CN109170144A - Method based on multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed - Google Patents
Method based on multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed Download PDFInfo
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- CN109170144A CN109170144A CN201811306209.5A CN201811306209A CN109170144A CN 109170144 A CN109170144 A CN 109170144A CN 201811306209 A CN201811306209 A CN 201811306209A CN 109170144 A CN109170144 A CN 109170144A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
- A23K10/38—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material from distillers' or brewers' waste
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention discloses a kind of methods based on multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed, specifically includes the following steps: (1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, incubation time: 18-24h.(2) liquid seeds are prepared: will be inoculated into the triangular flask equipped with liquid seed culture medium from slant medium by step (1) activated strain, be placed in constant-temperature table after 30 DEG C of 18~120h of culture be transferred to it is spare in refrigerator；(3) Cottonseed Meal ferments: the liquid seeds prepared in step (2) being seeded to Cottonseed Meal and are fermented, inoculum concentration is 3~30%, and fermentation temperature is 30 DEG C, and continuously ferment 24~72h；Fermented product is dried to obtain after fermentation at 60 DEG C, measures crude protein and free gossypol content in fermented product.Strain after activation is seeded to Cottonseed Meal to ferment, reduce the free gossypol in fermentation substrate using the effect of microorganism and improves amino acid and small peptide content.
Description
Technical field
The present invention relates to molecular biology fields, and in particular to one kind is based on multi-cultur es cooperative fermentation preparation yellow serofluid and cotton
The method of seed dregs of rice mixed type biology feed.
Background technique
Biological feedstuff is generally referred to using feed and feed addictive as object, with genetic engineering, protein engineering, fermentation work
The new and high technologies such as journey are that means utilize the beneficial microbes such as lactic acid bacteria, saccharomycete and bacillus under manual control condition
Anti-nutritional factors in raw material is decomposed or conversion, generation can more be searched for food by livestock and poultry, digestion, be inhaled by the growth metabolism activity of itself
It receives, nutrient is higher and the feed of nonhazardous effect.Currently, the market value of biological feedstuff reaches annual 3000000000 dollars, and with average annual
20% speed increase, it is contemplated that by 2025, the market volume of biological feedstuff was up to 20,000,000,000 dollars/year in world wide.Mesh
Preceding China has 1,000 Yu Jia enterprises to specialize in biological enzyme formulation, probiotic, plant extract species biological feedstuff and its addition
The production and processing of agent.But compared with foreign countries, 3% of biological feedstuff (fermented feed) popularity rate less than feed total amount, biological feedstuff
Research start late with industrialization, whole research and development lag behind developed country with industrialization level, and develop not perfectly flat weighing apparatus.Perhaps
The production of more key organism feeds lacks independent intellectual property right also in imitated horizontal or heavy dependence foreign technology.
With the continuous fast development of China's feed and aquaculture, protein word material shortage of resources oneself sent out as feed industry
The important restriction factor of exhibition.Presently mainly solved by protein additive amount in reduction feed and inlet part protein feed
Certainly, this will not only spend a large amount of foreign exchange, and the production cost of feedstuff industry is difficult to decrease.In addition, many resources in China
Puffed soybean protein feed abundant, cannot in animal diet due to containing anti-nutritional factors or nutritional ingredient imbalance etc.
It makes full use of.Under this new market condition, exploitation biological feedstuff raw material is a kind of solution protein feed resource shortage
One of effective way.
Yellow serofluid (Yellow serofluid) is the waste water of Bean Products Factory's discharge, is suitble to because of its nutrition rich in
The growth of microorganism.It was found that the content of general solid content is 1% or more in yellow serofluid, wherein protein content is about
0.3%, fat content is about 0.08%, content of reducing sugar is 0.15% or so；In addition, also containing isoflavones, soybean soapstock
The several functions compositions such as glycosides, yellow serofluid direct emission is not made full use of its nutrition not only by China's most enterprises at present
Value, and direct emission pollutes environment, is unfavorable for clean manufacturing and increases soybean processing added value.It is done in yellow serofluid waste liquid
Gu object form content is high, high concentrated organic wastewater is easily caused to pollute, difficulty of governance is big, and causes the serious waste of resource.Institute
Yellow serofluid is used agricultural resources economy with necessary, it is converted to a kind of good biological word material resource.
Cottonseed Meal (Cottonseed meal) is the byproduct extracted after cottonseed oil, and gross protein value may be up to 53%,
It is only second to dregs of beans, is important one of vegetable protein resource.China's Cottonseed Meal is resourceful, cheap, cottonseed protein
The approximate bean protein in quality, is a kind of important feed protein resource, is widely used in feed industry.But due to cotton
The anti-nutritional factors contained in the seed dregs of rice, especially free gossypol and amino acid ratio are unbalanced, simultaneously because cottonseed structure
Palatability difference and digestibility caused by densification are relatively low etc. to limit its feeding value.Currently, handling cottonseed using fermentation technique
The dregs of rice are one of the methods for effectively improving Cottonseed Meal quality, have researcher to mix soy meal and cotton seed meal and have been fermented with aspergillus oryzae
Afterwards, in addition to the toxicity that gossypol is effectively reduced, the free radical scavenging activity in fish tissues can also be improved.In addition, in fermentation process
In can also generate directly by animal absorb small peptide and other various bioactivators, this extremely has the growth of animal
Benefit.Taste compound can be generated during the fermentation simultaneously, this has apparent phagostimulating effect for young animal.Also, by
It is degraded in portion carbohydrate, Cottonseed Meal compact texture becomes loose, and palatability will significantly improve.
Currently, correlative study both at home and abroad is concentrated mainly on yellow serofluid, Cottonseed Meal list material fermentation and Cottonseed Meal feeding animals
Aspect is influenced caused by afterwards, for solid fermentation will be carried out after yellow serofluid and Cottonseed Meal combination, there is not yet related report.
Therefore, it is necessary to develop a kind of based on multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed
Method；On the basis of early period is about yellow serofluid and Cottonseed Meal fermentation research, yellow serofluid and Cottonseed Meal are mixed by a certain percentage
After conjunction, the fermentation of microbe in solid state method is carried out, produces multi-functional, environment protection biological feed；Fermentation is reduced using the effect of microorganism
Free gossypol and raising amino acid and small peptide content in substrate；Obtain ecological benefits, social benefit and Business Economic Benefit
It is preferable unified.
Summary of the invention
The technical problem to be solved by the present invention is to provide a kind of multi-functional environment-protection and the low-cost multi-cultur es that are based on cooperate with
The method of fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed.
In order to solve the above technical problems, the technical solution adopted by the present invention is that, it should be yellow based on the preparation of multi-cultur es cooperative fermentation
The method of pulp-water and Cottonseed Meal mixed type biology feed, specifically includes the following steps:
(1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, when culture
Between: 18-24h.
(2) liquid seeds are prepared: will be inoculated into from slant medium equipped with liquid strain by step (1) activated strain
In the triangular flask of sub- culture medium, be placed in constant-temperature table after 30 DEG C of 18~120h of culture be transferred to it is spare in refrigerator；
(3) Cottonseed Meal ferments: the liquid seeds prepared in step (2) are seeded to Cottonseed Meal and are fermented, inoculum concentration
It is 3~30%, fermentation temperature is 30 DEG C, and continuously ferment 24~72h；Fermented product, measurement hair are dried to obtain after fermentation at 60 DEG C
Crude protein and free gossypol content in ferment product.
By adopting the above technical scheme, the strain after activation is seeded to Cottonseed Meal to ferment, utilizes the effect of microorganism
It reduces the free gossypol in fermentation substrate and improves amino acid and small peptide content；Cottonseed Meal is carried out using microbe fermentation method
Detoxification, can influence to avoid chemical addition agent and physical method to cottonseed protein functional character, will not to its flavor, color with
And palatability constitutes damage, is also greatly improved by various digestive enzymes, amino acid, vitamin and small peptide etc. that fermentation generates
The nutritive value of Cottonseed Meal and the nutritional properties of albumen, greatly improve the economic value of Cottonseed Meal.
As the preferred technical solution of the present invention, the strain is mould or/and bacillus or/and saccharomycete.
As the preferred technical solution of the present invention, the mould is aspergillus niger and/or geotrichum candidum, and the bacillus is withered
One or more of careless bacillus, bafillus natto and bacillus licheniformis；The saccharomycete is Candida utilis ferment
Female, outstanding one or more of fourth Pichia pastoris and candida tropicalis.
As the preferred technical solution of the present invention, the strain is mould；Slant medium in the step (2) is beans
Bud juice culture medium, formula are as follows: bean sprouts 200g adds water 1000ml, filters after boiling 10min, and filtrate adds 2% glucose and 2% fine jade
Rouge；Liquid seed culture medium in the step (2) is bean sprout juice fluid nutrient medium, formula are as follows: bean sprouts 200g adds water
1000ml is filtered after boiling 10min, and filtrate adds 2% glucose.
As the preferred technical solution of the present invention, the strain is bacillus；Slant medium in the step (2)
For LB culture medium, formula are as follows: beef extract 5g, peptone 10g, sodium chloride 5g, 15~20g of agar, 1 000ml of water, pH value 7.0
~7.2；Liquid seed culture medium in the step (2) is LB liquid medium: beef extract 5g, peptone 10g, sodium chloride
5g, 1 000ml of water, pH value 7.0~7.2.
As the preferred technical solution of the present invention, the strain is saccharomycete；Slant medium in the step (2)
YPD culture medium, formula are as follows: yeast extract 10g, peptone 20g, glucose 20g, 15~20g of agar add water 1000ml；It is described
Liquid seed culture medium in step (2) is bean sprout juice fluid nutrient medium.
As the preferred technical solution of the present invention, step (3) the Cottonseed Meal fermentation is plate dibbling fermentation or/and triangle
The fermentation of bottle Cottonseed Meal or/and tray solid fermentation；The preparation of the primary dcreening operation culture medium of the plate dibbling fermentation: Cottonseed Meal 3%,
Agar 2%, pH value are 7.0 ± 0.5,121 DEG C of sterilizing 15min；The preparation of the culture medium of the triangular flask Cottonseed Meal fermentation: cotton
The seed dregs of rice 44%, wheat bran 10%, urea 2%, moisture 44% (press solid-liquid ratio 1:1, g/ml), then loading amount 50g/250ml, envelope
Mouthful, 121 DEG C of sterilizing 15min；The preparation of the tray solid fermentation culture medium: Cottonseed Meal 44%, wheat bran 10%, urea 2%,
Yellow serofluid 44% (presses solid-liquid ratio 1:1, g/ml), not sterilized；It is placed in 25cm × 45cm enamel tray, it is thin to be paved into about 3cm
Layer.It converts microprotein as far as possible by nonprotein nitrogen in yellow serofluid, improves protein content in feed；Improve yellow serofluid
And the protein quality of Cottonseed Meal fermented product, free gossypol content is reduced, reaches feeding standard, and be applied to animal
In raising.
As the preferred technical solution of the present invention, the Cottonseed Meal is dephenolization cottonseed protein, and wherein crude protein content is
49.90%, free gossypol content is 842mg/kg.
Compared with prior art, have the advantage that the strain after activation, which is seeded to Cottonseed Meal, to ferment, utilization is micro-
The effect of biology reduces the free gossypol in fermentation substrate and improves amino acid and small peptide content；Use microbe fermentation method pair
Cottonseed Meal carries out detoxification, can influence to avoid chemical addition agent and physical method to cottonseed protein functional character, will not be to its wind
Taste, color and palatability constitute damage, various digestive enzymes, amino acid, vitamin and the small peptide etc. generated by fermentation
The nutritive value of Cottonseed Meal and the nutritional properties of albumen are substantially increased, the economic value of Cottonseed Meal is greatly improved.
Detailed description of the invention
Fig. 1 is four kinds prepared in yellow serofluid and the method for Cottonseed Meal mixed type biology feed based on multi-cultur es cooperative fermentation
Photo figure after the yellow serofluid of single bacterium kind and Cottonseed Meal plate dibbling experiment culture；
Fig. 2 is to prepare tray in yellow serofluid and the method for Cottonseed Meal mixed type biology feed based on multi-cultur es cooperative fermentation to consolidate
Comparison photo figure before and after the fermentation of body fermentation test.
Specific embodiment
Embodiment 1: should be based on the method for multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed, tool
Body the following steps are included:
(1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, when culture
Between: 18-24h.
(2) liquid seeds are prepared: will be inoculated into from slant medium equipped with liquid strain by step (1) activated strain
In the triangular flask of sub- culture medium, be placed in constant-temperature table after 30 DEG C of 18~120h of culture be transferred to it is spare in refrigerator；
(3) Cottonseed Meal ferments: the liquid seeds prepared in step (2) are seeded to Cottonseed Meal and are fermented, inoculum concentration
It is 3~30%, fermentation temperature is 30 DEG C, and continuously ferment 24~72h；Fermented product, measurement hair are dried to obtain after fermentation at 60 DEG C
Crude protein and free gossypol content in ferment product；The strain is mould or/and bacillus or/and saccharomycete；It is described mould
Bacterium is aspergillus niger and/or geotrichum candidum, and the bacillus is bacillus subtilis, bafillus natto and bacillus licheniformis
One or more of；The saccharomycete be candida utili, outstanding one of fourth Pichia pastoris and candida tropicalis or
It is several；The strain is mould；Slant medium in the step (2) is bean sprouts medium, formula are as follows: bean sprouts 200g,
Add water 1000ml, filtered after boiling 10min, filtrate adds 2% glucose and 2% agar；Liquid seeds in the step (2)
Culture medium is bean sprout juice fluid nutrient medium, formula are as follows: bean sprouts 200g adds water 1000ml, filters after boiling 10min, filtrate adds
2% glucose；The strain is bacillus；Slant medium in the step (2) is LB culture medium, formula are as follows:
Beef extract 5g, peptone 10g, sodium chloride 5g, 15~20g of agar, 1 000ml of water, pH value 7.0~7.2；In the step (2)
Liquid seed culture medium be LB liquid medium: beef extract 5g, peptone 10g, sodium chloride 5g, 1 000ml of water, pH value
7.0~7.2；The strain is saccharomycete；Slant medium YPD culture medium in the step (2), formula are as follows: yeast extract
10g, peptone 20g, glucose 20g, 15~20g of agar add water 1000ml；Liquid seed culture medium in the step (2)
For bean sprout juice fluid nutrient medium；Step (3) the Cottonseed Meal fermentation is that plate dibbling fermentation or/and triangular flask Cottonseed Meal ferment
Or/and tray solid fermentation；The preparation of the primary dcreening operation culture medium of the plate dibbling fermentation: Cottonseed Meal 3%, agar 2%, pH value
It is 7.0 ± 0.5,121 DEG C of sterilizing 15min；The preparation of the culture medium of the triangular flask Cottonseed Meal fermentation: Cottonseed Meal 44%, wheat bran
10%, urea 2%, moisture 44%, then loading amount 50g/250ml, is sealed, 121 DEG C of sterilizing 15min；The tray solid fermentation
The preparation of culture medium: Cottonseed Meal 44%, wheat bran 10%, urea 2%, yellow serofluid 44% be not sterilized；Be placed on 25cm ×
In 45cm enamel tray, it is paved into about 3cm thin layer.It converts microprotein as far as possible by nonprotein nitrogen in yellow serofluid, improves feed
Middle protein content；The protein quality of yellow serofluid and Cottonseed Meal fermented product is improved, free gossypol content is reduced, reaches it
To feeding standard, and it is applied in animal feeding.
Embodiment 2: should be based on the method for multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed, tool
Body the following steps are included:
(1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, when culture
Between: 18-24h.
(2) liquid seeds are prepared: will be inoculated into from slant medium equipped with liquid strain by step (1) activated strain
In the triangular flask of sub- culture medium, be placed in constant-temperature table after 30 DEG C of 18~120h of culture be transferred to it is spare in refrigerator；The bacterium
Kind is mould；Slant medium in the step (2) is bean sprouts medium, formula are as follows: bean sprouts 200g adds water 1000ml,
It is filtered after boiling 10min, filtrate adds 2% glucose and 2% agar；Liquid seed culture medium in the step (2) is bean sprouts
Juice fluid nutrient medium, formula are as follows: bean sprouts 200g adds water 1000ml, filters after boiling 10min, and filtrate adds 2% glucose；
(3) Cottonseed Meal ferments: the liquid seeds prepared in step (2) being seeded to Cottonseed Meal and carry out plate dibbling hair
Ferment, inoculum concentration 15%, fermentation temperature are 30 DEG C, and continuously ferment 24~72h；Fermented product is dried to obtain after fermentation at 60 DEG C,
Measure the crude protein and free gossypol content in fermented product；The preparation of the primary dcreening operation culture medium of the plate dibbling fermentation: cottonseed
The dregs of rice 3%, agar 2%, pH value are 7.0 ± 0.5,121 DEG C of sterilizing 15min.
Embodiment 3: should be based on the method for multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed, tool
Body the following steps are included:
(1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, when culture
Between: 18-24h.
(2) liquid seeds are prepared: will be inoculated into from slant medium equipped with liquid strain by step (1) activated strain
In the triangular flask of sub- culture medium, be placed in constant-temperature table after 30 DEG C of 18~120h of culture be transferred to it is spare in refrigerator；The bacterium
Kind is bacillus；Slant medium in the step (2) is LB culture medium, formula are as follows: beef extract 5g, peptone 10g,
Sodium chloride 5g, 15~20g of agar, 1 000ml of water, pH value 7.0~7.2；Liquid seed culture medium in the step (2) is
LB liquid medium: beef extract 5g, peptone 10g, sodium chloride 5g, 1 000ml of water, pH value 7.0~7.2；
(3) Cottonseed Meal ferments: the liquid seeds prepared in step (2) being seeded to Cottonseed Meal and carry out plate dibbling hair
Ferment, inoculum concentration 15%, fermentation temperature are 30 DEG C, and continuously ferment 24~72h；Fermented product is dried to obtain after fermentation at 60 DEG C,
Measure the crude protein and free gossypol content in fermented product；The preparation of the primary dcreening operation culture medium of the plate dibbling fermentation: cottonseed
The dregs of rice 3%, agar 2%, pH value are 7.0 ± 0.5,121 DEG C of sterilizing 15min.
Embodiment 4: should be based on the method for multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed, tool
Body the following steps are included:
(1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, when culture
Between: 18-24h.
(2) liquid seeds are prepared: will be inoculated into from slant medium equipped with liquid strain by step (1) activated strain
In the triangular flask of sub- culture medium, be placed in constant-temperature table after 30 DEG C of 18~120h of culture be transferred to it is spare in refrigerator；The bacterium
Kind is saccharomycete；Slant medium YPD culture medium in the step (2), formula are as follows: yeast extract 10g, peptone 20g, Portugal
Grape sugar 20g, 15~20g of agar add water 1000ml；Liquid seed culture medium in the step (2) is bean sprout juice Liquid Culture
Base；Formula are as follows: bean sprouts 200g adds water 1000ml, filters after boiling 10min, and filtrate adds 2% glucose；
(3) Cottonseed Meal ferments: the liquid seeds prepared in step (2) being seeded to Cottonseed Meal and carry out plate dibbling hair
Ferment, inoculum concentration 15%, fermentation temperature are 30 DEG C, and continuously ferment 24~72h；Fermented product is dried to obtain after fermentation at 60 DEG C,
Measure the crude protein and free gossypol content in fermented product；The preparation of the primary dcreening operation culture medium of the plate dibbling fermentation: cottonseed
The dregs of rice 3%, agar 2%, pH value is naturally, 121 DEG C of sterilizing 15min.
By in embodiment 2, embodiment 3, embodiment 4 three kinds of strains and these three strain mixing dibblings contain to Cottonseed Meal
On the culture medium flat plate that amount is 3%, the preparation of the primary dcreening operation culture medium of the plate dibbling fermentation: Cottonseed Meal 3%, agar 2%,
PH value is naturally, 121 DEG C of sterilizing 15min；3 parallel tests are carried out, observes and records, is as a result illustrated in fig. 1 shown below.It can by lower Fig. 1
See, upgrowth situation slight difference of the different strain on the plating medium using Cottonseed Meal as sole substrate, but each bacterium of primary election
Strain can be grown on Cottonseed Meal culture medium.Meanwhile the Mixed Microbes of mould and bacillus, saccharomycete can also contain simultaneously
It is grown on the culture medium of Cottonseed Meal.
Embodiment 5: should be based on the method for multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed, tool
Body the following steps are included:
(1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, when culture
Between: 18-24h.
(2) liquid seeds are prepared: will be inoculated into from slant medium equipped with liquid strain by step (1) activated strain
In the triangular flask of sub- culture medium, be placed in constant-temperature table after 30 DEG C of 18~120h of culture be transferred to it is spare in refrigerator；The bacterium
Kind is mould；Slant medium in the step (2) is bean sprouts medium, formula are as follows: bean sprouts 200g adds water 1000ml,
It is filtered after boiling 10min, filtrate adds 2% glucose and 2% agar；Liquid seed culture medium in the step (2) is bean sprouts
Juice fluid nutrient medium, formula are as follows: bean sprouts 200g adds water 1000ml, filters after boiling 10min, and filtrate adds 2% glucose；
(3) Cottonseed Meal ferments: the liquid seeds prepared in step (2) being seeded to Cottonseed Meal and carry out triangular flask Cottonseed Meal
Fermentation, inoculum concentration 15%, fermentation temperature are 30 DEG C, and continuously ferment 24~72h；Fermentation production is dried to obtain after fermentation at 60 DEG C
Product measure crude protein and free gossypol content in fermented product；The preparation of the culture medium of the triangular flask Cottonseed Meal fermentation:
Cottonseed Meal 44%, wheat bran 10%, urea 2%, yellow serofluid 44%, then loading amount 50g/250ml, is sealed, 121 DEG C of sterilizings
15min。
Embodiment 6: should be based on the method for multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed, tool
Body the following steps are included:
(1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, when culture
Between: 18-24h.
(2) liquid seeds are prepared: will be inoculated into from slant medium equipped with liquid strain by step (1) activated strain
In the triangular flask of sub- culture medium, be placed in constant-temperature table after 30 DEG C of 18~120h of culture be transferred to it is spare in refrigerator；The bacterium
Kind is bacillus；Slant medium in the step (2) is LB culture medium, formula are as follows: beef extract 5g, peptone 10g,
Sodium chloride 5g, 15~20g of agar, 1 000ml of water, pH value 7.0~7.2；Liquid seed culture medium in the step (2) is
LB liquid medium: beef extract 5g, peptone 10g, sodium chloride 5g, 1 000ml of water, pH value 7.0~7.2；
(3) Cottonseed Meal ferments: the liquid seeds prepared in step (2) being seeded to Cottonseed Meal and carry out triangular flask Cottonseed Meal
Fermentation, inoculum concentration 15%, fermentation temperature are 30 DEG C, and continuously ferment 24~72h；Fermentation production is dried to obtain after fermentation at 60 DEG C
Product measure crude protein and free gossypol content in fermented product；The preparation of the culture medium of the triangular flask Cottonseed Meal fermentation:
Cottonseed Meal 44%, wheat bran 10%, urea 2%, moisture 44%, then loading amount 50g/250ml, is sealed, 121 DEG C of sterilizing 15min.
Embodiment 7: should be based on the method for multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed, tool
Body the following steps are included:
(1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, when culture
Between: 18-24h.
(2) liquid seeds are prepared: will be inoculated into from slant medium equipped with liquid strain by step (1) activated strain
In the triangular flask of sub- culture medium, be placed in constant-temperature table after 30 DEG C of 18~120h of culture be transferred to it is spare in refrigerator；The bacterium
Kind is saccharomycete；Slant medium YPD culture medium in the step (2), formula are as follows: yeast extract 10g, peptone 20g, Portugal
Grape sugar 20g, 15~20g of agar add water 1000ml；Liquid seed culture medium in the step (2) is bean sprout juice Liquid Culture
Base；Formula are as follows: bean sprouts 200g adds water 1000ml, filters after boiling 10min, and filtrate adds 2% glucose；
(3) Cottonseed Meal ferments: the liquid seeds prepared in step (2) being seeded to Cottonseed Meal and carry out triangular flask Cottonseed Meal
Fermentation, inoculum concentration 15%, fermentation temperature are 30 DEG C, and continuously ferment 24~72h；Fermentation production is dried to obtain after fermentation at 60 DEG C
Product measure crude protein and free gossypol content in fermented product；The preparation of the culture medium of the triangular flask Cottonseed Meal fermentation:
Cottonseed Meal 44%, wheat bran 10%, urea 2%, moisture 44%, then loading amount 50g/250ml, is sealed, 121 DEG C of sterilizing 15min.
Single bacterium triangular flask fermentation test: three kinds of strains in embodiment 5, embodiment 6, embodiment 7 are subjected to single bacterium respectively
Kind triangular flask Cottonseed Meal solid fermentation experiment, the results are shown in Table 1.
Crude protein content and free gossypol measurement result in Cottonseed Meal after the fermentation of 1 single bacterium of table
Note: data analysis uses 18.0 software one-way analysis of variance (ANOVA) of SPSS
--- indicate that difference is not significant (p > 0.05) compared with blank control (raw material)
* the significant difference (p < 0.05) compared with blank control (raw material) is indicated
As it can be seen from table 1 selected mould and saccharomycete can significantly improve the content of crude protein in Cottonseed Meal, together
When single bacterium can significantly reduce the content of free gossypol in feed.
Mixed bacterium triangular flask fermentation optimization test: by three kinds of strains in embodiment 5, embodiment 6, embodiment 7 by not year-on-year
Example mixing carries out mixed bacterium triangular flask Cottonseed Meal solid fermentation experiment, and the results are shown in Table 2.
Crude protein content and free cotton in Cottonseed Meal after 2 different proportion of table (mould, bacillus, saccharomycete) mixed fungus fermentation
Phenol measurement result
Note: data analysis uses 18.0 software one-way analysis of variance (ANOVA) of SPSS
* the significant difference (p < 0.05) compared with blank control (raw material) is indicated
* * indicates that difference is extremely significant (p < 0.001) compared with blank control (raw material).
As can be seen from the above table, bacteria solid fermentation cotton is mixed composed by mould and bacillus, saccharomycete different proportion
The seed dregs of rice can reduce the content of free gossypol in raw material, compared with Cottonseed Meal, mould and bacillus, saccharomycete extremely significantly
It is obvious to increase albumen effect in special ratios mixing.Triangular flask cotton is carried out with the Mixed Microbes that mould and bacillus, saccharomycete are
Seed dregs of rice fermentation test, zymotechnique are as follows: fermentation medium: Cottonseed Meal 44%, wheat bran 10%, urea 2%, yellow serofluid 44%
(pressing solid-liquid ratio 1:1, g/ml), loading amount 50g/250ml, sealing, 121 DEG C of sterilizing 15min；Inoculum concentration is 15%, fermentation temperature
It is 30 DEG C, fermentation time 72h.After fermented, in Cottonseed Meal crude protein compared to raw material have a degree of raising (from
54.78% to 59.41%), and wherein free gossypol content has extremely significant decline (p < 0.001) compared to raw material.
Embodiment 8: should be based on the method for multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed, tool
Body the following steps are included:
(1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, when culture
Between: 18-24h.
(2) liquid seeds are prepared: will be inoculated into from slant medium equipped with liquid strain by step (1) activated strain
In the triangular flask of sub- culture medium, be placed in constant-temperature table after 30 DEG C of 18~120h of culture be transferred to it is spare in refrigerator；The bacterium
Kind is aspergillus；Slant medium in the step (2) is bean sprouts medium, formula are as follows: bean sprouts 200g adds water 1000ml,
It is filtered after boiling 10min, filtrate adds 2% glucose and 2% agar；Liquid seed culture medium in the step (2) is bean sprouts
Juice fluid nutrient medium, formula are as follows: bean sprouts 200g adds water 1000ml, filters after boiling 10min, and filtrate adds 2% glucose；
(3) Cottonseed Meal ferments: the liquid seeds prepared in step (2) being seeded to Cottonseed Meal and carry out tray solid hair
Ferment, inoculum concentration 15%, fermentation temperature are 30 DEG C, and continuously ferment 24~72h；Fermented product is dried to obtain after fermentation at 60 DEG C,
Measure the crude protein and free gossypol content in fermented product；The preparation of the tray solid fermentation culture medium: Cottonseed Meal
44%, wheat bran 10%, urea 2%, yellow serofluid 44% is sterilized；It is placed in 25cm × 45cm enamel tray, is paved into about 3cm
Thin layer.
Embodiment 9: should be based on the method for multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed, tool
Body the following steps are included:
(1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, when culture
Between: 18-24h.
(2) liquid seeds are prepared: will be inoculated into from slant medium equipped with liquid strain by step (1) activated strain
In the triangular flask of sub- culture medium, be placed in constant-temperature table after 30 DEG C of 18~120h of culture be transferred to it is spare in refrigerator；The bacterium
Kind is bacillus；Slant medium in the step (2) is LB culture medium, formula are as follows: beef extract 5g, peptone 10g,
Sodium chloride 5g, 15~20g of agar, 1 000ml of water, pH value 7.0~7.2；Liquid seed culture medium in the step (2) is
LB liquid medium: beef extract 5g, peptone 10g, sodium chloride 5g, 1 000ml of water, pH value 7.0~7.2；
(3) Cottonseed Meal ferments: the liquid seeds prepared in step (2) being seeded to Cottonseed Meal and carry out tray solid hair
Ferment, inoculum concentration 15%, fermentation temperature are 30 DEG C, and continuously ferment 24~72h；Fermented product is dried to obtain after fermentation at 60 DEG C,
Measure the crude protein and free gossypol content in fermented product；The preparation of the tray solid fermentation culture medium: Cottonseed Meal
44%, wheat bran 10%, urea 2%, yellow serofluid 44% is sterilized；It is placed in 25cm × 45cm enamel tray, is paved into about 3cm
Thin layer.
Embodiment 10: the method that yellow serofluid and Cottonseed Meal mixed type biology feed should be prepared based on multi-cultur es cooperative fermentation,
Specifically includes the following steps:
(1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, when culture
Between: 18-24h.
(2) liquid seeds are prepared: will be inoculated into from slant medium equipped with liquid strain by step (1) activated strain
In the triangular flask of sub- culture medium, be placed in constant-temperature table after 30 DEG C of 18~120h of culture be transferred to it is spare in refrigerator；The bacterium
Kind is saccharomycete；Slant medium YPD culture medium in the step (2), formula are as follows: yeast extract 10g, peptone 20g, Portugal
Grape sugar 20g, 15~20g of agar add water 1000ml；Liquid seed culture medium in the step (2) is bean sprout juice Liquid Culture
Base；Formula are as follows: bean sprouts 200g adds water 1000ml, filters after boiling 10min, and filtrate adds 2% glucose；
(3) Cottonseed Meal ferments: the liquid seeds prepared in step (2) being seeded to Cottonseed Meal and carry out tray solid hair
Ferment, inoculum concentration 15%, fermentation temperature are 30 DEG C, and continuously ferment 24~72h；Fermented product is dried to obtain after fermentation at 60 DEG C,
Measure the crude protein and free gossypol content in fermented product；The preparation of the tray solid fermentation culture medium: Cottonseed Meal
44%, wheat bran 10%, urea 2%, yellow serofluid 44% is sterilized；It is placed in 25cm × 45cm enamel tray, is paved into about 3cm
Thin layer.
The test of tray solid fermentation: the strain in embodiment 8, embodiment 9, embodiment 10 is respectively put into and is consolidated with tray
In 25cm × 45cm enamel tray of body fermentation medium, it is paved into about 3cm thin layer, after 8 layers of gauze covering fermentation 2d, before fermentation
With the comparison photo after fermentation as shown in Fig. 2, Cottonseed Meal darkens, surface lumps have certain slot stink, illustrate cottonseed
The dregs of rice completely can not be sterilized and directly ferment.
Basic principles and main features and advantage of the invention have been shown and described above.The technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe originals of the invention
Reason, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these change and change
Into all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended claims and its equivalent
Object defines.
Claims (8)
1. a kind of method based on multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed, feature exist
In, specifically includes the following steps:
(1) activated spawn: strain being seeded in slant medium and is activated, cultivation temperature: 28-32 DEG C, incubation time:
18-24h。
(2) liquid seeds are prepared: will be inoculated by step (1) activated strain from slant medium and be trained equipped with liquid seeds
In the triangular flask for supporting base, it is spare in refrigerator to be placed in constant-temperature table 30 DEG C of cultures 18~be transferred to afterwards for 24 hours；
(3) Cottonseed Meal ferment: the liquid seeds prepared in step (2) are seeded to Cottonseed Meal and are fermented, inoculum concentration be 3~
30%, fermentation temperature is 30 DEG C, and continuously ferment 24~72h；Fermented product is dried to obtain after fermentation at 60 DEG C, measures fermented product
In crude protein and free gossypol content.
2. according to claim 1 based on multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed
Method, which is characterized in that the strain is mould or/and bacillus or/and saccharomycete.
3. according to claim 2 based on multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed
Method, which is characterized in that the mould is aspergillus niger and/or geotrichum candidum, and the bacillus is bacillus subtilis, natto bud
One or more of spore bacillus and bacillus licheniformis；The saccharomycete is candida utili, outstanding fourth Pichia pastoris and heat
One or more of band Candida.
4. according to claim 1 based on multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed
Method, which is characterized in that the strain is mould；Slant medium in the step (2) is bean sprouts medium, formula
Are as follows: bean sprouts 200g adds water 1000ml, filters after boiling 10min, and filtrate adds 2% glucose and 2% agar；In the step (2)
Liquid seed culture medium be bean sprout juice fluid nutrient medium, formula are as follows: bean sprouts 200g adds water 1000ml, boils mistake after 10min
Filter, filtrate add 2% glucose.
5. according to claim 1 based on multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed
Method, which is characterized in that the strain is bacillus；Slant medium in the step (2) is LB culture medium, formula
Are as follows: beef extract 5g, peptone 10g, sodium chloride 5g, 15~20g of agar, 1 000ml of water, pH value 7.0~7.2；The step (2)
In liquid seed culture medium be LB liquid medium: beef extract 5g, peptone 10g, sodium chloride 5g, 1 000ml of water, pH value
7.0~7.2.
6. according to claim 1 based on multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed
Method, which is characterized in that the strain is saccharomycete；Slant medium YPD culture medium in the step (2), formula are as follows: ferment
Female cream 10g, peptone 20g, glucose 20g, 15~20g of agar add water 1000ml；Liquid seeds training in the step (2)
Supporting base is bean sprout juice fluid nutrient medium.
7. according to claim 1 based on multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed
Method, which is characterized in that step (3) Cottonseed Meal fermentation be plate dibbling fermentation or/and the fermentation of triangular flask Cottonseed Meal or/and
Tray solid fermentation；The preparation of the primary dcreening operation culture medium of plate dibbling fermentation: Cottonseed Meal 3%, agar 2%, pH value is 7.0 ±
0.5,121 DEG C of sterilizing 15min；The preparation of the culture medium of the triangular flask Cottonseed Meal fermentation: Cottonseed Meal 44%, wheat bran 10%, urine
Element 2%, yellow serofluid 44%, then loading amount 50g/250ml, is sealed, 121 DEG C of sterilizing 15min；The tray solid fermentation culture medium
Preparation: Cottonseed Meal 44%, wheat bran 10%, urea 2%, yellow serofluid 44% be not sterilized；It is placed on 25cm × 45cm enamel tray
It is interior, it is paved into about 3cm thin layer.
8. according to claim 7 based on multi-cultur es cooperative fermentation preparation yellow serofluid and Cottonseed Meal mixed type biology feed
Method, which is characterized in that the Cottonseed Meal is dephenolization cottonseed protein, and wherein crude protein content is 49.90%, free gossypol content
For 842mg/kg.
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