CN102816807B - Production method of grifolan manganese compound - Google Patents
Production method of grifolan manganese compound Download PDFInfo
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- CN102816807B CN102816807B CN201110150890.0A CN201110150890A CN102816807B CN 102816807 B CN102816807 B CN 102816807B CN 201110150890 A CN201110150890 A CN 201110150890A CN 102816807 B CN102816807 B CN 102816807B
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Abstract
The invention relates to a production method of a grifolan manganese compound, and relates to the food microbiological application technology field. The method comprises the steps that: rice bran and wheat bran are leached in water with a temperature of 90 DEG C to 98 DEG C for 2.5-3h under normal pressure; residuals are removed, and a juice is obtained; the juice is used for fermentation, wherein the rice bran application amount is 30-120g/L culture media, the wheat bran application amount is 30-120g/L culture media, an addition amount of manganese is 100-300mg/L culture media, an addition amount of potassium dihydrogen phosphate is 1.0-1.5g/L, an addition amount of magnesium sulfate is 0.5-0.80g/L, a pH value is natural, and an applied strain is a grifola frondosa strain obtained by mutagenesis. Mycelium obtained by liquid culturing is subjected to centrifugal separation, and is washed by using distilled water; the mycelium is dried to constant weight, such that the mycelium is obtained; the mycelium is subjected to conventional water extraction, deproteinization, ethanol precipitation, and lyophilization, such that a intracellular polysaccharide manganese compound can be obtained. With the method provided by the invention, the obtained compound is novel, and is advantaged in high yield. With the method, cheap raw materials are applied with high efficiency and low cost.
Description
Technical field
The present invention relates to food microorganisms applied technical field, relate in particular to and use a strain to be adapted at the Grifola frondosa mutagenic strain of growing on substratum that rice bran and wheat bran mixture are Carbon and nitrogen sources, the production method of fermentation high yield grifolan manganese mixture.
Background technology
Scientific research both at home and abroad and domestic market show: edible fungi polysaccharide can strengthen the diseases such as body immunity, adjuvant therapy of tumors, has become the assistant product that knubble biological therapy is selected.The domestic medicine font size product of being made by fruit body of edible fungi has the ganoderma lucidum capsule of Wuhu, Zhejiang Qingyuan's Grifola frondosa capsule etc., and the non-medicine font size healthcare products of being made by fungi extracts also have sale.New Zealand produces and to have " GanoPoly " (Gan Nuobaoli) polyoses extract series product in the world, is on sale to America, the place such as Australia, China's (comprising Hong Kong and Taiwan).The contriver Gao Yihuai of this product is international well-known edible fungus exploitation expert, once obtains einstein's invention award." GanoPoly " series product of Gao Yihuai invention mainly form by the polysaccharide of extraction such as glossy ganoderma, rainbow conk, hedgehog hydnum is compound together with chitosan, for improving immunizing power, assisting antineoplaston etc.The New Zealand's peace hair-care and health present Fujian China of product company limited Quanzhou has the production base of edible fungi polysaccharide.In the research of various edible fungi polysaccharides, existing research explanation now, the polysaccharide of medicine-food two-purpose edible mushrooms Grifola frondosa also has good raising immunizing power, the effect of auxiliary antineoplaston etc., be worth causing enough attention, Ohno Naohito for example, the test of pesticide effectiveness that Suzuki Iwao etc. extracts grifolan from Grifola frondosa thalline shows, grifolan has antitumor action and immunoregulation effect, and (reference is shown in 1. Ohno N, Lino K, Suzuki I et al. Neutral and acidic antitumor polysaccharides extracted from cultured fruit bodies of
grifola Frondosa[j]. Chem. Pharm. Bull, 1985,33 (3): 1181-1186, 2. Naohito Ohno, Yoshiyuki Adachi, Iwao Suzuki, et al.Characterization of the antiumor glucan obtained from liquid-cultured
grifola frondosa[J] .Chem.pharm.Bull.1986,34 (4): 1709, 3. Iwao Suzuki, Koichi Hashimoto, Shozo Oikawa, et al.Antiumor and immunmodulating activity of a β-glucan obtained from liquid-cultureed
grifola frondosa[J] .chem.Pharm Bull, 1986,37 (2): 410.).
Grifola frondosa (
grifola frondosa) be Basidiomycotina, Hymenomycetes, Aphyllophorales, polyporaceae, Ramalina, having another name called strange fruit bacterium, polyporus frondosus, thousand Buddhist bacterium, lotus flower bacterium etc., is a kind of rare food and medicament dual-purpose fungi, its sporophore meat, handle is short and be coralliform, outward appearance likeness in form chrysanthemum, smell delicate fragrance overflows, and meat is tender and crisp good to eat.Wild Grifola frondosa is distributed in Japan, Europe etc., in provinces and regions such as China Heilungkiang, Jilin, Guangxi, Hebei, Sichuan, Yunnan, Fujian, also has distribution.The tame Grifola frondosa in Qianxi, Hebei province, its nutrition and taste have surpassed the mushroom of " king in mushroom ", can cook into multiple delicious food.1834, in the great < < bacterium spectrum > > of day written or printed documents slope, recorded Grifola frondosa and have the pharmaceutical use of " taste sweet, flat, nontoxic, can control hemorrhoid ", and China's traditional Chinese medical science thinks that it has the effect of " strengthening the body resistance to consolidate the constitution ".The research of recent domestic shows, grifolan has the effects such as immunomodulatory, antitumor, anti HIV-1 virus and liver protecting.Effects of Extracts of Grifola frondosa on Active, as a kind of high-class healthy food, has seen the markets such as Japan, Singapore.The solid-state cultivation production cycle of traditional Grifola frondosa is long, and labour intensity is large, be vulnerable to the infringement of disease and pest, and utilization rate of raw materials is low.The sporophore that the maitake mushroom mycelia nutritive value that research discovery liquid submerged fermentation obtains and medicinal effect and solid-state cultivation obtain is suitable, and therefore, people more and more pay attention to and obtained certain achievement in research to Grifola frondosa liquid state fermentation.The reported first such as Ohno N in 1986 Grifola frondosa liquid culture situation, Suzukil etc. also successfully turns out maitake mushroom mycelia by similar method subsequently.Zhuang C in 1992 etc. develop a set of liquid culturing apparatus, can gather in the crops mycelium and polysaccharide of fermentation broth simultaneously.The research of Chen Shiliang shows, the Crude polysaccharides content in Grifola frondosa fermentation mycelium is much higher than fruitbody polysaccharide, and the exocellular polysaccharide of fermented liquid is also active polysaccharide simultaneously.These researchs relate to the exploration of excellent species seed selection and substratum and culture process etc., and main purpose is reduce production costs and improve on this basis mycelia active polysaccharide output.Most process using glucose, grain class raw material are as the main Carbon and nitrogen sources as substratum such as starch, potato, use the byproduct of processing of farm products as rice bran or wheat bran, also be to add as submember, generally also need to use in a large number glucose or other grain class raw materials.Existing Grifola frondosa bacterial classification is difficult to grow not adding on glucose or the rice bran of other grain class raw materials or the substratum of wheat bran.The this patent designer Liu Wei people etc. once carried out research to the possibility of original Grifola frondosa bacterial classification liquid fermenting product grifolan on rice bran substratum, although find bacterial strain also to carry out the processing of preliminary ultraviolet mutagenesis, if do not added relatively large glucose, Grifola frondosa is undesirable in the top fermentation of rice bran substratum, so research of the Yang Suohua instructing at Liu Weimin and the Master's thesis of Gu Huimin, still added more glucose, so only take processing of farm products byproduct as raw material production grifolan with the imagination reducing costs can not obtain real realization (reference is shown in: Yang Suohua. Grifola frondosa ferment rice bran is prepared polysaccharide [D]. master thesis. Zhenjiang: the .2006 of Jiangsu University, Gu Huimin. Grifola frondosa is liquid research [D] of cultivating product polysaccharide and enrichment organoselenium in rice bran substratum. master thesis. Zhenjiang: the .2009. of Jiangsu University).The rear Liu Wei people instruct again Master degree candidate Zhang Jian original Grifola frondosa bacterial classification to be carried out to the processing of preliminary microwave-ultraviolet complex mutation, its mutagenic strain (does not add glucose) on rice bran, bran mass, and liquid fermenting product grifolan effect is better, the more original bacterial strain of mycelia dry weight and polysaccharide improves respectively 39.24% and 42.58%, and submitted Chinese invention patent application to, the title of applying for a patent is: for the Grifola frondosa strain of rice bran and wheat bran compound material production polysaccharide, application number is 201010579078.5.Although the polysaccharide that the polysaccharide that this bacterial strain is produced is produced compared with the original bacterial strain in laboratory is greatly improved, but mutagenesis bacterial classification can be degenerated, from the angle of stablizing mutagenesis bacterial classification, enhancing productivity, constantly mutagenesis bacterial classification, obtains more much higher candy output and is still the target that needs constantly the problem of research and the innovation and creation of Geng Gao.In addition, the Grifola frondosa strain that application number is 201010579078.5 is first to carry out the enzymolysis of vegetable fibre enzyme to rice bran and wheat bran than better suited zymotechnique, provides fermentation required carbon source, but introduce enzymolysis process, can increase production cost.If mutagenesis goes out efficient Grifola frondosa strain, not in advance enzymolysis in the situation that, also can Efficient Conversion rice bran and wheat bran, will reach the effect of innovation.Applicant and the present invention have submitted another application for a patent for invention to simultaneously, exercise question is: for the bacterial strain of high-efficiency fermenting rice bran and wheat bran extracting solution production grifolan, aim at above-mentioned target, carry out the new mutagenesis of Grifola frondosa bacterial classification, in the hope of obtaining the high efficient strain of new production grifolan.But < < does not relate to the method for producing grifolan manganese mixture with obtained strains for the patent of invention of the bacterial strain > > of high-efficiency fermenting rice bran and wheat bran extracting solution production grifolan.This method is described by patent of the present invention.
Manganese is quite wide in natural distribution, and vegetable food manganese content is more.Human body is less containing manganese, become in human body approximately containing manganese 10～
20 mg, are distributed in all tissues, more with liver, bone, brain, kidney, pancreas, hypophysis intensive amount.By intestinal absorption, because its solubleness is little, therefore specific absorption is low; By enteron aisle and kidney, discharged.
Manganese is core component or the activator of plurality of enzymes, and sulfatase, kinases, thioesterase, peptase, desaturase, decarboxylase, nucleotide cyclase and glucanotransferase etc. are had to activator or cofactor effect.
Manganese has material impact to people's growth, metabolism, blood formation, incretory gland, heart, kidney and neural system.Its concrete function may have: 1. promote fats oxidn in cell, reduce liver fat and store; 2. make nervus centralis and hypophysis keep good function; 3. promote the generation of steroid sexual hormoue; 4. promote the hematopoietic function of marrow; 5. the component as pyruvate carboxylase promotes glyconeogenesis, prevents the infringement of radical pair cardiac muscle as the component of superoxide-dismutase, at anti-cancer, anti-cardiac muscle, damages and plays a role; 6. stimulate antitoxic generation, improve the resistibility to transmissible disease.
Epidemiology survey shows, in Finland and Soviet Union's Alma-Ata Soils In The Region, manganese content and Cancer Mortality are negative correlation.China's Shunde, the High Phc Incidence Area Manganese In Soils content such as Chongming, Jiangsu are all lower, and in these regional farm crop and human hair manganese level also lower than or significantly lower than Di Fa district.Guangxi Si Huixian high risky area of nasopharyngeal carcinoma, in local soil, rice, manganese content is also significantly lower than Di Fa district.In addition, Sichuan Yanting, Taihang Mountain, Shanxi, Linxian, henan Province, Deng Di Esophageal Cancer in High Risk Areas, Hebei, in drinking-water and food, except molybdenum is low, manganese is also low.The most results of study of China confirm, cancer patients's body fluid manganese content lower than or significantly lower than normal people, also have similar report abroad.On the other hand, be that once report is controlled in river show, zingiberaceous plant has anti-tumor activity to Sar-coma A, and zingiberaceous plant manganese content is all higher, seemingly has inner link between the two, is the possible factor that some herbal medicine can suppress the growth of cancer knurl.The king of institute of oncology, Henan Province delivers along auspicious grade the content that research article report is measured 41 kinds of herbal medicine trace elements, wherein 25 kinds of herbal medicine are the traditional Chinese medical science conventional prescription drugs for cancer, through modern animal pharmacological experiment, also confirming has restraining effect in various degree to cancer cells, and the anticancer effective component of some drugs separating-purifying.Result of study shows that Mn, Zn, Si, Cu content are evident as height in 41 kinds of Chinese medicines measuring, and the antitumous effect of tentative confirmation and these medicines seemingly has certain relation.
Human body manganese deficiency also may cause other a series of diseases, children, can cause cessation of growth cessation, skeleton deformity and richets, and poor appetite, weight loss, sexual dysfunction, appear in adult's manganese deficiency, and magnesium-Deficiency of Pregnant Woman can cause infertile or foetal death.
Manganese intake too much will affect the redox processes of body, thereby causes a series of manganese poisoning symptom, the chronic toxic effect that main manifestations is neural psychiatric system, and the centrum of class parkinsonism of take is outward that nervous lesion is feature.The dominant mechanism of manganese poisoning relates to nerve cell apoptosis, neurotransmitter metabolism disorder, cynapse conduction function obstacle, also relevant with Gene Susceptibility and genetic polymorphism, have as symptoms such as headache, dizziness, fatigue and weak, aphasis, clumsy in one's movement, muscle spasm, palpitaition, tachycardia, hidrosis, salivations.
Use for reference edible mushrooms inorganic selenium is converted into the idea that organoselenium reduces toxicity and improves the service efficiency of selenium, and use for reference the higher fact of zingiberaceous plant manganese content, also available edible mushrooms is converted into Organic Manganese to improve the effect of Organic Manganese by inorganic manganese.It is 3.5mg/d that < < food science > > teaching material is recommended adult's Manganese intake, and the highest intake is 10mg/d.In polysaccharide, enrichment manganese need to be restricted the addition of manganese again.Consider that the polysaccharide amount that each grownup takes in every day is 0.5g～1g, wherein contained manganese is that 3.5mg～10mg is advisable.
Grifola frondosa is that this project is except improving another innovative point polysaccharide yield at rice bran bran mass top fermentation product polysaccharide enrichment Organic Manganese.
China is that to take paddy and wheat be main Chan Liang big country, and its processed side product rice bran, wheat bran source are abundant, and cheap.The material such as rich in starch, Mierocrystalline cellulose in rice bran, and wheat bran is rich in the materials such as albumen, Mierocrystalline cellulose.Theoretically, rice bran and wheat bran are compound has possessed the grow hyle of needed Carbon and nitrogen sources of Grifola frondosa.Under the effect of Grifola frondosa self cellulase and other enzymes, Grifola frondosa can change into rice bran and wheat bran the nutritive substance of self and grow, and produces grifolan.Therefore, use the cheap agricultural byproducts such as rice bran, wheat bran, as Carbon and nitrogen sources completely, and do not add other Carbon and nitrogen sources, provide Grifola frondosa liquid fermenting required Carbon and nitrogen sources nutritive substance, produce the Grifola frondosa manganese polysaccharide with auxiliary oncotherapy, will there is economic worth.The present invention is by a kind of effective ways of accomplished this target.Special instruction: rice bran bran mass of the present invention refers in particular to only take the substratum that rice bran and wheat bran mixture be Carbon and nitrogen sources material, no longer adds other Carbon and nitrogen sources materials in substratum.Organic Manganese and edible fungi polysaccharide is compound, can work in coordination with the effect of bringing into play two class materials, be combined innovation method, the grifolan manganese mixture obtaining is new product.In order to reduce raw materials cost, efficiently utilize processing of farm products byproduct rice bran and wheat bran, obtain the protective foods of high value, be necessary that research only take the new technology that rice bran wheat bran extract is Carbon and nitrogen sources.Due to Grifola frondosa strain, at rice bran wheat bran extract, for main carbon source nitrogenous source, do not add in the substratum of other carbon source nitrogenous sources and do not grow and be restricted, so be necessary to obtain efficiently growing bacterial strain by the means of mutagenesis, and use new strain fermentation to add the rice bran bran mass of manganese, produce grifolan manganese mixture as novel healthy food, thereby form new innovation and creation.
Realize one of the key issue of above-mentioned imagination for must obtain suitable Grifola frondosa strain of growing on rice bran and wheat bran complex medium, so must constantly carry out mutagenesis screening to existing bacterial strain, because existing Grifola frondosa strain is grown and is still wished to be greatly improved on rice bran bran mass, if filter out suitable Grifola frondosa strain, study and produce and will likely make a breakthrough.Another patent of invention of simultaneously submitting to the present invention, by the method for protoplastis mutation induced by laser, be take high polysaccharide as index, and directed Efficient Conversion rice bran wheat bran extracting solution be basic, and seed selection obtains high yield, Grifola frondosa strain cheaply.Key issue of the present invention is: utilize the resulting new mutagenic strain of another patent of invention of simultaneously submitting to the present invention, adding on the rice bran wheat bran complex medium of not using plant cellulose enzymolysis of manganese, fermentative production grifolan manganese mixture, obtains new grifolan manganese mixture and new production method thereof thus.Because dissociant is new bacterial strain, on produced polysaccharide material, change to some extent, the grifolan manganese of gained is a kind of new grifolan manganese mixture.New production method must be supported by new technique, the mutagenic strain that uses the present invention to adopt, on the rice bran wheat bran manganese addition complex medium without vegetable fibre enzymolysis, ferment, the technique of high yield grifolan manganese mixture is brand-new technique, and this research has no open report.So, the present invention adopting new mutagenic strain, using and aspect four of substratum, new grifolan manganese mixture, new grifolan manganese mixture production technique, there is novelty without the rice bran wheat bran manganese addition mixture of plant cellulose enzymolysis, established the basis of patent of the present invention.
Summary of the invention
The present invention by innovation a kind of new grifolan manganese mixture and production technology.In order to reduce production costs, the byproduct rice bran of the processing such as the consideration large agricultural-food paddy of employing and wheat and wheat bran are as the Carbon and nitrogen sources of substratum, no longer add other carbon sources as glucose and nitrogenous source, add manganous sulfate, carry out liquid fermenting, with the cost that economizes in raw materials, minimizing, use soil and reduce the production cycle, improving added value of product, but this needs mutagenesis screening to go out Grifola frondosa strain and the suitable producing and manufacturing technique of selection of suitable growth on rice bran wheat bran complex medium.Therefore, for achieving the above object, need to be outside natural seed selection, by animal nutrition, Grifola frondosa strain is carried out to mutagenesis, filter out suitable on rice bran bran mass the Grifola frondosa strain of high polysaccharide, study this mutagenic strain and produce the suitable fermentation technology of grifolan manganese mixture, to obtain grifolan manganese mixture, as protective foods.
The technical solution used in the present invention is as follows: the Grifola frondosa strain that utilizes new mutagenesis to obtain, it is Carbon and nitrogen sources that fermention medium only be take rice bran and wheat bran compound material, but need to add manganous sulfate, produce grifolan manganese mixture, according to following step, carry out: under (1) rice bran, wheat bran normal pressure, after 90-98 ℃ of flooding 2.5-3h, remove slag and get juice; (2) will obtain treatment solution as substratum for fermentation, according to rice bran usage quantity, it is 30-120 g/L substratum, the usage quantity of wheat bran is 30-120 g/L substratum, the addition of manganese (with the manganous sulfate conversion of adding) is 100-300mg/L substratum, add potassium primary phosphate 1.0-1.5g/L substratum, magnesium sulfate 0.5-0.80 g/L substratum, pH nature, bacterial strain uses therefor is the bacterial strain that new mutagenesis obtains; (3) by the mycelium of liquid culture gained after centrifugation, with distilled water wash, be dried to constant weight and obtain mycelium; (4) above-mentioned mycelium is got final product to obtain to intracellular polyse manganese mixture after conventional water extraction, deproteinated, ethanol precipitation and lyophilize.
The new bacterial strain of Grifola frondosa mutagenesis using in step in the present invention (2)
grifolasp. JSU 10-2 was deposited in the Chinese Typical Representative culture collection center (CCTCC) of the Wuhan University that is positioned at Wuhan, China on April 7th, 2011, and preservation strain is numbered CCTCC M2011113, and name is called
grifolasp. JSU 10-2.
In step in the present invention (2), the additive of manganese adopts manganous sulfate, and addition is converted by desired manganese amount.
Sample-loading amount while adopting shake flask fermentation in step in the present invention (2) is 40% of shaking flask volume, and inoculum size is 10% of dress sample volume, 26 ℃-28 ℃ of culture temperature, and incubation time shaking flask is 7-9d, rotating speed 150-180r/min.
While adopting upper tank fermentation in step in the present invention (2), sample-loading amount is 80% of fermentor tank volume, and during upper tank, ventilation is tinning fermentating liquid volume/min, 26 ℃-28 ℃ of culture temperature, stir speed (S.S.) 150-180r/min, upper tank fermentation 4-6 days.
beneficial effect of the present invention
The Grifola frondosa mutagenic strain of announcing in another patent of invention that the present invention adopts contriver to apply for simultaneously
grifolasp. JSU 10-2, do not add other Carbon and nitrogen sources without the rice bran wheat bran complex medium of vegetable fibre enzyme enzymolysis on cultivate to produce grifolan manganese mixture, compare Grifola frondosa Crude polysaccharides output with original strain higher, mycelia dry weight and mycelia polysaccharide have improved respectively 31.7% and 32.6%.Four kinds of innovations are merged in the present invention: (1) does not add the rice bran wheat bran complex medium technology without plant cellulose enzyme enzymolysis of other Carbon and nitrogen sources; (2) the new acquisition of employing is not adding the mutagenic strain that on the rice bran wheat bran complex medium of other Carbon and nitrogen sources, the speed of growth is faster, polysaccharide yield is higher
grifolasp. JSU 10-2 is as fermentation strain; (3) mutagenic strain
grifolasp. JSU 10-2 grows on without plant cellulose enzyme enzymolysis rice bran wheat bran complex medium, and produces the processing method of grifolan manganese; (4) new Grifola frondosa Crude polysaccharides manganese mixture.These four kinds of innovative combination make the present invention have obvious beneficial effect, can reduce production costs, economize in raw materials cost, minimizing use soil and reduction production cycle, produce the new grifolan manganese mixture protective foods of the auxiliary antineoplaston effect of having of high value.The present invention has used through protoplastis mutation induced by laser method mutagenesis the new bacterial strain of Grifola frondosa that filters out, has the mutant strain of high growth rates and high polysaccharide yield
grifolasp. JSU 10-2, obtained strains is adapted at without Fast Growth on the rice bran of vegetable fibre enzyme enzymolysis and wheat bran complex medium high yield grifolan manganese mixture.Grifolan has the value of auxiliary antineoplaston, makes Grifola frondosa become a kind of important medicinal fungi, but grifolan scale operation still high in the face of cost, the series of problems such as yield poorly.Based on this, the technical problem that the present invention solves is: provide do not add other Carbon and nitrogen sources without the rice bran wheat bran complex medium of vegetable fibre enzyme enzymolysis on the speed of growth is faster, polysaccharide yield is higher Grifola frondosa mutagenic strain
grifolasp. the production method of JSU 10-2 fermentative production grifolan manganese mixture.Described method, adopts Grifola frondosa mutagenic strain
grifolasp. JSU 10-2, on the rice bran wheat bran manganese addition complex medium without vegetable fibre enzyme enzymolysis, fermentative production grifolan manganese mixture, the method has high efficiency, low cost and has used cheap raw material, grifolan manganese mixture to be suitable as the beneficial effect that protective foods is used.
Embodiment
The invention provides a kind of Grifola frondosa strain
grifolasp. JSU 10-2( applies for a patent separately simultaneously), can with respect to original Grifola frondosa strain, there is high growth rates and high polysaccharide yield not adding on the rice bran wheat bran complex medium without plant cellulose enzyme enzymolysis of other Carbon and nitrogen sources; This Grifola frondosa strain
grifolasp. JSU 10-2 was deposited in the Chinese Typical Representative culture collection center (CCTCC) of the Wuhan University that is positioned at Wuhan, China on April 7th, 2011, and preservation strain is numbered CCTCC M2011113, and name is called
grifolasp. JSU 10-2.The invention provides the bacterial strain that adopts mutagenesis to obtain
grifolasp. JSU 10-2 produces grifolan manganese mixture as the production method of protective foods.
In embodiment, rice bran and wheat bran are carried out to 95 ℃ of left and right lixiviate 3h and process, obtain treatment solution as substratum for fermentation.
embodiment mono-
Grifola frondosa strain adopts mutagenesis to obtain
grifolasp. JSU 10-2 bacterial strain.Rice bran and wheat bran are carried out to 90 ℃ of lixiviate 2.5h and process, obtain treatment solution as substratum for fermentation, rice bran usage quantity is 30g/L substratum, the usage quantity of wheat bran is 30g/L substratum, the addition of manganese is 100mg/L substratum, adds potassium primary phosphate 1.0g/L, magnesium sulfate 0.5g/L, pH nature, the sample-loading amount of shaking flask is 40% volume, inoculum size 10%, 26 ℃ of culture temperature, rotating speed 150r/min, incubation time 7d.The mycelium of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 ℃ of conditions, is dried to constant weight, through weighing and obtain mycelia dry weight.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 80 ℃ of water-baths, 3000r/min centrifuging and taking supernatant liquor, after Sevage method deproteinated, then with 95% ethanolic soln alcohol precipitation extraction intracellular polyse of 3 times of volumes.Alcohol precipitation 24h in the refrigerator of 4 ℃, through centrifugal, lyophilize, measures mycelia intracellular polyse content by phenolsulfuric acid method.Obtaining mycelia dry weight is 3.52g/L nutrient solution, and mycelia intracellular polyse is 0.341g/L nutrient solution.The mycelia and the mycelia intracellular polyse powder that extract after polysaccharide oven dry are respectively taken to 0.500g micro-wave digestion respectively, adopt sodium periodate spectrophotometry manganese content, obtain original mycelia containing being respectively the dry mycelia of 2.34mg/g and the thick mycelia polysaccharide of 16.13mg/g containing Organic Manganese in Organic Manganese and mycelia polysaccharide extract, wherein said micro-wave digestion programming is as shown in subordinate list 1.This kind of mycelia polysaccharide adult usage quantity is for each person every day no more than 0.5g, can control adult's manganese amount that polysaccharide manganese mixture is taken in thus for each person every day and be no more than 9mg.
embodiment bis-
Grifola frondosa strain adopts mutagenesis to obtain
grifolasp. JSU 10-2 bacterial strain.Rice bran and wheat bran are carried out to 98 ℃ of lixiviate 3h and process, obtain treatment solution as substratum for fermentation, rice bran usage quantity is 120g/L substratum, the usage quantity of wheat bran is 120g/L substratum, the addition of manganese is 300mg/L substratum, adds potassium primary phosphate 1.5g/L, magnesium sulfate 0.8g/L, pH nature, the sample-loading amount of shaking flask is 40% volume, inoculum size 10%, 28 ℃ of culture temperature, rotating speed 180r/min, incubation time 9d.The mycelium of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 ℃ of conditions, is dried to constant weight, through weighing and obtain mycelia dry weight.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 80 ℃ of water-baths, 3000r/min centrifuging and taking supernatant liquor, after Sevage method deproteinated, then with 95% ethanolic soln alcohol precipitation extraction intracellular polyse of 3 times of volumes.Alcohol precipitation 24h in the refrigerator of 4 ℃, through centrifugal, lyophilize, measures polysaccharide content by phenolsulfuric acid method.Obtaining mycelia dry weight is 13.72g/L, and mycelia intracellular polyse is 1.43 g/L nutrient solutions.The mycelia and the mycelia intracellular polyse powder that extract after polysaccharide oven dry are respectively taken to 0.500g micro-wave digestion respectively, adopt sodium periodate spectrophotometry manganese content, obtain original mycelia containing being respectively the dry mycelia of 0.79mg/g and the thick mycelia polysaccharide of 5.24mg/g containing Organic Manganese in Organic Manganese and mycelia polysaccharide extract.Wherein said micro-wave digestion programming is as shown in subordinate list 1.This kind of mycelia polysaccharide adult usage quantity is for each person every day no more than 1g, can control adult's manganese amount that polysaccharide manganese mixture is taken in thus for each person every day and be no more than 6mg.
embodiment tri-
Grifola frondosa strain adopts mutagenesis to obtain
grifolasp. JSU 10-2 bacterial strain.Rice bran and wheat bran are carried out to 97 ℃ of lixiviate 3h and process, obtain treatment solution as substratum for fermentation, rice bran usage quantity is 120g/L substratum, the usage quantity of wheat bran is 50g/L substratum, the addition of manganese is 150mg/L substratum, adds potassium primary phosphate 1.4g/L, magnesium sulfate 0.75 g/L, pH nature, the sample-loading amount of shaking flask is 40% volume, inoculum size 10%, 27 ℃ of culture temperature, rotating speed 160r/min, incubation time 8d.The mycelium of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 ℃ of conditions, is dried to constant weight, through weighing and obtain mycelia dry weight.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 80 ℃ of water-baths, 3000r/min centrifuging and taking supernatant liquor, after Sevage method deproteinated, then with 95% ethanolic soln alcohol precipitation extraction intracellular polyse of 3 times of volumes.Alcohol precipitation 24h in the refrigerator of 4 ℃, through centrifugal, lyophilize, measures polysaccharide content by phenolsulfuric acid method.Obtaining mycelia dry weight is 9.38g/L, and in mycelia born of the same parents, intracellular polyse is 0.88g/L nutrient solution.The mycelia and the mycelia intracellular polyse powder that extract after polysaccharide oven dry are respectively taken to 0.500g micro-wave digestion respectively, adopt sodium periodate spectrophotometry manganese content, obtain original mycelia containing being respectively the dry mycelia of 0.97mg/g and the thick mycelia polysaccharide of 7.16 mg/g containing Organic Manganese in Organic Manganese and mycelia polysaccharide extract, wherein said micro-wave digestion programming is as shown in subordinate list 1.This kind of mycelia polysaccharide adult usage quantity is for each person every day no more than 1g, can control adult's manganese amount that polysaccharide manganese mixture is taken in thus for each person every day and be no more than 8mg.
embodiment tetra-
Grifola frondosa strain adopts mutagenesis to obtain
grifolasp. JSU 10-2 bacterial strain.Rice bran and wheat bran are carried out to 93 ℃ of lixiviate 2.9h and process, obtain treatment solution as substratum for fermentation, rice bran usage quantity is 50g/L substratum, the usage quantity of wheat bran is 120g/L substratum, the addition of manganese is 250mg/L substratum, adds potassium primary phosphate 1.2g/L substratum, magnesium sulfate 0.7g/L substratum, pH nature, the sample-loading amount of shaking flask is 40% volume, inoculum size 10%, 26 ℃ of culture temperature, rotating speed 170r/min, incubation time 9d.The mycelium of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 ℃ of conditions, is dried to constant weight, through weighing and obtain mycelia dry weight.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 80 ℃ of water-baths, 3000r/min centrifuging and taking supernatant liquor, after Sevage method deproteinated, then with 95% ethanolic soln alcohol precipitation extraction intracellular polyse of 3 times of volumes.Alcohol precipitation 24h in the refrigerator of 4 ℃, through centrifugal, lyophilize, measures polysaccharide content by phenolsulfuric acid method.Obtaining mycelia dry weight is 7.82g/L nutrient solution, and in mycelia born of the same parents, intracellular polyse is 0.91 g/L nutrient solution.The mycelia and the mycelia intracellular polyse powder that extract after polysaccharide oven dry are respectively taken to 0.500g micro-wave digestion respectively, adopt sodium periodate spectrophotometry manganese content, obtain original mycelia containing being respectively the dry mycelia of 1.17mg/g and 7.42 mg/g Crude polysaccharides containing Organic Manganese in Organic Manganese and mycelia polysaccharide extract.Wherein said micro-wave digestion programming is as shown in subordinate list 1.This kind of mycelia polysaccharide adult usage quantity is for each person every day no more than 1g, can control adult's manganese amount that polysaccharide manganese mixture is taken in thus for each person every day and be no more than 8mg.
embodiment five
Grifola frondosa strain adopts mutagenesis to obtain
grifolasp. JSU 10-2 bacterial strain.Rice bran and wheat bran are carried out to 95 ℃ of lixiviate 2.6h and process, obtain treatment solution as substratum for fermentation, the usage quantity of rice bran is 80g/L substratum, the usage quantity of wheat bran is 90g/L substratum, the addition of manganese is 270mg/L substratum, adds potassium primary phosphate 1.2g/L substratum, magnesium sulfate 0.5 g/L substratum, pH nature, the sample-loading amount of shaking flask is 40% volume, inoculum size 10%, 26 ℃ of culture temperature, rotating speed 180r/min, incubation time 7d.The mycelium of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 ℃ of conditions, is dried to constant weight, through weighing and obtain mycelia dry weight.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 80 ℃ of water-baths, 3000r/min centrifuging and taking supernatant liquor, after Sevage method deproteinated, then with 95% ethanolic soln alcohol precipitation extraction intracellular polyse of 3 times of volumes.Alcohol precipitation 24h in the refrigerator of 4 ℃, through centrifugal, lyophilize, measures polysaccharide content by phenolsulfuric acid method.Obtaining mycelia dry weight is 10.87g/L nutrient solution, and intracellular polyse is 1.13g/L nutrient solution.The mycelia and the mycelia intracellular polyse powder that extract after polysaccharide oven dry are respectively taken to 0.500g micro-wave digestion respectively, adopt sodium periodate spectrophotometry manganese content, obtain original mycelia containing being respectively the dry mycelia of 1.00mg/g and 6.45mg/g Crude polysaccharides containing Organic Manganese in Organic Manganese and mycelia polysaccharide extract.Wherein said micro-wave digestion programming is as shown in subordinate list 1.This kind of mycelia polysaccharide adult usage quantity is for each person every day no more than 1g, can control adult's manganese amount that polysaccharide manganese mixture is taken in thus for each person every day and be no more than 7mg.
embodiment six
Grifola frondosa strain adopts mutagenesis to obtain
grifolasp. JSU 10-2 bacterial strain.Rice bran and wheat bran are carried out to 94 ℃ of lixiviate 3h and process, obtain treatment solution as substratum for fermentation, the usage quantity of rice bran is 70g/L substratum, the usage quantity of wheat bran is 80g/L substratum, the addition of manganese is 220mg/L substratum, adds potassium primary phosphate 1.2g/L substratum, magnesium sulfate 0.6g/L substratum, pH nature, the sample-loading amount of shaking flask is 40% volume, inoculum size 10%, 28 ℃ of culture temperature, rotating speed 150r/min, incubation time 8d.The mycelium of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 ℃ of conditions, is dried to constant weight, through weighing and obtain mycelia dry weight.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 80 ℃ of water-baths, 3000r/min centrifuging and taking supernatant liquor, after Sevage method deproteinated, then with 95% ethanolic soln alcohol precipitation extraction intracellular polyse of 3 times of volumes.Alcohol precipitation 24h in the refrigerator of 4 ℃, through centrifugal, lyophilize, measures polysaccharide content by phenolsulfuric acid method.Obtaining mycelia dry weight is 8.71g/L nutrient solution, and intracellular polyse is 0.87g/L nutrient solution.The mycelia and the mycelia intracellular polyse powder that extract after polysaccharide oven dry are respectively taken to 0.500g micro-wave digestion respectively, adopt sodium periodate spectrophotometry manganese content, obtain original mycelia containing being respectively the dry mycelia of 1.17mg/g and 7.59mg/g Crude polysaccharides containing Organic Manganese in Organic Manganese and mycelia polysaccharide extract.Wherein said micro-wave digestion programming is as shown in subordinate list 1.This kind of mycelia polysaccharide adult usage quantity is for each person every day no more than 1g, can control adult's manganese amount that polysaccharide manganese mixture is taken in thus for each person every day and be no more than 8mg.
embodiment seven
Grifola frondosa strain adopts mutagenesis to obtain
grifolasp. JSU 10-2 bacterial strain.Rice bran and wheat bran are carried out to 95 ℃ of lixiviate 2.9h and process, obtain treatment solution as substratum for fermentation, the usage quantity of rice bran is 40g/L substratum, the usage quantity of wheat bran is 40g/L substratum, the addition of manganese is 120mg/L substratum, adds potassium primary phosphate 1.4g/L substratum, magnesium sulfate 0.6g/L substratum, pH nature, the sample-loading amount of shaking flask is 40% volume, inoculum size 10%, 26 ℃ of culture temperature, rotating speed 170r/min, incubation time 7d.The mycelium of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 ℃ of conditions, is dried to constant weight, through weighing and obtain mycelia dry weight.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 80 ℃ of water-baths, 3000r/min centrifuging and taking supernatant liquor, after Sevage method deproteinated, then with 95% ethanolic soln alcohol precipitation extraction intracellular polyse of 3 times of volumes.Alcohol precipitation 24h in the refrigerator of 4 ℃, through centrifugal, lyophilize, measures polysaccharide content by phenolsulfuric acid method.Obtaining mycelia dry weight is 8.53g/L nutrient solution, and intracellular polyse is 0.25g/L nutrient solution.The mycelia and the mycelia intracellular polyse powder that extract after polysaccharide oven dry are respectively taken to 0.500g micro-wave digestion respectively, adopt sodium periodate spectrophotometry manganese content, obtain original mycelia containing being respectively the dry mycelia of 0.63mg/g and 14.4mg/g Crude polysaccharides containing Organic Manganese in Organic Manganese and mycelia polysaccharide extract.Wherein said micro-wave digestion programming is as shown in subordinate list 1.This kind of mycelia polysaccharide adult usage quantity is for each person every day no more than 0.5g, can control adult's manganese amount that polysaccharide manganese mixture is taken in thus for each person every day and be no more than 8mg.
embodiment eight
Grifola frondosa strain adopts mutagenesis to obtain
grifolasp. JSU 10-2 bacterial strain.Rice bran and wheat bran are carried out to 94 ℃ of lixiviate 3h and process, obtain treatment solution as substratum for fermentation, the usage quantity of rice bran is 110g/L substratum, the usage quantity of wheat bran is 60g/L substratum, the addition of manganese is 180mg/L substratum, adds potassium primary phosphate 1.4g/L substratum, magnesium sulfate 0.8g/L substratum, pH nature, the sample-loading amount of shaking flask is 40% volume, inoculum size 10%, 26 ℃ of culture temperature, rotating speed 170r/min, incubation time 7d.The mycelium of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 ℃ of conditions, is dried to constant weight, through weighing and obtain mycelia dry weight.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 80 ℃ of water-baths, 3000r/min centrifuging and taking supernatant liquor, after Sevage method deproteinated, then with 95% ethanolic soln alcohol precipitation extraction intracellular polyse of 3 times of volumes.Alcohol precipitation 24h in the refrigerator of 4 ℃, through centrifugal, lyophilize, measures polysaccharide content by phenolsulfuric acid method.Obtaining mycelia dry weight is 8.59g/L nutrient solution, and intracellular polyse is 0.86g/L nutrient solution.The mycelia and the mycelia intracellular polyse powder that extract after polysaccharide oven dry are respectively taken to 0.500g micro-wave digestion respectively, adopt sodium periodate spectrophotometry manganese content, obtain original mycelia containing being respectively the dry mycelia of 1.04mg/g and 6.70mg/g Crude polysaccharides containing Organic Manganese in Organic Manganese and mycelia polysaccharide extract.Wherein said micro-wave digestion programming is as shown in subordinate list 1.This kind of mycelia polysaccharide adult usage quantity is for each person every day no more than 1g, can control adult's manganese amount that polysaccharide manganese mixture is taken in thus for each person every day and be no more than 7mg.
embodiment nine
Grifola frondosa strain adopts mutagenesis to obtain
grifolasp. JSU 10-2 bacterial strain.Rice bran and wheat bran are carried out to 95 ℃ of lixiviate 3h and process, obtain treatment solution as substratum for fermentation, the usage quantity of rice bran is 100g/L substratum, the usage quantity of wheat bran is 110g/L substratum, the addition of manganese is 100mg/L substratum, adds potassium primary phosphate 1.5g/L substratum, magnesium sulfate 0.6g/L substratum, pH nature, the sample-loading amount of shaking flask is 40% volume, inoculum size 10%, 27 ℃ of culture temperature, rotating speed 160r/min, incubation time 8d.The mycelium of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 ℃ of conditions, is dried to constant weight, through weighing and obtain mycelia dry weight.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 80 ℃ of water-baths, 3000r/min centrifuging and taking supernatant liquor, after Sevage method deproteinated, then with 95% ethanolic soln alcohol precipitation extraction intracellular polyse of 3 times of volumes.Alcohol precipitation 24h in the refrigerator of 4 ℃, through centrifugal, lyophilize, measures polysaccharide content by phenolsulfuric acid method.Obtaining mycelia dry weight is 10.01g/L nutrient solution, and intracellular polyse is 1.18g/L nutrient solution.The mycelia and the mycelia intracellular polyse powder that extract after polysaccharide oven dry are respectively taken to 0.500g micro-wave digestion respectively, adopt sodium periodate spectrophotometry manganese content, obtain original mycelia containing being respectively the dry mycelia of 0.77mg/g and 4.66mg/g Crude polysaccharides containing Organic Manganese in Organic Manganese and mycelia polysaccharide extract.Wherein said micro-wave digestion programming is as shown in subordinate list 1.This kind of mycelia polysaccharide adult usage quantity is for each person every day no more than 1g, can control adult's manganese amount that polysaccharide manganese mixture is taken in thus for each person every day and be no more than 5mg.
embodiment ten
Grifola frondosa strain adopts mutagenesis to obtain
grifolasp. JSU 10-2 bacterial strain.Rice bran and wheat bran are carried out to 98 ℃ of lixiviate 3h processing, obtain treatment solution as substratum for fermentation, the usage quantity of rice bran is 120g/L substratum, the usage quantity of wheat bran is 120g/L substratum, the addition of manganese is 300mg/L substratum, add potassium primary phosphate 1.5g/L substratum, magnesium sulfate 0.8g/L substratum, pH nature, sample-loading amount is 80% of fermentor tank volume, culture temperature is 28 ℃, ventilation is tinning fermentating liquid volume/min, stirring velocity 180r/min, tank gauge pressure 0.05MPa, inoculum size 10%, incubation time 6d.The mycelium of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 ℃ of conditions, is dried to constant weight, through weighing and obtain mycelia dry weight.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 80 ℃ of water-baths, 3000r/min centrifuging and taking supernatant liquor, after Sevage method deproteinated, then with 95% ethanolic soln alcohol precipitation extraction intracellular polyse of 3 times of volumes.Alcohol precipitation 24h in the refrigerator of 4 ℃, through centrifugal, lyophilize, measures polysaccharide content by phenolsulfuric acid method.Obtaining mycelia dry weight is 15.53g/L nutrient solution, and intracellular polyse is 1.50g/L nutrient solution.The mycelia and the mycelia intracellular polyse powder that extract after polysaccharide oven dry are respectively taken to 0.500g micro-wave digestion respectively, adopt sodium periodate spectrophotometry manganese content, obtain original mycelia containing being respectively the dry mycelia of 0.69mg/g and 4.92mg/g Crude polysaccharides containing Organic Manganese in Organic Manganese and mycelia polysaccharide extract.Wherein said micro-wave digestion programming is as shown in subordinate list 1.This kind of mycelia polysaccharide adult usage quantity is for each person every day no more than 1g, can control adult's manganese amount that polysaccharide manganese mixture is taken in thus for each person every day and be no more than 5mg.
embodiment 11
Grifola frondosa strain adopts mutagenesis to obtain
grifolasp. JSU 10-2 bacterial strain.Rice bran and wheat bran are carried out to 90 ℃ of lixiviate 2.5h processing, obtain treatment solution as substratum for fermentation, the usage quantity of rice bran is 30g/L substratum, the usage quantity of wheat bran is 30g/L substratum, the addition of manganese is 100mg/L substratum, add potassium primary phosphate 1.0g/L substratum, magnesium sulfate 0.5g/L substratum, pH nature, sample-loading amount is 80% of fermentor tank volume, culture temperature is 26 ℃, ventilation is tinning fermentating liquid volume/min, stirring velocity 150r/min, tank gauge pressure 0.05MPa, inoculum size 10%, incubation time 4d.The mycelium of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 ℃ of conditions, is dried to constant weight, through weighing and obtain mycelia dry weight.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 80 ℃ of water-baths, 3000r/min centrifuging and taking supernatant liquor, after Sevage method deproteinated, then with 95% ethanolic soln alcohol precipitation extraction intracellular polyse of 3 times of volumes.Alcohol precipitation 24h in the refrigerator of 4 ℃, through centrifugal, lyophilize, measures polysaccharide content by phenolsulfuric acid method.Obtaining mycelia dry weight is 3.64g/L nutrient solution, and intracellular polyse is 0.37g/L nutrient solution.The mycelia and the mycelia intracellular polyse powder that extract after polysaccharide oven dry are respectively taken to 0.500g micro-wave digestion respectively, adopt sodium periodate spectrophotometry manganese content, obtain original mycelia containing being respectively the dry mycelia of 2.34mg/g and 14.86mg/g Crude polysaccharides containing Organic Manganese in Organic Manganese and mycelia polysaccharide extract.Wherein said micro-wave digestion programming is as shown in subordinate list 1.This kind of mycelia polysaccharide adult usage quantity is for each person every day no more than 0.5g, can control adult's manganese amount that polysaccharide manganese mixture is taken in thus for each person every day and be no more than 8mg.
embodiment 12
Grifola frondosa strain adopts mutagenesis to obtain
grifolasp. JSU 10-2 bacterial strain.Rice bran and wheat bran are carried out to 95 ℃ of lixiviate 3h processing, obtain treatment solution as substratum for fermentation, the usage quantity of rice bran is 70g/L substratum, the usage quantity of wheat bran is 80g/L substratum, the addition of manganese is 200mg/L substratum, add potassium primary phosphate 1.3g/L substratum, magnesium sulfate 0.7g/L substratum, pH nature, sample-loading amount is 80% of fermentor tank volume, culture temperature is 27 ℃, ventilation is tinning fermentating liquid volume/min, stirring velocity 170r/min, tank gauge pressure 0.05MPa, inoculum size 10%, incubation time 5d.The mycelium of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 ℃ of conditions, is dried to constant weight, through weighing and obtain mycelia dry weight.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 80 ℃ of water-baths, 3000r/min centrifuging and taking supernatant liquor, after Sevage method deproteinated, then with 95% ethanolic soln alcohol precipitation extraction intracellular polyse of 3 times of volumes.Alcohol precipitation 24h in the refrigerator of 4 ℃, through centrifugal, lyophilize, measures polysaccharide content by phenolsulfuric acid method.Obtaining mycelia dry weight is 8.77g/L nutrient solution, and intracellular polyse is 0.94g/L nutrient solution.The mycelia and the mycelia intracellular polyse powder that extract after polysaccharide oven dry are respectively taken to 0.500g micro-wave digestion respectively, adopt sodium periodate spectrophotometry manganese content, obtain original mycelia containing being respectively the dry mycelia of 1.09mg/g and 6.38mg/g Crude polysaccharides containing Organic Manganese in Organic Manganese and mycelia polysaccharide extract.Wherein said micro-wave digestion programming is as shown in subordinate list 1.This kind of mycelia polysaccharide adult usage quantity is for each person every day no more than 1g, can control adult's manganese amount that polysaccharide manganese mixture is taken in thus for each person every day and be no more than 7mg.
Claims (4)
1. a production method for grifolan manganese mixture, is characterized in that carrying out according to following step: under (1) rice bran, wheat bran normal pressure, after 90-98 ℃ of flooding 2.5-3h, remove slag and get juice; (2) will obtain treatment solution as substratum for fermentation, rice bran usage quantity is 30-120 g/L substratum, the usage quantity of wheat bran is 30-120 g/L substratum, the addition of manganese is 100-300mg/L substratum, add potassium primary phosphate 1.0-1.5g/L substratum, magnesium sulfate 0.50-0.80 g/L substratum, pH nature, bacterial strain uses therefor is the Grifola frondosa strain that new mutagenesis obtains; (3) by the mycelium of liquid culture gained after centrifugation, with distilled water wash, be dried to constant weight and obtain mycelium; (4) above-mentioned mycelium is got final product to obtain to intracellular polyse manganese mixture after conventional water extraction, deproteinated, ethanol precipitation and lyophilize;
Wherein bacterial strain uses therefor be Grifola frondosa strain (
grifolasp.) JSU 10-2, preservation strain is numbered CCTCC NO:M2011113.
2. the production method of a kind of grifolan manganese mixture according to claim 1, is characterized in that described in step (2), manganese adds in substratum with the form of manganous sulfate, and addition is converted by desired manganese amount.
3. the production method of a kind of grifolan manganese mixture according to claim 1, it is characterized in that fermentation described in step (2) is for shake flask fermentation, sample-loading amount is 40% of shaking flask volume, inoculum size is 10% of dress sample volume, the shake-flask culture time is 7-9d, 26 ℃-28 ℃ of culture temperature, rotating speed 150-180r/min.
4. the production method of a kind of grifolan manganese mixture according to claim 1, it is characterized in that fermentation described in step (2) is for ferment tank, during upper tank fermentation, sample-loading amount is 80% of fermentor tank volume, during upper tank, ventilation is tinning liquid volume/min, 26 ℃-28 ℃ of culture temperature, stirring velocity 150-180r/min, upper tank fermentation 4-6d.
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