CN101228830A - Method of liquid fermentation culturing flammulina velutipes mycelium rich in Se and Zn - Google Patents
Method of liquid fermentation culturing flammulina velutipes mycelium rich in Se and Zn Download PDFInfo
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Abstract
A method for cultivating golden mushroom mycelia abundant in Se and Zn elements through liquid fermentation belongs to the field of biotechnology. The invention substantially comprises cultivation of strains, cultivation of liquid seed, namely the domestication of two elements, and enrichment, fermentation and cultivation of two elements in a deep liquid, and collecting, drying and smashing of mycelia. The dried golden mushroom mycelia abundant in organic Se and organic Zn obtained with the method of the invention has over 35Mug/g organic Se and 4.25mg/g organic Zn. The powder of the mycelia with suitable food can be used as food additives and made into functional food, which has great health care function for the people in sub-health state resulted from deficient intake of microelements of Se and Zn. The invention is high in technological content, high content in effective components of the products, high additive value, which is suitable for industrial production.
Description
Technical field
The method of the golden mushroom mycelium of the rich selenium of a kind of liquid fermentation and culture, zinc element belongs to technical field of bioengineering.Relate to fermentation method and cultivate golden mushroom mycelium, be that the Asparagus spore inoculating is cultivated to fresh solid inclined-plane, treat to be seeded to the cultivation of carrying out seed in the liquid nutrient medium that contains low concentration inorganic selenium and inorganic zinc after the inclined-plane is grown well, promptly bacterial classification is tamed; Insert again and carry out the deep fermentation cultivation in the fermentation medium that has added an amount of selenium element, zinc element, utilize the enrichment and the transformation function of Asparagus, make the organic selenium and the organic zinc of accumulation high concentration in the mycelium.After the fermentation ends,, be rich in the golden mushroom mycelium powder of organic selenium element, organic zinc element simultaneously by collection, oven dry to thalline.
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 Cobastab in the bright Asparagus of every hectogram
253.2 milligram, 10.9 milligrams of vitamin Cs.Contain protein 13 grams in the dried Asparagus of every hectogram, carbohydrate 52 grams, mineral matter 7.56 grams also contain carotin, 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, but enhancing body is to the resist ability of cancer cell.The flammulina velutipes that Asparagus contains has cholesterol reducing, 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.Studies show that, often, in their mycelium, also contain effective component identical and similar active constituent content with fruit body with the fungi of fruit body medication.This produces golden mushroom mycelium just for the liquid fermentation method theoretical foundation is provided.
Selenium is a kind of metalloid element; it is one of essential trace element of organism; it has anti-oxidant, anti-aging effect in human body; can protect the 26S Proteasome Structure and Function of cell membrane; enhancing body immunity; for cardiovascular function, fertility, eyesight etc. material impact is arranged, and what really bring into play biological action is organic selenium.Have more than 40 countries and regions to lack selenium in the world, China 72% area is for lacking selenium, low selenium area, and the deficiency of selenium intake is having a strong impact on the healthy of people in the meals.In the Se content of wholefood, the inorganic selenium of sodium selenite etc. can only be used for medicine because of toxicity is higher can not be used for food, moreover the availability of inorganic selenium is also very low.
Zinc also is one of metallic element that function of human body is played an important role.Zinc is the composition of a lot of metalloenzyme or the activator of enzyme.The nearly 70-100 kind of the enzyme relevant with zinc.Zinc can't synthesize in human body, can only be from external acquisition.Zinc deficiency is ubiquity in Chinese population, and it is especially serious that baby, children, pregnant woman and the women of child-bearing age lack zinc.Replenish organic zinc and can eliminate inorganic zinc to the side effect of human body with to the stimulation of stomach and intestine, thus make zinc can be more efficient by human body, more safely absorb.
Asparagus has confirmed to have selenium and zinc organic degree height in certain rich zinc ability and the selenium rich ability mycelium.Selenium is present in the compound that contains element sulphur, in large biological molecule materials such as protein, amino acid, polysaccharide, ribonucleic acid.And the organic zinc in the Asparagus is 50% or more and protein combination, 34% and sugar and contaminated with lipid, and be absorbed by the body especially easily and utilize.
Summary of the invention
The purpose of this invention is to provide a kind of deep fermentation method and cultivate the method for the golden mushroom mycelium powder that is rich in organic selenium element, organic zinc element simultaneously.The present invention adopts advanced biotechnology, and utilize higher fungi to have the characteristics of the gathering trace element ability of convertibility, carry out purebred liquid deep layer fermenting and cultivate golden mushroom mycelium, creativeness makes a kind of edible fungus become the functional food additive that can replenish two kinds of trace elements simultaneously.
Technical scheme of the present invention: the Asparagus spore inoculating is cultivated to fresh solid inclined-plane, treat that the inclined-plane grows in the liquid seed culture medium that is seeded to the inorganic selenium that added low concentration and zinc ion after good, cultivate when enlarging, bacterial classification is tamed, again the inorganic selenium of an amount of concentration and inorganic zinc are joined in the deep fermentation medium and cultivate, utilize the enrichment and the transformation function of golden mushroom, make the organic selenium and the organic zinc of accumulation higher concentration in the filament.After the fermentation ends,, be rich in the golden mushroom mycelium powder of organic selenium element, organic zinc element simultaneously by collection, oven dry to thalline.
Step is:
1) stand-by culture of strains:
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. slant culture:
Culture medium prescription is in g/L: wheat bran 40, sucrose 20, potassium dihydrogen phosphate 3.0, magnesium sulfate 1.5, agar 20.0, 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. the one-level shake-flask seed is cultivated, liquid amount 150mL/500mL:
Culture medium prescription is in g/L: glucose 20, analysis for soybean powder 10, corn flour 25, 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. the secondary shake-flask seed is cultivated, liquid amount 400mL/1000mL:
Culture medium prescription: identical with one-level shake-flask seed culture medium prescription;
Cultural method: cultured first order seed is seeded to shaking in the bottle of prior sterilization with 10% inoculum concentration, and at 26 ± 1 ℃, enlarged culture is 2 days under the rotating speed 110rpm, stand-by bacterial classification;
3) 50L level liquid seed culture, liquid amount 25-30L:
1. level liquid seed culture based formulas is in g/L: glucose 20, analysis for soybean powder 10, corn flour 25, wheat bran 50, water intaking dissolubility wheat bran juice behind the wheat bran poach, yeast extract 3.0, Cobastab
10.05, magnesium sulfate 0.5, potassium dihydrogen phosphate 1.0, sodium selenite 0.01, zinc sulphate 0.2, soya-bean oil 2.0, pH nature;
2. control technology: sterilization is 30 minutes under 0.1Mpa, and stand-by bacterial classification is inserted by inoculum concentration 5%-10%, 26 ± 1 ℃ of temperature, tank pressure 0.05Mpa, throughput 1: 0.3, V/V, speed of agitator 100rpm in the cooling back;
3. change a jar standard: fermentation time 30-35 hour, mycelium pellet come-up 1/3rd, microscopy has clamp connection, does not have assorted bacterium;
3) 500L secondary liquid seeds is cultivated, liquid amount 250-300L:
1. culture medium prescription is identical with level liquid seed prescription:
2. control technology: sterilization is 30 minutes under 0.1Mpa, and first order seed is inserted by inoculum concentration 10%-15% in the cooling back, 26 ± 1 ℃ of temperature, and tank pressure 0.05Mpa, throughput 1: 0.3, V/V, speed of agitator 100rpm,
3. change a jar standard: fermentation time 20-25 hour, mycelium pellet come-up 1/3rd, microscopy has clamp connection, does not have assorted bacterium;
4) the 3000L deep fermentation is cultivated, liquid amount 1800L:
1. fermentative medium formula is in g/L: glucose 30, soya bean 30 is got juice, corn flour 10, 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, yeast extract 3.0, soya-bean oil 2.0 and sodium selenite 0.05, zinc sulphate 0.7, pH nature;
2. control technology: sterilization is 30 minutes under 0.1Mpa, and secondary seed is inserted by inoculum concentration 10%-15% in the cooling back, 26 ± 1 ℃ of temperature, and tank pressure 0.05Mpa, throughput 1: 0.3, V/V, speed of agitator 100rpm,
3. put a jar standard: fermentation time 40-45 hour, pH＞5, dull and stereotyped do not have an assorted bacterium, mycelium pellet come-up 1/2nd, microscopy has clamp connection, and mycelium pellet can not self-dissolving;
5) mycelial extraction: cultured zymotic fluid is handled with the centrifuge of 5000-6000rpm, collected mycelium;
6) mycelial oven dry: the mycelium of collecting is dried down at 50-60 ℃ with vacuum drying chamber:
7) mycelial pulverizing: both got product more than 80 orders with the mycelium powder of drying being broken to freezing cracker.
8) check: test stone is as follows:
I. product appearance: color is the buff powder, and fineness 80 orders have Asparagus fragrance, free from extraneous odour.
Ii. polyoses content 〉=6%.
Iii. protein content 〉=8%.
Iv. organic selenium content 〉=20 microgram/grams.
V. organic zinc content 〉=4.0mg/g
Vi. moisture≤8%.
Vii. total plate count≤1000cfu/g, coliform≤40npn/100g, yeast≤25cfu/g,, mould≤25cfu/g, pathogenic bacteria must not detect.LD50 is negative.
Viii. 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 thick thalline powder of Asparagus that product is rich in organic selenium, organic zinc after packing with the aluminium film package bag after the assay was approved, can be used as food additives, as the Main Ingredients and Appearance of functional food.
Beneficial effect of the present invention: the present invention adopts advanced biotechnology, and utilize higher fungi 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 fruit body 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.Be rich in the thick thalline powder of Asparagus of organic selenium, organic zinc, and have the distinctive fragrance of Asparagus, can with the numerous food compatibility, make various tastes, become and have additional organic selenium and organic zinc function, and be easy to the functional food of absorption of human body and utilization.
Embodiment
1, stand-by culture of strains: 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 sterile water vibration 10 minutes, makes bacteria suspension.Inhale this bacteria suspension of 4ml (by the medium of above-mentioned one-level shake-flask seed formulated, liquid amount is 150m1) 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 in 7 days; In the triangular flask with the 5000mL (liquid amount is 1500mL) of each the one bottle above-mentioned secondary shake-flask seed of the usefulness formulated of under sterile working, pouring the bacterium of having gone out in advance into of cultured one-level shake-flask seed, at 26 ℃, enlarged culture is 2 days under the rotating speed 110r/min, long good after with two bottles of seed liquor merge stand-by bacterial classification.
2, level liquid seed culture: by above-mentioned level liquid seed culture based formulas, shake-flask seed is inserted in 30 minutes cooling backs of sterilization under 0.1Mpa.Cultivate by above-mentioned level liquid seed culture parameter.Adopt the 50L automatic fermenter, liquid amount is 25-30L.
3, the secondary liquid seeds is cultivated: by above-mentioned secondary liquid seed 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, deep fermentation is cultivated: adopt the 3000L fermentation tank, liquid amount is 1800L.By above-mentioned fermentative medium formula, the secondary liquid seeds is inserted in the same terms sterilization back.Culture parameters by above-mentioned fermentation tank is cultivated.
5, mycelial extraction: collect mycelium by above-mentioned thalline extracting method, obtain 288 kilograms of wet myceliums.
6, be rich in the acquisition of organic selenium, organic zinc Asparagus thalline powder: oven dry and breaking method by above-mentioned thalline obtain 47 kilograms of products.
Claims (1)
1. the method for the golden mushroom mycelium of the rich selenium of a liquid fermentation and culture, zinc element, it is characterized in that the Asparagus spore inoculating is cultivated to fresh solid inclined-plane, treat to be seeded to the cultivation of carrying out seed in the liquid nutrient medium that contains low concentration inorganic selenium and inorganic zinc after the inclined-plane is grown well, promptly bacterial classification is tamed; Insert again and carry out the deep fermentation cultivation in the fermentation medium that has added an amount of selenium element, zinc element, utilize the enrichment and the transformation function of golden mushroom, make the organic selenium and the organic zinc of accumulation high concentration in the filament.After the fermentation ends,, be rich in the golden mushroom mycelium powder of organic selenium element, organic zinc element simultaneously by collection, oven dry, pulverizing to thalline.
Step is:
1) stand-by culture of strains:
Starting strain is Asparagus (Flammulina Valutipes) F12;
1. slant culture:
Culture medium prescription is in g/L: wheat bran 40, sucrose 20, potassium dihydrogen phosphate 3.0, magnesium sulfate 1.5, agar 20.0, 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. the one-level shake-flask seed is cultivated, liquid amount 150mL/500mL:
Culture medium prescription is in g/L: glucose 20, analysis for soybean powder 10, corn flour 25, 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. the secondary shake-flask seed is cultivated, liquid amount 400mL/1000mL:
Culture medium prescription: identical with one-level shake-flask seed culture medium prescription;
Cultural method: cultured first order seed is seeded to shaking in the bottle of prior sterilization with 10% inoculum concentration, and at 26 ± 1 ℃, enlarged culture is 2 days under the rotating speed 110rpm, stand-by bacterial classification;
2) 50L level liquid seed culture, liquid amount 25-30L:
1. level liquid seed culture based formulas is in g/L: glucose 20, analysis for soybean powder 10, corn flour 25, wheat bran 50, water intaking dissolubility wheat bran juice behind the wheat bran poach, yeast extract 3.0, Cobastab
10.05, magnesium sulfate 0.5, potassium dihydrogen phosphate 1.0, sodium selenite 0.01, zinc sulphate 0.2, soya-bean oil 2.0, pH nature;
2. control technology: sterilization is 30 minutes under 0.1Mpa, and stand-by bacterial classification is inserted by inoculum concentration 5%-10%, 26 ± 1 ℃ of temperature, tank pressure 0.05Mpa, throughput 1: 0.3, V/V, speed of agitator 100rpm in the cooling back;
3. change a jar standard: fermentation time 30-35 hour, mycelium pellet come-up 1/3rd, microscopy has clamp connection, does not have assorted bacterium;
3) 500L secondary liquid seeds is cultivated, liquid amount 250-300L:
1. culture medium prescription is identical with level liquid seed prescription:
2. control technology: sterilization is 30 minutes under 0.1Mpa, and first order seed is inserted by inoculum concentration 10%-15% in the cooling back, 26 ± 1 ℃ of temperature, and tank pressure 0.05Mpa, throughput 1: 0.3, V/V, speed of agitator 100rpm,
3. change a jar standard: fermentation time 20-25 hour, mycelium pellet come-up 1/3rd, microscopy has clamp connection, does not have assorted bacterium;
4) the 3000L deep fermentation is cultivated, liquid amount 1800L:
1. fermentative medium formula is in g/L: glucose 30, and soya bean 30 is got juice with the colloid mill defibrination after centrifugal after the soybean soaking, corn flour 10, wheat bran 50, water intaking dissolubility wheat bran juice behind the wheat bran poach, vitaminB10 .05, magnesium sulfate 0.5, potassium dihydrogen phosphate 1.0, yeast extract 3.0, soya-bean oil 2.0 and sodium selenite 0.05, zinc sulphate 0.7, the pH nature;
2. control technology: sterilization is 30 minutes under 0.1Mpa, and secondary seed, 26 ± 1 ℃ of temperature, tank pressure 0.05Mpa, throughput 1: 0.3, V/V, speed of agitator 100rpm are inserted by inoculum concentration 10%-15% in the cooling back;
3. put a jar standard: fermentation time 40-45 hour, pH＞5, dull and stereotyped do not have an assorted bacterium, mycelium pellet come-up 1/2nd, microscopy has clamp connection, and mycelium pellet can not self-dissolving;
5) mycelial extraction: cultured zymotic fluid is handled with the centrifuge of 5000-6000rpm, collected mycelium;
6) mycelial oven dry: the mycelium of collecting is dried down at 50-60 ℃ with vacuum drying chamber;
7) mycelial pulverizing: both got product more than 80 orders with the mycelium powder of drying being broken to freezing cracker.
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