WO2019148418A1 - Procédé d'augmentation du rendement de polysaccharides extracellulaires de champignon comestible et médicinal par ajout de phénylalanine - Google Patents

Procédé d'augmentation du rendement de polysaccharides extracellulaires de champignon comestible et médicinal par ajout de phénylalanine Download PDF

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Publication number
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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WIPO (PCT)
Prior art keywords
fermentation
phenylalanine
yield
medicinal
edible
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PCT/CN2018/074935
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English (en)
Chinese (zh)
Inventor
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 PCT/CN2018/074935 priority Critical patent/WO2019148418A1/fr
Priority to JP2020535638A priority patent/JP6995206B2/ja
Publication of WO2019148418A1 publication Critical patent/WO2019148418A1/fr

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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

Definitions

  • 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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  • Chemical & Material Sciences (AREA)
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Abstract

La présente invention concerne un procédé d'augmentation du rendement des polysaccharides extracellulaires d'un champignon comestible et médicinal par ajout de phénylalanine, qui concerne le domaine de la fermentation microbienne. Le présent procédé comprend l'ajout exogène d'une certaine quantité de phénylalanine pendant un processus de culture par fermentation d'un liquide de champignon comestible et médicinal pour augmenter le rendement des polysaccharides extracellulaires du champignon comestible et médicinal de 40 %, les polysaccharides du champignon comestible et médicinal pouvant être synthétisés de façon efficace et stable, ce qui constitue une nouvelle direction pour l'étude des polysaccharides de champignons comestibles et médicinaux et est avantageux pour application en production industrielle.
PCT/CN2018/074935 2018-02-01 2018-02-01 Procédé d'augmentation du rendement de polysaccharides extracellulaires de champignon comestible et médicinal par ajout de phénylalanine WO2019148418A1 (fr)

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PCT/CN2018/074935 WO2019148418A1 (fr) 2018-02-01 2018-02-01 Procédé d'augmentation du rendement de polysaccharides extracellulaires de champignon comestible et médicinal par ajout de phénylalanine
JP2020535638A JP6995206B2 (ja) 2018-02-01 2018-02-01 フェニルアラニンの添加により食用・薬用真菌の細胞外多糖の収量を高める方法

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100021426A1 (en) * 2003-10-03 2010-01-28 Wang ya-chun Methods for making and compositions comprising fermentation products of cordyceps sinensis
CN102311469A (zh) * 2011-09-16 2012-01-11 陕西科技大学 一种利用黄芩毛状根生产黄芩苷的方法

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Publication number Priority date Publication date Assignee Title
NO20014256D0 (no) * 2001-09-03 2001-09-03 Bjoern Kristiansen Fremstilling av immunstimulerende forbindelse

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100021426A1 (en) * 2003-10-03 2010-01-28 Wang ya-chun Methods for making and compositions comprising fermentation products of cordyceps sinensis
CN102311469A (zh) * 2011-09-16 2012-01-11 陕西科技大学 一种利用黄芩毛状根生产黄芩苷的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JING LIUPING ET AL.: "Effect of eight kinds of precursor and nutrient on cordycepin and polysaccharide production", JOURNAL OF NORTHWEST A & F UNIVERSITY (NATURAL SCIENCE EDITION), vol. 38, no. 11, 10 November 2010 (2010-11-10), pages 156 - 160, ISSN: 1671-9387 *

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