 The anti-oxidation properties and antioxidant components of 70% ethanol extracts were studied for the mycelium with different content of ergot sulfur in the coral mushroom fruit body, HEPM and LEPM. The coral mushroom fruit body, HEPM and LEPM mycelium were extracted with 70% ethanol, extracted and concentrated to dryness under reduced pressure, and directly diluted with antioxidants or 70% ethanol to different concentrations (0.1~10.0 mg/mL). Used as an analysis of antioxidant properties. The antioxidant properties analyzed in this study include: antioxidant capacity, reducing power, scavenging DPPH free radicals, chelated ferrous ions and TEAC antioxidant capacity, and determining the content of antioxidants in 70% ethanol extract. Further, the evaluation results of various antioxidant properties obtained by the experiment are converted by the internal and external difference method of linear regression, and expressed by EC 50 (Effective concentration) value, and the coral mushroom fruit body, HEPM and LEPM can be better understood. The anti-oxidation properties of the 70% ethanol extract of the mycelium; and the conversion of 70% ethanol extraction rate will better understand how much the coral mushroom fruit body and mycelium powder can exhibit the EC 50 effect. And using the correlation coefficient (r) to assess the correlation between antioxidants and EC 50 values of antioxidant capacity.
抗氧化力的測定是以共軛雙烯化合物之生成做為指標，實驗的原理是利用不飽和脂肪酸(如亞麻油酸)氧化初期時會因脫氫作用而形成自由基，此自由基再經分子內重排而產生共軛雙烯鍵，共軛雙烯化合物可經由測定最大吸收波長234nm之吸光值而得其生成量，用以判定測試樣品是否與自由基結合，而降低共軛雙烯化合物生成，吸光值愈低表示其抗氧化能力愈強，或以抑制過氧化百分比表示，即其抑制率愈高抗氧化效果愈佳。 The determination of antioxidant capacity is based on the formation of conjugated diene compounds. The principle of the experiment is to use unsaturated fatty acids (such as linoleic acid) to form free radicals due to dehydrogenation during the initial oxidation. The intramolecular rearrangement produces a conjugated diene bond, and the conjugated diene compound can be obtained by measuring the absorbance of the maximum absorption wavelength of 234 nm to determine whether the test sample binds to the radical and lowers the conjugated diene. When the compound is formed, the lower the absorbance value, the stronger the antioxidant capacity, or the percentage of inhibition of peroxidation, that is, the higher the inhibition rate, the better the oxidation resistance.
由圖6可知，珊瑚菇子實體、HEPM及LEPM兩種菌絲體之70%乙醇萃取物之抗氧化力，會隨萃取物濃度之增加而提高。當濃度為0.1mg/mL時，珊瑚菇子實體與HEPM及LEPM兩種菌絲體之抗氧化力皆不具有顯著性差異，其抗氧化力為45.75~50.65%，皆較BHA(93.09%)及α-生育酚(90.02%)差，但較抗壞血酸的抗氧化能力(3.02%)好。當濃度為0.5mg/mL時，子實體之抗氧化力為77.91%，顯著高於HEPM菌絲體(63.11%)及LEPM(61.72%)。當濃度為5.0mg/mL時，HEPM菌絲體(76.52%)之抗氧化力則高於LEPM菌絲體(71.44%)，而珊瑚菇子實體之抗氧化力可高達97.87%與標準品BHA(98.46%)相當。當濃度提高為10.0mg/mL時，HEPM菌絲體之抗氧化能力為82.47%，顯著高於LEPM(79.17%)。 It can be seen from Fig. 6 that the antioxidant activity of the 70% ethanol extract of the coral mushroom fruit body, HEPM and LEPM mycelium increases with the concentration of the extract. When the concentration was 0.1mg/mL, there was no significant difference in the antioxidant power between the coral mushroom fruit body and the HEPM and LEPM mycelium. The antioxidant capacity was 45.75~50.65%, which was better than BHA (93.09%). And α-tocopherol (90.02%) is poor, but better than the ascorbic acid antioxidant capacity (3.02%). When the concentration was 0.5 mg/mL, the antioxidant capacity of the fruiting body was 77.91%, which was significantly higher than that of HEPM mycelium (63.11%) and LEPM (61.72%). When the concentration is 5.0mg/mL, the antioxidant capacity of HEPM mycelium (76.52%) is higher than that of LEPM mycelium (71.44%), while the antioxidant capacity of coral mushroom fruit body can be as high as 97.87% and standard BHA. (98.46%) is equivalent. When the concentration was increased to 10.0 mg/mL, the antioxidant capacity of HEPM mycelium was 82.47%, which was significantly higher than LEPM (79.17%).
還原力的測定是以普魯士藍(Fe4[Fe(CN)6]3)之生成量做為指標，其主要的原理將赤血鹽(potassium ferricyanide,K3Fe(CN)6)還原成黃血鹽(K4Fe(CN)6)後，再與三價鐵離子作用生成普魯士藍，並以分光光度計在最大吸收波長700nm下測定普魯士藍含量，用以檢測樣品之還原力。 樣品若將鐵離子還原成亞鐵離子，再與三價鐵生成普魯士藍而使吸光值上升，則表示樣品之還原力愈強。由圖7可知，在還原力方面，以HEPM菌絲體之還原力效果最佳。當樣品濃度為1.0mg/mL時，HEPM菌絲體還原力可達0.59，顯著高於子實體(0.34)和LEPM菌絲體(0.35)。而當濃度提高為5.0mg/mL時，HEPM菌絲體還原力即高達1.14，顯著高於子實體(1.02)。 The reduction power is measured by the amount of Prussian blue (Fe 4 [Fe(CN) 6 ] 3 ). The main principle is to reduce the potassium ferricyanide (K 3 Fe(CN) 6 ) to yellow. After the blood salt (K 4 Fe(CN) 6 ), it reacts with ferric ions to form Prussian blue, and the Prussian blue content is measured by a spectrophotometer at a maximum absorption wavelength of 700 nm to detect the reducing power of the sample. If the sample reduces iron ions to ferrous ions and then generates Prussian blue with ferric iron to increase the absorbance, it means that the reducing power of the sample is stronger. It can be seen from Fig. 7 that the reducing power of the HEPM mycelium is optimal in terms of reducing power. When the sample concentration is 1.0mg/mL, the HEPM mycelium reducing power can reach 0.59, which is significantly higher than the fruiting body (0.34) and LEPM mycelium (0.35). When the concentration was increased to 5.0 mg/mL, the reducing power of HEPM mycelium was as high as 1.14, which was significantly higher than that of the fruiting body (1.02).
此外，在清除DPPH自由基能力方面，以HEPM菌絲體之清除DPPH自由基能效果最佳，其EC50濃度為0.29mg/mL。在螯合亞鐵離子能力方面，珊瑚菇子實體之70%乙醇萃取物其EC50濃度為0.29mg/mL，顯著低於HEPM和LEPM菌絲體。在TEAC抗氧化力方面，珊瑚菇子實體之70%乙醇萃取物其EC50濃度為3.14mg/mL，顯著低於HEPM和LEPM菌絲體。萃取物物抗氧化成分方面，HEPM菌絲體之70%乙醇萃取物中麥角硫因含量為61.53mg/g，顯著高於珊瑚菇子實體(17.04mg/g)和LEPM菌絲體(3.84mg/g)。整體而言，HEPM菌絲體之70%乙醇萃取物其抗氧化性質優於珊瑚菇子實體和LEPM菌絲體，整合結果參見下表4。 In addition, in the ability to scavenge DPPH free radicals, HEPM mycelium was the best to remove DPPH free radicals with an EC 50 concentration of 0.29 mg/mL. In terms of the ability to chelate ferrous ions, the 70% ethanol extract of the coral mushroom fruiting body has an EC 50 concentration of 0.29 mg/mL, which is significantly lower than that of HEPM and LEPM mycelium. In terms of TEAC antioxidant capacity, the 70% ethanol extract of the coral mushroom fruiting body had an EC 50 concentration of 3.14 mg/mL, which was significantly lower than that of HEPM and LEPM mycelium. In terms of the antioxidant content of the extract, the content of ergot in the 70% ethanol extract of HEPM mycelium was 61.53 mg/g, which was significantly higher than that of the coral mushroom fruiting body (17.04 mg/g) and LEPM mycelium (3.84). Mg/g). Overall, the 70% ethanol extract of HEPM mycelium has better antioxidant properties than coral mushroom fruit body and LEPM mycelium. The integration results are shown in Table 4 below.
以上結果可知，HEPM菌絲體之70%乙醇萃取物其抗氧化力、還原力及清除DPPH自由基能力方面皆有不錯的效果，當70%乙醇萃取物濃度為0.08mg/mL時，即可達50%的抗氧化力，與珊瑚菇子實體(0.09mg/mL)沒有顯著差異。整體而言，HEPM菌絲體之70%乙醇萃取物的抗氧化性質優於珊瑚菇子實體和LEPM菌絲體。 The above results show that the 70% ethanol extract of HEPM mycelium has a good effect on antioxidant capacity, reducing power and DPPH free radical scavenging ability. When the concentration of 70% ethanol extract is 0.08 mg/mL, Up to 50% of the antioxidant power was not significantly different from the coral mushroom fruiting body (0.09 mg/mL). Overall, the 70% ethanol extract of HEPM mycelium has superior antioxidant properties to coral mushroom fruit bodies and LEPM mycelia.
抗氧化成分組成方面，HEPM菌絲體之70%乙醇萃取物中總多酚含量為89.54mg/g，顯著高於珊瑚菇子實體(35.76mg/g)和LEPM(30.38mg/g)；換算成粉末，HEPM菌絲體樣品中總多酚含量(21.20mg/g)>珊瑚菇子實體(8.17mg/g)>LEPM菌絲體(2.97mg/g)，參見下表5。 In terms of antioxidant composition, the total polyphenol content in 70% ethanol extract of HEPM mycelium was 89.54 mg/g, which was significantly higher than that of coral mushroom fruit body (35.76 mg/g) and LEPM (30.38 mg/g). Powdered, total polyphenol content in HEPM mycelium samples (21.20 mg/g) > coral mushroom fruit body (8.17 mg/g) > LEPM mycelium (2.97 mg/g), see Table 5 below.
麥角硫因為真菌之代謝產物，主要存在於植物及動物組織中，但由於人體與脊椎動物無法合成麥角硫因，因此僅能藉由攝取食物來吸收。研究指出，菇類(每克乾重約含0.1-1mg麥角硫因)與藍綠藻(cyanobacteria)是麥角硫因最好的食物來源。Lin(International Journal of Medicinal Mushrooms. 15：315-323,2013)分析49種食藥用菇類麥角硫因含量，包括9種子實體、39種菌絲體和一種營養細胞，其中以側耳屬(Pleurotus)所得麥角硫因含量較高。本發明係揭示於最適培養基條件下培養後所得含高麥角硫因之HEPM菌絲體，本發明方法所得之HEPM菌絲體之70%乙醇萃取物中麥角硫因含量可達61.53mg/g，顯著高於珊瑚菇子實體(17.04mg/g)和LEPM菌絲體(3.84mg/g)；換算成粉末乾重，HEPM菌絲體樣品中麥角硫因含量(14.57mg/g dw)>珊瑚菇子實體(3.89mg/g dw)>LEPM菌絲體(0.37mg/g dw)。 Because of the metabolites of fungi, ergot sulfur is mainly found in plant and animal tissues. However, since humans and vertebrates cannot synthesize ergot sulfur, they can only be absorbed by ingesting food. Studies have shown that mushrooms (about 0.1-1 mg ergothione per gram dry weight) and cyanobacteria are the best food sources for ergot sulfur. Lin ( International Journal of Medicinal Mushrooms. 15 : 315-323, 2013) analyzed the content of ergot sulfur in 49 kinds of edible and medicinal mushrooms, including 9 seed entities, 39 mycelia and a vegetative cell, of which Pleurotus Pleurotus ) has a high content of ergot sulfur. The present invention discloses a HEPM mycelium containing high ergot sulfur obtained after culturing under optimum medium conditions, and the content of ergothione in the 70% ethanol extract of the HEPM mycelium obtained by the method of the invention can reach 61.53 mg/ g, significantly higher than the coral mushroom fruit body (17.04mg / g) and LEPM mycelium (3.84mg / g); converted to powder dry weight, HEPM mycelium sample ergot sulfur content (14.57mg / g dw > Coral mushroom fruiting body (3.89 mg/g dw) > LEPM mycelium (0.37 mg/g dw).
統合以上結果可知，經過最適培養條件培養以及添加胺基酸前驅物誘導下，珊瑚菇菌絲體中麥角硫因含量可提高39倍，故本發明之深層液體培養方法可獲得含高麥角硫因之珊瑚菇菌絲體，且該菌絲體具有優異的抗氧化力、還原力及清除DPPH自由基能力，可用於生產麥角硫因之發酵菌種酛。再者，本發明培養方法所製得之菌絲體不僅具有高含量麥角硫因，並具有良好的抗氧化性質及抗氧化成分，且不影響珊瑚菇原有的鮮味，故可做為高營養成分的食品來源或添加物。 According to the above results, it can be seen that the ergot sulfur content in the mycelium of the coral mushroom can be increased by 39 times under the optimum culture condition and the addition of the amino acid precursor, so the deep liquid culture method of the present invention can obtain the high ergot angle. Sulfuric acid mycelium mycelium, and the mycelium has excellent antioxidant, reducing power and DPPH free radical scavenging ability, and can be used for producing fermenting bacteria of ergot sulfur. Furthermore, the mycelium obtained by the culture method of the invention not only has high content of ergothione, but also has good antioxidant and anti-oxidation components, and does not affect the original umami taste of the coral mushroom, so it can be used as High-nutrient food source or supplement.
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