CN101143002B - Deep liquid fermentation method for preparing selenium-rich gold needle mushroom crude polysaccharide powder - Google Patents
Deep liquid fermentation method for preparing selenium-rich gold needle mushroom crude polysaccharide powder Download PDFInfo
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Abstract
A method for preparing golden mushroom thick polyoses original powder full of selenium by deep liquid fermentation method belongs to biological project technical field. The invention mainly contains cultivation of strain, fermentation technique, richness of selenium, collection of mycelium, drying, smashing, and then golden mushroom thick polyoses original powder full of selenium in the cell is obtained. Besides, fermentation liquor is filtrated and concentrated. The concentrated liquor is frozen and dried after being extracted and then golden mushroom thick polyoses original powder full of selenium outside the cell is obtained. The two combines to get the product of golden mushroom thick polyoses original powder full of selenium. The organic selenium content of each gram of thick polyoses original powder achieves more than twenty micrograms. The golden mushroom thick polyoses original powder full of selenium has the health care functions of anti-tumor, enhancing human immunity, anti-fatigue, and promoting the brain development of children and so on, and is an excellent health care product. The method of the invention of manufacturing golden mushroom thick polyoses original powder full of selenium has a short cycle, a low cost, convenient techniques, high technology content, high effective elements, and high additional value, and is not easy to be imitated and is suitable for industrialization manufacture.
Description
Technical field
A kind of deep fermentation legal system is equipped with the method for selenium-rich gold needle mushroom crude polysaccharide powder, belongs to technical field of bioengineering.Relate to fermentation method and cultivate the Asparagus thalline, in fermentation medium, add the inorganic selenium element step by step, make organic selenium of accumulation high concentration in the golden mushroom mycelium.Therefrom obtain crude polysaccharide powder in the selenium-rich gold needle mushroom born of the same parents.The selenium element that higher concentration is also arranged in the active ingredient of zymotic fluid simultaneously.Again zymotic fluid is carried out the concentrated former powder of selenium-rich gold needle mushroom crude extracellular polysaccharide that obtains of ultrafiltration.The two merging is obtained the product selenium-rich gold needle mushroom crude polysaccharide powder.
Background technology
Asparagus (Flammulina velutipes) is one of world-renowned edible mushroom, belongs to Basidiomycotina, Hymenomycetes, and Agaricales, Tricholomataceae, dried mushroom belongs to (Flammulina) or money Pseudomonas (Collybia).Asparagus fine and tender taste, soft cunning, taste is pleasant.Its protein content height.Show according to one's analysis, contain 253.2 milligrams of Cobastabs, 10.9 milligrams of vitamin Cs in the bright Asparagus of every hectogram.Contain protein 13 grams in the dried Asparagus of every hectogram, carbohydrate 52 grams, mineral matter 7.56 grams also contain carrotene, several amino acids and nucleic acid.It is more complete that Asparagus contains the essential amino acid composition, and especially lysine and arginic content are high especially, are of value to the growth of children brain cell, therefore abroad is called " increasing the intelligence mushroom ".Also contain a kind of material that is Flammulin in the Asparagus, can strengthen the resist ability of body cancer cell.The flammulina velutipes that Asparagus contains has the cholesterol of reduction, antibacterial activity, immunocompetence and antitumor action.It can regulate the organism metabolism level, improves the liver detoxification ability, promotes nucleic acid and protein biosynthesis, help the reparation and the renewal of damaging cells, thereby delaying cell aging is of value to and promotes longevity.In addition, flammulina velutipes can promote the synthetic of protein, nucleic acid, improves body biological immune power, be mainly medicinal, flammulina velutipes has anti-inflammatory, protects liver, nourishes heart and anti-aging effects, can stop the effect of tumor growth simultaneously effectively, is the best ancillary drug of cancer patient chemotherapy.
Studies show that, often, in their mycelium, also contain effective component identical and similar active constituent content with fructification with the fungi of fructification medication.This produces flammulina velutipes just for the liquid fermentation method theoretical foundation is provided.
Selenium is the trace element of needed by human, is the essential composition of glutathione peroxidase in vivo, and that selenium has in human body is anti-oxidant, the effect of protection cell membrane.Asparagus has confirmed to have certain anti-selenium ability and selenium rich ability, and the organic degree height of rich selenium mycelium selenium, selenium are present in the compound that contains element sulphur, in large biological molecule materials such as protein, amino acid, polysaccharide, ribonucleic acid.The selenium of finite concentration scope all has facilitation to the formation of the growth of Asparagus mycelia and soluble protein, amino acid and polysaccharide.Selenium-rich gold needle mushroom has the effect of removing free radical, anti-inflammatory, antitumor, anti-ageing and anti peroxidation of lipid.
Along with improving constantly of people's living standard, people to the demand of nutrient and healthcare products from before blindness consumption change to reasonable best quality.The selenium-rich gold needle mushroom polysaccharide will become high-quality health products with its remarkable nutritional quality and mouthfeel.
Summary of the invention
The purpose of this invention is to provide a kind of deep fermentation method and produce the method for selenium-rich gold needle mushroom crude polysaccharide powder, adopt advanced biotechnology, and utilize higher fungus to have the characteristics of the selenium rich ability of convertibility, carry out purebred liquid deep layer fermenting and cultivate the Asparagus thalline, shortened the production cycle greatly, the production cycle of the present invention is 5 days (cycle of traditional solid culture fructification is generally about 30-50 days).The present invention all adopts grain raw material, pollutes for a short time, with high content of technology, and selenium-rich gold needle mushroom polysaccharide yield height helps absorption of human body, the added value of product height.
Technical scheme of the present invention: the Asparagus spore inoculating is cultivated to fresh solid inclined-plane, treated to move to after the inclined-plane is grown well and carry out culture of seed liquid in the fluid nutrient medium; By three grade fermemtation, go to the 3000L fermentation tank at last and carry out the deep fermentation cultivation again.In fermentation medium, add the inorganic selenium element step by step, and increase concentration step by step, make organic selenium of accumulation high concentration in the golden mushroom mycelium, the selenium element of higher concentration is also arranged in the active ingredient in the zymotic fluid simultaneously.After the fermentation ends, by gold needle mushroom crude polysaccharide powder in the born of the same parents that mycelial collection, oven dry, pulverizing obtained be rich in the selenium element, and it is concentrated that zymotic fluid is carried out ultrafiltration, alcohol extracting, freeze drying obtain the outer gold needle mushroom crude polysaccharide powder of born of the same parents, both are merged finally obtain selenium-rich gold needle mushroom crude polysaccharide powder.
Step is:
1) bacterial classification is cultivated:
Starting strain is Asparagus (Flammulina Valutipes) F12; This Asparagus bacterial strain is in 2002 06 phases of " biotechnology " magazine, and in December, 2002, exercise question is open in " edible fungi submerged fermentation technical study and functional food exploitation ".
1. the inclined-plane is cultivated:
Culture medium prescription is in g/L: glucose 10, potassium dihydrogen phosphate 1.0, magnesium sulfate 0.5, potato juice 200, agar 20, pH nature;
Cultural method: will be kept at bacterial classification inoculation on the wood chip to fresh blank inclined-plane, and cultivate 7-10 days down at 26 ± 1 ℃;
2. 500mL one-level shake-flask seed is cultivated, liquid amount 150mL:
Culture medium prescription is in g/L: glucose 20, analysis for soybean powder 10, corn flour 15-20, potassium dihydrogen phosphate 1.0, magnesium sulfate 0.5 and Cobastab
10.05, the pH nature;
Cultural method: cultured inclined-plane is seeded to shaking in the bottle of prior sterilization, and at 26 ± 1 ℃, rotating speed 110rpm cultivated 7 days down;
3. 5000mL secondary shake-flask seed is cultivated, liquid amount 1500mL:
Culture medium prescription is in g/L: glucose 20, peptone 10, corn flour 15-20, potassium dihydrogen phosphate 1.0, magnesium sulfate 0.5 and vitamin B1 0.05, pH nature;
Cultural method: cultured first order seed is seeded to shaking in the bottle of prior sterilization with 10% inoculum concentration, and at 26 ± 1 ℃, rotating speed 110rpm enlarges down and cultivated 2 days, shake-flask seed liquid;
3) three grade fermemtation is cultivated:
1. 50L one grade fermemtation seed tank culture, liquid amount 25-30L:
The first class seed pot culture medium prescription is in g/L: glucose 20, soya bean 20 is got juice, corn flour 5.0, wheat bran 50, water intaking dissolubility wheat bran juice behind the wheat bran poach, yeast extract 3.0, Cobastab with the colloid mill defibrination after centrifugal after the soybean soaking
10.05, magnesium sulfate 0.5, potassium dihydrogen phosphate 1.0, soya-bean oil 2.0 and sodium selenite 20 μ g/L, pH nature; Sterilization is 30 minutes under 0.1Mpa, shake-flask seed is inserted by inoculum concentration 5%-10% in the cooling back, 26 ± 1 ℃ of temperature, tank pressure 0.05Mpa, throughput 1: 0.3, V/V, speed of agitator 100rpm, change a jar standard: fermentation time 30-35 hour, mycelium pellet come-up 1/3rd, microscopy has clamp connection, does not have assorted bacterium;
2. 500L second order fermentation seed tank culture, liquid amount 250-300L:
Secondary seed jar culture medium prescription is in g/L: glucose 20, soya bean 20 is got juice, corn flour 5.0, wheat bran 50, water intaking dissolubility wheat bran juice behind the wheat bran poach, yeast extract 3.0, Cobastab with the colloid mill defibrination after centrifugal after the soybean soaking
10.05, magnesium sulfate 0.5, potassium dihydrogen phosphate 1.0, soya-bean oil 2.0 and sodium selenite 40 μ g/L, pH nature; Sterilization is 30 minutes under 0.1Mpa, first order seed is inserted by inoculum concentration 10%-15% in the cooling back, 26 ± 1 ℃ of temperature, tank pressure 0.05Mpa, throughput 1: 0.3, V/V, speed of agitator 100rpm, change a jar standard: fermentation time 20-25 hour, mycelium pellet come-up 1/3rd, microscopy has clamp connection, does not have assorted bacterium;
3. the cultivation of 3000L fermentation tank, liquid amount 1800L:
Fermentative medium formula is in g/L: glucose 30, soya bean 20 is got juice, corn flour 5.0, wheat bran 50, water intaking dissolubility wheat bran juice behind the wheat bran poach, Cobastab with the colloid mill defibrination after centrifugal after the soybean soaking
10.05, magnesium sulfate 0.5, potassium dihydrogen phosphate 1.0, soya-bean oil 2.0 and sodium selenite 50 μ g/L, pH nature; Sterilization is 30 minutes under 0.1Mpa, and secondary seed, 26 ± 1 ℃ of temperature are inserted by inoculum concentration 10%-1 5% in the cooling back, tank pressure 0.05Mpa, throughput 1: 0.3, V/V, speed of agitator 100rpm, put a jar standard: fermentation time 40-45 hour, pH＞5, the assorted bacterium of dull and stereotyped nothing, mycelium pellet come-up 1/2nd, microscopy has clamp connection, and mycelium pellet can not self-dissolving;
3) extraction of product: cultured zymotic fluid is handled with the centrifuge of 5000-6000rpm, collected mycelium, zymotic fluid is collected in the extractor;
1. the obtaining of crude polysaccharide powder in the born of the same parents: with the mycelium collected several times with the pure water washing, treat the cleaning solution clarification after, put into vacuum drying chamber and dry, dry by the fire to moisture and pulverizing below 6%, promptly get the interior crude polysaccharide powder of Asparagus born of the same parents;
2. obtaining of the former powder of crude extracellular polysaccharide: will collect the zymotic fluid in the extractor and merge the mycelium cleaning solution that comes is that 5000 hollow fiber ultrafiltration membrane equipment carries out ultrafiltration and concentrates with holding back relative molecular mass, simmer down to stoste volume 1/5th after, use 95% food grade ethanol alcohol extracting of 3 times of amounts of volume after 12-16 hour concentrate, with the centrifugal 20-30 of 5000-6000rpm minute, collect solid, after the solid collected redissolved with a small amount of pure water, freeze drying can obtain the former powder of Asparagus crude extracellular polysaccharide after the pulverizing;
3. the acquisition of product:, promptly get the product gold needle mushroom crude polysaccharide powder with crude polysaccharide powder in the Asparagus born of the same parents and the two merging of the former powder of Asparagus crude extracellular polysaccharide.
4) check: test stone is as follows:
I. product appearance: color is the buff powder, and fineness 40 orders have Asparagus fragrance, free from extraneous odour.
Ii. polyoses content 〉=6%.
Iii. protein content 〉=8%.
Iv. organic selenium content 〉=20 microgram/grams.
V. moisture≤8%.
Vi. total plate count≤1000cfu/g, coliform≤40npn/100g, yeast≤25cfu/g,, mould≤25cfu/g, pathogenic bacteria must not detect.LD50 is negative.
Vii. lead≤1.5mg/kg, arsenic≤1.0mg/kg.
Product detects by health and epidemic prevention department and can sell after qualified.Packing: promptly get the product gold needle mushroom crude polysaccharide powder after packing with the aluminium film package bag after the assay was approved, can be used as the intermediate of health products.
Beneficial effect of the present invention: the present invention adopts advanced biotechnology, and utilize higher fungus to have the characteristics of the selenium rich ability of convertibility, carry out purebred liquid deep layer fermenting and cultivate the Asparagus thalline, shortened the production cycle greatly, production cycle of the present invention is 5 days, and the cycle of traditional solid culture fructification is generally about 30-50 days.The present invention all adopts grain raw material, pollutes for a short time, and cost is low, and technology is easy, and is high in technological content, is difficult for by counterfeit active constituent content height, added value height, suitability for industrialized production.Selenium-rich gold needle mushroom polysaccharide yield height helps absorption of human body.The product selenium-rich gold needle mushroom crude polysaccharide powder, organic selenium content reaches 20 micrograms/more than the gram in every gram crude polysaccharide powder.The selenium-rich gold needle mushroom polysaccharide has antitumor, improves body immunity, and antifatigue promotes health-care efficacies such as children's brain development, is first-class health products active ingredient.
The specific embodiment
1, the preparation of seed liquor: with the wood chip spore inoculating to a blank eggplant bottle inclined-plane of the above-mentioned inclined-plane of the usefulness formulated of the bacterium of having gone out in advance, after 26 ℃ are cultivated 7-10 days down, the thalline on the inclined-plane is scraped in the 250ml triangular flask that bead is housed of the bacterium of having gone out in advance, other adds 30ml sterilized water vibration 10 minutes, makes bacteria suspension.Inhale this bacteria suspension of 4ml (shake the culture medium of bottle formulated by above-mentioned one-level, liquid amount is 150ml) in 2 500ml triangular flasks of the bacterium of having gone out in advance, at 26 ℃, rotating speed 110r/min cultivates down and got the one-level shake-flask seed liquid in 7 days; Each one bottle above-mentioned secondary of usefulness of pouring the bacterium of having gone out in advance under sterile working into of cultured one-level shake-flask seed liquid is shaken in the triangular flask of 5000mL (liquid amount is 1500mL) of bottle formulated, at 26 ℃, rotating speed 110r/min enlarges down and cultivated 2 days, long good after with two bottles of seed liquor merge shake-flask seed liquid.
2, first class seed pot is cultivated: by above-mentioned first class seed pot culture medium prescription, shake-flask seed is inserted in 30 minutes cooling backs of sterilization under 0.1Mpa.Cultivate by above-mentioned first class seed pot culture parameters.Adopt the 50L automatic fermenter, liquid amount is 25-30L.
3, the secondary seed jar is cultivated: by above-mentioned secondary seed jar culture medium prescription, first order seed is inserted in 30 minutes cooling backs of sterilization under 0.1Mpa.Cultivate by above-mentioned secondary seed jar culture parameters.Adopt the 500L fermentation tank, liquid amount is 250-300L.
4, the cultivation of fermentation tank: adopt the 3000L fermentation tank, liquid amount is 1800L.By above-mentioned fermentative medium formula, secondary seed is inserted in the same terms sterilization back.Culture parameters by above-mentioned fermentation tank is cultivated.
5, obtaining of the interior crude polysaccharide powder of born of the same parents: with the extracting method of cultured zymotic fluid, collect mycelium, obtain 260 kilograms of wet thallus, after drying and crushing, obtain crude polysaccharide powder in 30 kilograms of born of the same parents with the said goods.
6, obtaining of the former powder of crude extracellular polysaccharide: the acquisition methods by the former powder of above-mentioned crude extracellular polysaccharide, obtain the 300L concentrate, after the freeze drying, pulverize and obtain 6 kilograms in the former powder of Asparagus crude extracellular polysaccharide.
7, the two is merged 36 kilograms of product gold needle mushroom crude polysaccharide powders.
Claims (1)
1. a deep fermentation legal system is equipped with the method for selenium-rich gold needle mushroom crude polysaccharide powder, it is characterized in that the Asparagus spore inoculating is cultivated to fresh solid inclined-plane, treats that the inclined-plane is grown to move to after good to carry out culture of seed liquid in the fluid nutrient medium; Pass through three grade fermemtation again, go to the 3000L fermentation tank at last and carry out the deep fermentation cultivation, in fermentation medium, add the inorganic selenium element step by step, and increase concentration step by step, make organic selenium of accumulation high concentration in the golden mushroom mycelium, in the active ingredient certain density selenium element is arranged also in the zymotic fluid simultaneously; After the fermentation ends, obtain being rich in gold needle mushroom crude polysaccharide powder in the born of the same parents of selenium element by collection, oven dry, pulverizing to thalline, and to zymotic fluid carry out that ultrafiltration concentrates, alcohol extracting, freeze drying obtain the outer gold needle mushroom crude polysaccharide powder of born of the same parents, both are merged finally obtain selenium-rich gold needle mushroom crude polysaccharide powder; Step is:
1) bacterial classification is cultivated:
Starting strain is Asparagus (Flammulina Valutipes) F12;
1. the inclined-plane is cultivated:
Culture medium prescription is in g/L: glucose 10, potassium dihydrogen phosphate 1.0, magnesium sulfate 0.5, potato juice 200, agar 20, pH nature;
Cultural method: will be kept at bacterial classification inoculation on the wood chip to fresh blank inclined-plane, and cultivate 7-10 days down at 26 ± 1 ℃;
2. 500mL one-level shake-flask seed is cultivated, liquid amount 150mL:
Culture medium prescription is in g/L: glucose 20, analysis for soybean powder 10, corn flour 15-20, potassium dihydrogen phosphate 1.0, magnesium sulfate 0.5 and Cobastab
10.05, the pH nature;
Cultural method: cultured inclined-plane is seeded to shaking in the bottle of prior sterilization, and at 26 ± 1 ℃, rotating speed 110rpm cultivated 7 days down;
3. 5000mL secondary shake-flask seed is cultivated, liquid amount 1500mL:
Culture medium prescription is in g/L: glucose 20, peptone 10, corn flour 15-20, potassium dihydrogen phosphate 1.0, magnesium sulfate 0.5 and Cobastab
10.05, the pH nature;
Cultural method: cultured first order seed is seeded to shaking in the bottle of prior sterilization with 10% inoculum concentration, and at 26 ± 1 ℃, rotating speed 110rpm enlarges down and cultivated 2 days, shake-flask seed liquid;
2) three grade fermemtation is cultivated:
1. 50L one grade fermemtation seed tank culture, liquid amount 25-30L:
The first class seed pot culture medium prescription is in g/L: glucose 20, soya bean 20 is got juice, corn flour 5.0, wheat bran 50, water intaking dissolubility wheat bran juice behind the wheat bran poach, yeast extract 3.0, Cobastab with the colloid mill defibrination after centrifugal after the soybean soaking
10.05, magnesium sulfate 0.5, potassium dihydrogen phosphate 1.0, soya-bean oil 2.0 and sodium selenite 20 μ g/L, pH nature; Sterilization is 30 minutes under 0.1Mpa, shake-flask seed is inserted by inoculum concentration 5%-10% in the cooling back, 26 ± 1 ℃ of temperature, tank pressure 0.05Mpa, throughput 1: 0.3, V/V, speed of agitator 100rpm, change a jar standard: fermentation time 30-35 hour, mycelium pellet come-up 1/3rd, microscopy has clamp connection, does not have assorted bacterium;
2. 500L second order fermentation seed tank culture, liquid amount 250-300L:
Secondary seed jar culture medium prescription is in g/L: glucose 20, soya bean 20 is got juice, corn flour 5.0, wheat bran 50, water intaking dissolubility wheat bran juice behind the wheat bran poach, yeast extract 3.0, Cobastab with the colloid mill defibrination after centrifugal after the soybean soaking
10.05, magnesium sulfate 0.5, potassium dihydrogen phosphate 1.0, soya-bean oil 2.0 and sodium selenite 40 μ g/L, pH nature; Sterilization is 30 minutes under 0.1Mpa, first order seed is inserted by inoculum concentration 10%-15% in the cooling back, 26 ± 1 ℃ of temperature, tank pressure 0.05Mpa, throughput 1: 0.3, V/V, speed of agitator 100rpm, change a jar standard: fermentation time 20-25 hour, mycelium pellet come-up 1/3rd, microscopy has clamp connection, does not have assorted bacterium;
3. the cultivation of 3000L fermentation tank, liquid amount 1800L:
Fermentative medium formula is in g/L: glucose 30, soya bean 20 is got juice, corn flour 5.0, wheat bran 50, water intaking dissolubility wheat bran juice behind the wheat bran poach, Cobastab with the colloid mill defibrination after centrifugal after the soybean soaking
10.05, magnesium sulfate 0.5, potassium dihydrogen phosphate 1.0, soya-bean oil 2.0 and sodium selenite 50 μ g/L, pH nature; Sterilization is 30 minutes under 0.1Mpa, and secondary seed, 26 ± 1 ℃ of temperature are inserted by inoculum concentration 10%-15% in the cooling back, tank pressure 0.05Mpa, throughput 1: 0.3, V/V, speed of agitator 100rpm, put a jar standard: fermentation time 40-45 hour, pH＞5, the assorted bacterium of dull and stereotyped nothing, mycelium pellet come-up 1/2nd, microscopy has clamp connection, and mycelium pellet can not self-dissolving;
3) extraction of product: cultured zymotic fluid is handled with the centrifuge of 5000-6000rpm, collected mycelium, zymotic fluid is collected in the extractor;
1. the obtaining of crude polysaccharide powder in the born of the same parents: with the mycelium collected several times with the pure water washing, treat the cleaning solution clarification after, put into vacuum drying chamber and dry, dry by the fire to moisture and pulverizing below 6%, promptly get the interior crude polysaccharide powder of Asparagus born of the same parents;
2. obtaining of the former powder of crude extracellular polysaccharide: will collect the zymotic fluid in the extractor and merge the mycelium cleaning solution that comes is that 5000 hollow fiber ultrafiltration membrane equipment carries out ultrafiltration and concentrates with holding back relative molecular mass, simmer down to stoste volume 1/5th after, use 95% food grade ethanol alcohol extracting of 3 times of amounts of volume after 12-16 hour concentrate, with the centrifugal 20-30 of 5000-6000rpm minute, collect solid, after the solid collected redissolved with a small amount of pure water, freeze drying can obtain the former powder of Asparagus crude extracellular polysaccharide after the pulverizing;
3. the acquisition of product:, promptly get the product gold needle mushroom crude polysaccharide powder with crude polysaccharide powder in the Asparagus born of the same parents and the two merging of the former powder of Asparagus crude extracellular polysaccharide.
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| CN105713849A (en) * | 2016-05-05 | 2016-06-29 | 盐城工学院 | Liquid culture medium for culturing Flammulina velutipes mycelia and culture method using liquid culture medium |
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| CN109797107B (en) * | 2019-01-29 | 2021-06-04 | 西北大学 | Preparation method of fungus mycelium fine powder |
| CN111518842A (en) * | 2019-10-10 | 2020-08-11 | 江南大学 | Method for biosynthesizing nano-selenium by utilizing needle mushrooms |
| CN110484363A (en) * | 2019-10-10 | 2019-11-22 | 广州方中化工有限公司 | A kind of preparation method of Anti-oxidant agent and flammulina velutipes |
| CN111887101A (en) * | 2020-07-22 | 2020-11-06 | 湖北民族大学 | Preparation method and application of selenium-rich pleurotus citrinopileatus mycelium |
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