CN1101855C - Liquid fermentation process for preparing both ganoderic polyose and ganoderic acid - Google Patents
Liquid fermentation process for preparing both ganoderic polyose and ganoderic acid Download PDFInfo
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- CN1101855C CN1101855C CN00111953A CN00111953A CN1101855C CN 1101855 C CN1101855 C CN 1101855C CN 00111953 A CN00111953 A CN 00111953A CN 00111953 A CN00111953 A CN 00111953A CN 1101855 C CN1101855 C CN 1101855C
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Abstract
The present invention discloses a technology for simultaneously producing ganoderma polysaccharide and ganoderma acid by a liquid fermentation method. In the present invention, microbes, namely Ganoderma lucidum (LeyssexFr.)Karst. strains, are used to simultaneously produce extracellular ganoderma polysaccharide, intracellular ganoderma polysaccharide and ganoderma acid by an aerobic liquid fermentation method, an aerobic liquid fermentation-statical liquid culture method or a static liquid culture method. The content of the ganoderma acid can reach 2.8 mg/100 mg, and the total content of the extracellular ganoderma polysaccharide and the intracellular ganoderma polysaccharide can reach 2.34 g/L. The method of the present invention is simple in a culture technology, and is a production method with industrialization prospects.
Description
The invention belongs to biotechnology and technical field.The production technique that relates to a kind of ganoderan and Ganodenic acid relates in particular to the technology of a kind of fermentative Production ganoderan and Ganodenic acid.
Glossy ganoderma (Ganoderma lucidum (Leyss ex Fr.) Karst.) is Basidiomycetes, polyporaceae, Ganoderma fungi.Since ancient times, just that it is edible as medicine or tonic the East Asia Region people.Modern medicine study finds that contained effective constituent mainly contains polyose (that is: ganoderan), triterpenes (being mainly the glossy ganoderma acids), sterols, alkaloids, furan derivative, protein, polypeptide and organic germanium nucleosides etc. in the glossy ganoderma.When the glossy ganoderma pharmaceutical use was extensively disclosed, the output of glossy ganoderma became the bottleneck that suppresses the glossy ganoderma application, because very rare the wild glossy ganoderma of occurring in nature, and wherein ganoderic acid content is 1 milligram/100 milligrams dry weights, far can not satisfy people's needs.Compare with the glossy ganoderma artificial culture, fermentative Production glossy ganoderma effective ingredient since with short production cycle, labor force economize and be subjected to advantages such as external environment influence is little to be considered to a kind of more efficient methods.
Relevant deep glossy ganoderma fermenting research starts from the seventies, and how research at that time guarantees that Ganoderma mycelium successfully ferments and how to obtain more biomass if laying particular emphasis on.Since the eighties, people begin to note how obtaining its biologically active substance, wherein mainly concentrate on the fermentative production research aspect of ganoderan (particularly exocellular polysaccharide), as:
Document: Li Pingzuo, Xu Rou, Zhang Kechang.The optimization of glossy ganoderma exocellular polysaccharide submerged fermentation culture medium; Wuxi Light Industry Univ.'s journal 1998,17 (4): 26-30 has reported a kind of method, but this method can only be produced ganoderan;
Document: Luo Lixin, Yao Ruhua, Zhou Shaoqi.The research of glossy ganoderma deep layer culture process; South China Science ﹠ Engineering University's journal (natural science edition) 1997,25 (5): 67-70 has reported a kind of method, but this method ganoderan output only is about the 1.0-2.1 grams per liter;
Japanese Patent: openly specially permit the communique spy and open flat 4-304890,5pp discloses the fermentation manufacturing technique of another kind of major physiological active substance-Ganodenic acid, but its not mentioned ganoderan production, and the content of Ganodenic acid only is 0.46-1.0 milligram/100 milligram dry weight in the product of gained;
Also have many patents and document also open and reported the whole bag of tricks, still, its major physiological active substance is all lower, has also only reported the production of ganoderan simultaneously.
It should be noted that simultaneously, because ganoderan and Ganodenic acid are respectively primary metabolite and secondary metabolic product, both fermentative production conditions differ, and need in addition integrated survey to optimize the production process of ganoderan (comprising the outer and intracellular polyse of born of the same parents) and Ganodenic acid simultaneously.Therefore do not appear in the newspapers so far about the research of producing ganoderan and Ganodenic acid with solution fermentation simultaneously.
In sum, still do not have the technology that sophisticated solution fermentation is produced ganoderan and Ganodenic acid so far, therefore, a kind of technology that can produce ganoderan and Ganodenic acid simultaneously by solution fermentation of development research is that people institute is very expected as early as possible.
The objective of the invention is to disclose the technology that a kind of solution fermentation is produced ganoderan and Ganodenic acid simultaneously, to satisfy people's needs.
Design of the present invention is such:
The contriver thinks, as long as adopt suitable bacterial classification, in same fermenting process, adopt suitable culture condition, just can from Ganoderma fermentation liquid, separate the glossy ganoderma exocellular polysaccharide that obtains high yield, extracting obtains the glossy ganoderma intracellular polyse of high yield and the Ganodenic acid of high yield from Ganoderma mycelium, thereby lays the foundation for industrialized economy production ganoderan and Ganodenic acid.
According to above-mentioned design, the contriver has proposed to realize the technical scheme as described below of the object of the invention by a large amount of experiments:
The microorganism of using:
The microorganism that is used to produce described ganoderan and Ganodenic acid is the bacterial classification of Ganoderma Ganoderma lucidum (Leyss ex Fr.) Karst., and concrete bacterial strain is as follows:
Straight clinic of 67 armies of army finds that in China Committee for Culture Collection of Microorganisms's preservation, preserving number is the bacterial strain of CGMCC No.5.75;
Middle school, Wusong, Shanghai finds that in China Committee for Culture Collection of Microorganisms's preservation, preserving number is the bacterial strain of CGMCC No.5.110;
Japan's capital great discovery, in China Committee for Culture Collection of Microorganisms's preservation, preserving number is the bacterial strain of CGMCCNo.5.533;
China finds that in China Committee for Culture Collection of Microorganisms's preservation, preserving number is the bacterial strain of CGMCCNo.5.616 in Guizhou;
Institute of microbiology of the Chinese Academy of Sciences finds that in China Committee for Culture Collection of Microorganisms's preservation, preserving number is the bacterial strain of CGMCC No.5.644;
The Chinese Academy of Agricultural Sciences finds that in China Committee for Culture Collection of Microorganisms's preservation, preserving number is the bacterial strain of CGMCC No.5.653.
The related properties of above-mentioned bacterial classification can be consulted Chinese common micro-organisms DSMZ (CGMCC) bacterial classification catalogue, and the present invention repeats no more.
Cultural method:
Bacterial classification with above-mentioned Pseudomonas adopts liquid aerobic fermentation method, liquid aerobic fermentation-liquid leaves standstill culture method or liquid leaves standstill culture method, with sucrose or glucose is that carbon source, peptone and yeast extract paste are that nitrogenous source is as seed culture medium, and add an amount of inorganic salt and VITAMIN, bacterial classification inoculation is arrived liquid nutrient medium, obtain the Ganoderma mycelium that contains the fermented liquid of glossy ganoderma exocellular polysaccharide and contain glossy ganoderma intracellular polyse and Ganodenic acid by liquid fermenting, above-mentioned cultural method there is no strict demand to temperature, generally carries out in room temperature.Now be described below respectively:
1. liquid aerobic fermentation method:
Inoculation 10-1000 milligram dry weight/rise seed is in there being the shaking in the bottle of substratum, and 20-35 ℃, the 80-180 rev/min of cultivation of circling round fermented 4-12 days;
2. liquid aerobic fermentation-liquid leaves standstill culture method: above-mentioned tunning is left standstill cultivation, left standstill 6-20 days, finish to cultivate results mycelium and fermented liquid.Increase is also arranged leaving standstill the cultivation stage cell concentration, it is synthetic also to help Ganodenic acid simultaneously;
3. leave standstill culture method:
Inoculation 10-1000 milligram dry weight/rise seed 20-35 ℃, leaves standstill and cultivated 10～20 days in the shaking in the bottle of substratum arranged;
Substratum can adopt the conventional substratum that contains compositions such as carbon source, nitrogenous source, inorganic salt and VITAMIN.Wherein: carbon source comprises glucose, sucrose, fructose, maltose, lactose, starch, molasses or murphy juice etc.; Nitrogenous source has yeast extract paste, peptone, casein or soybean cake powder etc.; Inorganic salt comprise KH
2PO
4And MgSO
4Wherein will more help cell when adding murphy juice in the carbon source and grow fast, it is synthetic that glucose, sucrose help exocellular polysaccharide, and maltose helps obtaining high cell concentration, and it is synthetic that lactose helps intracellular polyse.Organic nitrogen source helps cell and absorbs.
Exocellular polysaccharide obtains by ethanol precipitation.Intracellular polyse is by behind the alkali dissolution mycelium, and ethanol precipitation obtains.Ganodenic acid obtains through preliminary purification after extracting by aqueous ethanolic solution.More than said method be prior art, in many documents, be described, the present invention repeats no more.
Adopt conventional analytical procedure that product is carried out analytical test, the result shows, described method can obtain glossy ganoderma exocellular polysaccharide, intracellular polyse and Ganodenic acid simultaneously, ganoderic acid content can reach 2.8 milligrams/100 milligrams, in the born of the same parents and the exocellular polysaccharide total amount up to 2.34 grams per liters, apparently higher than report and commercially available glossy ganoderma in the past.
In sum, the ganoderic acid content that the present invention obtains is apparently higher than the reported in literature level, intracellular polyse content a little more than the reported in literature level exocellular polysaccharide content be on close level with the document report.Can in the glossy ganoderma fermentation process, obtain Ganodenic acid and these two kinds of main pharmaceutical components of glossy ganoderma of ganoderan simultaneously by present method with higher yields.For existing employing similar liquids fermentation means are only produced a kind of ganoderan, present method is easy owing to not increasing complicated optional equipment and culture technique, as in industrial realization Ganodenic acid and these two kinds high value effective constituents of ganoderan, producing, the economic benefit of production process will be increased substantially, also considerable social benefit can be brought.These products all have higher pharmaceutical use, can be widely used in industries such as medicine and health care of food.This shows that method of the present invention really is a kind of production method with industrial prospect.
Below will relevant details of the present invention be further described by embodiment.
Embodiment 1
The bacterial classification that adopts; CGMCC No.5.616;
Contain glucose 35 grams per liters in the fermentation broth, peptone 5 grams per liters, yeast extract paste 5 grams per liters, KH
2PO
41 grams per liter, MgSO
40.5 grams per liter, VB1 0.05 grams per liter.Leavening temperature is 30 ℃, inoculates 15 milligrams of mycelium and cultivates 8 days in 50 milliliters of substratum/250 ml shake flasks.Finish fermentation, results mycelium and fermented liquid, and carry out following assay determination:
Centrifugation obtains mycelium 15.4 grams per liters and fermented liquid under 15000rpm;
95% of 4 times of volumes of adding ethanol in the fermented liquid, mixing is spent the night, and centrifugation gets ganoderan outside the born of the same parents under 10000rpm.Through 1 mol NaOH dissolving, adopting the vitriol oil-phynol method to measure polysaccharide content is 0.8 grams per liter;
Mycelium is got 100 milligrams 45 ℃ of dryings, adds 3 milliliter of 50% extraction using alcohol Ganodenic acid twice, centrifugal under 4000rpm, 50 ℃ of dryings of supernatant liquor are dissolved with 2 ml waters, and, get the chloroform phase with 2 milliliters of chloroform extractions, use 2 milliliters of extractions of 5% sodium bicarbonate again, the water intaking phase adds 2 mol hydrochloric acid to pH=3, adds chloroform, get the chloroform phase, with ultraviolet colorimetric method for determining Ganodenic acid behind the dissolve with ethanol, calculating content is 1.43 milligrams/100 milligrams dry weights after volatilizing, and output is 214 mg/litre;
100 milligrams of mycelium add 1 mol NaOH dissolving, and it is 8 milligrams/100 milligrams dry weights that the vitriol oil-phynol method is measured intracellular polyse, and output is 1.1 grams per liters.
Embodiment 2
Aerobic fermentation condition ferments after 4 days with example 1, stops to shake the bottle vibration, transfers to and leaves standstill cultivation.Leave standstill and cultivate after 8 days, finish to cultivate, results mycelium and fermented liquid carry out the effective ingredient analysis.Measuring method is with example 1, as a result, dry cell weight, exocellular polysaccharide, intracellular polyse and Ganodenic acid output are respectively 20.9 grams per liters, 0.47 grams per liter, 1.4 grams per liter (6.6 milligrams/100 milligrams dry weights of born of the same parents' intensive amount), 581 mg/litre (2.8 milligrams/100 milligrams dry weights of born of the same parents' intensive amount).
Embodiment 3
The carbon source of liquid fermentation medium is a lactose, and fermentation condition is cultivated after 10 days with example 1, finishes fermentation and results thalline and fermented liquid.Analytical results, dry cell weight, exocellular polysaccharide, intracellular polyse and Ganodenic acid output are respectively 16.81 grams per liters, 0.35 grams per liter, 1.99 grams per liters (11.83 milligrams/100 milligrams of born of the same parents' intensive amounts), 207 mg/litre (1.40 milligrams/100 milligrams dry weights of born of the same parents' intensive amount).
Embodiment 4
The bacterial classification that adopts: CGMCC No.5.75;
Contain 0.6 liter of 200 gram potato extracting solution in every liter of fermented liquid substratum, sucrose 30 grams per liters, peptone 2 grams per liters, KH
2PO
41.5 grams per liter, MgSO
40.75 grams per liter, VB
10.001 grams per liter.Leavening temperature is 30 ℃, inoculates 1 milligram of mycelium in 50 milliliters of substratum/250 ml shake flasks, cultivates 12 days.Finish fermentation, results mycelium and fermented liquid, analysis test method is with embodiment 1, analytical results shows, dry cell weight, intracellular polyse and Ganodenic acid output are respectively 8 grams per liters, 0.36 grams per liter (4.5 milligrams/100 milligrams of born of the same parents' intensive amounts), 112 mg/litre (1.40 milligrams/100 milligrams dry weights of born of the same parents' intensive amount).
Embodiment 5～10
Adopt bacterial classification to be respectively a kind of among CGMCC No.5.75, CGMCC No.5.110, CGMCC No.5.533, CGMCCCC No.5.616, CGMCC No.5.644 or the CGMCC No.5.653; Cultural method is identical with embodiment 4, the analytical test result:
Dry cell weight and intracellular polyse content are respectively 9 grams per liters and 3.5 milligrams/100 milligrams dry weights, 5.5 grams per liters and 4.4 milligrams/100 milligrams dry weights, 2.8 grams per liters and 4.7 milligrams/100 milligrams dry weights, 8.5 grams per liters and 4.8 milligrams/100 milligrams dry weights, 2.8 grams per liters and 5.0 milligrams/100 milligrams dry weights and 2.7 grams per liters and 4.2 milligrams/100 milligrams dry weights.
Claims (4)
1. method that solution fermentation is produced ganoderan and Ganodenic acid simultaneously, it is characterized in that, use the bacterial classification of Ganoderma Ganoderma lucidum (Leyss ex Fr.) Karst., adopt following steps to carry out: inoculation 10-1000mg dry weight/L bacterial classification is in the shaking in the bottle of substratum arranged, the cultivation of circling round, fermented 4～12 days, and from tunning, collected ganoderan and Ganodenic acid then;
Said bacterial classification is a kind of among CGMCC No.5.75, CGMCC No.5.110, CGMCC No.5.533, CGMCC No.5.616, CGMCC No.5.644 or the CGMCC No.5.653, substratum is the substratum that contains carbon source, nitrogenous source, inorganic salt and vitamin ingredients, culture temperature is 20～35 ℃, and the rotating speed of cultivating that circles round is 80-180 rev/min.
2. the method for claim 1 is characterized in that, leaves standstill after the fermentation to cultivate 6-20 days again, finishes to cultivate results mycelium and fermented liquid.
3. method that solution fermentation is produced ganoderan and Ganodenic acid simultaneously, it is characterized in that, use the bacterial classification of Ganoderma Ganoderma lucidum (Leyss ex Fr.) Karst., adopt following steps to carry out: inoculation 10-1000mg dry weight/L bacterial classification is in the shaking in the bottle of substratum arranged, leave standstill and cultivated 10～20 days, from tunning, collect ganoderan and Ganodenic acid then;
Said bacterial classification is a kind of among CGMCC No.5.75, CGMCC No.5.110, CGMCC No.5.533, CGMCC No.5.616, CGMCC No.5.644 or the CGMCC No.5.653, substratum is the substratum that contains carbon source, nitrogenous source, inorganic salt and vitamin ingredients, and culture temperature is 20～35 ℃.
4. as the described method of the arbitrary claim of claim 1-3, it is characterized in that the carbon source in the said substratum is glucose or lactose.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101326954B (en) * | 2008-07-30 | 2011-11-16 | 江苏大学 | Method for producing feed containing Ganoderma lucidum acid from wheat straw transformed by Ganoderma lucidum and uses therefor |
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|---|---|---|---|---|
| CN100335504C (en) * | 2003-04-14 | 2007-09-05 | 中国科学院上海药物研究所 | FB1 polyose and preparaton method and application |
| CN1307314C (en) * | 2004-04-28 | 2007-03-28 | 中国食品发酵工业研究院 | Enzymolytic preparation method for glossy ganoderma amylose |
| CN101376904B (en) * | 2007-08-29 | 2013-06-19 | 上海医药工业研究院 | Intra-polysaccharides from mycelia of ganoderma sinensis, and preparation and use thereof |
| CN101492645B (en) * | 2008-01-21 | 2010-12-22 | 湖北工业大学 | Glossy ganoderma cell culture method for accelerating biosynthesis of ganoderic acid and ganoderma iucidum polysaccharide |
| CN101353689B (en) * | 2008-09-02 | 2011-09-07 | 中南林业科技大学 | Method for enhancing yield of ganoderma triterpene in ganoderma lucidum fermentation liquor |
| 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 |
| CN101880700B (en) * | 2010-07-20 | 2012-09-05 | 山东省农业科学院土壤肥料研究所 | Liquid fermentation method capable of improving yield of ganoderma iucidum polysaccharide |
| CN102643884A (en) * | 2012-05-04 | 2012-08-22 | 苏州百趣食品有限公司 | Method for producing polysaccharide by utilizing fermentation of ganoderma |
| CN105378056B (en) * | 2013-08-09 | 2018-03-13 | 双鹤生物科技股份有限公司 | The technical grade cultural method of ganoderma lucidum mycelium |
| CN106755182B (en) * | 2016-12-16 | 2020-11-06 | 江南大学 | Method for promoting ganoderma lucidum liquid fermentation to produce extracellular polysaccharide |
| CN114836492A (en) * | 2021-02-02 | 2022-08-02 | 中粮生物科技（北京）有限公司 | Method for producing ganoderan and ganoderic acid by fermentation |
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|---|---|---|---|---|
| CN1101860A (en) * | 1993-10-21 | 1995-04-26 | 谭礼 | Process for producing biochemical raw material of spore powder of glossy ganoderma |
| CN1181271A (en) * | 1996-10-31 | 1998-05-13 | 高益槐 | Water extracting process for high activity glossy ganoderma holoside and high content glossy ganoderma acid |
| CN1235196A (en) * | 1998-05-11 | 1999-11-17 | 封守业 | Manufacture of Sanzhen health-care products |
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2000
- 2000-03-10 CN CN00111953A patent/CN1101855C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1101860A (en) * | 1993-10-21 | 1995-04-26 | 谭礼 | Process for producing biochemical raw material of spore powder of glossy ganoderma |
| CN1181271A (en) * | 1996-10-31 | 1998-05-13 | 高益槐 | Water extracting process for high activity glossy ganoderma holoside and high content glossy ganoderma acid |
| CN1235196A (en) * | 1998-05-11 | 1999-11-17 | 封守业 | Manufacture of Sanzhen health-care products |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101326954B (en) * | 2008-07-30 | 2011-11-16 | 江苏大学 | Method for producing feed containing Ganoderma lucidum acid from wheat straw transformed by Ganoderma lucidum and uses therefor |
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