CN105779299B - Paecilomyces hepiali strain for producing adenosine and mannitol substances and application thereof - Google Patents
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Abstract
The invention discloses a Paecilomyces hepiali strain for producing adenosine and mannitol substances, which is separated from wild cordyceps sinensis in Qinghai-Tibet plateau and an application thereof, wherein the Paecilomyces hepiali (Paecilomyces hepiali) JNPF-PH01 strain is currently stored in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the collection number of CGMCC No. 11803. The strain is obtained by separation and screening, the optimal fermentation medium disclosed by the invention is applied to production in a 500L fermentation tank, the biomass can reach 36.1g/L, the adenosine content and the yield are respectively 0.61% and 220.1mg/L, and the mannitol substance content and the yield are respectively 12.8% and 4.6g/L, which are higher than the highest adenosine (110.2mg/L) and mannitol substance (1.4g/L) yield of the Paecilomyces hepiali strain reported at present.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a paecilomyces hepiali strain for producing adenosine and mannitol substances and application thereof.
Background
The cordyceps sinensis is a high-grade nourishing and rare traditional Chinese medicinal material, and is recorded in 'materia medica assistant new' at an early application history in folk, and comprises the following components: "Cordyceps sinensis has the effects of protecting lung, tonifying kidney, stopping bleeding and resolving phlegm, and has cough … … just like the folk reproduced tonic edible bird's nest. The cordyceps sinensis is a complex formed by fruiting bodies of cordyceps sinensis and batryticated sclerotium (larva corpse of hepialus), mainly contains cordycepin, cordycepic acid, adenosine, polysaccharide, ergosterol and other components, can inhibit the growth of various pathogenic bacteria, increase the cell number of an immune system, promote the generation of antibodies, has good regulation effect on an endocrine system and a nervous system, has anti-tumor active substances, and has extremely high health care and medicinal values.
The natural cordyceps sinensis has rare resources, special growth environment and long growth period, and in addition, the ecological environment is seriously damaged by excessive digging, and the artificial cultivation difficulty is very high. In order to alleviate the contradiction between supply and demand in the market, researchers try to separate strains from cordyceps sinensis, and produce mycelia as a substitute of cordyceps sinensis medicinal materials through fermentation. But the ministry of health stipulates that only 2 mycelia produced by cordyceps sinensis strains can be used for health-care food, and one of the mycelia is paecilomyces hepiali.
The mycelium obtained by liquid fermentation of Paecilomyces hepiali (Paecilomyces hepiali) is very similar to the main chemical components and pharmacological action of Cordyceps sinensis. The nucleoside components contained in the bacterial powder have the drug effect of expanding heart and peripheral blood vessels, wherein the adenosine has the most obvious effect, and can relieve stress and tension, improve sleep, enhance physical strength and resist fatigue. The contained mannitol has the effects of relieving cough and asthma, reducing respiratory tract problems, reducing intracranial pressure and relieving cerebral hemorrhage and cerebral thrombosis. The fermented cordyceps powder has the advantages of lower price than natural cordyceps, easy control of growth environment and heavy metal content of mycelium, and the like. Therefore, the large-scale and high-quality industrial fermentation production of the paecilomyces hepiali has important significance for promoting the health of people and improving the quality of life.
The patent CN101690463B provides a mutant strain of Cordyceps militaris and a cultivation method thereof, a new strain for producing Cordyceps militaris is obtained by chemical mutagenesis, the yield of adenosine is 70.0mg/L, and the yield of mannitol substances is 1.4 g/L; the patent CN104911109A provides a mutant strain for producing paecilomyces hepiali and a cultivation method thereof, wherein the yield of adenosine is 110.2mg/L, and the yield of mannitol substances is 1.4 g/L. The invention discloses a paecilomyces hepiali strain for producing adenosine and mannitol substances, wherein the yield of the adenosine and the mannitol substances respectively reaches 220.1mg/L and 4.6g/L after fermentation optimization.
Disclosure of Invention
The invention aims to solve the technical problem of providing a paecilomyces hepiali strain for producing adenosine and mannitol substances and a fermentation process thereof, so as to improve the yield of the adenosine and mannitol substances produced by the paecilomyces hepiali industry.
In order to solve the technical problems, the specific technical scheme provided by the invention is as follows:
strain separation: the method comprises the steps of disinfecting the surface of wild cordyceps sinensis in Qinghai-Tibet plateau, cutting the wild cordyceps sinensis into small blocks with the size of 2mm multiplied by 2mm, inoculating the small blocks into a PDA culture medium, culturing at 25 ℃, when aerial hyphae grow out around tissues, picking hyphae from the edges of bacterial colonies by using an inoculating loop, inoculating the hyphae into a new culture medium, separating and purifying the strains by a method of picking hyphae at the edges of the strains for multiple times, and finally obtaining 11 filamentous fungus strains.
And (3) screening in a shaking flask: inoculating the 11 filamentous fungus seed solutions into a medium containing 100mL of fermentation medium (grape) at an inoculation amount of 5% (v/v)2-4% of sugar, 1-3% of soybean cake powder and KH2PO40.01～0.5％，MgSO40.01-0.5%) in a 500mL shake flask, culturing at 25 ℃ and 150rpm for 6d, centrifuging at 6000rpm, collecting mycelia, drying, determining biomass, and obtaining bacterial powder. The content of adenosine and mannitol substances in the bacterial powder is measured according to a method described in Chinese pharmacopoeia 2010, and the No. 9 bacterial strain is found to grow rapidly, have high biomass and have the highest content of adenosine and mannitol substances, so the No. 9 bacterial strain is selected for further research.
And (3) strain identification: the No. 9 strain hypha is white velvet, dense, oval in colony shape, slightly bulged in the middle, neat in edge, provided with a diaphragm and oval or rod-shaped spores, and the diameter of the spores is 2.0-5.0 mu M. Extracting total DNA of the No. 9 strain, amplifying by using universal primers of fungus 18SrDNA and ITS to obtain a corresponding gene sequence, sequencing and comparing, and determining that the No. 9 strain is Paecilomyces hepialid (Paecilomyces hepiali) JNPF-PH01 by combining morphological characteristics of the strain, wherein the strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, No. 3 of No.1 Hopkins of West Lu 1 of the sunward area in Beijing at 12.2.2015, and the preservation number is CGMCC No. 11803.
Optimizing a fermentation medium: the fermentation medium and the fermentation conditions of the strain are optimized by utilizing a single-factor experiment and an orthogonal experiment, and the optimal culture medium formula is obtained as follows: 1-4% of glucose, 1-3% of yeast powder, 1-3% of peptone, 1-3% of soybean cake powder and KH2PO40.1～1.0％，MgSO40.01-0.5% and VB 10.01-0.5%. The culture conditions were: the inoculation amount is 5% (v/v), the temperature is 25 ℃, the rotating speed is 150r/min, the liquid loading amount is 100mL/500mL, and the fermentation period is 6 d.
Carrying out amplification culture in a 100L fermentation tank: inoculating the seed solution into a 100L fermentation tank filled with 50L of optimal fermentation medium according to the inoculation amount of 5% (v/v), wherein the temperature is 25 ℃, the speed is 150r/min, and the ventilation volume is 8-14 m3And h, the tank pressure is 0.06-0.14 MPa, and the culture lasts for 5 days.
And (3) performing amplification culture in a 500L fermentation tank: inoculating the seed liquid into a 500L fermentation tank filled with 400L optimal fermentation medium according to the inoculation amount of 5% (v/v), wherein the temperature is 25 ℃, the speed is 150r/min, and the ventilation volume is 8-14 m3And h, the tank pressure is 0.06-0.14 MPa, and the culture lasts for 4 days.
The invention has the beneficial effects that: a Paecilomyces hepiali strain for producing adenosine and mannitol substances and its fermentation process are provided. The strain and the optimal fermentation medium are applied to the production in a 500L fermentation tank, the final biomass can reach 36.1g/L, the adenosine content and the yield are respectively 0.61 percent and 220.1mg/L, the mannitol substance content and the yield are respectively 12.8 percent and 4.6g/L, and the yield is higher than the highest adenosine (110.2mg/L) and mannitol substance (1.4g/L) yield of the Paecilomyces hepiali strain reported at present.
Detailed Description
Example 1 isolation of endophytic fungus strains of Cordyceps sinensis
1. Culture medium
PDA culture medium: 1.0L of potato extract, 20.0g of glucose and 15.0g of agar.
Seed culture medium: glucose 3%, peptone 1%, KH2PO40.1％，MgSO40.05％。
2. The method comprises the steps of disinfecting the surface of wild cordyceps sinensis in Qinghai-Tibet plateau, cutting the wild cordyceps sinensis into small blocks with the size of 2mm multiplied by 2mm, inoculating the small blocks into a PDA culture medium, culturing at 25 ℃, picking hyphae from the edge of a bacterial colony by using an inoculating loop when aerial hyphae grow out around tissues, inoculating the hyphae into a new culture medium, and separating and purifying the strains by a method of picking hyphae at the edge of the strains for multiple times. And performing primary separation and purification to obtain 29 strains of fungi, preliminarily determining 25 strains of fungi, 3 strains of bacteria and 1 strain of actinomycetes according to morphological observation, and selecting the strains with better growth for continuous culture to finally obtain 11 strains of filamentous fungi.
Example 2 Shake flask screening and Strain identification
And (3) carrying out shake flask fermentation on the 11 separated filamentous fungi by using a fermentation medium, detecting indexes such as biomass, adenosine content, mannitol substance content and the like, and screening out strains with good growth vigor and high adenosine and mannitol substance yield for strain identification.
1. Culture medium
Fermentation medium: 3% of glucose, 1% of peptone, 2% of bean cake powder and KH2PO40.1％，MgSO40.05％。
2. Detection method
(1) Biomass detection
Centrifuging the fermentation liquid at 6000r/min for 10min, removing the supernatant, drying the precipitate in an oven at 105 ℃ to constant weight, and calculating the biomass. The mycelium is crushed and then used for detecting the content of adenosine and mannitol substances.
(2) Adenosine content detection
And determining the adenosine content in the bacterial powder by high performance liquid chromatography with reference to appendix VD of the second part of the Chinese pharmacopoeia 2000 edition. The operation steps are as follows: accurately weighing 0.25g of bacterial powder, placing the bacterial powder into a 50mL measuring flask, adding a proper amount of 0.01mol/L sodium phosphate solution, fully shaking, ultrasonically extracting for 20 minutes, cooling, adding 0.01mol/L sodium phosphate solution to scale, shaking uniformly, filtering, and filtering the subsequent filtrate by a 0.22 mu m membrane for adenosine content detection. Chromatographic conditions are as follows: a chromatographic column: waters Xbridge C18 Column, 4.6X 250mm Column (5 μm); a detector: an ultraviolet detector; mobile phase: water-methanol (85: 15); flow rate: 0.5 mL/min; detection wavelength: 260 nm; column temperature: at 30 ℃.
(3) Mannitol-based substance detection
Referring to appendix V D of the second part of the Chinese pharmacopoeia 2000 edition, the content of mannitol substances in the fungus powder is determined by a titration method. The operation steps are as follows: accurately weighing 0.5g of bacterial powder, placing the bacterial powder into a 100mL measuring flask, adding a proper amount of 0.01mol/L sodium phosphate solution, shaking for 30 minutes, diluting to scale, shaking uniformly, filtering, accurately taking 10mL of subsequent filtrate, placing the subsequent filtrate into an iodine flask, and accurately adding sodium (potassium) periodate solution [ 90mL of sulfuric acid solution (1 → 20) and 110mL of sodium (potassium) periodate solution (2.3 → 1000) to mix to prepare the compound preparation]50mL, placing on a water bath for heating for 15 minutes, cooling, adding 10mL of potassium iodide solution, placing for 5 minutes, titrating with sodium thiosulfate solution (0.02mol/L), adding 1mL of starch indicator when the end point is reached, continuing to drip until the blue color disappears, and correcting the titration result by using a blank test. Each 1mL of sodium thiosulfate titration solution (0.02mol/L) is equivalent to 0.3643mg of mannitol (C)6H14O6)。
3. Shake flask fermentation
The 11 filamentous fungal strain seed solutions obtained in example 1 were inoculated into a fermentation medium (100 mL/500mL in volume) at an inoculum size of 5% (v/v), and cultured at 150rpm for 6 days at 25 ℃. The content detection of biomass, adenosine and mannitol substances shows that the biomass (26.9g/L) of the strain No. 9 is high, and the content of adenosine (0.47%) and mannitol substances (0.86%) is highest, so the strain No. 9 is selected for further research.
Table 19 strain shake flask fermentation results
Note: the fermentation conditions were 5% (v/v) inoculum size, 100mL/500mL liquid loading, 25 ℃ temperature, 150rpm rotation speed, and 6d culture.
4. Strain identification
Extracting total DNA of the No. 9 strain, amplifying by using universal primers of fungi 18S rDNA and ITS to obtain a corresponding gene sequence, determining the No. 9 strain as Paecilomyces hepiali (Paecilomyces hepiali) JNPF-PH01 by sequencing comparison and combining morphological characteristics of the strain, and storing the strain in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms No. 3 of No.1 Hosieboldian, Navy, in 2015 12-2 days, with the collection number of CGMCC No. 11803.
Example 3 Paecilomyces hepiali fermentation Medium optimization
The seed culture medium is used as a basic culture medium, and carbon sources, nitrogen sources, inorganic salts and growth factors are subjected to species screening and addition amount optimization. The culture conditions were: the liquid loading amount is 100mL/500mL, the inoculation amount is 5% (v/v), the temperature is 25 ℃, the rotation speed is 150r/min, and the culture is carried out for 6 d.
1. Carbon source screening
The influence of glucose, fructose, sucrose, maltose and lactose on the fermentation biomass of the paecilomyces hepiali, the yield of adenosine and mannitol is respectively inspected, and the best effect of 3% of glucose is found.
2. Nitrogen source screening
The influence of nitrate, bean cake powder, corn steep liquor, yeast powder, tryptone and beef extract on the yield of the fermented biomass, adenosine and mannitol substances of the paecilomyces hepiali is respectively inspected by taking 3% glucose as an optimal carbon source, and three parallel experiments are set to find that the effect of adding 1% peptone, 1% yeast powder and 2% bean cake powder is optimal.
3. Screening of inorganic salts
The influence of the addition of monopotassium phosphate, magnesium sulfate, ferrous sulfate, ferric chloride, zinc chloride and ammonium chloride on the fermentation biomass of paecilomyces hepialid, adenosine and mannitol substances is respectively considered by taking 3% of glucose as an optimal carbon source and 1% of yeast powder, 1% of peptone and 2% of bean cake powder as nitrogen sources, and the effect of the addition of 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate and 0.025% of zinc chloride is found to be optimal.
4. Growth factor screening
On the basis of adding 3% of glucose, 1% of yeast powder, 1% of peptone, 2% of bean cake powder, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate and 0.025% of zinc chloride, the influence of vitamin B1, biotin, nicotinic acid and riboflavin on paecilomyces hepiali fermentation biomass, adenosine and mannitol substances is respectively considered, and the effect of adding 0.01% of vitamin B1 is found to be optimal.
Example 3100L and 500L fermentor scale-up
1.100L fermenter for scale-up cultivation
Inoculating the seed solution into a 100L fermentation tank filled with 50L of optimal fermentation medium according to the inoculation amount of 5% (v/v), wherein the temperature is 25 ℃, the speed is 150r/min, and the ventilation volume is 8-14 m3And h, the tank pressure is 0.06-0.14 MPa, and the culture lasts for 5 days. The final biomass was 36.7g/L, the adenosine content and yield were 0.59% and 216.5mg/L, respectively, and the mannitol content and yield were 12.28% and 4.5g/L, respectively.
2.500L fermenter culture
Inoculating the seed liquid into a 500L fermentation tank filled with 400L optimal fermentation medium according to the inoculation amount of 5% (v/v), wherein the temperature is 25 ℃, the speed is 150r/min, and the ventilation volume is 8-14 m3And h, the tank pressure is 0.06-0.14 MPa, and the culture lasts for 4 days. The final biomass was 36.1g/L, the adenosine content and yield were 0.61% and 220.2mg/L, respectively, and the mannitol content and yield were 12.76% and 4.6g/L, respectively.
Claims (5)
1. Paecilomyces hepiali strain for producing adenosine and mannitol substancesPaecilomyces hepiali) JNPF-PH01 with the preservation number of CGMCCNo.11803.
2. The use of the paecilomyces hepialid strain of claim 1 in the preparation of adenosine and mannitol substances.
3. The fermentation method of the paecilomyces hepiali with the preservation number of CGMCC No.11803 is characterized in that a fermentation culture medium contains 3% of glucose, 1% of yeast powder, 1% of peptone, 2% of bean cake powder, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.025% of zinc chloride and 0.01% of vitamin B1 in mass-volume ratio; the culture conditions were: the liquid loading amount is 100mL/500mL, the inoculation amount is 5% (v/v), the temperature is 25 ℃, the rotating speed is 150r/min, and the ventilation volume is 8-14 m3The pot pressure is 0.06-0.14 MPa.
4. The fermentation method of the paecilomyces hepiali with the preservation number of CGMCC No.11803 is characterized in that a fermentation culture medium contains 3% of glucose, 1% of yeast powder, 1% of peptone, 2% of bean cake powder, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.025% of zinc chloride and 0.01% of vitamin B1 in mass-volume ratio; the culture conditions were: the liquid loading amount is 100mL/500mL, the inoculation amount is 5% (v/v), the temperature is 25 ℃, the rotating speed is 150r/min, and the ventilation volume is 8-14 m3The pressure of the tank is 0.06-0.14 Mpa, the volume of the fermentation tank is 100L or 400L, and the culture time is 4 days or 5 days or 6 days.
5. Fermentation process according to claims 3-4, characterized in that the seed medium is formulated with 3% glucose, 1% peptone, 0.1% KH2PO40.05% of MgSO4。
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| KR101935195B1 (en) * | 2018-03-26 | 2019-01-03 | 이원재 | Paecilomyces hepiali CS4 strain having improved capability of producing adenosine isolated from Cordyceps sinensis |
| CN108504621B (en) * | 2018-05-25 | 2021-01-12 | 江西国药有限责任公司 | Culture medium for paecilomyces hepiali Cs-4 and preparation method thereof |
| CN112746022A (en) * | 2020-11-27 | 2021-05-04 | 江苏神华药业有限公司 | Method for preventing adenosine loss in paecilomyces hepiali fermentation mycelia |
| CN112795607B (en) * | 2020-12-31 | 2023-06-23 | 河南巨龙生物工程股份有限公司 | Method for improving adenosine fermentation yield |
| CN117187076A (en) * | 2023-07-24 | 2023-12-08 | 江苏神华药业有限公司 | Paecilomyces hepialid mutagenesis strain with high adenosine yield and preparation method and application thereof |
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