CN103155785B - Establishment method for brown mushroom three-level strain propagation system - Google Patents

Establishment method for brown mushroom three-level strain propagation system Download PDF

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CN103155785B
CN103155785B CN201110408698.7A CN201110408698A CN103155785B CN 103155785 B CN103155785 B CN 103155785B CN 201110408698 A CN201110408698 A CN 201110408698A CN 103155785 B CN103155785 B CN 103155785B
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wheat
sterilizing
peptone
glucose
boil
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CN103155785A (en
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班立桐
黄亮
王玉
杨丽维
孙茜
孙少起
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TIANJIN JINSANNONG AGRICULTURAL TECHNOLOGY DEVELOPMENT Co Ltd
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TIANJIN JINSANNONG AGRICULTURAL TECHNOLOGY DEVELOPMENT Co Ltd
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Abstract

The invention discloses an establishment method for a brown mushroom three-level strain propagation system. The establishment method for the brown mushroom three-level strain propagation system comprises three-level strain propagation of stock cultures, liquid strains and cultispecies, wherein the formula of culture mediums of the stock cultures comprises, by weight (g/L), 600g of turfy soil, 20g of glucose, 100g of wheat grains, 1.5g of magnesium sulfate, 1g of monopotassium phosphate, 2g of peptone and 1000mL of water, the formula of the liquid strains comprises, by weight (g/L), 200g of potatoes, 20g of glucose, 2g of peptone, 2g of monopotassium phosphate, 1g of magnesium sulfate and 10.01g of VB, the pH (potential of hydrogen) is natural, and the cultispecies comprise, by weight (%), 92% of wheat grains, 5% of gypsum and 3% of turfy soil. Compared with a traditional solid state three-level strain, through using the propagation system, the propagation cycle is shortened by 38 days. The establishment method for the brown mushroom three-level strain propagation system is an optimum scheme, and has a certain guiding significance to the mass industrialized production of brown mushrooms.

Description

A kind of brown mushroom three-class strain expands the method for building up of traditional font system
Technical field
The invention provides the cultivation method that relates to brown mushroom, say that more specifically a kind of brown mushroom three-class strain expands numerous method.
Background technology
Brown mushroom ( agaricus brunnescenspeck), be the rare veriety in mushroom, belong to Basidiomycetes (Basidiomycotina), Agaricales (Agaritales), Agaricus edibilis (Agaricaceae), Agaricus ( garicus).Brown mushroom is commonly called as brown mushroom, is Bo Ximei subspecies.This bacterial strain output is high, good mushroom quality is good, mouthfeel is fresh and tender, and its mushroom lid is nature sepia, stem white, the fine and close consolidation of meat.All white mushroom kinds are the white mutation of brown mushroom now, that is to say that brown mushroom is the wild ancestors of white mushroom.Brown mushroom, another name white mushroom, Mushroom, belong to Basidiomycetes, Agaricales, Agaricus edibilis, Agaricus.Brown mushroom is nutritious, delicious flavour, and fresh mushroom contains protein 3%-4% according to reports, fat 0.2%-0.3%, and carbohydrate 2.4%-3.8%, can digestibility reach 70%-90%, enjoys the title of " Vegetable meat ".The amino matter acid composition of mushroom is more comprehensive, is especially rich in the lysine of needed by human etc.Brown mushroom also contains the mineral elements such as abundant iron, potassium, calcium.Also contain multivitamin and the enzymes such as thiamine, vitamin b3, nicotinic acid, ascorbic acid.Owing to containing higher unsaturated fatty acid, therefore many edible mushrooms have obvious effect to reducing blood fat, to curing metastatic hepatitis, chronic hepatitis, hepatomegaly, early-phase hepatocirrhosis etc., all there is significant curative effect with the canned mushroom processing medicine that liquid makes of precooking.Along with people pursue arrival natural, that return uncut jade and return straight trend, brown mushroom is subject to pursuing of consumers in general on market, is a good commercial variety.
At present, brown mushroom is cultivated the main solid culture method that adopts, and fabrication cycle is long, adds that its Production Time is on the occasion of high temperature season, causes production cost to rise.And liquid culture is to adopt fluid matrix matter to ferment to carry out the production of fermented and cultured bacterial classification, its principle is: in fermentation tank or triangular flask, adopt liquid culture matrix, passing into filtrated air is stirred or is vibrated, increase the dissolved oxygen content in culture matrix, provide mycelium respiratory metabolism needed oxygen, and control suitable external condition, make thalline in the breeding growth of liquid depths, obtain a large amount of mycelium or metabolite.
Because brown mushroom has good market prospects, but at present less to the research of its cultivation method, especially than agaricus bisporus three-class strain, breed the time longer, the present invention is intended to by the making of the preparation to brown liquid fungus culture and cultivated species, the best that works out brown mushroom three-class strain expands numerous scheme, and the large-scale industrialized production to brown mushroom is had to certain directive significance.
Summary of the invention
Technology contents disclosed by the invention is as follows:
Brown mushroom three-class strain expands a method for building up for traditional font system, it is characterized in that it comprises that female kind, the three-class strain of liquid spawn, cultivated species expand numerous combining, and the one of foundation expands traditional font system, and its method is as follows:
(1) female preparation of planting:
1) Mother culture based formulas, i.e. turfy soil medium (g/L): turfy soil 600g, glucose 20g, wheat 100g, magnesium sulfate 1.5g, potassium dihydrogen phosphate 1g, peptone 2g, water 1000mL;
2) preparation medium: get appropriate turfy soil, wheat, add in the clear water of 1000ml and boil 20~30min, filtrate; In filtrate, add magnesium sulfate, potassium dihydrogen phosphate, peptone, glucose, agar, after medicine dissolves, supply moisture, packing test tube;
3) sterilizing: 121 ℃, sterilizing 20min, after sterilizing finishes, takes out pendulum inclined-plane;
4) inoculation, cultivation: brown mushroom mycelium is inoculated in to middle part, inclined-plane in strict accordance with sterile working; 25 ℃ of constant temperature culture are to full packages;
5) choose assorted, preservation: after inclined-plane not good to microbiological contamination, growth conditions is removed, residue inclined-plane is placed in to 4 ℃ of Refrigerator stores, standby;
(2) preparation of liquid spawn:
1) liquid spawn formula, i.e. seed culture medium (g/L): potato 200, glucose 20, peptone 2, potassium dihydrogen phosphate 2, magnesium sulfate 1, VB1 0.01, pH nature; Wheat bran fermentation medium is (g/L): wheat bran 120, glucose 15, peptone 2, pH nature;
2) preparation seed liquor medium: take after potato processing, add water boil 30 minutes, filter, then add glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate 1, VB1, filters packing, 50mL/250mL triangular flask while hot after fully dissolving;
3) sterilizing, 121 ℃, sterilizing 20min;
4) prepare seed liquor: after sterilizing finishes, taking-up cools, in strict accordance with sterile working inoculation, get 5 0.5 cm 2fresh slant strains is inoculated in seed culture medium, and 25 ℃, 150rpm shaking table is cultivated 7 days;
5) preparation fermentation medium: get wheat bran, add clear water to boil 20-30min, filtrate, boils in liquid and add glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate to wheat bran, after medicine dissolves, adds clear water to supply moisture, packing, 100mL/250mL triangular flask;
6) sterilizing: 121 ℃, sterilizing 20min;
7) inoculation, cultivation: the seed liquor preparing is moved into inoculum concentration 8% in the 250mL triangular flask of dress fermentation medium, 25 ℃, 150rpm cultivates 7 days, obtains brown liquid fungus culture;
8) choose assorted: whether observe zymotic fluid by outward appearance every day muddy, has tart flavour, and explanation is contaminated by bacterial, and removes;
(3) making of cultivated species:
1) wheat soil medium (%): wheat 92, gypsum 5, turfy soil 3;
2) boil wheat: select the wheat of free from insect pests, pour in boiling water and boil 20~25 minutes, and constantly stir, boil to wheat and during without the white heart, cease fire wheat is pulled out immediately, the anhydrous mark to surface dries in the air;
3) batching: gypsum, turfy soil are added in proportion, and turned evenly, can bottle;
4) bottling sterilizing: 121 ℃, sterilizing 2~2.5 hours;
5) inoculation: by prepared liquid spawn, the inoculum concentration with 15% evenly sprays in cultivated species, requires operation in strict accordance with sterile working;
6) cultivate: postvaccinal cultivated species is placed in to 25 ℃ of constant temperature culture chamber shadings and cultivates, to full bottle;
7) choose assorted: after not good to microbiological contamination, growth conditions cultivated species is removed, residue bacterial classification is saved backup.
In order to determine best culture matrix, the present invention has carried out a large amount of experimental research work, finally take the lead in, in brown mushroom expansion is numerous, having created the three-class strain multiplication technique system of female kind of (slant strains) → original seed (liquid spawn) → cultivated species (wheat kind).Adopting this expansion traditional font is than traditional solid-state three-class strain, expands numerous cycle to have shortened 38 days.
It is method for building up that bacterial classification disclosed by the invention expands traditional font, is a kind of foundation of innovation system.Because tradition is separately carried out mother's kind, original seed, three grades of kinds, the present invention combines three, and innovative point is changing traditional wheat original seed into liquid original seed, and the growth cycle of bacterial classification has been shortened in the foundation of this individual system greatly, and strain quality is excellent.
The main excellent feature of brown mushroom MC441 bacterial strain that the present invention selects is:
1. this bacterial strain mushroom cap surface is nature sepia, mushroom handle, bacterial context white; Good mushroom quality, fruit body rounding, mushroom lid is thick, solid, not concave crown; Delicious flavour, mouthfeel is fresh and tender, long fresh-keeping period.Mycelial growth rate is fast.Output is high, under the same conditions, than white mushroom kind, does sth. in advance 2-3 days fruitings, and mushroom tide is neat, and output reaches 9-10 kilogram/square.3. fruiting temperature range is wide, and especially low temperature ability is stronger than conventional white mushroom, and at 5 ℃-8 ℃, yield increased 2796 is high by 70%.Culture technique is simple and easy, culturing raw material source is wide.Its culture material formula is take paddy and wheat grass as primary raw material, and composts or fertilisers of cultivating and fermentation reactor system method are identical with conventional mushroom.
The good effect that brown mushroom three-class strain expanding propagation method disclosed by the invention compared with prior art had is:
(1) bacterial classification is purer, and liquid spawn produces through screening, purifies, and belongs to pure culture.
(2) cycle shorter, adopt liquid spawn from the production of hybrid seeds to mycelia, to cover with cultivation bag and only need 30 days.
(3) pollute still less, within after employing liquid-spawn inoculation 24 hours, sprout, bacterial classification sprouting speed exceedes varied bacteria growing speed.
(4) fruiting neatly goes on the market early, adopts the brown mushroom of liquid spawn plantation, and fruiting is neat, can shift to an earlier date listing in 2 months.
Embodiment
Below in conjunction with embodiment, the present invention is described, the scheme of embodiment described here, do not limit the present invention, one of skill in the art can make improvements and change according to spirit of the present invention, these described improvement and variation all should be considered as within the scope of the invention, and scope of the present invention and essence are limited by claim.Wherein brown mushroom MC441 draws from Fujian Province's mushroom externally on sale.
Embodiment 1
Brown mushroom three-class strain expands the foundation of traditional font system
1.1 female preparations of planting:
(1) Mother culture based formulas, i.e. turfy soil medium (g/L): turfy soil 600g, glucose 20g, wheat 100g, magnesium sulfate 1.5g, potassium dihydrogen phosphate 1g, peptone 2g, water 1000mL.
(2) preparation medium.Get appropriate turfy soil, wheat, add in a certain amount of clear water and boil 20~30min, by 4 layers of filtered through gauze, obtain filtrate.In filtrate, add magnesium sulfate, potassium dihydrogen phosphate, peptone, glucose, agar, after medicine dissolves, supply moisture, packing test tube.
(3) sterilizing.121 ℃, sterilizing 20min.After sterilizing finishes, take out pendulum inclined-plane.
(4) inoculation, cultivation.In strict accordance with sterile working, brown mushroom mycelium is inoculated in to middle part, inclined-plane.25 ℃ of constant temperature culture are to full packages.
(5) choose assorted, preservation.After inclined-plane not good to microbiological contamination, growth conditions is removed, residue inclined-plane is placed in to 4 ℃ of Refrigerator stores, standby.
The preparation of 1.2 liquid spawns:
(1) liquid spawn formula, i.e. seed culture medium (g/L): potato 200, glucose 20, peptone 2, potassium dihydrogen phosphate 2, magnesium sulfate 1, VB1 0.01, pH nature.Wheat bran fermentation medium is (g/L): wheat bran 120, glucose 15, peptone 2, pH nature.
(2) preparation seed liquor medium.Take appropriate potato, clean peeling, trachoma, chopping, adds suitable quantity of water and boils half an hour, filtered through gauze, then add glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate 1, VB1, filtered through gauze while hot after fully dissolving, packing, 50mL/250mL triangular flask.
(3) sterilizing.121 ℃, sterilizing 20min.
(4) prepare seed liquor.After sterilizing finishes, taking-up cools.In strict accordance with sterile working inoculation, get 5 0.5 cm 2fresh slant strains is inoculated in seed culture medium, and 25 ℃, 150rpm shaking table is cultivated 7 days.
(5) preparation fermentation medium.Get appropriate wheat bran, add a certain amount of clear water to boil 20-30min, by 4 layers of filtered through gauze, obtain filtrate.To wheat bran, boil in liquid and add glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate, after medicine dissolves, add a certain amount of clear water to supply moisture, packing, 100mL/250mL triangular flask.
(6) sterilizing.121 ℃, sterilizing 20min.
(7) inoculation, cultivation.The seed liquor preparing is moved into inoculum concentration 8% in the 250mL triangular flask of dress fermentation medium, 25 ℃, 150rpm cultivates 7 days, obtains brown liquid fungus culture.
(8) choose assorted.Whether observe zymotic fluid by outward appearance every day muddy, has tart flavour, and explanation is contaminated by bacterial, and removes.After fermentation ends, 1. outward appearance is observed, zymotic fluid clarification, and a fairly large number of diameter 2-3mm mycelium pellet is arranged at bottom, or in zymotic fluid thickness, has more mycelia, has illustrated that polysaccharide produces, and has the distinctive fragrant of mushroom, without tart flavour.2. microscopic examination, whether the zymotic fluid that takes a morsel, by sarranine or violet staining, have living contaminants at oily Microscopic observation.Normal fermentation liquid spawn is continued to employ.
The making of 1.3 cultivated speciess:
(1) wheat soil medium (%): wheat 92, gypsum 5, turfy soil 3.
(2) boil wheat.Select the wheat of free from insect pests, pour in boiling water and boil 20~25 minutes, during boiling drag for the floating foreign material such as wheat is flat, and constantly stir, boil to wheat and during without the white heart, cease fire immediately wheat is pulled out, must guard against wheat epidermis is boiled brokenly.Anhydrous mark dries in the air in cement flooring to surface.
(3) batching.Gypsum, turfy soil are added in proportion, and turned evenly, can bottle.  
(4) bottling sterilizing.121 ℃, sterilizing 2~2.5 hours.
(5) inoculation.By prepared liquid spawn, the inoculum concentration with 15% evenly sprays in cultivated species, in strict accordance with sterile working, requires operation.
(6) cultivate.Postvaccinal cultivated species is placed in to 25 ℃ of constant temperature culture chamber shadings and cultivates, to full bottle.
(7) choose assorted.After cultivated species not good to microbiological contamination, growth conditions is removed, residue bacterial classification is saved backup.
Embodiment 2
A kind of brown mushroom three-class strain expands the method for building up of traditional font system
(1) female preparation of planting:
1) Mother culture based formulas, i.e. turfy soil medium (g/L): turfy soil 600g, glucose 20g, wheat 100g, magnesium sulfate 1.5g, potassium dihydrogen phosphate 1g, peptone 2g, water 1000mL;
2) preparation medium: get appropriate turfy soil, wheat, add in the clear water of 1000ml and boil 30min, filtrate; In filtrate, add magnesium sulfate, potassium dihydrogen phosphate, peptone, glucose, agar, after medicine dissolves, supply moisture, packing test tube;
3) sterilizing: 121 ℃, sterilizing 20min, after sterilizing finishes, takes out pendulum inclined-plane;
4) inoculation, cultivation: brown mushroom mycelium is inoculated in to middle part, inclined-plane in strict accordance with sterile working; 25 ℃ of constant temperature culture are to full packages;
5) choose assorted, preservation: after inclined-plane not good to microbiological contamination, growth conditions is removed, residue inclined-plane is placed in to 4 ℃ of Refrigerator stores, standby;
(2) preparation of liquid spawn:
1) liquid spawn formula, i.e. seed culture medium (g/L): potato 200, glucose 20, peptone 2, potassium dihydrogen phosphate 2, magnesium sulfate 1, VB1 0.01, pH nature; Wheat bran fermentation medium is (g/L): wheat bran 120, glucose 15, peptone 2, pH nature;
2) preparation seed liquor medium: take after potato processing, add water boil 30 minutes, filter, then add glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate 1, VB1, filters packing, 50mL/250mL triangular flask while hot after fully dissolving;
3) sterilizing, 121 ℃, sterilizing 20min;
4) prepare seed liquor: after sterilizing finishes, taking-up cools, in strict accordance with sterile working inoculation, get 5 0.5 cm 2fresh slant strains is inoculated in seed culture medium, and 25 ℃, 150rpm shaking table is cultivated 7 days;
5) preparation fermentation medium: get wheat bran, add clear water to boil 20min, filtrate, boils in liquid and add glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate to wheat bran, after medicine dissolves, adds clear water to supply moisture, packing, 100mL/250mL triangular flask;
6) sterilizing: 121 ℃, sterilizing 20min;
7) inoculation, cultivation: the seed liquor preparing is moved into inoculum concentration 8% in the 250mL triangular flask of dress fermentation medium, 25 ℃, 150rpm cultivates 7 days, obtains brown liquid fungus culture;
8) choose assorted: whether observe zymotic fluid by outward appearance every day muddy, has tart flavour, and explanation is contaminated by bacterial, and removes;
(3) making of cultivated species:
1) wheat soil medium (%): wheat 92, gypsum 5, turfy soil 3;
2) boil wheat: select the wheat of free from insect pests, pour in boiling water and boil 20~25 minutes, and constantly stir, boil to wheat and during without the white heart, cease fire wheat is pulled out immediately, the anhydrous mark to surface dries in the air;
3) batching: gypsum, turfy soil are added in proportion, and turned evenly, can bottle;
4) bottling sterilizing: 121 ℃, sterilizing 2.5 hours;
5) inoculation: by prepared liquid spawn, the inoculum concentration with 15% evenly sprays in cultivated species, requires operation in strict accordance with sterile working;
6) cultivate: postvaccinal cultivated species is placed in to 25 ℃ of constant temperature culture chamber shadings and cultivates, to full bottle;
7) choose assorted: after not good to microbiological contamination, growth conditions cultivated species is removed, residue bacterial classification is saved backup.

Claims (1)

1. brown mushroom three-class strain expands a method for building up for traditional font system, it is characterized in that it comprises that female kind, the three-class strain of liquid spawn, cultivated species expand numerous combining, and the one of foundation expands traditional font system, and its method is as follows:
(1) female preparation of planting:
1) Mother culture based formulas, i.e. turfy soil medium (g/L): turfy soil 600, glucose 20, wheat 100, magnesium sulfate 1.5, potassium dihydrogen phosphate 1, peptone 2, water 1000mL;
2) preparation medium: get appropriate turfy soil, wheat, add in the clear water of 1000ml and boil 20~30min, filtrate; In filtrate, add magnesium sulfate, potassium dihydrogen phosphate, peptone, glucose, agar, after medicine dissolves, supply moisture, packing test tube;
3) sterilizing: 121 ℃, sterilizing 20min, after sterilizing finishes, takes out pendulum inclined-plane;
4) inoculation, cultivation: brown mushroom mycelium is inoculated in to middle part, inclined-plane in strict accordance with sterile working; 25 ℃ of constant temperature culture are to full packages;
5) choose assorted, preservation: after inclined-plane not good to microbiological contamination, growth conditions is removed, residue inclined-plane is placed in to 4 ℃ of Refrigerator stores, standby;
(2) preparation of liquid spawn:
1) liquid spawn formula, i.e. seed culture medium (g/L): potato 200, glucose 20, peptone 2, potassium dihydrogen phosphate 2, magnesium sulfate 1, VB1 0.01, pH nature; Wheat bran fermentation medium is (g/L): wheat bran 120, glucose 15, peptone 2, pH nature;
2) preparation seed liquor medium: take after potato processing, add water boil 30 minutes, filter, then add glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate, VB1, filter while hot packing, 50mL/250mL triangular flask after fully dissolving;
3) sterilizing, 121 ℃, sterilizing 20min;
4) prepare seed liquor: after sterilizing finishes, taking-up cools, in strict accordance with sterile working inoculation, get 5 0.5 cm 2fresh slant strains is inoculated in seed culture medium, and 25 ℃, 150rpm shaking table is cultivated 7 days;
5) preparation fermentation medium: get wheat bran, add clear water to boil 20-30min, filtrate, boils in liquid and add glucose, peptone, magnesium sulfate, potassium dihydrogen phosphate to wheat bran, after medicine dissolves, adds clear water to supply moisture, packing, 100mL/250mL triangular flask;
6) sterilizing: 121 ℃, sterilizing 20min;
7) inoculation, cultivation: the seed liquor preparing is moved into inoculum concentration 8% in the 250mL triangular flask of dress fermentation medium, 25 ℃, 150rpm cultivates 7 days, obtains brown liquid fungus culture;
8) choose assorted: whether observe zymotic fluid by outward appearance every day muddy, has tart flavour, and explanation is contaminated by bacterial, and removes;
(3) making of cultivated species:
1) wheat soil medium (%): wheat 92, gypsum 5, turfy soil 3;
2) boil wheat: select the wheat of free from insect pests, pour in boiling water and boil 20~25 minutes, and constantly stir, boil to wheat and during without the white heart, cease fire wheat is pulled out immediately, the anhydrous mark to surface dries in the air;
3) batching: gypsum, turfy soil are added in proportion, and turned evenly, can bottle;
4) bottling sterilizing: 121 ℃, sterilizing 2~2.5 hours;
5) inoculation: by prepared liquid spawn, the inoculum concentration with 15% evenly sprays in cultivated species, requires operation in strict accordance with sterile working;
6) cultivate: postvaccinal cultivated species is placed in to 25 ℃ of constant temperature culture chamber shadings and cultivates, to full bottle;
7) choose assorted: after not good to microbiological contamination, growth conditions cultivated species is removed, residue bacterial classification is saved backup.
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