CN101353689B - Method for enhancing yield of ganoderma triterpene in ganoderma lucidum fermentation liquor - Google Patents
Method for enhancing yield of ganoderma triterpene in ganoderma lucidum fermentation liquor Download PDFInfo
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Abstract
The invention discloses a method for improving the yield of Ganoderma triterpenes in Ganoderma fermentation liquor. The steps are as follows: the Ganoderma lucidum with the stain preservation number of CCGMC 5.616 and preserved in the CGMCC is adopted to inoculate a Ganoderma lucidum strain; rotary submerged fermentation is carried out for 1 to 5 days under the temperature of 26 to 30 DEG C and at the rotating speed of 130 to 180r/min; after being added with 1.5-3.0g/L of supplementary palmitinic acid, continuous fermentation is ended in 3 to 6 days under the temperature of 25 to 30 DEG C andat the rotating speed of 100 to 180r/min. The Ganoderma triterpene yield of the method of the invention is up to 396.2mg/L.
Description
Technical field
The invention belongs to technical field of biological fermentation, be specifically related to a kind of zymotechnique that improves yield of ganoderma lucidum triterpenoid.
Background technology
Glossy ganoderma (Ganoderma lucidum) is the famous medicinal fungi of China.Because it can prevent and treat numerous disease, once be called as " celestial grass " in ancient times.Pharmacopoeia of the People's Republic of China regulation, red sesame (Ganoderma lucidum) and purple sesame (Ganoderma sinense) are the glossy ganoderma certified products.Comprise that in recent years the country in Southeast Asia of China and the U.S. and Canadian many scholars have carried out deep research to glossy ganoderma, modern pharmacology studies have shown that the crucial effective component of glossy ganoderma is ganoderan and Ganoderma triterpenoids.Wherein Ganoderma triterpenoids have anti-inflammatory analgetic, calmness, detoxify, protect the liver, AIDS virus resisting, function such as antitumor, be the important substance basis of glossy ganoderma performance drug effect.
Present stage, the output of glossy ganoderma thalline and crucial effective component thereof becomes the bottleneck that hinders the ganoderma lucidum prod application.Since the wild Ganoderma rareness, and traditional cultivating method cycle is long, and output far can not satisfy market demand.Because producing glossy ganoderma, the liquid submerged fermentation technology has characteristics such as with short production cycle, product active substance and wild Ganoderma basically identical, the present most effectual way of obtaining glossy ganoderma and active substance thereof that become.In recent years, the liquid fermentation technology of glossy ganoderma has been obtained remarkable progress, but the output that how further to improve glossy ganoderma thalline or crucial effective component has become and accelerated ganoderma lucidum prod industrialization process problem demanding prompt solution to reduce production costs, to increase economic efficiency.
The fermentation technique of crucial effective component-Ganoderma triterpenoids of glossy ganoderma mainly is divided into two big classes, the first kind mainly is by the optimization to glossy ganoderma fermentation matrix and fermenting process, as optimize substratum and form, optimize Fermentation Process of Parameter, or adopt strategy such as feed supplement-batch fermentation to improve the output of Ganoderma triterpenoids, as:
Document: Li Pingzuo, Xu Rou, the summer knot is red, Zhang Kechang. and deep glossy ganoderma fermenting is produced the research of tetracyclic triterpene acid. industrial microorganism, 2000,30 (1): 15-17.Reported the basic technology of a kind of submerged fermentation production tetracyclic triterpene acid, but the output of tetracyclic triterpene acid is lower.
Document: Tang YJ, Zhong JJ.Fed-batch fermentation of Ganoderma lucidumfor hyperproduction of polysaccharide and anoderic acid.Enzyme andMicrobial Technology, 2002,31:20-28.Reported that a kind of employing adds the stream of lactose and add fermentation process, the output that obtains Ganodenic acid (Ganoderma triterpenoids) is 297.9-367.1mg/L.
Chinese patent: clock Kien Giang etc., application number 00111953.2 has proposed the technology that solution fermentation is produced ganoderan and Ganoderma triterpenoids simultaneously.Adopt liquid aerobic fermentation, liquid aerobic fermentation-leave standstill to cultivate or liquid leaves standstill the fermentation process of cultivation, Ganodenic acid (Ganoderma triterpenoids) content in the glossy ganoderma thalline of its production can reach 2.8mg/100mg.The fermentation strategy of this technology is better, output also can improve, but that shortcoming is the fermentation period of Ganoderma triterpenoids is oversize, and the liquid aerobic fermentation adds that the total fermentation time that leaves standstill cultivation has surpassed 20 days, and the fermentation time of general ganoderma lucidum prod is 4-8 days, so its integral production efficient is lower.
Another kind of is on the basis of fermentation technology optimization, promotes the biosynthesizing of Ganoderma triterpenoids by the interpolation allogenic material, thereby improves the output of Ganoderma triterpenoids.As:
Document: Wang Yuhong, fourth Double Ninth Festival, Zhang Kechang, Xu Rou. bitter buckwheat is produced the influence of Ganodenic acid to glossy ganoderma fermentation. food and fermentation industries, 2003,29 (9): 97-99.After having reported the bitter buckwheat of interpolation Chinese medicine, the output of Ganodenic acid (Ganoderma triterpenoids) increases, and the production peak of Ganodenic acid is 310mg/L in its fermented liquid.Document: Liu Gaoqiang, fourth Double Ninth Festival, Zhang Kechang. dung beetle is to the influence of fermented hyphostroma of Ganoderma growth and the formation of triterpene product. the fungus journal, 2008,27 (5). after having reported interpolation insect dung beetle, the output of Ganoderma triterpenoids increases, and the production peak of triterpene in its fermented liquid (the outer triterpene of born of the same parents) is 304.3mg/L.Though above method can make yield of ganoderma lucidum triterpenoid increase, the cost of Chinese medicine is higher, and has the hidden danger of toxic side effect aspect.
We are by a large amount of screening experiments, found a kind of external source substance that can significantly improve yield of ganoderma lucidum triterpenoid, simultaneously ferment strategy and liquid nutrient medium have been carried out optimization design, formed and a kind ofly can significantly improve yield of ganoderma lucidum triterpenoid and the simple Ganoderma triterpenoids production technique of production technique in the Ganoderma fermentation liquid.
Summary of the invention
The objective of the invention is to all deficiencies of existing at existing Ganoderma triterpenoids fermentation technique, provide a kind of and can significantly improve yield of ganoderma lucidum triterpenoid in the Ganoderma fermentation liquid and production technique is simple, additive is with low cost, fermentation time is relatively short Ganoderma triterpenoids production technique.
For realizing the object of the invention, technical scheme of the present invention is:
Adopt the bacterial classification of Ganoderma Ganodermalucidum (the Leyss ex Fr.) Karst. of Chinese common micro-organisms culture presevation management committee preservation, the bacterial strain preserving number is CCGMC5.616.The related properties of bacterial classification can be consulted Chinese common micro-organisms culture presevation management committee (CCGMC) bacterial classification catalogue, and the present invention no longer describes in detail.
After Ganderma lucidum strain inoculated liquid nutrient medium, be that 26-30 ℃, rotating speed are the rotatable next time submerged fermentation 1-5 of 130-180r/min condition days in temperature; After adding palmitinic acid 1.5-3.0g/L, be that 25-30 ℃, rotating speed are to finish in the rotatable next time submerged fermentation 3-6 of 100-180r/min condition days, from fermented liquid, extract Ganoderma triterpenoids in temperature.
Liquid nutrient medium of the present invention consists of: with Semen Maydis powder, glucose is carbon source, and preferred content is respectively 5-10g/L, 25-38g/L; With peptone, soybean cake powder is nitrogenous source, and preferred content is respectively 3-4g/L, 2-3g/L; With KH2PO4 and MgSO4 is mineral element, and preferred content is respectively 1.5g/L, 0.75g/L.
Treat to carry out after the fermentation ends extraction of Ganoderma triterpenoids, extracting method is: earlier fermented liquid is concentrated, the industrial spirit that adds 4 times of volumes is with precipitation macromolecular polysaccharide and protein, after getting supernatant liquor and boiling off water, with 95% ethanolic soln refluxing extraction 2 hours, extract extracted 3 hours 50 ℃ of following joltings with propyl carbinol again, boiled off propyl carbinol under 60 ℃, obtained the Ganoderma triterpenoids product.
Compare with existing glossy ganoderma fermentation technology, the present invention has following advantage:
1) by adding an amount of palmitinic acid after for some time at glossy ganoderma fermentation, can significantly improve the output of Ganoderma triterpenoids in the Ganoderma fermentation liquid, its output is higher than existing bibliographical information output up to 396.2mg/L.
2) zymotechnique of the present invention is simple, and the total fermentation time of Ganoderma triterpenoids is shorter, and production efficiency obviously improves, and is fit to suitability for industrialized production.
What 3) the present invention added is edible palmitinic acid, have no side effect, and addition seldom, and to compare cost lower with existing Chinese medicine additive.
Embodiment
Embodiment 1:
Adopt the Ganderma lucidum strain (Ganodermalucidum) of Chinese common micro-organisms culture presevation administrative center preservation, preserving number: CCGMC5.616.
Liquid nutrient medium is formed: Semen Maydis powder 10g/L, glucose 25g/L, peptone 3g/L, soybean cake powder 3g/L, KH
2PO
41.5g/L, MgSO
40.75g/L.The 10mL seed is inoculated in the 150mL substratum, places 500mL to shake bottle and ferment.Leavening temperature is 26 ℃, and rotating speed is 130r/min, fermentation earlier 1 day, and the disposable then 1.5g/L palmitinic acid of adding continues fermentation 6 days in fermented liquid, results fermented liquid and related products.
With fermented liquid under 8000r/min centrifugal 8 minutes, obtain supernatant liquor and concentrated, the industrial spirit that adds 4 times of volumes, precipitation macromolecular polysaccharide and protein etc., get the 200mL supernatant liquor, after boiling off water, with 400mL, 95% ethanolic soln refluxing extraction 2 hours, extract extracted 3 hours 50 ℃ of following joltings with propyl carbinol again, boiled off propyl carbinol under 60 ℃, obtain extract, with behind the dissolve with methanol as sample, after color reaction, measure absorbancy in the 250nm place, the output that calculates Ganoderma triterpenoids is 351.3mg/L.
Embodiment 2:
Adopt the Ganderma lucidum strain (Ganodermalucidum) of Chinese common micro-organisms culture presevation administrative center preservation, preserving number: CCGMC5.616.
Liquid nutrient medium is formed: glucose 30g/L, Semen Maydis powder 10g/L, peptone 4g/L, soybean cake powder 4g/L, KH
2PO
41.5g/L, MgSO
40.75g/L.Inoculation 10mL seed shakes in the bottle in 150mL substratum/500mL.Leavening temperature is 28 ℃, and rotating speed is 150r/min, fermentation earlier 2 days, and the disposable then 2.0g/L palmitinic acid of adding continues fermentation 5 days in fermented liquid.Finish fermentation, results fermented liquid and related products.The extraction of product and analytical procedure are with case 1, and the output of Ganoderma triterpenoids is 359.8mg/L as a result.
Embodiment 3:
Adopt the Ganderma lucidum strain (Ganodermalucidum) of Chinese common micro-organisms culture presevation administrative center preservation, preserving number: CCGMC5.616.
Substratum composition and inoculum size are with embodiment 2.Leavening temperature is 30 ℃, and rotating speed is 180r/min, fermentation earlier 2 days, and the disposable then 2.0g/L palmitinic acid of adding continues fermentation 5 days in fermented liquid.Finish fermentation, results fermented liquid and related products.The extraction of product and analytical procedure are with embodiment 1, and the output of Ganoderma triterpenoids is 353.8mg/L as a result.
Embodiment 4:
Adopt the Ganderma lucidum strain (Ganodermalucidum) of Chinese common micro-organisms culture presevation administrative center preservation, preserving number: CCGMC5.616.
Fermentation broth is formed: glucose 35g/L, Semen Maydis powder 8g/L, peptone 5g/L, soybean cake powder 3g/L, KH
2PO
41.5g/L, MgSO
40.75g/L.Inoculation 10mL seed shakes in the bottle in 100mL substratum/500mL.Leavening temperature is 30 ℃, and rotating speed is 140r/min, fermentation earlier 2 days, and the disposable then 2.0g/L palmitinic acid of adding continues fermentation 5 days in fermented liquid.Finish fermentation, results fermented liquid and related products.The extraction of product and analytical procedure are with embodiment 1, and the output of Ganoderma triterpenoids is 369.5mg/L as a result.
Embodiment 5:
Adopt the Ganderma lucidum strain (Ganodermalucidum) of Chinese common micro-organisms culture presevation administrative center preservation, preserving number: CCGMC5.616.
Fermentation broth is formed: glucose 38g/L, Semen Maydis powder 5g/L, peptone 4g/L, soybean cake powder 2g/L, KH
2PO
41.5g/L, MgSO
40.75g/L.Inoculation 10mL seed shakes in the bottle in 100mL substratum/500mL.Leavening temperature is 30 ℃, and rotating speed is 160r/min, fermentation earlier 3 days, and the disposable then 3.0g/L palmitinic acid of adding continues fermentation 5 days in fermented liquid.Finish fermentation, results fermented liquid and related products.The extraction of product and analytical procedure are with embodiment 1, and the output of Ganoderma triterpenoids is 378.5mg/L as a result.
Embodiment 6:
Adopt the Ganderma lucidum strain (Ganodermalucidum) of Chinese common micro-organisms culture presevation administrative center preservation, preserving number: CCGMC5.616.
Substratum composition, inoculum size, leavening temperature and rotating speed are with embodiment 5.Fermentation earlier 4 days, the disposable then 2.5g/L palmitinic acid of adding continue fermentation 4 days in fermented liquid.Finish fermentation, results fermented liquid and related products.The extraction of product and analytical procedure are with embodiment 1, and the output of Ganoderma triterpenoids is 383.6mg/L as a result.
Embodiment 7:
Adopt the Ganderma lucidum strain (Ganodermalucidum) of Chinese common micro-organisms culture presevation administrative center preservation, preserving number: CCGMC5.616.
Substratum composition, inoculum size, leavening temperature and rotating speed are with embodiment 5.Fermentation earlier 5 days, the disposable then 2.8g/L palmitinic acid of adding continue fermentation 3 days in fermented liquid.Finish fermentation, results fermented liquid and related products.The extraction of product and analytical procedure are with embodiment 1, and the output of Ganoderma triterpenoids is 396.2mg/L as a result.
Embodiment 8:
Adopt the Ganderma lucidum strain (Ganodermalucidum) of Chinese common micro-organisms culture presevation administrative center preservation, preserving number: CCGMC5.616.
Substratum composition, inoculum size, leavening temperature and rotating speed are with embodiment 5.Fermentation earlier 4 days, the disposable then 3.0g/L palmitinic acid of adding continue fermentation 4 days in fermented liquid.Finish fermentation, results fermented liquid and related products.The extraction of product and analytical procedure are with embodiment 1, and the output of Ganoderma triterpenoids is 380.3mg/L as a result.
Claims (2)
1. method that improves yield of ganoderma lucidum triterpenoid in the Ganoderma fermentation liquid is characterized in that: use Ganderma lucidum strain [Ganoderma lucidum (Leyss ex Fr.) Karst.], the bacterial strain preserving number is CCGMC 5.616, carries out as follows:
1) Ganderma lucidum strain inoculation being contained the substratum of carbon source, nitrogenous source, mineral element composition, is that 26-30 ℃, rotating speed are the rotatable next time submerged fermentation 1-5 of 130-180r/min condition days in temperature; It is to be carbon source with Semen Maydis powder, glucose that substratum is formed, and is nitrogenous source with peptone, soybean cake powder, with KH
2PO
4And MgSO
4Be mineral element; Semen Maydis powder content is that 5-10g/L, glucose content are 25-40g/L in the substratum; Peptone content is that 3-5g/L, soybean cake powder content are 2-4g/L in the substratum; KH in the substratum
2PO
4Content is 1.5g/L, MgSO
4Content is 0.75g/L;
2) add palmitinic acid 1.5-3.0g/L after, be that 25-30 ℃, rotating speed are to finish in the rotatable next time submerged fermentation 3-6 of 100-180r/min condition days in temperature;
3) from fermented liquid, extract Ganoderma triterpenoids.
2. a kind of method that improves yield of ganoderma lucidum triterpenoid in the Ganoderma fermentation liquid according to claim 1, it is characterized in that, the Ganoderma triterpenoids extracting method is in the fermented liquid: fermented liquid is through concentrating, the industrial spirit that adds 4 times of volumes is with precipitation macromolecular polysaccharide and protein, after getting supernatant liquor and boiling off water, with 95% ethanolic soln refluxing extraction 2 hours, extract extracted 3 hours 50 ℃ of following joltings with propyl carbinol again, the Ganoderma triterpenoids product.
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| CN101892282B (en) * | 2010-07-20 | 2012-09-19 | 山东省农业科学院土壤肥料研究所 | Liquid fermentation method for improving yield of ganoderma lucidum triterpenoid by using rare earth element |
| CN106852496A (en) * | 2015-12-09 | 2017-06-16 | 黄建钧 | The method of the triterpenes synthesis contained by induction mushroom gill fungus |
| CN105506049B (en) * | 2016-02-02 | 2018-11-27 | 华中农业大学 | A method of improving the ganodenic acid content intracellular of ganoderma lucidum mycelium liquid fermentation |
| CN106262807A (en) * | 2016-07-29 | 2017-01-04 | 江苏中祥高科技实业有限公司 | A kind of liquid fermentation height Ganoderma triterpenoids Ganoderma ferment and preparation technology thereof |
| CN115634241A (en) * | 2022-11-16 | 2023-01-24 | 青岛农业大学 | Preparation method of Shandong ganoderma lucidum extract and application of Shandong ganoderma lucidum extract in preparation of hypoglycemic drugs |
| CN118562914A (en) * | 2024-05-17 | 2024-08-30 | 中科药创(青岛)发酵工程有限公司 | A Ganoderma fermentation product containing selenium-enriched Ganoderma triterpene and polysaccharide for repairing liver injury, and its fermentation method |
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