CN104893992A - Submerged fermentation method for ganoderma lucidum - Google Patents
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Abstract
The invention discloses a submerged fermentation method for ganoderma lucidum. Natural Chinese herb raw materials such as ginseng, bee pollen, ganoderma lucidum spore powder and indian buead serve as a submerged fermentation culture medium for ganoderma lucidum. The submerged fermentation method has the characteristics of high target product yield, stable quality, good mycelium growth, short fermentation period, simple production process, high extraction yield, rich nutrient, low cost and the like, is an environment-friendly submerged medicinal fungi fermentation technology with remarkable economic benefits, and has a remarkable development and application prospect.
Description
Technical field
The invention belongs to medicinal fungi submerged fermentation production technology and biomedicine field, be specifically related to a kind of submerged fermentation method of Ganderma lucidum.
Background technology
Glossy ganoderma (Ganoderma lucidum) is the general name of Basidiomycetes polyporaceae (Polyporaceae) Ganoderma (Ganodelma) fungi red sesame G.lucidumkarst and purple sesame G.japonicrnLloyd, there is effects such as strengthening the body resistance to consolidate the constitution, be called top grade by " herbal classic ".In recent years, to the chemical composition of Ganoderma fungi and clinical study a lot of, to the sexual cell of glossy ganoderma---the research of spore pharmaceutical use is also quite paid attention to.
Ganoderma spore brown, avette, one end is truncate, (8.5 ~ 11.2) μm × (5.2 ~ 6.9) μm, tool double wall.Produce many needle-like pimples at the brown internal layer of each spore, stretch into the transparent outer layer of conidial cell wall dearly, outer sporoderm is smooth.The two layers of walls of Ganoderma spore parcel is very tough and tensile, general chemistry, and physical method is difficult to be broken, therefore limits the research to its chemical composition, bioactive ingredients.
The virgin forest that glossy ganoderma is maximum in the whole nation, Changbai Mountain and the growth of Natural Reserves in Hainan optimum.But due to the excessive harvesting of the mankind for many years, the storage of wild Ganoderma is fewer and feweri.Glossy ganoderma has provide protection to the liver injury that multiple physics and chemistry and biotic factor cause.No matter before liver damage occurs or after generation, take glossy ganoderma and all can protect liver, alleviate liver injury.Glossy ganoderma can promote that liver is to the metabolism of medicine, poisonous substance, has definite curative effect for toxic hepatitis.Especially chronic hepatitis, glossy ganoderma obviously can eliminate the symptoms such as dizziness, weak, nauseating, uncomfortable liver area, and effectively can improve liver function, makes indices be tending towards normal.So glossy ganoderma can be used for treating chronic poisoning, all kinds of chronic hepatitis, liver cirrhosis, hepatic insufficiency.Glossy ganoderma can coronary artery dilator effectively, increases coronary flow, improves Myocardial Microcirculation, strengthen the supply of myocardium oxygen and energy, therefore, have provide protection to myocardial ischemia, can be widely used in treatment and the prevention of coronary heart diseases and angina pectoris etc.In addition, Regular Insulin can be replaced after taking glossy ganoderma and suppress disengaging of lipid acid, the symptom such as blood sugar, glucose in urine can be improved.Water-soluble polysaccharide in glossy ganoderma, can alleviate the occurring degree of non insulin dependent diabetes.
The chemical composition of Ganoderma is comparatively complicated, and different because bacterial classification used, the bacterial classification place of production, cultivating method, extraction process, formulation method are different.Containing polyose, ucleosides, furan derivative, steroid ferment class, alkaloids, protein, polypeptide, amino acids, triterpenes, sesquiterpene, organic germanium, inorganic salt etc. in the sporophore of Ganoderma, mycelium and spore.Ganoderan is one of principle active component of glossy ganoderma.
Glossy ganoderma generally grows in the mountain forest that humidity is high and rather dark, and primary growth is at corruption tree or the root of its trees.Glossy ganoderma is not plant, and self can not carry out photosynthesis, can only absorb nutriment from other organism or rotten tree.Glossy ganoderma is the macro fungi of a kind of hard, many spore and micro-band bitterness.When the glossy ganoderma reaching the ripening stage will spray the spore of powdery, thus breed.Now wild glossy ganoderma is rarely found, and quality is not easy to control.
Glossy ganoderma and glossy ganoderma biologically active substance become novel protective foods and the study hotspot of medicine field because of the physicochemical property of its uniqueness and the biological function of mystery.But the growth cycle of Ganoderma Lucidum long (being greater than 2 ~ 3 months), greatly affected by environment, yield poorly, quality is unstable, production cost is high, greatly have impact on glossy ganoderma and the development of biologically active substance in health industry thereof.Therefore, use biotechnology in recent years, to the research of Fermentation Process of Ganoderma Lucidum Mycelium Submerged fermentation, obtain Ganoderma mycelium and Active Growth meta-bolites (Ganodenic acid, ganoderan etc.) thereof, and by the control of fermentation condition, shorten the production cycle, reduce production cost, make glossy ganoderma and biologically active substance thereof be applied in food and medicine and promote.
Ginseng (formal name used at school: Panax ginseng) is the root with meat, pharmaceutically acceptable.Ginseng belongs to Araliaceae, primary growth in East Asia, particularly cold district.Ginseng is common medicinal material, for recovery more afterwards, physical strength reinforcing, adjustment hormone, reduces blood sugar and controls blood pressure, the health care of control liver exponential sum liver function etc.Saponin(e contained by ginseng root is its effective constituent, and Changbai Mountain in China open country ginseng saponin constituent is higher, but obtains not easily, and price is high.Ginseng nutrient component content (every 100 grams) heat 80.0 kilocalorie carbohydrate 21.2 grams of fat 0.7 gram of protein 0.6 gram of Mierocrystalline cellulose 3.5 grams, be the active substance producing fermention medium Carbon and nitrogen sources, the meta-bolites of derivative nutritive ingredient can be promoted again.
Bee pollen (formal name used at school POLLEN), pollen contains multiple nutrients material, comprising 22 seed amino acids, 14 kinds of VITAMIN and 30 various trace elements and a large amount of active proteases, nucleic acid, flavonoid compound and other active substance.The bak protein 20%-25% that pollen is rich in, total amino acid content more than 20%, total free aminoacids 1%-2%, carbohydrate 40%-50%, fatty 5%-10%; Also has the abundant multiple nutrients material such as natural complex, trace element, enzyme, hormone, biologically active substance.Particularly the content of vitamin B group is very abundant.Every 100 grams of pollen contain VitB1 600 microgram, riboflavin 1670 microgram, pyridoxol 900 microgram, pantothenic acid 2700 microgram, nicotinic acid 10000 microgram.It contains several mineral materials, in these mineral substance, the content of some elements respectively: potassium 20 ~ 40%, magnesium 1 ~ 20%, calcium 1 ~ 15%, iron 1 ~ 12%, silicon 2 ~ 10%, phosphorus 1 ~ 20%.And a large amount of active protease, nucleic acid, flavonoid compound and other active substance etc.When carrying out fermentation culture as compounded carbons nitrogenous source, be conducive to mycelial growth.
Ganoderma spore powder (English name: Ganoderma spore).Ganoderma spore powder crude protein content is 18.12%, and aminoacids content is 5.869%, and crude fat content is 20.39%.Unsaturated fatty acids is advantage lipid acid. with oleic acid, linolic acid is main.Also containing abundant triterpene compound, multiple beneficial mineral element.With one of raw material of Ganoderma spore powder deep glossy ganoderma fermenting substratum, the precursor inductor of mycelium transposon mutagenesis can be promoted and impel a large amount of synthesis of exocellular polysaccharide.Its biomass, Crude polysaccharides and thick ganoderic acid content are all higher, are conducive to the formation of ganoderma active material (Ganodenic acid) and the raising of ganoderic acid content.
Poria cocos (formal name used at school Wolfiporia cocos).Tuckahoe nutrient enriches, the nutritive ingredient of every 100 grams of Poria cocos: energy 16 kilocalories, 1.2 grams, protein, 0.5 gram, fat, 82.6 grams, carbohydrate, food fibre 80.9 grams, 0.12 gram, riboflavin, 58 milligrams, potassium, 1 milligram, sodium, calcium 2 milligrams, 8 milligrams, magnesium, iron 9.4 milligrams, 1.39 milligrams, manganese, zinc 0.44 milligram of copper 0.23 milligram of P 32 milligram, selenium 4.55 microgram, 0.4 milligram, nicotinic acid etc.Using Poria powder as Chinese-medicinal culture medium raw material, the synthesis of ganoderan can be promoted, be conducive to the raising of ganoderma polyoses content.
Summary of the invention
The object of the present invention is to provide a kind of submerged fermentation method of Ganderma lucidum, the output with object product is high, steady quality, mycelial growth are good, fermentation period is short, production technique is simple, extract the features such as yield is high, nutritive substance abundant, cost is low, be the medicinal fungi submerged fermentation technology of the environment-friendly type of a remarkable in economical benefits, there is significant development prospect.
For achieving the above object, the present invention adopts following technical scheme:
A submerged fermentation method for Ganderma lucidum, is characterized in that: comprise the following steps:
(1) 5-7d is cultivated at the inclined-plane of inoculation being put in 26-28 DEG C;
(2) will cover with mycelial inclined plane inoculating in liquid nutrient medium, in shaking speed 80-100r/min, stroke 7-8cm back and forth shakes, and 26-28 DEG C of shaking culture 2-3d, makes seed suspension liquid;
(3) seeding tank seed culture, by kind of amount 15%-20%, a temperature 27-30 DEG C, illumination condition: continuous darkness condition, stirring velocity 160-180r/min, sterile air air flow is per minute 2.5m
3, tank pressure 0.04MPa, fermentation culture 48-72h; PH is down to 4.5-5.0;
(4) ferment tank is cultivated: inoculum size 25-30%, temperature 27-30 DEG C, stirring velocity is 150-200r/min, and sterile air air flow is per minute 2m
3, tank pressure 0.05MPa, cultivates 48-72h; Fermented liquid retrogradation, thalline starts self-dissolving, and pH is down to 3.0-3.5, and residual sugar amount is down to 0.2-0.3%;
(5) enzymolysis: add compound protease 0.5%, amylase 0.75%, cellulase 0.75%; Proteolytic enzyme is 2 × 10
5u/kg, amylase is 1.0 × 10
5u/kg, cellulase is 1.5 × 10
5u/kg; Temperature 55-60 DEG C, time 3-4h.
Slant medium: potato liquor 1000mL, skim-milk 30g, glucose 30g, agar 30g, peptone 30g, yeast extract paste 5g, magnesium sulfate 0.2g, VITMAIN B1 0.1g, pH nature;
Submerged fermentation culture medium, percentage: ginseng powder 2.0%, bee pollen 2.0%, Ganoderma spore powder 1.0%, Poria powder 1.0%, soyflour 2.0%, yeast powder 0.5%, glucose 3.0%, magnesium sulfate 0.2%, dipotassium hydrogen phosphate 0.3%, sodium-chlor 0.2%, VITMAIN B1 0.1%, Camellia oil 0.3%, surplus is water, pH=6-7, fineness of powder: 80-100 order.
The extracting method of tunning comprises the following steps:
(1) high-pressure homogeneous process: the fermented liquid through enzymolysis processing is first through colloidal mill homogeneous, rotating grinding rotating speed 800-1000r/min, makes hyphostoma particle disintegration, reaches homogeneous, again through high pressure homogenizer, make fermented liquid under 40MPa-60MPa High Pressure by 200 order membrane filtrations;
(2) supercritical CO
2extraction: in the supercritical state, contacted by supercritical co with material to be separated, makes it selectively the boiling point height composition different with molecular size range be extracted precipitation effective active matter successively;
(3) fermented liquid concentrates: fermented liquid is concentrated into concentration Brix=28-32%;
(4) adopt vacuum lyophilization, condenser temperature≤-60 DEG C, obtain tunning ganoderan and Ganoderma triterpenoids active substance dry powder goods.
Remarkable advantage of the present invention is: select the natural Chinese medicinal herb raw materials such as ginseng, bee pollen, Ganoderma spore powder and Poria cocos as Ganderma lucidum submerged fermentation culture medium, the output with object product is high, steady quality, mycelial growth are good, fermentation period is short, production technique is simple, extract the features such as yield is high, nutritive substance abundant, cost is low, be the medicinal fungi submerged fermentation technology of the environment-friendly type of a remarkable in economical benefits, there is significant development prospect.
Adopt the herbal medicine such as natural phant ginseng powder, bee pollen, Ganoderma spore powder, Poria powder as the raw material of Ganderma lucidum submerged fermentation culture medium, Optimal Medium nutritive substance, make full use of nitrogenous source, carbon source, promote Ganoderma lucidum mycelium bulk-growth, improve and obtain a large amount of thalline and meta-bolites, significantly improve the content of the effective active such as ganoderan and triterpene composition.Adopt the herbal medicine such as natural phant ginseng powder, bee pollen, Ganoderma spore powder, Poria powder as Ganderma lucidum submerged fermentation culture medium, and adopt supercritical CO
2extraction, enzymolysis process, extract glossy ganoderma polysaccharide and triterpene there is not yet relevant report.
Embodiment
Embodiment 1:
Bacterial classification: by the bacterial strain bacterial classification of Changbai Mountain in China wild Ganoderma separate tissue seed selection.
The submerged culturing method for making of Ganderma lucidum and tunning dry powder extract preparation method:
1, slant medium is prepared: potato liquor 1000ml, skim-milk 30g, glucose 30g, agar 30g, peptone 30g, yeast extract paste 5g, magnesium sulfate 0.2g, VITMAIN B1 0.1g, pH nature.
2, slant strains is cultivated: be seeded in by good quality strain on solid slant culture base, cultivate 5-7d at being put in 26-28 DEG C, mycelium covers with inclined-plane, and micro-band white, growth is vigorous.
3, triangular flask shake-flask culture: slant strains moved in the triangular flask containing 200ml liquid nutrient medium, adopt shaking speed 80-100r/min, stroke 7-8cm back and forth shakes, 26-28 DEG C of shaking culture 2-3d, covers with the mycelia of white, makes seed suspension liquid.
4, submerged fermentation culture medium is prepared.By following weight percent obtaining liq substratum: submerged fermentation culture medium (by weight percentage): ginseng powder 2.0%, bee pollen 2.0%, Ganoderma spore powder 1.0%, Poria powder 1.0%, soyflour 2.0%, yeast powder 0.5%, glucose 3.0%, magnesium sulfate 0.2%, dipotassium hydrogen phosphate 0.3%, sodium-chlor 0.2%, VITMAIN B1 0.1%, Camellia oil 0.3%, surplus: water, pH:6.5, fineness of powder: 80-100 order.(bee pollen selects Flos Camelliae Japonicae pollen)
By above-mentioned weight percent, get ginseng powder 6000g, bee pollen 6000g, Ganoderma spore powder 3000g, Poria powder 3000g, soyflour 6000g, yeast powder 1500g, glucose 9000g, magnesium sulfate 600g, dipotassium hydrogen phosphate 900g, sodium-chlor 600g, VITMAIN B1 300g, Camellia oil 900g, surplus: water, pH:6.5, fineness of powder: 80-100 order.Obtaining liq substratum.Substratum is sterilizing 30min under 120-125 DEG C of condition, for subsequent use after cooling.(bee pollen selects Flos Camelliae Japonicae pollen)
5, seeding tank seed enlarged culturing (one grade fermemtation): seeding tank adopts the stainless cylinder of steel of 500L, and feed intake 300L.Seed suspension liquid is moved into seeding tank, carries out seed enlarged culturing, by kind of amount 17.5%, a temperature 27-30 DEG C, illumination condition: continuous darkness condition, stirring velocity 160-180r/min, sterile air air flow is per minute 1:0.4m
3v/v, min, tank pressure 0.04MPa, fermentation culture 56h; PH is down to 4.7, and seeding tank spawn culture is ripe.
6, fermentor tank Submerged fermentation (second order fermentation): fermentor tank adopts the stainless cylinder of steel of 5000L, and feed intake 3000L.By kind of amount 27.5%, a temperature 27-30 DEG C, illumination condition: continuous darkness condition, stirring velocity 160-180r/min, sterile air air flow is per minute 1:0.4m
3v/v, min, tank pressure 0.04MPa, fermentation culture 60h; PH is down to 3.3, and residual sugar amount is down to 0.25%, fermentation ends, forms submerged fermentation mycelia liquid.
7, enzymolysis: by weight percentage, adds compound protease 0.5%, amylase 0.75%, cellulase 0.75%, and proteolytic enzyme is 2 × 10
5u/kg, amylase is 1.0 × 10
5u/kg, cellulase is 1.5 × 10
5u/kg.Temperature remains on 55-60 DEG C, enzymolysis time 3.5h.
8, high-pressure homogeneous process: the fermented liquid through enzymolysis processing is first through colloidal mill homogeneous, rotating grinding rotating speed 800-1000r/min.Make the material particles disintegrations such as mycelium, reach homogeneous, then through high pressure homogenizer (40MPa-60MPa), make fermented liquid under High Pressure by 200 order membrane filtrations.
9, supercritical CO
2extraction, utilize the dissolving power of supercritical co and the relation of its density, in the supercritical state, supercritical co is contacted with material to be separated, make it selectively the boiling point height composition different with molecular size range be extracted precipitation effective active matter successively.
10, fermented liquid concentrates, and adopt dual-effect concentrator, vaporization temperature 55-70 DEG C, vapor pressure 0.25MPa, is concentrated into concentration Brix=30% by fermentation broth liquor.
11, vacuum lyophilization, adopts working vacuum degree≤130Pa, the vacuum freeze of condenser temperature≤-60 DEG C, the tunning active substance dry powder obtained.
12, obtain tunning active substance dry powder meter 106.5kg through measuration, yield is 35.5g/L.(dry powder observed watercut is 5.0%).
By above-mentioned implementation method and proportioning duplication of production again, embodiment 2 and embodiment 3:
Embodiment 2, obtain tunning active substance dry powder meter 108.5kg through measuration, yield is 36.2g/L.(dry powder observed watercut is 5.0%).
Embodiment 3, obtain tunning active substance dry powder meter 107.8kg through measuration, yield is 35.9g/L.(dry powder observed watercut is 5.0%).
Table 1 Ganderma lucidum fermentation dry powder main component and content (g/100g dry weight)
Above-mentioned main component examination criteria and method:
1, polysaccharide body (with glucose meter), phend-sulphuric acid.
2, total triterpene (in ursolic acid), ultraviolet spectrophotometry.
3, protein (in butt, N × 6.25), adopts GB5009-2010 standard detection.
4, amino acid adopts GB/T 5009.124-2003 automatic analyzer for amino acids to measure amino acid whose method mensuration in food.
5, soluble dietary fibre, adopts GB/T5009.88 standard determination method.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (3)
1. a submerged fermentation method for Ganderma lucidum, is characterized in that: comprise the following steps:
(1) 5-7d is cultivated at the inclined-plane of inoculation being put in 26-28 DEG C;
(2) will cover with mycelial inclined plane inoculating in liquid nutrient medium, in shaking speed 80-100r/min, stroke 7-8cm back and forth shakes, and 26-28 DEG C of shaking culture 2-3d, makes seed suspension liquid;
(3) seeding tank seed culture, by kind of amount 15%-20%, a temperature 27-30 DEG C, illumination condition: continuous darkness condition, stirring velocity 160-180r/min, sterile air air flow is per minute 2.5m
3, tank pressure 0.04MPa, fermentation culture 48-72h; PH is down to 4.5-5.0;
(4) ferment tank is cultivated: inoculum size 25-30%, temperature 27-30 DEG C, stirring velocity is 150-200r/min, and sterile air air flow is per minute 2m
3, tank pressure 0.05MPa, cultivates 48-72h; Fermented liquid retrogradation, thalline starts self-dissolving, and pH is down to 3.0-3.5, and residual sugar amount is down to 0.2-0.3%;
(5) enzymolysis: add compound protease 0.5%, amylase 0.75%, cellulase 0.75%; Proteolytic enzyme is 2 × 10
5u/kg, amylase is 1.0 × 10
5u/kg, cellulase is 1.5 × 10
5u/kg; Temperature 55-60 DEG C, time 3-4h.
2. the submerged fermentation method of Ganderma lucidum according to claim 1, it is characterized in that: slant medium: potato liquor 1000mL, skim-milk 30g, glucose 30g, agar 30g, peptone 30g, yeast extract paste 5g, magnesium sulfate 0.2g, VITMAIN B1 0.1g, pH nature;
Submerged fermentation culture medium, percentage: ginseng powder 2.0%, bee pollen 2.0%, Ganoderma spore powder 1.0%, Poria powder 1.0%, soyflour 2.0%, yeast powder 0.5%, glucose 3.0%, magnesium sulfate 0.2%, dipotassium hydrogen phosphate 0.3%, sodium-chlor 0.2%, VITMAIN B1 0.1%, Camellia oil 0.3%, surplus is water, pH=6-7, fineness of powder: 80-100 order, makes liquid nutrient medium.
3. the submerged fermentation method of Ganderma lucidum according to claim 1, is characterized in that: the extracting method of tunning comprises the following steps:
(1) high-pressure homogeneous process: the fermented liquid through enzymolysis processing is first through colloidal mill homogeneous, rotating grinding rotating speed 800-1000r/min, makes hyphostoma particle disintegration, reaches homogeneous, again through high pressure homogenizer, make fermented liquid under 40MPa-60MPa High Pressure by 200 order membrane filtrations;
(2) supercritical CO
2extraction: in the supercritical state, contacted by supercritical co with material to be separated, makes it selectively the boiling point height composition different with molecular size range be extracted precipitation effective active matter successively;
(3) fermented liquid concentrates: fermented liquid is concentrated into concentration Brix=28-32%;
(4) adopt vacuum lyophilization, condenser temperature≤-60 DEG C, obtain tunning ganoderan and Ganoderma triterpenoids active substance dry powder goods.
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| CN105851326A (en) * | 2016-04-27 | 2016-08-17 | 佛山市泓丞生物科技有限公司 | Bio-transformed high-selenium polysaccharide moringa oleifera leaf health preservation tea and production technology thereof |
| CN105935143A (en) * | 2016-06-29 | 2016-09-14 | 浙江蕴本生物科技有限公司 | Manufacturing method of lucid ganoderma ferment |
| CN106852496A (en) * | 2015-12-09 | 2017-06-16 | 黄建钧 | The method of the triterpenes synthesis contained by induction mushroom gill fungus |
| CN107460132A (en) * | 2017-08-31 | 2017-12-12 | 山东恒发食用菌有限公司 | A kind of method of liquid state fermentation production lucidum strain |
| CN107557306A (en) * | 2017-10-11 | 2018-01-09 | 浙江海洋大学 | A kind of bottom mud microbe with bacteriostatic activity |
| CN107950752A (en) * | 2017-12-26 | 2018-04-24 | 福建光阳蛋业股份有限公司 | A kind of lotus leaf feed for improving laying rate |
| CN111304125A (en) * | 2020-03-02 | 2020-06-19 | 广州金塞迩生物医学科技有限公司 | A strain compound for high-efficiency Chinese medicinal fermentation, its preparation method, application, and Chinese medicinal fermented product fermented by the same |
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Cited By (10)
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| CN106852496A (en) * | 2015-12-09 | 2017-06-16 | 黄建钧 | The method of the triterpenes synthesis contained by induction mushroom gill fungus |
| CN105851326A (en) * | 2016-04-27 | 2016-08-17 | 佛山市泓丞生物科技有限公司 | Bio-transformed high-selenium polysaccharide moringa oleifera leaf health preservation tea and production technology thereof |
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| CN107460132A (en) * | 2017-08-31 | 2017-12-12 | 山东恒发食用菌有限公司 | A kind of method of liquid state fermentation production lucidum strain |
| CN107557306A (en) * | 2017-10-11 | 2018-01-09 | 浙江海洋大学 | A kind of bottom mud microbe with bacteriostatic activity |
| CN107950752A (en) * | 2017-12-26 | 2018-04-24 | 福建光阳蛋业股份有限公司 | A kind of lotus leaf feed for improving laying rate |
| CN107950752B (en) * | 2017-12-26 | 2021-05-07 | 福建光阳蛋业股份有限公司 | Lotus leaf feed for improving laying rate |
| CN111304125A (en) * | 2020-03-02 | 2020-06-19 | 广州金塞迩生物医学科技有限公司 | A strain compound for high-efficiency Chinese medicinal fermentation, its preparation method, application, and Chinese medicinal fermented product fermented by the same |
| CN113303419A (en) * | 2021-06-15 | 2021-08-27 | 山东效峰生物科技股份有限公司 | Preparation process of medicinal and edible fungus mixed fermentation mycelium powder |
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