CN106978465B - Fermentation method for improving fermentation yield of total triterpenoids in inonotus obliquus - Google Patents
Fermentation method for improving fermentation yield of total triterpenoids in inonotus obliquus Download PDFInfo
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Abstract
The invention discloses a fermentation method for improving the fermentation yield of total triterpenoids in inonotus obliquus, which comprises the following steps: (1) preparing seed liquid; (2) and (5) inoculating and fermenting. The method optimizes the formulas of the seed culture medium and the fermentation culture medium, has the advantages of easily obtained raw materials and low cost, adds 2.5-3.5 g/L of soybean oil into the fermentation culture medium before fermentation, optimizes and improves fermentation process parameters, has simple steps, strong operability and short fermentation period, and can obviously improve the yield of mycelium and the yield of total triterpenoids.
Description
Technical Field
The invention relates to a method for producing a macro-fungus active metabolite by utilizing a liquid submerged fermentation technology, in particular to a fermentation method for improving the fermentation yield of total triterpenoids in inonotus obliquus.
Background
Inonotus obliquus is a precious medicinal large-scale fungus growing on white birch, and is mainly distributed in northern Russia, northern Europe, China Heilongjiang, Jilin and other places. Inonotus obliquus has a long history of use as a folk herb in countries such as eastern Europe, Russia, etc. Modern medical research shows that the inonotus obliquus has good effects of treating various cancers of digestive systems, improving immunity and treating diabetes, and also has the functions of resisting aging and viruses. Long-term animal experiments and clinical experiments show that the inonotus obliquus has no toxic or side effect. The magical effects of inonotus obliquus need to be researched and developed from both economic value and social value.
Inonotus obliquus polysaccharide and triterpene are important effective components. The triterpene compound has the biological activities of resisting tumor, resisting virus, resisting inflammation, improving immunity and the like, and has very high medicinal development prospect. Currently, up to dozens of triterpenoids of inonotus obliquus are found, and most of the triterpenoids are tetracyclic triterpenes and pentacyclic triterpenes. At present, the triterpenoids of the inonotus obliquus are mainly separated from wild sclerotium, but the wild inonotus obliquus has rare resources and high price. The artificial cultivation technology is still in the research stage at present, and the artificial cultivation sporocarp has short growth cycle and low content of effective components, so that the medicinal requirement of the artificial cultivation sporocarp cannot be met. Therefore, the liquid submerged fermentation method becomes a hot spot for the production research of the functional components of the inonotus obliquus.
However, the current research on submerged fermentation of inonotus obliquus mainly focuses on optimization of fermentation medium and fermentation process by taking mycelium and polysaccharide as target products. In the invention patent of "a submerged fermentation medium of inonotus obliquus and a submerged fermentation method of inonotus obliquus" (publication number is CN102115350A) disclosed in 2011, the yields of mycelia and intracellular and extracellular polysaccharides are improved by optimizing the fermentation medium and developing a two-stage fermentation process. In the 'method for producing inonotus obliquus bacterial powder by rapid submerged liquid fermentation' (publication number is CN105670944A) published in 2016, a semi-continuous fermentation process is developed to rapidly obtain a fermentation liquid rich in mycelia, and then the fermentation liquid is treated and separated by a microfiltration membrane to obtain inonotus obliquus mycelia. In order to achieve higher total triterpene output of inonotus obliquus, the triterpene content in the mycelium and the yield of fermentation mycelium need to be improved simultaneously. However, in the current research on fermentation of the inonotus obliquus triterpenes, the mycelium yield is low, the triterpene content is high, but the total triterpene content is not high. If the content of the triterpene is improved by adding the methyl jasmonate and the fatty acid, the content of the total triterpene is limited to be further improved due to low yield of mycelium, and the cost of the added methyl jasmonate and the fatty acid is higher.
Disclosure of Invention
The invention aims to solve the problem of low fermentation yield of the total triterpenoids of inonotus obliquus in the prior art, and provides a fermentation method for improving the fermentation yield of the total triterpenoids of the inonotus obliquus, which has the advantages of simple steps, strong operability and short fermentation period, and can obviously improve the mycelium yield and the total triterpenoids content.
In order to achieve the purpose, the invention adopts the following technical scheme:
a fermentation method for improving the fermentation yield of total triterpenes of inonotus obliquus comprises the following steps:
(1) preparation of seed liquid
Scraping inonotus obliquus from a flat plate under an aseptic condition, inoculating the inonotus obliquus into a shake flask filled with 100ml of seed culture medium, and culturing for 3-5 days at the temperature of 28-30 ℃ and the stirring rotation speed of 150-180 rpm to obtain a first-grade seed solution; pouring the primary seed solution into a sterilized medicine bottle filled with glass beads under the aseptic condition, shaking uniformly, scattering inonotus obliquus mycelia, taking the volume of a seed culture medium as a reference, inoculating the primary seed solution with the inoculation amount of 9-10% into a shake flask filled with 1L of the seed culture medium, and culturing for at least 2 days at the temperature of 28-30 ℃ and the stirring speed of 150-180 rpm to obtain a secondary seed solution.
(2) Inoculating fermentation
And (2) after a fermentation medium is filled in a fermentation tank and sterilized, inoculating 9-10% of second-level seed liquid into the fermentation tank under an aseptic condition by taking the volume of the fermentation medium as a reference, simultaneously adding 2.5-3.5 g/L of soybean oil into the fermentation medium, continuously fermenting for at least 9 days at the temperature of 28-30 ℃, the stirring speed of 150-180 rpm and the ventilation volume of 5-7L/min, and stopping the tank to finish the whole fermentation process. The key point of the invention is the addition of the soybean oil, the addition time and the addition amount of the soybean oil, and the mycelium yield and the total triterpene content can be improved only by adding the soybean oil before fermentation and controlling the addition amount to be 2.5-3.5 g/L, otherwise, the effect is greatly reduced.
Preferably, in step (1), the formulation of the seed culture medium is: 35g/L of glucose, 7.5g/L of yeast powder, 1g/L of monopotassium phosphate, 0.5g/L of anhydrous magnesium sulfate and 5.5-6.0 of pH.
Preferably, in the step (2), the volume of the fermentation medium accounts for 60-70% of the total volume of the fermentation tank.
Preferably, the specific formula of the fermentation medium is as follows: 45g/L of glucose, 10g/L of yeast powder, 0.5g/L of anhydrous magnesium sulfate, 1g/L of monopotassium phosphate, 0.4g/L of calcium chloride and pH of 5.5-6.0.
Preferably, in step (2), the sterilization conditions are: sterilizing at 120-125 deg.C for 30-40 min.
Therefore, the invention has the following beneficial effects: the formula of the seed culture medium and the fermentation culture medium is optimized, the raw materials are easy to obtain, the cost is low, 2.5-3.5 g/L of soybean oil is added into the fermentation culture medium before fermentation, and meanwhile, the fermentation process parameters are optimized and improved, the method steps are simple, the operability is strong, the fermentation period is short, and the mycelium yield and the total triterpene yield can be obviously improved.
Detailed Description
The invention is further described below by means of specific embodiments.
The Inonotus obliquus strain (Inonotus obliquus) used in the following examples and comparative examples was deposited in the china forestry microbial strain preservation management center (strain code cfcc 60260).
Example 1
(1) Preparation of seed liquid
Scraping inonotus obliquus from a flat plate under aseptic condition, inoculating into a shake flask containing 100ml of seed culture medium, and culturing at 28 deg.C and stirring speed of 150rpm for 5 days to obtain first-stage seed solution; pouring the primary seed solution into a sterilized medicine bottle filled with glass beads under the aseptic condition, shaking uniformly, scattering inonotus obliquus mycelia, taking the volume of a seed culture medium as a reference, inoculating the primary seed solution with the inoculation amount of 9% into a shake flask filled with 1L of the seed culture medium, and culturing at the temperature of 28 ℃ and the stirring speed of 150rpm for at least 2 days to obtain a secondary seed solution; wherein, the formula of the seed culture medium is as follows: 35g/L glucose, 7.5g/L yeast powder, 1g/L potassium dihydrogen phosphate, 0.5g/L anhydrous magnesium sulfate and pH of 5.5-6.0
(2) Inoculating fermentation
Filling a fermentation medium accounting for 60% of the total volume of the fermentation tank into the fermentation tank, sterilizing at 120 ℃ for 30min, inoculating 9% of second-stage seed liquid into the fermentation tank under an aseptic condition by taking the volume of the fermentation medium as a reference, simultaneously adding 2.5g/L of soybean oil into the fermentation medium, continuously fermenting for at least 9 days at 28-30 ℃ and a stirring rotation speed of 150rpm under the condition of an air flow of 5L/min, and stopping the tank to finish the whole fermentation process, wherein the specific formula of the fermentation medium is as follows: 45g/L of glucose, 10g/L of yeast powder, 0.5g/L of anhydrous magnesium sulfate, 1g/L of monopotassium phosphate, 0.4g/L of calcium chloride and pH of 5.5-6.0.
Samples were taken after fermentation for analysis of mycelium yield and total triterpene content, and the results are shown in Table 1.
Examples 2 to 3
Examples 2 to 3 are different from example 1 in that: the addition amounts of soybean oil were 3.0g/L and 3.5g/L, respectively, and the rest was the same.
Samples were taken after fermentation for analysis of mycelium yield and total triterpene content, and the results are shown in Table 1.
Comparative examples 1 to 6
Comparative examples 1 to 6 are different from example 1 in that: the addition amounts of soybean oil were 0g/L, 0.5g/L, 1.0g/L, 1.5g/L, 2.0g/L, and 4.0g/L, respectively, and the rest were the same. Samples were taken after fermentation for analysis of mycelium yield and total triterpene content, and the results are shown in Table 1.
Comparative examples 7 to 9
Comparative examples 7 to 9 differ from example 1 in that: the adding time of the soybean oil is respectively 2 days, 4 days and 6 days after fermentation, and the rest is the same. Samples were taken after fermentation for analysis of mycelium yield and total triterpene content, and the results are shown in Table 1.
TABLE 1 measurement results of the mycelium yield and the total triterpene content in examples 1 to 3 and comparative examples 1 to 9
As can be seen from the data in Table 1, the addition of 2.5-3.5 g/L soybean oil to the fermentation medium before fermentation can significantly improve the yield of mycelia and the total triterpene content.
Example 4
This example is different from example 1 in that: (1) the first-stage seed liquid culture conditions are as follows: culturing at 29 deg.C and stirring speed of 160rpm for 4 days; the secondary seed liquid culture conditions are as follows: the temperature is 29 ℃, and the stirring speed is 160 rpm; (2) the fermentation process conditions are as follows: the fermentation tank was filled with a fermentation medium accounting for 65% of the total volume of the fermentation tank and sterilized at 122 ℃ for 35min, and then the second-stage seed liquid in an amount of 9.5% was inoculated into the fermentation tank under aseptic conditions based on the volume of the fermentation medium, and continuously fermented at 29 ℃ and a stirring speed of 160rpm with an aeration rate of 6L/min for at least 9 days, with the remainder being exactly the same as in example 1.
Example 5
The present embodiment is different from the embodiments in that: (1) the first-stage seed liquid culture conditions are as follows: culturing at 30 deg.C and stirring speed of 180rpm for 3 days; the secondary seed liquid culture conditions are as follows: the temperature is 30 ℃, and the stirring speed is 180 rpm; (2) the fermentation process conditions are as follows: the fermentation tank was filled with 70% by volume of a fermentation medium based on the total volume of the fermentation tank and sterilized at 125 ℃ for 40min, and then the second-stage seed liquid in an amount of 10% was inoculated into the fermentation tank under aseptic conditions based on the volume of the fermentation medium, and continuously fermented at 30 ℃ and a stirring speed of 180rpm for at least 9 days with a ventilation of 7L/min, and the rest was completely the same as in example 1.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Claims (5)
1. A fermentation method for improving the fermentation yield of total triterpenes of inonotus obliquus is characterized by comprising the following steps:
(1) preparation of seed liquid
Scraping inonotus obliquus from a flat plate under an aseptic condition, inoculating the inonotus obliquus into a shake flask filled with 100ml of seed culture medium, and culturing for 3-5 days at the temperature of 28-30 ℃ and the stirring rotation speed of 150-180 rpm to obtain a first-grade seed solution; pouring the primary seed solution into a sterilized medicine bottle filled with glass beads under the aseptic condition, shaking uniformly, scattering inonotus obliquus mycelia, taking the volume of a seed culture medium as a reference, inoculating the primary seed solution with the inoculation amount of 9-10% into a shake flask filled with 1L of the seed culture medium, and culturing for at least 2 days at the temperature of 28-30 ℃ and the stirring speed of 150-180 rpm to obtain a secondary seed solution;
(2) inoculating fermentation
And (2) after a fermentation medium is filled in a fermentation tank and sterilized, inoculating 9-10% of second-level seed liquid into the fermentation tank under an aseptic condition by taking the volume of the fermentation medium as a reference, simultaneously adding 2.5-3.5 g/L of soybean oil into the fermentation medium, continuously fermenting for at least 9 days at the temperature of 28-30 ℃, the stirring speed of 150-180 rpm and the ventilation volume of 5-7L/min, and stopping the tank to finish the whole fermentation process.
2. The fermentation method for improving the fermentation yield of the total triterpenes in inonotus obliquus according to claim 1, wherein in the step (1), the formula of the seed culture medium is as follows: 35g/L of glucose, 7.5g/L of yeast powder, 1g/L of monopotassium phosphate, 0.5g/L of anhydrous magnesium sulfate and 5.5-6.0 of pH.
3. The fermentation method for improving the fermentation yield of the total triterpenes in inonotus obliquus according to claim 1, wherein in the step (2), the volume of the fermentation medium accounts for 60-70% of the total volume of the fermentation tank.
4. The fermentation method for improving the fermentation yield of the total triterpenes of inonotus obliquus according to claim 1 or 3, wherein the specific formula of the fermentation medium is as follows: 45g/L of glucose, 10g/L of yeast powder, 0.5g/L of anhydrous magnesium sulfate, 1g/L of monopotassium phosphate, 0.4g/L of calcium chloride and pH of 5.5-6.0.
5. The fermentation method for improving the fermentation yield of the total triterpenes in inonotus obliquus according to claim 1, wherein in the step (2), the sterilization conditions are as follows: sterilizing at 120-125 deg.C for 30-40 min.
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| CN107841522A (en) * | 2017-10-19 | 2018-03-27 | 浙江大学 | The method for extracting betulic acid from Inonotus obliquus using jasmonate induction |
| CN107841523A (en) * | 2017-10-19 | 2018-03-27 | 浙江大学 | Triterpene substance method is extracted from Inonotus obliquus using the induction of quorum sensing molecule |
| CN108715870B (en) * | 2018-04-19 | 2021-05-14 | 浙江理工大学 | Method for increasing yield of triterpene produced by fermentation of inonotus obliquus |
| CN109337953B (en) * | 2018-11-01 | 2021-11-26 | 黑龙江中医药大学 | Inonotus obliquus D extract and preparation method and detection method thereof |
| CN110195083B (en) * | 2019-07-15 | 2021-01-29 | 江南大学 | Method for increasing yield of inonotus obliquus alcohol through liquid fermentation of inonotus obliquus |
| CN110734932B (en) | 2019-07-29 | 2021-10-22 | 浙江养生堂天然药物研究所有限公司 | Fermented birch juice and its production method |
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| WO2003045161A1 (en) * | 2001-11-29 | 2003-06-05 | Nc Biotec Co., Ltd. | A preparation method of feed and feed additive using mushroom mycelium |
| CN1456670A (en) * | 2002-03-15 | 2003-11-19 | 林忠平 | Genes of resveratrol stilbene synthase in parthenocissus and their use |
| CN104099385A (en) * | 2014-07-15 | 2014-10-15 | 江苏阜丰生物科技有限公司 | Method for producing inonotus obliquus exopolysaccharides through submerged fermentation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2003045161A1 (en) * | 2001-11-29 | 2003-06-05 | Nc Biotec Co., Ltd. | A preparation method of feed and feed additive using mushroom mycelium |
| CN1456670A (en) * | 2002-03-15 | 2003-11-19 | 林忠平 | Genes of resveratrol stilbene synthase in parthenocissus and their use |
| CN104099385A (en) * | 2014-07-15 | 2014-10-15 | 江苏阜丰生物科技有限公司 | Method for producing inonotus obliquus exopolysaccharides through submerged fermentation |
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