CN110904156A - Method for increasing yield of triterpenoids in phellinus igniarius liquid state fermentation - Google Patents
Method for increasing yield of triterpenoids in phellinus igniarius liquid state fermentation Download PDFInfo
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Abstract
The invention discloses a method for improving the yield of triterpenoids in phellinus igniarius liquid state fermentation, and belongs to the technical field of microbial fermentation. The method for improving the yield of the triterpene in the liquid fermentation of the phellinus igniarius is to add a mulberry wood chip extract in the fermentation process of phellinus igniarius, wherein the mulberry wood chip extract comprises a mulberry wood chip water extract, a petroleum ether extract, an ethyl acetate extract or a methanol extract. On the basis, the ethyl acetate extract of mulberry sawdust and the methanol extract of mulberry sawdust are found to have better effects as the exogenous factors, so that the yield of phellinus linteus triterpenes can reach 299.5 mg/L. The method has the advantages of less equipment investment, simple post-treatment, basically no pollution, low cost and high resource utilization rate, and can obviously improve the yield of the phellinus igniarius liquid fermentation triterpenoid.
Description
Technical Field
The invention relates to a method for improving the yield of triterpenoids in phellinus igniarius liquid state fermentation, and belongs to the technical field of microbial fermentation.
Background
Phellinus igniarius is a precious fungus used as both medicine and food, and the host is mulberry, so that the mulberry wine has the efficacy of losing weight and prolonging life after long-time taking. Phellinus linteus contains triterpene, flavone, polysaccharide, polyphenol and other chemical components with pharmacological activity, and has anticancer, antioxidant, blood sugar lowering, and immunity regulating effects. The phellinus linteus triterpenes mainly take lanoline alkane type as a main active ingredient, and are anti-inflammatory and anti-tumor main active ingredients. Therefore, the development of new drugs using phellinus linteus triterpenes as raw materials will show great development potential and economic benefits.
Because the number of wild phellinus igniarius sporocarp is extremely small and artificial predatory exploitation is difficult to become a stable industrial product source, people carry out more intensive research on artificial culture of phellinus igniarius to relieve the situation, at present, the artificial culture mode of phellinus igniarius mainly comprises three categories of cut-log culture, substitute material culture and liquid state fermentation, but the artificial propagation and domestication culture technology of the wild phellinus igniarius is not mature so far. Although the reports show that the phellinus igniarius sporocarp can be successfully obtained by artificial cultivation, the obtained phellinus igniarius sporocarp has low yield, long production period and poor reproducibility, and large-scale production has great difficulty. Liquid fermentation technology is a promising reliable route for obtaining more high-value secondary metabolites. Compared with the traditional solid fermentation method, the liquid fermentation technology has the advantages of short production period, high yield, easy control of the production process, high yield, easy extraction and separation and the like, and has wide application prospect in the production of edible and medicinal fungi.
Due to the influence of factors such as low extraction rate, low yield and low purity of phellinus linteus triterpenoids, the development and utilization of medicinal values of phellinus linteus triterpenoids are seriously insufficient, and the low yield becomes a limiting factor of research and practical application of phellinus linteus triterpenoids. Currently, few studies are being made on the yield of phellinus linteus triterpenes.
Yaoqiang et al, induced by adding rare earth element cerium, make the content of triterpenes in phellinus linteus intracellular up to 106.7 mg/L. The content of the intracellular triterpene reaches 339.3mg/L by adding oleic acid for induction. However, in the above studies, the cost of cerium and oleic acid is too high for industrial production due to problems such as the price of cerium and oleic acid.
Disclosure of Invention
In order to make up the defect of narrow selection range of exogenous factors in the prior literature, the invention improves the yield of phellinus linteus triterpenes by using exogenous factors (comprising mulberry sawdust aqueous extract, petroleum ether extract, ethyl acetate extract and methanol extract). On the basis, screening of different solvent extracts of mulberry sawdust shows that the ethyl acetate extract and the methanol extract have better effects as exogenous factors, and the yield of phellinus linteus triterpenes can reach 299.5 mg/L.
In addition, whether the yield of the triterpenoids in the liquid fermentation phellinus linteus mycelium can be improved through a fermentation process optimization experiment is particularly important. However, only one literature report on optimizing and improving the total triterpene content of phellinus igniarius through liquid state fermentation exists at present, the final optimization effect is not obvious, and the triterpene content is only 67.6mg/L and cannot meet the requirement of industrial production. Therefore, the invention also aims at optimizing the culture medium and culture conditions for liquid fermentation of substances with anticancer activity, such as phellinus linteus triterpenes.
The first purpose of the invention is to provide a method for improving the yield of triterpenoids in liquid fermentation of phellinus igniarius, which is to add a mulberry wood chip extract in the fermentation process of phellinus igniarius.
In one embodiment, the mulberry sawdust extract is any one or more of the following: mulberry sawdust water extract, mulberry sawdust petroleum ether extract, mulberry sawdust ethyl acetate extract and mulberry sawdust methanol extract.
In one embodiment, the method for preparing the mulberry sawdust extract comprises the following steps: adding solvent and mulberry sawdust (dry and crushed mulberry branches), ultrasonic extracting, filtering, and spin-drying the solvent to obtain the mulberry sawdust extract.
In one embodiment, the method for preparing the aqueous extract of mulberry sawdust comprises the following steps: adding a certain amount of deionized water, performing ultrasonic extraction, performing suction filtration, and performing rotary drying by using a rotary evaporator to obtain the crude mulberry sawdust water extract. The specific method comprises the following steps: adding 150ml deionized water into 15g of sawdust, performing ultrasonic treatment (160W and 40 ℃) for 20min, performing suction filtration and spin drying to obtain the crude mulberry sawdust water extract.
In one embodiment, the method for preparing the mulberry sawdust petroleum ether extract comprises the following steps: adding a certain amount of petroleum ether, performing ultrasonic extraction, performing suction filtration, and performing rotary drying by using a rotary evaporator to obtain the crude extract of the mulberry sawdust petroleum ether. The specific method comprises the following steps: adding 150ml petroleum ether into 15g sawdust, performing ultrasonic treatment (160W and 40 ℃) for 20min, performing suction filtration and spin drying to obtain the crude petroleum ether extract of mulberry sawdust.
In one embodiment, the method for preparing the mulberry sawdust ethyl acetate extract comprises the following steps: adding a certain amount of ethyl acetate, carrying out ultrasonic extraction, carrying out suction filtration, and carrying out rotary drying by a rotary evaporator to obtain the mulberry sawdust ethyl acetate crude extract. The specific method comprises the following steps: adding 150ml ethyl acetate into 15g sawdust, performing ultrasonic treatment (160W and 40 ℃) for 20min, performing suction filtration and spin drying to obtain the crude extract of the mulberry sawdust ethyl acetate.
In one embodiment, the method for preparing the methanol extract of mulberry sawdust comprises the following steps: adding a certain amount of methanol, performing ultrasonic extraction, performing suction filtration, and performing rotary drying by a rotary evaporator to obtain the crude methanol extract of mulberry sawdust. The specific method comprises the following steps: taking 15g of sawdust, adding 150ml of methanol, performing ultrasonic treatment (160W and 40 ℃) for 20min, performing suction filtration and spin drying to obtain the methanol crude extract of the mulberry sawdust.
In one embodiment, the wood chip extract is water extract, petroleum ether extract, ethyl acetate extract, methanol extract, and the added concentration is 0.001% -0.1% (w/v) (i.e. the weight of the added extract is the percentage of the volume of the fermentation liquid, and 0.001-0.1g of the extract is added to 100mL of the fermentation liquid).
In one embodiment, the wood chip extract is an ethyl acetate extract, and the addition concentration is more than 0.02%.
In one embodiment, the wood chip extract is methanol extract, and the addition concentration is more than 0.02%.
In one embodiment, the method comprises inoculating phellinus linteus into a fermentation medium for fermentation, and adding a mulberry wood chip extract during the fermentation.
In one embodiment, the mulberry sawdust extract is added in the 3 rd to 4 th day of the middle stage of fermentation.
In one embodiment, the carbon source of the fermentation medium used for the fermentation is one of glucose, sucrose, maltose, rice flour, potato starch, corn starch, and soluble starch; the nitrogen source of the fermentation medium is one of corn steep liquor powder, yeast extract, beef extract, fish meal peptone, ammonium sulfate, ammonium chloride, ammonium tartrate and urea.
In one embodiment, the carbon source is glucose. Optionally, the addition amount of glucose is 20-70 g/L. Preferably 60 g/L.
In one embodiment, the nitrogen source is corn steep liquor. Optionally, the corn steep liquor powder is added in an amount of 4-14 g/L. Preferably, the addition amount of the corn steep liquor powder is 10 g/L.
In one embodiment, the pH of the fermentation broth is in the range of 3 to 8, preferably, the pH of the fermentation broth is 4.
In one embodiment, the seed solution is inoculated in an amount of 5 to 25%, preferably 15%.
In one embodiment, the seed liquid age is 2-6 days, preferably, the seed liquid age is the fourth day.
In one embodiment, the culture period of the fermentation broth is 2-11 days, preferably 8 days.
In one embodiment, the fermentation medium: 60g/L glucose, 10g/L corn starch and MgSO4·7H2O0.5g/L，KH2PO41g/L, initial pH 4.
In one embodiment, the inoculum size is 5-25%, the initial pH is 3-8, and the seed age is 2-6 days.
In one embodiment, the inoculation is inoculation of phellinus linteus seed solution; the seed solution is obtained by inoculating the cut Phellinus linteus cultured by plate culture to a seed culture medium, culturing at 25-30 ℃ and 130-170rpm for 2-6 days.
In one embodiment, the seed medium is: 15-25g/L glucose, 15-25ml/L soybean hydrolysate, 0.5-1.5g/L yeast extract powder, MgSO4·7H2O 0.2-0.8g/L，KH2PO40.5-1.5g/L，CaCl20.05-0.2g/L, and the initial pH is natural.
The invention also provides the application of the phellinus linteus triterpene prepared by the method in the fields of medicine preparation and food.
The invention has the beneficial effects that:
according to the invention, the exogenous factor is added as a regulation and control means for increasing the yield of the triterpene, and a large amount of exogenous factors are screened, so that the yield of the phellinus linteus sawdust extract can be effectively increased, wherein the effects of the mulberry sawdust ethyl acetate extract and the mulberry sawdust methanol extract are optimal, and the yield of the triterpene can be increased by 31.2% and 30.2% respectively. The mulberry sawdust extract has rich raw material sources and low cost, and is suitable for industrial application.
Furthermore, the culture medium and culture conditions of liquid fermentation are optimized aiming at the phellinus linteus triterpene which is a substance with anticancer activity, and on the basis, the yield of the polyterpene is up to 299.5 mg/L.
The method has the advantages of less equipment investment, simple post-treatment, basically no pollution, low cost and high resource utilization rate, and can obviously improve the yield of the phellinus igniarius liquid fermentation triterpenoid.
Biological material
The strains adopted by the invention are respectively Phellinus baumii (CGMCC5.1265), Phellinus vannamei (CGMCC5.891) and Phellinus igniarius (CGMCC5.864) which are purchased from China general microbiological culture Collection center; phellinus linteus (CFCC 84388) purchased from China center for forestry culture Collection of microorganisms.
Drawings
FIG. 1: triterpene spectrophotometer standard curve.
FIG. 2: the growth curve of triterpene is produced by liquid fermentation of phellinus igniarius.
Detailed Description
The detection method of the triterpene comprises the following steps:
(1) pretreatment of liquid fermentation phellinus igniarius mycelium
Filtering Phellinus linteus pellet obtained by liquid fermentation with 4 layers of gauze, washing off surface floating substances with deionized water, oven drying in oven at 45 deg.C to constant weight, weighing, and grinding the dried thallus into powder with mortar. Placing 0.15g of bacterial powder in a colorimetric tube, adding 10mL of absolute ethyl alcohol, weighing the weight of an initial system, carrying out ultrasonic treatment (power of 160W) for 20min, cooling, shaking up, filtering, and taking a filtrate as a triterpene compound test sample solution.
(2) Determination of triterpene content
Measuring the content of triterpene compounds in Phellinus linteus fruiting body and mycelium by vanillin-glacial acetic acid method, sucking 1mL of sample solution, volatilizing solvent in 70 deg.C metal bath, adding 0.4mL of 5% vanillin-glacial acetic acid solution and 1.6mL perchloric acid, mixing, heating in 70 deg.C water bath for 15min, taking out, cooling in cold water bath to room temperature, adding 5mL glacial acetic acid, shaking thoroughly, standing, cooling to room temperature, measuring absorbance at 546nm wavelength, and calculating triterpene content in the sample solution according to triterpene standard curve (shown in figure 1, measuring total triterpene content by oleanolic acid as standard is a common total triterpene quantitative method).
(3) Calculation of triterpene content
c ═ absorbance +0.0001)/2.4132
Triterpene content (mg/L) ═ c 10 m/0.15
In the formula, c: triterpene concentration (mg/ml) in the extract;
m dry weight of the cells (g/L)
10: volume of extract (ml)
0.15 measurement of Dry weight (g) of sample cells
Mulberry wood chip extract:
mulberry sawdust aqueous extract: adding 150ml deionized water into 15g of sawdust, performing ultrasonic treatment (160W and 40 ℃) for 20min, performing suction filtration and spin drying to obtain the crude mulberry sawdust water extract.
Mulberry wood chip petroleum ether extract: adding 150ml petroleum ether into 15g sawdust, performing ultrasonic treatment (160W and 40 ℃) for 20min, performing suction filtration and spin drying to obtain the crude petroleum ether extract of mulberry sawdust.
Mulberry sawdust ethyl acetate extract: adding 150ml ethyl acetate into 15g sawdust, performing ultrasonic treatment (160W and 40 ℃) for 20min, performing suction filtration and spin drying to obtain the mulberry sawdust ethyl acetate crude extract.
Methanol extract of mulberry sawdust: taking 15g of sawdust, adding 150ml of methanol, performing ultrasonic treatment (160W and 40 ℃) for 20min, performing suction filtration and spin drying to obtain the methanol crude extract of the mulberry sawdust.
Example 1: method for improving yield of triterpene produced by fermenting different Phellinus linteus strains by using exogenous factor (Phellinus linteus sawdust extract)
Seed liquid culture medium: 20g/L glucose, 20ml/L soybean hydrolysate, 1g/L yeast extract powder and MgSO4·7H2O0.5g/L，KH2PO41g/L，CaCl20.1g/L, initial pH is natural.
Culture medium of fermentation liquid: 30g/L glucose, 3g/L fish meal peptone, 2.5g/L yeast extract powder, (NH)4)2SO42g/L，MgSO4·7H2O 1g/L，KH2PO41g/L, initial pH is natural.
(1) Preparation of seed liquid
Activating Phellinus baumii (CGMCC5.1265), Phellinus vannamei (CGMCC5.891), Phellinus igniarius (CGMCC5.864) and Phellinus linteus (CFCC 84388), inoculating to seed liquid culture medium, and shake-flask fermentation culturing at 150rpm and 28 deg.C for 4 d.
(2) Direct fermentation culture
Inoculating the liquid seeds of Phellinus baumii (CGMCC5.1265), Phellinus vannamei (CGMCC5.891), Phellinus igniarius (CGMCC5.864) and Phellinus linteus (CFCC 84388) in logarithmic growth phase obtained in the step (1) into a fermentation culture medium according to the inoculation amount of 10% by volume, and performing fermentation culture for 7d at the temperature of 28 ℃ and the rotation speed of 150 rpm. After completion, the triterpene content was found to be 71.3mg/L, 63.2mg/L, 52.7mg/L, 54.3mg/L, respectively (see Table 1).
(3) Fermentation culture with exogenous extract
Inoculating the liquid seeds of phellinus baumannii (CGMCC5.1265), phellinus igniarius (CGMCC5.891), phellinus igniarius (CGMCC5.864) and phellinus linteus (CFCC 84388) which are obtained in the step (1) and are in the logarithmic growth phase into a fermentation culture medium according to the inoculation amount of 10% by volume ratio, performing fermentation culture for 3 days at the temperature of 28 ℃ and the rotation speed of 150rpm, and then respectively adding 0.005% (m/v) of mulberry sawdust aqueous extract, 0.005% (m/v) of mulberry sawdust petroleum ether extract, 0.02% (m/v) of mulberry sawdust ethyl acetate extract and 0.02% (m/v) of mulberry sawdust methanol extract, and culturing for 4 days.
The results show that the addition of aqueous extract, petroleum ether extract, ethyl acetate extract, methanol extract:
the triterpene contents of the fermentation liquor of the phellinus baumannii are respectively 81.4mg/L, 83.5mg/L, 93.1mg/L and 89.9mg/L, and the yield is respectively improved by 14.1 percent, 17.1 percent, 30.4 percent and 25.0 percent compared with the yield of direct fermentation.
The triterpene contents of the fermentation liquor of the Waningphellinus igniarius are respectively 70.2mg/L, 74.5mg/L, 69.8mg/L and 78.3mg/L, and the yield is respectively improved by 11.1 percent, 17.3 percent, 10.4 percent and 23.9 percent compared with the yield of direct fermentation.
The triterpene contents of the fermentation liquor of the phellinus igniarius are respectively 62.4mg/L, 61.7mg/L, 61.9mg/L and 59.3mg/L, and the yield is respectively improved by 18.4 percent, 17.1 percent, 17.6 percent and 12.5 percent compared with the yield of direct fermentation.
The triterpene contents of the fermentation liquor of the phellinus linteus are respectively 61.6mg/L, 60.3mg/L, 67.0mg/L and 62.4mg/L, which are respectively improved by 15.3 percent, 11.0 percent, 23.4 percent and 14.9 percent compared with the yield of direct fermentation.
TABLE 1 results of fermentation of different species
Example 2: influence of different extract concentrations on triterpene production by liquid fermentation of Phellinus linteus
Compared with (3) in example 1, the effect of the concentration of the extract on the production of triterpene by fermentation of Phellinus baumii (CGMCC5.1265) was compared by changing the concentration of the exogenous factor extract and keeping the other culture conditions unchanged.
TABLE 2 fermentation results for different extract concentrations
Example 3: influence of different carbon sources on triterpene production by liquid fermentation of phellinus igniarius
Fermentation medium: carbon source (glucose, sucrose, maltose, rice flour, potato starch, corn starch, and optionallyOne of soluble starch) 30g/L, fish meal peptone 3g/L, yeast extract 2g/L, (NH)4)2SO42g/L，MgSO4·7H2O 0.5g/L，KH2PO41g/L, initial pH is natural.
The Phellinus linteus (CGMCC5.1265) seed solution in logarithmic growth phase obtained in example 1 was inoculated into fermentation medium containing 30g/L of different carbon source species at an inoculum size of 10% by volume, and fermentation-cultured at 28 ℃ and 150rpm for 7 days. The carbon source types are glucose, sucrose, maltose, rice flour, potato starch, corn starch and soluble starch respectively. And after the fermentation is finished, measuring the content of the triterpenoids in the phellinus igniarius cells. The optimal carbon source is glucose, and the yield of triterpene is 78.1 mg/L.
TABLE 3 fermentation results for different carbon sources
Example 4: influence of different nitrogen sources on triterpene production by liquid fermentation of phellinus igniarius
Fermentation medium: nitrogen source (one of corn steep liquor powder, yeast extract, beef extract, fish meal peptone, ammonium sulfate, ammonium chloride, ammonium tartrate and urea) 7g/L, glucose 30g/L, MgSO4·7H2O 0.5g/L，KH2PO41g/L, initial pH is natural.
The Phellinus linteus (CGMCC5.1265) seed solution in logarithmic growth phase obtained in example 1 was inoculated into fermentation medium containing 7g/L of different nitrogen sources at an inoculum size of 10% by volume, and fermentation-cultured at 28 ℃ and 150rpm for 7 d. The nitrogen source is selected from corn steep liquor powder, yeast extract, beef extract, fish meal peptone, ammonium sulfate, ammonium chloride, ammonium tartrate and urea. And after the fermentation is finished, measuring the content of the triterpenoids in the phellinus igniarius cells. The optimal nitrogen source type is corn steep liquor powder, and the triterpene yield is 126.7 mg/L.
TABLE 4 fermentation results for different nitrogen source species
Example 5: influence of different carbon source addition amounts on triterpene production by Phellinus linteus liquid fermentation
Fermentation medium: glucose (20, 30, 40, 50, 60, 70g/L respectively), corn steep liquor powder 7g/L, MgSO4·7H2O0.5g/L，KH2PO41g/L, initial pH is natural.
The Phellinus linteus (CGMCC5.1265) seed solution in logarithmic growth phase obtained in example 1 was inoculated into carbon source fermentation medium containing glucose in different addition amounts at an inoculum size of 10% by volume, and fermentation-cultured at 28 deg.C and 150rpm for 7 d. The amounts of carbon source (glucose) added were 20, 30, 40, 50, 60 and 70g/L, respectively. And after the fermentation is finished, measuring the content of the triterpenoids in the phellinus igniarius cells. The optimal glucose adding amount is 60g/L, and the triterpene output is 157.8 mg/L.
TABLE 5 fermentation results with different carbon source additions
Example 6: influence of different nitrogen source addition amounts on triterpene production by phellinus igniarius liquid fermentation
Fermentation medium: corn starch (4, 6, 8, 10, 12, 14g/L, respectively), glucose 60g/L, MgSO4·7H2O0.5g/L，KH2PO41g/L, initial pH is natural.
The Phellinus linteus (CGMCC5.1265) seed solution in logarithmic growth phase obtained in example 1 was inoculated into nitrogen source fermentation medium containing corn steep liquor powder in different addition amounts at an inoculum size of 10% by volume, and fermentation-cultured at 28 deg.C and 150rpm for 7 d. The addition amounts of the nitrogen source (corn steep liquor powder) were 4, 6, 8, 10, 12 and 14g/L, respectively. And after the fermentation is finished, measuring the content of the triterpenoids in the phellinus igniarius cells. The optimal addition amount of the corn steep liquor powder is 10g/L, and the yield of the triterpene is 187.8 mg/L.
TABLE 6 results of fermentation with different amounts of carbon sources added
Example 7: influence of different pH values on triterpene production by liquid fermentation of phellinus igniarius
Fermentation medium: 60g/L glucose, 10g/L corn starch and MgSO4·7H2O 0.5g/L，KH2PO41g/L, and the initial pH is one of 3, 4, 5, 6 and 7.
The Phellinus linteus (CGMCC5.1265) seed solution in logarithmic growth phase obtained in example 1 was inoculated into a fermentation medium at an inoculum size of 10% by volume, and fermentation-cultured at a temperature of 28 ℃ and a rotation speed of 150rpm for 7 days. The pH values were 3, 4, 5, 6, 7, respectively. And after the fermentation is finished, measuring the content of the triterpenoids in the phellinus igniarius cells. The optimum pH is 4 and the triterpene yield is 190.3 mg/L.
TABLE 7 results of fermentation at different pH
Example 8: influence of different inoculation amounts on triterpene production by liquid fermentation of phellinus igniarius
Fermentation medium: 60g/L glucose, 10g/L corn starch and MgSO4·7H2O 0.5g/L，KH2PO41g/L, initial pH is natural.
The Phellinus linteus (CGMCC5.1265) seed solution in logarithmic growth phase obtained in example 1 was inoculated into fermentation medium at different volume ratio, and fermentation-cultured at 28 deg.C and 150rpm for 7 d. The inoculation amounts were 5%, 10%, 15%, 20%, 25%, respectively. And after the fermentation is finished, measuring the content of the triterpenoids in the phellinus igniarius cells. The optimal inoculation amount is 15%, and the triterpene yield is 220.2 mg/L.
TABLE 8 fermentation results for different Phellinus igniarius inoculation amounts
Example 9: influence of different ages on triterpene production by liquid fermentation of Phellinus linteus
Seed liquid culture medium: 20g/L glucose, 20ml/L soybean hydrolysate, 1g/L yeast extract powder and MgSO4·7H2O0.5g/L，KH2PO41g/L，CaCl20.1g/L, initial pH is natural.
Fermentation medium: 60g/L glucose, 10g/L corn starch and MgSO4·7H2O 0.5g/L，KH2PO41g/L, initial pH 4.
Preparation of seed liquid
Activating Phellinus Linteus (CGMCC5.1265), inoculating to seed liquid culture medium, and shake-flask fermentation culturing at 28 deg.C at rotation speed of 150r/min for 2-6 d.
Inoculating Phellinus linteus (CGMCC5.1265) liquid seeds with the age of 2, 3, 4, 5, 6 days into fermentation culture medium at 15% volume ratio, and fermenting at 28 deg.C and 150rpm for 7 d. And after the fermentation is finished, measuring the content of the triterpenoids in the phellinus igniarius cells. The optimal seed age is 4 days, and the yield of the triterpene is 229.7 mg/L.
TABLE 9 fermentation results of Phellinus linteus of different ages
Example 10: influence of different culture periods on triterpene production by liquid fermentation of phellinus igniarius
Fermentation medium: 60g/L glucose, 10g/L corn starch and MgSO4·7H2O 0.5g/L，KH2PO41g/L, initial pH 4.
The four-day-old Phellinus linteus (CGMCC5.1265) seed solution obtained in example 9 was inoculated into fermentation medium at 15% and fermented at 28 deg.C and 150rpm for 2-11 days (the fermentation curve is shown in FIG. 2). And after the fermentation is finished, measuring the content of the triterpenoids in the phellinus igniarius cells. The optimal culture time is 8 days, and the triterpene yield is 236.8 mg/L.
TABLE 10 fermentation results of Phellinus linteus with different cultivation time
Example 11: influence of four mulberry sawdust extracts on triterpene production by liquid fermentation of phellinus igniarius
Fermentation medium: 60g/L glucose, 10g/L corn starch and MgSO4·7H2O 0.5g/L，KH2PO41g/L, initial pH 4.
The seed solution of Phellinus linteus (CGMCC5.1265) in logarithmic growth phase obtained in example 1 was inoculated into the fermentation medium at an inoculum size of 15% by volume. Adding 0.005% (m/v) of mulberry sawdust aqueous extract, 0.005% (m/v) of mulberry sawdust petroleum ether extract, 0.02% (m/v) of mulberry sawdust ethyl acetate extract and 0.02% (m/v) of mulberry sawdust methanol extract at 28 deg.C and 150rpm on the 3 rd day of fermentation, and fermenting and culturing for 5 days. The results show that the triterpene contents of the phellinus igniarius fermentation liquor added with the water extract, the petroleum ether extract, the ethyl acetate extract and the methanol extract are respectively 256.4mg/L, 262.7mg/L, 299.5mg/L and 297.1mg/L, and the yield is respectively 12.4%, 15.1%, 31.2% and 30.2% higher than that of direct fermentation (comparison, no additional mulberry wood chip extract is added, and other conditions are the same).
TABLE 11 fermentation results of different wood chip extracts
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for improving yield of triterpene in liquid fermentation of phellinus igniarius is characterized in that a mulberry sawdust extract is added in the fermentation process of phellinus igniarius.
2. The method according to claim 1, wherein the mulberry sawdust extract is any one or more of the following: mulberry sawdust water extract, mulberry sawdust petroleum ether extract, mulberry sawdust ethyl acetate extract and mulberry sawdust methanol extract.
3. The method according to claim 1, wherein the mulberry sawdust extract is prepared by: adding solvent and mulberry sawdust, ultrasonic extracting, suction filtering, and spin drying solvent to obtain mulberry sawdust extract.
4. The method as claimed in claim 2, wherein the wood chip extract is added at a concentration of 0.001-0.1% w/v.
5. The method according to claim 1, wherein the wood chip extract is an ethyl acetate extract, and the added concentration is 0.02% or more.
6. The method according to claim 1, wherein the fermentation is carried out by inoculating Phellinus linteus into fermentation medium, and adding mulberry sawdust extract during fermentation; optionally, the addition time of the mulberry sawdust extract is 3-4 days in the middle stage of fermentation.
7. The method of claim 1, wherein the carbon source of the fermentation medium used for the fermentation is one of glucose, sucrose, maltose, rice flour, potato starch, corn starch, soluble starch; the nitrogen source of the fermentation medium is one of corn steep liquor powder, yeast extract, beef extract, fish meal peptone, ammonium sulfate, ammonium chloride, ammonium tartrate and urea.
8. The method of claim 7, wherein the carbon source is glucose; optionally, the nitrogen source is corn steep liquor; optionally, the pH of the fermentation broth ranges from 3 to 8; optionally, the seed solution inoculation amount is 5-25%; optionally, the seed liquid is 2-6 days old; optionally, the culture period of the fermentation broth is 2-11 days.
9. A fermentation medium for liquid fermentation of phellinus igniarius, which is characterized by comprising the following components in percentage by weight: 60g/L glucose, 10g/L corn starch and MgSO4·7H2O 0.5g/L，KH2PO41g/L, initial pH 4.
10. Use of the method of claim 1 in the fields of pharmaceutical preparation and food products containing phellinus linteus triterpenes.
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