WO2019148418A1 - Method for increasing yield of extracellular polysaccharides of edible and medicinal fungus by adding phenylalanine - Google Patents

Method for increasing yield of extracellular polysaccharides of edible and medicinal fungus by adding phenylalanine Download PDF

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WO2019148418A1
WO2019148418A1 PCT/CN2018/074935 CN2018074935W WO2019148418A1 WO 2019148418 A1 WO2019148418 A1 WO 2019148418A1 CN 2018074935 W CN2018074935 W CN 2018074935W WO 2019148418 A1 WO2019148418 A1 WO 2019148418A1
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fermentation
phenylalanine
yield
medicinal
edible
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PCT/CN2018/074935
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French (fr)
Chinese (zh)
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丁重阳
马忠宝
徐萌萌
王琼
许正宏
赵丽婷
刘高强
顾正华
石贵阳
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江南大学
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Priority to JP2020535638A priority Critical patent/JP6995206B2/en
Priority to PCT/CN2018/074935 priority patent/WO2019148418A1/en
Publication of WO2019148418A1 publication Critical patent/WO2019148418A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

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  • the invention relates to a method for adding phenylalanine to increase the yield of extracellular polysaccharides of edible medicinal fungi, and belongs to the field of microbial fermentation.
  • Edible fungus is one of the important food sources. It is rich in vitamins, minerals and dietary fiber. It is a high-protein, low-fat, low-calorie, food for all ages. It is also a rare green food in human food in the 21st century. .
  • the fungal polysaccharides, peptides and other extremely rich biological active ingredients contained in the edible and medicinal fungi have various medicinal values such as anti-tumor, hypolipidemic, regulating immunity, and protecting cardiovascular and cerebrovascular diseases.
  • Ganoderma lucidum is a valuable medicinal and medicinal fungus with good nutritional and medicinal value.
  • Ganoderma lucidum polysaccharide is one of the main biological active substances of Ganoderma lucidum, and has activities such as immune regulation, anti-tumor, anti-oxidation, blood sugar lowering and liver protection. These biological activities also make Ganoderma lucidum polysaccharides have great application potential in the fields of food, medicine, cosmetics and the like.
  • a first object of the present invention is to provide a method for increasing the yield of fungal extracellular polysaccharides by adding phenylalanine at the beginning of the fungal fermentation or during the fermentation.
  • the fungus is a medicinal fungus.
  • the medicinal and medicinal fungi include, but are not limited to, Ganoderma lucidum, fungus, shiitake mushroom, Swine fever, white fungus, ash tree flower, medlar, Yunzhi, Hericium erinaceus, Cordyceps sinensis and common organisms thereof.
  • the fungus includes, but is not limited to, Ganoderma lucidum, Pleurotus ferulae, Coriolus hisutus, Schizophyllum commune.
  • the method is the addition of phenylalanine at 0 to 96 h of fermentation.
  • the final concentration of the phenylalanine is from 0.05 to 3.5 g/L.
  • the method is to inoculate a fungus into a fermentation medium to add phenylalanine to the fermentation medium.
  • the fermentation medium is a liquid fermentation medium commonly used for medicinal fungi.
  • the liquid fermentation medium contains: glucose 20g, tryptone 5g, amino-free yeast (YNB) 5g, potassium dihydrogen phosphate 4.5g, magnesium sulfate heptahydrate 2g, natural pH per L. .
  • a second object of the present invention is to provide a method for producing a fungal extracellular polysaccharide by inoculating a fungus into a fermentation medium, and adding phenylalanine to the fermentation medium at the beginning of the fungal fermentation or during the fermentation process. , fermentation at 25 ⁇ 33 ° C, 150 ⁇ 200r ⁇ min -1 5 ⁇ 7d.
  • inoculation is carried out in an inoculum amount of 3 to 6 g of wet weight mycelium/L medium.
  • the invention also provides for the use of the method in the preparation of a product comprising an exopolysaccharide.
  • the present invention significantly increases the yield of extracellular polysaccharides of edible and medicinal fungi by adding phenylalanine in the fermentation process without increasing the original fermentation cycle, and the maximum increase is over 40%, which is greatly reduced.
  • the production cost of edible and medicinal fungal polysaccharides is beneficial to industrial production and product application.
  • Polysaccharide extraction take 100mL fermentation broth filtrate, add 4 times of 95% alcohol, stir for 20min, centrifuge at 4000r ⁇ min -1 for 5min, remove protein, add 2.25 times 95% alcohol, stir for 20min, then let stand at 4 °C overnight. The solution was centrifuged at 10,000 r ⁇ min -1 for 5 min, the supernatant was removed, and 30 mL of distilled water was added to the precipitate to dissolve and dissolve. The solution was centrifuged at 10,000 r ⁇ min -1 for 10 min, and the clear solution was a solution of water-soluble polysaccharide.
  • the content of polysaccharide was determined by using a phenol sulfuric acid method, and the measurement system was 2 mL of a sample solution, 1 mL of 6% phenol, and 5 mL of concentrated sulfuric acid. After cooling, the OD value was measured at a wavelength of 490 nm.
  • Ganoderma lucidum seed culture Take 0.5cm 2 size of the bacteria, inoculate the seed culture medium with 80mL/250mL flask, and incubate at 150r ⁇ min -1 and 30°C for 7d.
  • Ganoderma lucidum fermentation culture 150 mL of flask was added to 150 mL of fermentation medium, and sterilized at 115 ° C for 20 minutes. The inoculum was 0.5 g wet ganoderma mycelium, cultured at 150 r ⁇ min -1 and 30 ° C for 7 days.
  • the seed and fermentation medium contained 20 g of glucose, 5 g of tryptone, 5 g of amino yeast (YNB), 4.5 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate heptahydrate, and natural pH.
  • the medium and the culture method were the same as those in Example 1, except that phenylalanine was added to the fermentation medium at 0 h to make the final concentration of phenylalanine in the medium 0.05 g/L.
  • the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.400 g/L, which was 25% higher than that of Example 1.
  • the medium and the culture method were the same as in Example 1, except that phenylalanine was added to the fermentation medium at a final concentration of 3.5 g/L.
  • the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.384 g/L, which was 20% higher than that of Example 1.
  • the medium and the culture method were the same as in Example 1, except that phenylalanine was added to the fermentation medium at a final concentration of 1 g/L.
  • the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.432 g/L, which was 35% higher than that of Example 1.
  • the medium and the culture method were the same as in Example 1, except that phenylalanine was added at 48 h of fermentation. The final concentration was 2 g/L.
  • the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.448 g/L, which was 40% higher than that of Example 1.
  • the medium and the culture method were the same as in Example 1, except that phenylalanine was added at 96 h of fermentation. The final concentration was 1 g/L.
  • the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.416 g/L, which was 30% higher than that of Example 1.
  • Ganoderma lucidum seed culture Take 0.5cm 2 size of the bacteria, inoculate the seed culture medium with 80mL/250mL flask, and incubate at 150r ⁇ min -1 and 30°C for 7d.
  • Ganoderma lucidum fermentation culture 150 mL of flask was added to 150 mL of fermentation medium, and sterilized at 115 ° C for 20 minutes. The inoculum was 0.5 g wet ganoderma mycelium, cultured at 150 r ⁇ min -1 and 30 ° C for 7 days.
  • the seed and fermentation medium contained 20 g of glucose, 10 g of corn flour, 10 g of bran, 3 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate heptahydrate, and natural pH.
  • the yield of extracellular polysaccharide was measured during the fermentation. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.368 g/L after 7 d of fermentation.
  • the medium and the culture method were the same as in Example 7, except that phenylalanine was added to the fermentation medium at a final concentration of 1 g/L.
  • the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.442 g/L, which was 20% higher than that of Example 7.
  • Ganoderma lucidum seed culture Take 0.5cm 2 size of the bacteria, inoculate the seed culture medium with 80mL/250mL flask, and incubate at 150r ⁇ min -1 and 30°C for 7d.
  • Ganoderma lucidum fermentation culture 3 L fermentation medium was added to a 5 L fermentor, and sterilized at 115 ° C for 20 minutes.
  • the inoculation amount was 10 g wet weight ganoderma lucidum mycelium, 150 r ⁇ min -1 , the aeration was 1.5 L ⁇ min -1 , and cultured at 30 ° C for 7 d.
  • the seed and fermentation medium contained 20 g of glucose, 10 g of corn flour, 10 g of bran, 3 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate heptahydrate, and natural pH.
  • Seed culture of Pleurotus ostreatus Take 0.5cm 2 size of the bacteria, inoculate the seed culture medium with 80mL/250mL flask, and incubate at 150r ⁇ min -1 and 28°C for 7d.
  • Fermentation culture of Pleurotus ostreatus 150 mL of fermentation flask was added to a 500 mL flask and sterilized at 115 ° C for 20 minutes.
  • the inoculum amount was 0.5 g wet weight mycelium, cultured at 150 r ⁇ min -1 and 28 ° C for 7 days.
  • the seed and fermentation medium contained 20 g of glucose, 5 g of tryptone, 5 g of amino yeast (YNB), 4.5 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate heptahydrate, and natural pH.
  • the medium and the culture method were the same as those in Example 10 except that phenylalanine was added to the fermentation medium at a final concentration of 1 g/L.
  • the yield of the extracellular polysaccharide of Pleurotus ostreatus was measured, and the results showed that the yield of the exopolysaccharide of Pleurotus ostreatus was 0.264 g/L, which was 31% higher than that of Example 10.
  • the medium and the culture method were the same as those in Example 10, except that the experimental strain was selected from the genus Coriolus versicolor. During the fermentation process, the yield of extracellular polysaccharide of C. versicolor was determined. The results showed that the yield of extracellular polysaccharide of C. versicolor was 0.280 g/L.
  • the medium and the culture method were the same as those in Example 10. The difference was that the experimental strain was selected from the genus Coriolus versicolor, and phenylalanine was added to the fermentation medium at a final concentration of 2 g/L. During the fermentation, the yield of the extracellular polysaccharide of C. versicolor was determined, and the results showed that the yield of the extracellular polysaccharide of C. versicolor was 0.322 g/L, which was 15% higher than that of Example 12.
  • the medium and the culture method were the same as those in Example 10, except that the experimental strain was selected from Schizophyllum.
  • the yield of the extracellular polysaccharide of the bacterium was determined, and the yield of the extracellular polysaccharide of the bacterium was 0.452 g/L.
  • the medium and the culture method were the same as those in Example 10, except that the experimental strain was selected from Schizophyllum sp., and phenylalanine was added to the fermentation medium at a final concentration of 3 g/L.
  • the yield of the extracellular polysaccharide of Schizophyllum sp. was measured, and the results showed that the yield of the extracellular polysaccharide of Schizophyllum sp. was 0.563 g/L, which was 25% higher than that of Example 14.
  • phenylalanine was added to the fermentation process of edible fungi such as fungus, shiitake mushroom, hog, white fungus, alfalfa, Yunzhi, Hericium erinaceus, and Cordyceps sinensis, and the results showed that The yield of extracellular polysaccharides has increased to varying degrees.
  • the medicinal and medicinal fungi referred to in the present application is not limited to fungi which are known to be medicinal and medicinal to produce extracellular polysaccharides at the present stage, and fungi of known or unknown species having similarities with known medicinal and medicinal fungi are suitable.
  • the method of the present application is capable of achieving an approximate effect of an increase in the production of extracellular polysaccharides.

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Abstract

A method for increasing the yield of the extracellular polysaccharides of an edible and medicinal fungus by adding phenylalanine, which relates to the field of microbial fermentation. The present method comprises exogenously adding a certain amount of phenylalanine during a fermentation culture process of an edible and medicinal fungus liquid to increase the yield of the extracellular polysaccharides of the edible and medicinal fungus by 40%, wherein the polysaccharides of the edible and medicinal fungus may be efficiently and stably synthesized, providing a new direction for the study of the polysaccharides of edible and medicinal fungi and being beneficial for application in industrial production.

Description

一种添加苯丙氨酸提高食药用真菌胞外多糖产量的方法Method for adding phenylalanine to increase the yield of extracellular polysaccharide of edible and medicinal fungi 技术领域Technical field
本发明涉及一种添加苯丙氨酸提高食药用真菌胞外多糖产量的方法，属于微生物发酵领域。The invention relates to a method for adding phenylalanine to increase the yield of extracellular polysaccharides of edible medicinal fungi, and belongs to the field of microbial fermentation.
背景技术Background technique
食用菌是重要的食物来源之一,富含多种维生素、矿物质和膳食纤维，是高蛋白、低脂肪、低热量、老少皆宜的食品，也是21世纪人类食物中不可多得的绿色食品。食药用真菌所含的真菌多糖、多肽类以及其他极为丰富的生物活性成分，具有抗肿瘤、降血脂、调节机体免疫、保护心脑血管等多种药用价值。例如，灵芝(Ganoderma lucidum)是一种名贵的食药用真菌，具有很好的营养功能和药用价值。灵芝多糖是灵芝主要的生物活性物质之一，具有免疫调节、抗肿瘤、抗氧化、降血糖、保护肝脏等活性。这些生物活性也使得灵芝多糖在食品、医药、化妆品等领域具有巨大的应用潜力。Edible fungus is one of the important food sources. It is rich in vitamins, minerals and dietary fiber. It is a high-protein, low-fat, low-calorie, food for all ages. It is also a rare green food in human food in the 21st century. . The fungal polysaccharides, peptides and other extremely rich biological active ingredients contained in the edible and medicinal fungi have various medicinal values such as anti-tumor, hypolipidemic, regulating immunity, and protecting cardiovascular and cerebrovascular diseases. For example, Ganoderma lucidum is a valuable medicinal and medicinal fungus with good nutritional and medicinal value. Ganoderma lucidum polysaccharide is one of the main biological active substances of Ganoderma lucidum, and has activities such as immune regulation, anti-tumor, anti-oxidation, blood sugar lowering and liver protection. These biological activities also make Ganoderma lucidum polysaccharides have great application potential in the fields of food, medicine, cosmetics and the like.
目前，通过液体深层发酵技术培养食药用真菌已经成为获取食药用真菌多糖的主要发酵方式，相较于固态发酵，其发酵周期短，提取成本降低，但是，其多糖产量相对较低，如何大幅提高食药用真菌多糖的发酵产量，以进一步降低生产成本，以实现在上述领域的广泛应用已成为食药用真菌液体发酵急需解决的问题。At present, the cultivation of edible and medicinal fungi by liquid submerged fermentation technology has become the main fermentation mode for obtaining edible and medicinal fungal polysaccharides. Compared with solid fermentation, the fermentation cycle is short and the extraction cost is reduced. However, the polysaccharide yield is relatively low. It is an urgent problem to solve the liquid fermentation of edible and medicinal fungi by greatly increasing the fermentation yield of edible and medicinal fungal polysaccharides to further reduce the production cost, so as to achieve wide application in the above fields.
发明内容Summary of the invention
本发明的第一个目的是提供一种提高真菌胞外多糖产量的方法，是在真菌发酵初始时或发酵过程中加入苯丙氨酸。A first object of the present invention is to provide a method for increasing the yield of fungal extracellular polysaccharides by adding phenylalanine at the beginning of the fungal fermentation or during the fermentation.
在本发明的一种实施方式中，所述真菌为食药用真菌。In one embodiment of the invention, the fungus is a medicinal fungus.
在本发明的一种实施方式中，所述食药用真菌包括但不限于灵芝、木耳、香菇、猪苓、银耳、灰树花、茯苓、云芝、猴头菇、冬虫夏草及其常见的生物分类学上亲缘关系相近或性状相近的食药用真菌子实体或菌丝体。In one embodiment of the present invention, the medicinal and medicinal fungi include, but are not limited to, Ganoderma lucidum, fungus, shiitake mushroom, Swine fever, white fungus, ash tree flower, medlar, Yunzhi, Hericium erinaceus, Cordyceps sinensis and common organisms thereof. A medicinal fungal fruiting body or mycelium of similar phylogenetic relationship or similar traits.
在本发明的一种实施方式中，所述真菌包括但不限于灵芝(Ganoderma lucidum)、阿魏蘑(Pleurotus ferulae)、彩绒革盖菌(Coriolus hisutus)、裂褶菌(Schizophyllum commune)。In one embodiment of the invention, the fungus includes, but is not limited to, Ganoderma lucidum, Pleurotus ferulae, Coriolus hisutus, Schizophyllum commune.
在本发明的一种实施方式中，所述方法是在发酵0～96h添加苯丙氨酸。In one embodiment of the invention, the method is the addition of phenylalanine at 0 to 96 h of fermentation.
在本发明的一种实施方式中，所述苯丙氨酸的终浓度为0.05～3.5g/L。In one embodiment of the invention, the final concentration of the phenylalanine is from 0.05 to 3.5 g/L.
在本发明的一种实施方式中，所述方法是将真菌接种至发酵培养基中，向发酵培养基中添加苯丙氨酸。In one embodiment of the invention, the method is to inoculate a fungus into a fermentation medium to add phenylalanine to the fermentation medium.
在本发明的一种实施方式中，发酵培养基为食药用真菌常用液体发酵培养基。In one embodiment of the invention, the fermentation medium is a liquid fermentation medium commonly used for medicinal fungi.
在本发明的一种实施方式中，所述液体发酵培养基每L含有：葡萄糖20g，胰蛋白胨5g，无氨基酵母(YNB)5g，磷酸二氢钾4.5g，七水硫酸镁2g，自然pH。In one embodiment of the present invention, the liquid fermentation medium contains: glucose 20g, tryptone 5g, amino-free yeast (YNB) 5g, potassium dihydrogen phosphate 4.5g, magnesium sulfate heptahydrate 2g, natural pH per L. .
本发明的第二个目的是提供一种真菌胞外多糖的生产方法，所述方法是将真菌接种至发酵培养基中，在真菌发酵初始时或发酵过程向发酵培养基中加入苯丙氨酸，于25～33℃，150～200r·min -1发酵5～7d。 A second object of the present invention is to provide a method for producing a fungal extracellular polysaccharide by inoculating a fungus into a fermentation medium, and adding phenylalanine to the fermentation medium at the beginning of the fungal fermentation or during the fermentation process. , fermentation at 25 ~ 33 ° C, 150 ~ 200r · min -1 5 ~ 7d.
在本发明的一种实施方式中，按3～6g湿重菌丝体/L培养基的接种量进行接种。In one embodiment of the present invention, inoculation is carried out in an inoculum amount of 3 to 6 g of wet weight mycelium/L medium.
本发明还提供所述方法在制备含有胞外多糖的产品方面的应用。The invention also provides for the use of the method in the preparation of a product comprising an exopolysaccharide.
有益效果：本发明在发酵过程中通过添加苯丙氨酸，在不增加原有发酵周期的基础上，显著提高了食药用真菌胞外多糖产量，最大提高幅度达40％以上，大大降低了食药用真菌多糖的生产成本，有利于工业化生产和产品应用。Advantageous Effects: The present invention significantly increases the yield of extracellular polysaccharides of edible and medicinal fungi by adding phenylalanine in the fermentation process without increasing the original fermentation cycle, and the maximum increase is over 40%, which is greatly reduced. The production cost of edible and medicinal fungal polysaccharides is beneficial to industrial production and product application.
具体实施方式Detailed ways
多糖提取：取100mL发酵液滤液，加4倍的95％酒精，搅拌20min，4000r·min -1离心5min，去除蛋白，上清液加2.25倍95％酒精，搅拌20min后，于4℃静置过夜。溶液经10000r·min -1离心5min，去上清，沉淀加30mL蒸馏水振荡溶解，10000r·min -1离心10min，清液即为水溶性多糖的溶液。 Polysaccharide extraction: take 100mL fermentation broth filtrate, add 4 times of 95% alcohol, stir for 20min, centrifuge at 4000r·min -1 for 5min, remove protein, add 2.25 times 95% alcohol, stir for 20min, then let stand at 4 °C overnight. The solution was centrifuged at 10,000 r·min -1 for 5 min, the supernatant was removed, and 30 mL of distilled water was added to the precipitate to dissolve and dissolve. The solution was centrifuged at 10,000 r·min -1 for 10 min, and the clear solution was a solution of water-soluble polysaccharide.
多糖测定：测定多糖的含量，采用苯酚硫酸法，测定体系为2mL的样品溶液，1mL的6％的苯酚，5mL的浓硫酸。冷却后在490nm的波长下测定OD值。Determination of polysaccharide: The content of polysaccharide was determined by using a phenol sulfuric acid method, and the measurement system was 2 mL of a sample solution, 1 mL of 6% phenol, and 5 mL of concentrated sulfuric acid. After cooling, the OD value was measured at a wavelength of 490 nm.
实施例1Example 1
灵芝种子培养：取0.5cm 2大小的菌块，接种到装液量80mL/250mL三角瓶的种子培养基中，150r·min -1、30℃培养7d。 Ganoderma lucidum seed culture: Take 0.5cm 2 size of the bacteria, inoculate the seed culture medium with 80mL/250mL flask, and incubate at 150r·min -1 and 30°C for 7d.
灵芝发酵培养：500mL三角瓶中加入150mL发酵培养基，115℃灭菌20分钟。接种量为0.5g湿重灵芝菌丝体，150r·min -1、30℃培养7d。 Ganoderma lucidum fermentation culture: 150 mL of flask was added to 150 mL of fermentation medium, and sterilized at 115 ° C for 20 minutes. The inoculum was 0.5 g wet ganoderma mycelium, cultured at 150 r·min -1 and 30 ° C for 7 days.
所述种子与发酵培养基每L含有葡萄糖20g，胰蛋白胨5g，无氨基酵母(YNB)5g，磷酸二氢钾4.5g，七水硫酸镁2g，自然pH。The seed and fermentation medium contained 20 g of glucose, 5 g of tryptone, 5 g of amino yeast (YNB), 4.5 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate heptahydrate, and natural pH.
发酵过程中，测定灵芝胞外多糖的产量。结果显示，培养7d的灵芝多糖的产量为0.320g/L。During the fermentation, the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of the Ganoderma lucidum polysaccharide cultured for 7 days was 0.320 g/L.
实施例2Example 2
培养基和培养方法同实施例1，区别在于，0h在发酵培养基中添加苯丙氨酸，使培养基中苯丙氨酸的终浓度为0.05g/L。发酵过程中，测定灵芝胞外多糖的产量。结果显示，灵芝多 糖的产量为0.400g/L，相对于实施例1提高了25％。The medium and the culture method were the same as those in Example 1, except that phenylalanine was added to the fermentation medium at 0 h to make the final concentration of phenylalanine in the medium 0.05 g/L. During the fermentation, the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.400 g/L, which was 25% higher than that of Example 1.
实施例3Example 3
培养基和培养方法同实施例1，区别在于，0h在发酵培养基中添加苯丙氨酸，使其终浓度为3.5g/L。发酵过程中，测定灵芝胞外多糖的产量。结果显示，灵芝多糖的产量为0.384g/L，相对于实施例1提高了20％。The medium and the culture method were the same as in Example 1, except that phenylalanine was added to the fermentation medium at a final concentration of 3.5 g/L. During the fermentation, the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.384 g/L, which was 20% higher than that of Example 1.
实施例4Example 4
培养基和培养方法同实施例1，区别在于，0h在发酵培养基中添加苯丙氨酸，使其终浓度为1g/L。发酵过程中，测定灵芝胞外多糖的产量。结果显示，灵芝多糖的产量为0.432g/L，相对于实施例1提高了35％。The medium and the culture method were the same as in Example 1, except that phenylalanine was added to the fermentation medium at a final concentration of 1 g/L. During the fermentation, the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.432 g/L, which was 35% higher than that of Example 1.
实施例5Example 5
培养基和培养方法同实施例1，区别在于，在发酵48h时添加苯丙氨酸。使其终浓度为2g/L。发酵过程中，测定灵芝胞外多糖的产量。结果显示，灵芝多糖的产量为0.448g/L，相对于实施例1提高了40％。The medium and the culture method were the same as in Example 1, except that phenylalanine was added at 48 h of fermentation. The final concentration was 2 g/L. During the fermentation, the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.448 g/L, which was 40% higher than that of Example 1.
实施例6Example 6
培养基和培养方法同实施例1，区别在于，在发酵96h时添加苯丙氨酸。使其终浓度为1g/L。发酵过程中，测定灵芝胞外多糖的产量。结果显示，灵芝多糖的产量为0.416g/L，相对于实施例1提高了30％。The medium and the culture method were the same as in Example 1, except that phenylalanine was added at 96 h of fermentation. The final concentration was 1 g/L. During the fermentation, the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.416 g/L, which was 30% higher than that of Example 1.
实施例7Example 7
灵芝种子培养：取0.5cm 2大小的菌块，接种到装液量80mL/250mL三角瓶的种子培养基中，150r·min -1、30℃培养7d。 Ganoderma lucidum seed culture: Take 0.5cm 2 size of the bacteria, inoculate the seed culture medium with 80mL/250mL flask, and incubate at 150r·min -1 and 30°C for 7d.
灵芝发酵培养：500mL三角瓶中加入150mL发酵培养基，115℃灭菌20分钟。接种量为0.5g湿重灵芝菌丝体，150r·min -1、30℃培养7d。 Ganoderma lucidum fermentation culture: 150 mL of flask was added to 150 mL of fermentation medium, and sterilized at 115 ° C for 20 minutes. The inoculum was 0.5 g wet ganoderma mycelium, cultured at 150 r·min -1 and 30 ° C for 7 days.
所述种子与发酵培养基每L含有葡萄糖20g，玉米粉10g，麸皮10g，磷酸二氢钾3g，七水硫酸镁2g，自然pH。The seed and fermentation medium contained 20 g of glucose, 10 g of corn flour, 10 g of bran, 3 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate heptahydrate, and natural pH.
发酵过程中，测定胞外多糖的产量。结果显示，发酵7d，灵芝多糖的产量为0.368g/L。The yield of extracellular polysaccharide was measured during the fermentation. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.368 g/L after 7 d of fermentation.
实施例8Example 8
培养基和培养方法同实施例7，区别在于，0h在发酵培养基中添加苯丙氨酸，使其终浓度为1g/L。发酵过程中，测定灵芝胞外多糖的产量。结果显示，灵芝多糖的产量为0.442g/L，相对于实施例7提高了20％。The medium and the culture method were the same as in Example 7, except that phenylalanine was added to the fermentation medium at a final concentration of 1 g/L. During the fermentation, the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.442 g/L, which was 20% higher than that of Example 7.
实施例9Example 9
灵芝种子培养：取0.5cm 2大小的菌块，接种到装液量80mL/250mL三角瓶的种子培养基中，150r·min -1、30℃培养7d。 Ganoderma lucidum seed culture: Take 0.5cm 2 size of the bacteria, inoculate the seed culture medium with 80mL/250mL flask, and incubate at 150r·min -1 and 30°C for 7d.
灵芝发酵培养：5L发酵罐中加入3L发酵培养基，115℃灭菌20分钟。接种量为10g湿重灵芝菌丝体，150r·min -1，通气量为1.5L·min -1，30℃培养7d。 Ganoderma lucidum fermentation culture: 3 L fermentation medium was added to a 5 L fermentor, and sterilized at 115 ° C for 20 minutes. The inoculation amount was 10 g wet weight ganoderma lucidum mycelium, 150 r·min -1 , the aeration was 1.5 L·min -1 , and cultured at 30 ° C for 7 d.
所述种子与发酵培养基每L含有葡萄糖20g，玉米粉10g，麸皮10g，磷酸二氢钾3g，七水硫酸镁2g，自然pH。The seed and fermentation medium contained 20 g of glucose, 10 g of corn flour, 10 g of bran, 3 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate heptahydrate, and natural pH.
在24～96小时的发酵过程中连续流加浓度为20g/L的苯丙氨酸，发酵7d，共计流加160mL，使其终浓度为1g/L。发酵过程中，测定灵芝胞外多糖的产量。结果显示，灵芝多糖的产量为0.471g/L，相对于实施例7提高了28％。During the fermentation period of 24 to 96 hours, a concentration of 20 g/L of phenylalanine was continuously added, and the fermentation was carried out for 7 days, and a total of 160 mL was added to make a final concentration of 1 g/L. During the fermentation, the yield of Ganoderma lucidum extracellular polysaccharide was determined. The results showed that the yield of Ganoderma lucidum polysaccharide was 0.471 g/L, which was 28% higher than that of Example 7.
实施例10Example 10
阿魏蘑种子培养：取0.5cm 2大小的菌块，接种到装液量80mL/250mL三角瓶的种子培养基中，150r·min -1、28℃培养7d。 Seed culture of Pleurotus ostreatus: Take 0.5cm 2 size of the bacteria, inoculate the seed culture medium with 80mL/250mL flask, and incubate at 150r·min -1 and 28°C for 7d.
阿魏蘑发酵培养：500mL三角瓶中加入150mL发酵培养基，115℃灭菌20分钟。接种量为0.5g湿重菌丝体，150r·min -1、28℃培养7d。 Fermentation culture of Pleurotus ostreatus: 150 mL of fermentation flask was added to a 500 mL flask and sterilized at 115 ° C for 20 minutes. The inoculum amount was 0.5 g wet weight mycelium, cultured at 150 r·min -1 and 28 ° C for 7 days.
所述种子与发酵培养基每L含有葡萄糖20g，胰蛋白胨5g，无氨基酵母(YNB)5g，磷酸二氢钾4.5g，七水硫酸镁2g，自然pH。The seed and fermentation medium contained 20 g of glucose, 5 g of tryptone, 5 g of amino yeast (YNB), 4.5 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate heptahydrate, and natural pH.
发酵过程中，测定阿魏蘑胞外多糖的产量，结果显示，阿魏蘑胞外多糖的产量为0.201g/L。During the fermentation, the yield of extracellular polysaccharides of Pleurotus ostreatus was determined. The results showed that the yield of extracellular polysaccharides of Pleurotus ostreatus was 0.201 g/L.
实施例11Example 11
培养基和培养方法同实施例10，区别在于，0h在发酵培养基中添加苯丙氨酸，使其终浓度为1g/L。发酵过程中，测定阿魏蘑胞外多糖的产量，结果显示，阿魏蘑胞外多糖的产量为0.264g/L，相对于实施例10提高了31％。The medium and the culture method were the same as those in Example 10 except that phenylalanine was added to the fermentation medium at a final concentration of 1 g/L. During the fermentation, the yield of the extracellular polysaccharide of Pleurotus ostreatus was measured, and the results showed that the yield of the exopolysaccharide of Pleurotus ostreatus was 0.264 g/L, which was 31% higher than that of Example 10.
实施例12Example 12
培养基和培养方法同实施例10，区别在于，实验菌株选用彩绒革盖菌。发酵过程中，测定彩绒革盖菌胞外多糖的产量，结果显示，彩绒革盖菌胞外多糖的产量为0.280g/L。The medium and the culture method were the same as those in Example 10, except that the experimental strain was selected from the genus Coriolus versicolor. During the fermentation process, the yield of extracellular polysaccharide of C. versicolor was determined. The results showed that the yield of extracellular polysaccharide of C. versicolor was 0.280 g/L.
实施例13Example 13
培养基和培养方法同实施例10，区别在于，实验菌株选用彩绒革盖菌，而且0h在发酵培养基中添加苯丙氨酸，使其终浓度为2g/L。发酵过程中，测定彩绒革盖菌胞外多糖的产量，结果显示，彩绒革盖菌胞外多糖的产量为0.322g/L，相对于实施例12提高了15％。The medium and the culture method were the same as those in Example 10. The difference was that the experimental strain was selected from the genus Coriolus versicolor, and phenylalanine was added to the fermentation medium at a final concentration of 2 g/L. During the fermentation, the yield of the extracellular polysaccharide of C. versicolor was determined, and the results showed that the yield of the extracellular polysaccharide of C. versicolor was 0.322 g/L, which was 15% higher than that of Example 12.
实施例14Example 14
培养基和培养方法同实施例10，区别在于，实验菌株选用裂褶菌。发酵过程中，测定裂 褶菌胞外多糖的产量，结果显示，裂褶菌胞外多糖的产量为0.452g/L。The medium and the culture method were the same as those in Example 10, except that the experimental strain was selected from Schizophyllum. During the fermentation, the yield of the extracellular polysaccharide of the bacterium was determined, and the yield of the extracellular polysaccharide of the bacterium was 0.452 g/L.
实施例15Example 15
培养基和培养方法同实施例10，区别在于，实验菌株选用裂褶菌，而且0h在发酵培养基中添加苯丙氨酸，使其终浓度为3g/L。发酵过程中，测定裂褶菌胞外多糖的产量，结果显示，裂褶菌胞外多糖的产量为0.563g/L，相对于实施例14提高了25％。The medium and the culture method were the same as those in Example 10, except that the experimental strain was selected from Schizophyllum sp., and phenylalanine was added to the fermentation medium at a final concentration of 3 g/L. During the fermentation, the yield of the extracellular polysaccharide of Schizophyllum sp. was measured, and the results showed that the yield of the extracellular polysaccharide of Schizophyllum sp. was 0.563 g/L, which was 25% higher than that of Example 14.
实施例16Example 16
采用与实施例1～15相同的策略，分别在木耳、香菇、猪苓、银耳、茯苓、云芝、猴头菇、冬虫夏草等食药用真菌的发酵过程中添加苯丙氨酸，结果显示，胞外多糖的产量具有不同程度的提高。Using the same strategy as in Examples 1 to 15, phenylalanine was added to the fermentation process of edible fungi such as fungus, shiitake mushroom, hog, white fungus, alfalfa, Yunzhi, Hericium erinaceus, and Cordyceps sinensis, and the results showed that The yield of extracellular polysaccharides has increased to varying degrees.
对照例1Comparative Example 1
按照实施例1相同的方式，区别在于，分别向发酵液中加入终浓度为1g/L的氨基酸，测定胞外多糖产量，结果如下表所示。In the same manner as in Example 1, the difference was that amino acids having a final concentration of 1 g/L were separately added to the fermentation liquid, and the yield of extracellular polysaccharide was measured. The results are shown in the following table.
表1 添加不同氨基酸后的灵芝胞外多糖产量变化Table 1 Changes in the production of Ganoderma lucidum exopolysaccharide after adding different amino acids
Figure PCTCN2018074935-appb-000001
Figure PCTCN2018074935-appb-000001
注：“-”表示产量降低。Note: “-” indicates a decrease in production.
本申请所指的食药用真菌不局限于现阶段为人们所知食药用产胞外多糖的真菌，与已知的食药用真菌存在相似性的已知或未知种属的真菌皆适用于本申请的方法，并能够达到胞外多糖产量提高的近似效果。The medicinal and medicinal fungi referred to in the present application is not limited to fungi which are known to be medicinal and medicinal to produce extracellular polysaccharides at the present stage, and fungi of known or unknown species having similarities with known medicinal and medicinal fungi are suitable. The method of the present application is capable of achieving an approximate effect of an increase in the production of extracellular polysaccharides.
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed in the above preferred embodiments, the present invention is not limited thereto, and various modifications and changes can be made thereto without departing from the spirit and scope of the invention. The scope of the invention should be determined by the scope of the claims.

Claims (10)

  1. 一种提高真菌胞外多糖产量的方法，其特征在于，是在真菌发酵初始时或发酵过程中加入苯丙氨酸。A method for increasing the yield of fungal extracellular polysaccharides, characterized in that phenylalanine is added at the beginning of the fungal fermentation or during the fermentation.
  2. 根据权利要求1所述的方法，其特征在于，所述真菌为食药用真菌。The method of claim 1 wherein the fungus is a medicinal medicinal fungus.
  3. 根据权利要求1或2所述的方法，其特征在于，所述真菌包括灵芝(Ganoderma lucidum)、阿魏蘑(Pleurotus ferulae)、彩绒革盖菌(Coriolus hisutus)或裂褶菌(Schizophyllum commune)。The method according to claim 1 or 2, wherein the fungus comprises Ganoderma lucidum, Pleurotus ferulae, Coriolus hisutus or Schizophyllum commune. .
  4. 根据权利要求1～3任一所述的方法，其特征在于，苯丙氨酸的终浓度为0.05～3.5g/L。The method according to any one of claims 1 to 3, wherein the final concentration of phenylalanine is 0.05 to 3.5 g/L.
  5. 根据权利要求4所述的方法，其特征在于，在发酵0～96h添加苯丙氨酸。The method according to claim 4, wherein phenylalanine is added at 0 to 96 hours of fermentation.
  6. 根据权利要求1～5所述的方法，其特征在于，所述方法通过分批补加或连续流加的方式加入苯丙氨酸。Process according to any of claims 1 to 5, characterized in that the method adds phenylalanine by means of batch addition or continuous addition.
  7. 一种食药用真菌的胞外多糖生产方法，其特征在于，将真菌接种至发酵培养基中进行发酵，并在发酵的第0～96h向发酵培养基中添加苯丙氨酸。A method for producing an extracellular polysaccharide of a medicinal fungus, characterized in that a fungus is inoculated into a fermentation medium for fermentation, and phenylalanine is added to the fermentation medium at 0 to 96 h of the fermentation.
  8. 根据权利要求7所述的方法，其特征在于，在真菌发酵初始时或发酵过程向发酵培养基中加入终浓度为0.05～3.5g/L的苯丙氨酸，于25～33℃，150～200r·min -1发酵5～7d。 The method according to claim 7, wherein a phenylalanine having a final concentration of 0.05 to 3.5 g/L is added to the fermentation medium at the beginning of the fungal fermentation or during the fermentation, at 25 to 33 ° C, 150 °. 200 r·min -1 fermentation for 5-7 days.
  9. 根据权利要求7或8所述的方法，其特征在于，按3～6g湿重菌丝体/L培养基的接种量接种食药用真菌。The method according to claim 7 or 8, wherein the medicinal fungus is inoculated in an amount of 3 to 6 g of the wet weight mycelium/L medium.
  10. 权利要求1～9任一所述方法在制备含有胞外多糖的产品方面的应用。Use of the method according to any one of claims 1 to 9 for the preparation of a product containing an exopolysaccharide.
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CN102311469A (en) * 2011-09-16 2012-01-11 陕西科技大学 Method for producing scutelloside by use of Scutellaria baicalensis Georgi hairy roots

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