CN104996722B - A kind of method of the step combined ferment feed of multi-cultur es two - Google Patents

A kind of method of the step combined ferment feed of multi-cultur es two Download PDF

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CN104996722B
CN104996722B CN201510498368.XA CN201510498368A CN104996722B CN 104996722 B CN104996722 B CN 104996722B CN 201510498368 A CN201510498368 A CN 201510498368A CN 104996722 B CN104996722 B CN 104996722B
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culture
parts
seed
pure culture
fermentation
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CN104996722A (en
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赵建新
刘桂香
毛丙永
闫博文
田丰伟
范大明
赵国忠
王刚
陈卫
张灏
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Jiangnan University
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Abstract

The invention discloses a kind of method of the step combined ferment feed of multi-cultur es two, belong to field of feed.The present invention is using corn flour, palm kernel meal, dregs of beans, wheat bran, wheat-middlings as raw material, using bread mold, aspergillus oryzae, bacillus subtilis, Lactobacillus plantarum, saccharomyces cerevisiae as fermented bacterium, by the fermented feed of two steps acquisitions of aerobic fermentation and anaerobic fermentation.The dietary protein level to be fermented using this method is up to 30%~34%, 45%~62% is improved than raw material, alpha amylase content is up to 90~110U/g, and acid protease activity is up to 110~130U/g, is the feed of rich protein, rich probiotics and enzymatic activity high.The inventive method has workable, the features such as suitable for large-scale industrial production, has good market prospects.

Description

A kind of method of the step combined ferment feed of multi-cultur es two
Technical field
The present invention relates to a kind of method of the step combined ferment feed of multi-cultur es two, belong to field of feed.
Background technology
In recent years, fermented feed turns into study hotspot, and the research of a variety of fermented feeds has obtained certain development.Fermentation is raised Material is the effect using microorganism, and feedstuff is converted into microbial bacteria body protein, bioactive micro peptide amino acid, micro- life The biological fermentation feed that thing active probiotic and complex enzyme formulation are integrated.Fermented feed, which can be not only made up in conventional feed, to be held The amino acid easily lacked, and roughage material composition can be made to degrade, improve food conversion ratio, the antigen egg in dregs of beans of degrading In vain, in addition, fermented feed can also reduce the diarrhea rate of pig and significantly reduce thickness of fat, lactobacillus-fermented feed Also reduce the effect of pig house ammonia content.
Fermented feed divides from fermentation raw material can be divided under ensilage fermentation, concentrated feed fermentation, processing of farm products Heel fermentation etc., is divided into solid fermentation, liquid fermentation and semisolid fermentation from the physical state row of fermentation substrate.With corn flour, Brown dregs of rice etc. for primary raw material solid fermentation feed relative to fruits and vegetables slag, soybean whey liquid, dining room hogwash, ensilage etc., Product quality is more stable, is more appropriate to storage and transport, commercialization large-scale production is advantageously implemented, mainly including following several Kind production method:(1) there was only the fermentation process of anaerobic fermentation stage, after strain mixes with feed, direct envelope carries out anaerobism training Support, for the method for this directly pack fermentation because of oxygen deficiency, aerobic bacteria growing is limited, causes hydrolytic enzyme activities in raw material low, no Macromolecular substances in energy hydrolysate feed are small-molecule substance, and then supporting for abundance can not be provided for the probiotics of anaerobic fermentation Point.Protein content improves not notable in the feed finally obtained, and hydrolytic enzyme activities are low, and probiotics total plate count is low.(2) it is aerobic Add anaerobism two-part fermentation process, make to be produced by the aerobic fermentation process being combined with Solid anaerobic two ways of solid Fermented feed producing organic acid and while lactic acid bacteria, containing a high proportion of small molecular protein by degraded, Yi Jigeng Low content of cellulose.
Some fermented feeds only take bacterium, saccharomycete and lactic acid bacteria to carry out anaerobic fermentation, and this kind combination needs outer add Enzyme preparation or outer addition monose to provide nutrition for the early growth of flora.Some only take yeast and mold or withered grass gemma Bacillus etc. carries out aerobic fermentation and feed is significantly improved on protein content is improved without anaerobic fermentation, this kind fermentation.
The present invention, for raw material, is combined using mould, saccharomycete, bacterium and lactic acid bacteria and sent out with palm kernel meal, dregs of beans, corn etc. Ferment, fermentation process are divided to good support to be carried out with two stages of anaerobism.In the aerobic fermentation stage, mould, bacillus subtilis vigorous growth, Produce amylase, protease, a variety of biology enzymes such as cellulase, the macromolecular such as starch, protein, cellulose in hydrolysis material Material is small molecule nutriment, and nutriment is provided for saccharomycete, bacterium and the growth of lactic acid bacteria;It is mould in anaerobic stages Bacterium is dissolved, and intracellular hydrolase is discharged into feed, and the macro-nutrients in further hydrolysate feed are sought for small molecule Material is supported, bacillus subtilis, saccharomycete and lactic acid bacteria continued growth, the lactic acid and anaerobic condition of generation can preferably suppress The growth of harmful bacteria, the activity for ensureing probiotics, the fragrance for finally obtaining rich protein, rich probiotics and enzymatic activity high are fitted The feed of mouth.
The content of the invention
It is an object of the invention to provide a kind of method of the step combined ferment feed of multi-cultur es two, it is therefore an objective to passes through a variety of micro- lifes Aerobic, the anaerobism two-step fermentation of thing, obtain the fragrant agreeable to the taste feed of rich protein, rich probiotics and enzymatic activity high.
Technical solution of the present invention mainly includes the following steps that:
(1) activated spawn, and respectively prepare head mold, aspergillus oryzae, bacillus subtilis, Lactobacillus plantarum, brewer's yeast Pure culture;
(2) fermentation raw material is pre-processed, including raw material crushed, mixes after water mixes and sterilizes;
(3) aerobic fermentation:It is inoculated with head mold, aspergillus oryzae, bacillus subtilis, Lactobacillus plantarum, brewer's yeast seed culture Thing is into fermentation raw material, and example is inoculated with mass ratio, material after every 100 parts of sterilizings, inoculation rhizopus wheat bran seed pure culture 0.2 ~0.7 part, 0.2~0.7 part of aspergillus oryzae wheat bran seed pure culture, 0.8~1.2 part of bacillus subtilis pure culture, plant 0.5~1.5 part of lactobacillus pure culture, 2~8 parts of brewer's yeast pure culture;Mixed thoroughly after inoculation, it is good to be placed in 28~30 DEG C of moisturizings Oxygen 36~48h of culture, 37 DEG C of constant incubator aerlbic culture 24h are placed in after temperature rise, control relative humidity 85%~95%;
(4) anaerobic fermentation:After aerobic stage terminates, break up, mix fermented feed thoroughly, load one-way exhaust hermetic bag, be placed in 4~8d of Anaerobic culturel at room temperature.
In one embodiment of the invention, the fermentation raw material pretreatment includes:(1) crushing of fermentation raw material:Press Mass ratio takes 10~15 parts of corn, 10~15 parts of wheat bran, 15~25 parts of dregs of beans, 35~45 parts of palm kernel meal, 10~15 parts of wheat-middlings, urine 1~2 part of element, 0.5~1 part of glucose, mix, crush, sieving, fineness is 20~40 mesh;(2) fermentation raw material mixes water, expects water Than for 1:0.6~1.0, mix thoroughly, sabot;(3) sterilizing of fermentation raw material:High pressure steam sterilization is carried out to fermentation raw material, sterilizing Condition is 0.1Mpa~0.2Mpa, 15~30min.
In one embodiment of the invention, material after every 100 parts of sterilizings, is inoculated with rhizopus wheat bran seed pure culture 0.2~0.7 part, 0.2~0.7 part of aspergillus oryzae wheat bran seed pure culture, the inoculum concentration of bacillus subtilis pure culture is 1 Part, the inoculum concentration of Lactobacillus plantarum pure culture is 1 part, and the inoculum concentration of brewer's yeast pure culture is 6 parts.
In one embodiment of the invention, the activation of head mold is the spore for being inoculated with rhizopus to potato glucose On slant tube culture medium, 28~30 DEG C of incubated 24~48h, picking new life mycelium, it is oblique to be transferred to potato glucose Interview on pipe culture medium, 28~30 DEG C of incubated 24~48h.
In one embodiment of the invention, the activation of aspergillus oryzae is the spore for being inoculated with aspergillus oryzae to potato grape On sugared slant tube culture medium, 28~30 DEG C of incubated 24~48h, picking new life mycelium, potato glucose is transferred to On slant tube culture medium, 28~30 DEG C of incubated 24~48h.
In one embodiment of the invention, the activation of bacillus subtilis, it is that bacillus subtilis is seeded to LB On slant tube culture medium, 37 DEG C of incubated 20~24h, then be transferred on LB slant tube culture mediums, 37 DEG C are incubated 20~24h.
In one embodiment of the invention, the activation of Lactobacillus plantarum, it is that Lactobacillus plantarum is seeded to MRS liquid On culture medium, 37 DEG C of incubated 20~24h, then transfer once on MRS fluid nutrient mediums, 37 DEG C of incubated 20~24h.
In one embodiment of the invention, the activation of brewer's yeast, it is by brewer's yeast, is seeded to the training of YPD liquid Support on base, 37 DEG C of incubated 20~24h, then transfer once on YPD fluid nutrient mediums, 37 DEG C of incubated 20~24h.
In one embodiment of the invention, the preparation of head mold seed pure culture:Head mold mycelia after inoculation activation Body is on dregs of beans bran mass solid medium, 28~30 DEG C of incubated 3~5d.
In one embodiment of the invention, the preparation of aspergillus oryzae seed pure culture:Aspergillus oryzae after inoculation activation Filament is on dregs of beans bran mass solid medium, 28~30 DEG C of incubated 3~5d.
In one embodiment of the invention, the preparation of bacillus subtilis seed pure culture:It is withered after inoculation activation Careless bacillus is to 50ml LB seed culture fluids, 37 DEG C of 10~18h of constant-temperature shaking culture.
In one embodiment of the invention, the preparation of Lactobacillus plantarum seed pure culture:1ml~the 2ml that transfers is activated Lactobacillus plantarum liquid medium afterwards is to 50ml MRS seed culture fluids, 37 DEG C of 10~18h of quiescent culture.
In one embodiment of the invention, the preparation of brewer's yeast seed pure culture:After the 1ml~2ml that transfers is activated Brewer's yeast liquid nutrient solution to 50ml YPD seed culture fluids, 28~30 DEG C of 12~18h of quiescent culture.
The present invention has advantages below compared to classical production process:
1st, the activity of amylase and protease in feed is greatly improved.The aerobic training of multi-cultur es two-step method fermented feed Support process, beneficial to mould and bacillus subtilis vigorous growth breed, produce abundant hydrolysis enzyme system, especially amylase and Protease.Solve the problems, such as merely with addition enzyme preparation outside being needed when saccharomycete and bacterial fermentation.Alphalise starch in fermentation process Enzymatic activity up to reaches more than 160U/g, acid protease activity up to more than 140U/g;At the end of anaerobic stages, alphalise starch Enzymatic activity still may remain in 90~110U/g, and acid protease activity is maintained at 110~130U/g.These hydrolases can Using the macromolecular substances in hydrolysis material as small molecule nutrients.
2nd, the content of feed protein is greatly improved.The dietary protein level to be fermented using this method is up to 30% ~34%, improve 45%~62% than raw material.Produce a variety of hydrolases in aerobic incubation, big point in hydrolysis material Sub- material is small molecule nutrients, continuously provides nutrition for the growth of saccharomycete, bacillus subtilis, lactic acid bacteria, obtains Obtain a large amount of mycoproteins.This is solved only causes protein to contain with bacterium and yeast anaerobic fermentation feed because of hydrolase deficiency The problem of amount increase is little.
3rd, the content of probiotics in feed is greatly improved.By the culture of aerobic stage and anaerobic stages, aspergillus oryzae, Head mold, brewer's yeast, bacillus subtilis and the substantial amounts of growth and breeding of lactic acid bacteria, abundant benefit is contained in the feed finally obtained Raw flora.After fermentation ends, lactic acid bacteria is 5.0 × 1010~5.0 × 1011Cfu/g, bacillus subtilis are 2.0 × 109cfu/g ~2.0 × 1010Cfu/g, saccharomycete are 1.5 × 104~2.0 × 103cfu/g。
The inventive method has workable, the features such as suitable for large-scale industrial production, has good market Prospect.
Brief description of the drawings
The content of thick protein in Fig. 1 feeds
Alpha-amylase activity in Fig. 2 feeds
Acid protease activity in Fig. 3 feeds
Lactic acid bacteria total plate count logarithm value in Fig. 4 feeds
Embodiment
The content of protein determines according to standard GB/T 5009.5-2010 methods.
The content of α-amylase determines according to GB/T5521-2008 methods.
Lactic acid bacteria bacterium colony counts to be determined according to GB4789.35-2010 methods.
Dregs of beans bran mass solid medium composition is 35~45g of bean cake powder, 30~40g of wheat bran, 40~50mL of water, is filled Expect 1~2cm of thickness.
Potato dextrose agar is formulated as follows:200 parts of potato, add 500mL water 30min is boiled, is filtered, is taken filtrate, boil, adds 20 parts of agar strips, stirring adds 20 parts of glucose, moisturizing to being completely dissolved To 1000 parts, 115~121 DEG C of moist heat sterilization 20min.
LB culture mediums are formed by the preparation of reagents of following parts by weight:3 parts of beef extract, 10 parts of peptone, sodium chloride 5 Part, 1000 parts of deionized water, pH to 7.0~7.2,115~121 DEG C of moist heat sterilization 20min are adjusted after mixing reagent.
YPD culture mediums are formed by the preparation of reagents of following parts by weight:10 parts of dusty yeast, 20 parts of peptone, glucose 20 parts, 1000 parts of deionized water, mix, 115~121 DEG C of moist heat sterilization 20min.
MRS broth bouillons are formed by the preparation of reagents of following parts by weight:Peptone:10 parts;Beef extract:10 parts; Yeast extract:5 parts;Glucose:20 parts;Dipotassium hydrogen phosphate:2 parts;Diammonium hydrogen citrate:2 parts;Anhydrous sodium acetate:5 parts;Sulphur Sour magnesium:0.58 part;Manganese sulfate:0.25 part;Tween 80:1 part;Deionized water:1000 parts;Mix reagent after adjust pH to 6.2~ 6.4,115~121 DEG C of moist heat sterilization 20min.
1 liang of strain of embodiment is aerobic, anaerobism two-step fermentation feed
Specific implementation step is as follows:
A. the culture of seed
A. the preparation of bacillus subtilis seed pure culture:Bacillus subtilis liquid medium after switching activation is to LB Seed culture fluid, 37 DEG C of 10~18h of constant-temperature shaking culture.
B. the preparation of Lactobacillus plantarum seed pure culture:Lactobacillus plantarum liquid medium after switching activation is to MRS kinds Son training
Nutrient solution, 37 DEG C of 10~18h of quiescent culture.
B. the crushing of fermentation raw material
Take 10~15 parts of corn, 10~15 parts of wheat bran, 15~25 parts of dregs of beans, 35~45 parts of palm kernel meal, 10~15 parts of wheat-middlings, 1~2 part of urea, 0.5~1 part of glucose, mix, crush, sieving, fineness is 20~40 mesh.
D. fermentation raw material mixes water
Material-water ratio is 1:0.6~1.0, mix thoroughly, sabot.
E. the sterilizing of fermentation raw material
High pressure steam sterilization is carried out to fermentation raw material, the condition of sterilizing is 0.1Mpa~0.2Mpa, 15~30min.
F. aerobic fermentation
Bacillus subtilis inoculum is inoculated with into fermentation raw material, inoculative proportion bacillus subtilis:The matter of raw material Amount is than being 1.5~2.5:100, mixed thoroughly after inoculation, be placed in 28~30 DEG C of aerobic cultures of incubator moisturizing, 37 are placed in after temperature rise DEG C constant incubator aerlbic culture, control relative humidity 85%~95%.
After aerobic fermentation 2d terminates, inoculated plant lactobacillus inoculum, inoculative proportion is Lactobacillus plantarum:Raw material Mass ratio is 4~6:100, mix thoroughly, fill one-way exhaust hermetic bag, be placed in Anaerobic culturel 6d at room temperature.Determine protein in feed Content, the content of α-amylase, the content of acid protease and the viable count of lactic acid bacteria.
The multi-cultur es anaerobic fermentation feed of embodiment 2
Specific implementation step is as follows:
A. the preparation of seed liquor
A. the preparation of bacillus subtilis seed pure culture:Bacillus subtilis bacteria liquid after the 1ml~2ml that transfers is activated is trained Nutrient solution is to 50mlLB seed culture fluids, 37 DEG C of 10~18h of constant-temperature shaking culture.
B. the preparation of Lactobacillus plantarum seed pure culture:Lactobacillus plantarum liquid medium after switching 1ml~2ml activation To 50mlMRS seed culture fluids, 37 DEG C of 10~18h of quiescent culture.
C. the preparation of brewer's yeast seed pure culture:Brewer's yeast liquid nutrient solution after switching 1ml~2ml activation is extremely 50mlYPD seed culture fluids, 28~30 DEG C of 12~18h of quiescent culture.
B. the crushing of fermentation raw material
Take 10~15 parts of corn, 10~15 parts of wheat bran, 15~25 parts of dregs of beans, 35~45 parts of palm kernel meal, 10~15 parts of wheat-middlings, 1~2 part of urea, 0.5~1 part of glucose, mix, crush, sieving, fineness is 20~40 mesh.
C. fermentation raw material mixes water
Material-water ratio is 1:0.6~1.0, mix thoroughly, sabot.
D. the sterilizing of fermentation raw material
High pressure steam sterilization is carried out to fermentation raw material, the condition of sterilizing is 0.1Mpa~0.2Mpa, 15~30min.
E. anaerobic fermentation
Bacillus subtilis, Lactobacillus plantarum, brewer's yeast inoculum are inoculated with into fermentation raw material, inoculation quality ratio Under such as, bacillus subtilis:Raw material 0.5~2:100, Lactobacillus plantarum:Raw material 0.5~2:100:Brewer's yeast:Raw material 2~ 8:100.Mixed thoroughly after inoculation, fill one-way exhaust hermetic bag, anaerobic fermentation.Ferment determine after 6d the content of protein, α in feed- The viable count of the content of amylase, the content of acid protease and lactic acid bacteria.
The method of 3 Fodder making of the present invention of embodiment
Specific implementation step is as follows:
A. the activation of strain
A. the activation of head mold:From the spore for being stored in picking head mold on the test tube slant of 0-4 DEG C of refrigerator, potato is seeded to On glucose slant tube culture medium, 28~30 DEG C of incubated 24~48h, picking new life mycelium, potato Portugal is transferred to On grape sugar slant tube culture medium, 28~30 DEG C of incubated 24~48h.
B. the activation of aspergillus oryzae:From the spore for being stored in picking aspergillus oryzae on the test tube slant of 0-4 DEG C of refrigerator, horse is seeded to On bell potato glucose slant tube culture medium, 28~30 DEG C of incubated 24~48h, picking new life mycelium, Ma Ling is transferred to On potato glucose slant tube culture medium, 28~30 DEG C of incubated 24~48h.
C. the activation of bacillus subtilis:From the ring bacillus subtilis of test tube slant picking one of 0-4 DEG C of refrigerators, inoculation To LB slant tube culture mediums, 37 DEG C of incubated 20~24h, then be transferred on LB slant tube culture mediums, 37 DEG C of constant temperature Cultivate 20~24h.
D. the activation of Lactobacillus plantarum:From the ring Lactobacillus plantarum of test tube slant picking one of 0-4 DEG C of refrigerators, MRS is seeded to On fluid nutrient medium, 37 DEG C of incubated 20~24h, then transfer once on MRS fluid nutrient mediums, 37 DEG C incubated 20~ 24h。
E. the activation of brewer's yeast:From the ring brewer's yeast of test tube slant picking one of 0~4 DEG C of refrigerator, YPD liquid is seeded to On culture medium, 37 DEG C of incubated 20~24h, then transfer once on YPD fluid nutrient mediums, 37 DEG C of incubated 20~24h.
B. the preparation of strain pure culture
A. the preparation of head mold seed pure culture:The head mold mycelium after activation is inoculated with to dregs of beans bran mass solid culture On base, 28~30 DEG C of incubated 3~5d.
B. the preparation of aspergillus oryzae seed pure culture:Head mold mycelium after inoculation activation is trained to dregs of beans bran mass solid Support on base, 28~30 DEG C of incubated 3~5d.
C. the preparation of bacillus subtilis seed pure culture:Brewer's yeast liquid nutrient solution after switching 1ml~2ml activation To 50mlLB seed culture fluids, 37 DEG C of 10~18h of constant-temperature shaking culture.
D. the preparation of Lactobacillus plantarum seed pure culture:Brewer's yeast liquid nutrient solution after switching 1ml~2ml activation is extremely 50mlMRS seed culture fluids, 37 DEG C of 10~18h of quiescent culture.
E. the preparation of brewer's yeast seed pure culture:Brewer's yeast liquid nutrient solution after switching 1ml~2ml activation is extremely 50mlYPD seed culture fluids, 28~30 DEG C of 12~18h of quiescent culture.
C. the crushing of fermentation raw material
Take 10~15 parts of corn, 10~15 parts of wheat bran, 15~25 parts of dregs of beans, 35~45 parts of palm kernel meal, 10~15 parts of wheat-middlings, 1~2 part of urea, 0.5~1 part of glucose, mix, crush, sieving, fineness is 20~40 mesh.
D. fermentation raw material mixes water
Material-water ratio is 1:0.6~1.0, mix thoroughly, sabot.
E. the sterilizing of fermentation raw material
High pressure steam sterilization is carried out to fermentation raw material, the condition of sterilizing is 0.1Mpa~0.2Mpa, 15~30min.
F. aerobic fermentation
It is inoculated with head mold, aspergillus oryzae, bacillus subtilis, Lactobacillus plantarum, brewer's yeast inoculum to fermentation raw material In, inoculation quality ratio is as follows, rhizopus:Raw material 0.25~1.5:100, aspergillus oryzae:Raw material 0.25~1.5:100, withered grass bud Spore bacillus:Raw material 0.5~2:100, Lactobacillus plantarum:Raw material 0.5~2:100:Brewer's yeast:Raw material 2~8:100.After inoculation Mix thoroughly, be placed in the aerobic culture 48h of 28~30 DEG C of incubator moisturizings, 37 DEG C of constant incubator aerlbic cultures are placed in after temperature rise, Control relative humidity 85%~95%.
G. anaerobic fermentation
After aerobic stage terminates, break up, mix fermented feed thoroughly, fill one-way exhaust hermetic bag, be placed in Anaerobic culturel at room temperature.
After anaerobic fermentation 6d, the content of protein, the content of α-amylase, the content of acid protease in feed are determined And the viable count of lactic acid bacteria.
The additional enzyme preparation anaerobic fermentation feed method of embodiment 4
Specific implementation step is as follows:
A. the preparation of seed liquor
A. the preparation of bacillus subtilis seed pure culture:Bacillus subtilis bacteria liquid after the 1ml~2ml that transfers is activated is trained Nutrient solution is to 50mlLB seed culture fluids, 37 DEG C of 10~18h of constant-temperature shaking culture.
B. the preparation of Lactobacillus plantarum seed pure culture:Lactobacillus plantarum liquid medium after switching 1ml~2ml activation To 50mlMRS seed culture fluids, 37 DEG C of 10~18h of quiescent culture.
C. the preparation of brewer's yeast seed pure culture:Brewer's yeast liquid nutrient solution after switching 1ml~2ml activation is extremely 50mlYPD seed culture fluids, 28~30 DEG C of 12~18h of quiescent culture.
B. the crushing of fermentation raw material
Take 10~15 parts of corn, 10~15 parts of wheat bran, 15~25 parts of dregs of beans, 35~45 parts of palm kernel meal, 10~15 parts of wheat-middlings, 1~2 part of urea, 0.5~1 part of glucose, mix, crush, sieving, fineness is 20~40 mesh.
C. fermentation raw material mixes water
Material-water ratio is 1:0.6~1.0, mix thoroughly, sabot.
D. the sterilizing of fermentation raw material
High pressure steam sterilization is carried out to fermentation raw material, the condition of sterilizing is 0.1Mpa~0.2Mpa, 15~30min.
E. it is inoculated with
Bacillus subtilis, Lactobacillus plantarum, brewer's yeast inoculum are inoculated with into fermentation raw material, inoculation quality ratio Under such as, bacillus subtilis:Raw material 0.5~2:100, Lactobacillus plantarum:Raw material 0.5~2:100:Brewer's yeast:Raw material 2~ 8:100.
F. additional enzyme preparation
Fermented feed inoculation α-amylase 100U/g after inoculation, acid protease 100U/g, mixes thoroughly, loads unidirectional row Airtight envelope, ferments at room temperature.
Ferment 6d after, determine feed in the content of thick protein, the content of α-amylase, the content of acid protease with And saccharomycete, the viable count of lactic acid bacteria.
Fermentation raw material species and proportioning are identical in each example above, and material protein content is 20.11% (with over dry material Meter), α-amylase, acid protease content and lactic acid bacteria viable count are all 0.
To the fermented feed gross protein value in embodiment 1, embodiment 2, embodiment 3, embodiment 4, α-amylase, The content of acid protease, the viable count of lactic acid bacteria carry out correlation analysis, and analysis result is listed in Fig. 1, Fig. 2, Fig. 3 and Fig. 4.From Fig. 1 can be seen that in 4 kinds of embodiments, protein content highest in 3 final tunning of embodiment, be 32.00%, than Protein content improves 59.12% in former feed, and the final fermented feed protein content time of embodiment 4 is high, is 27.10%, carries It is high by 34.26%.It can be seen that raising of the zymotechnique of embodiment 3 most beneficial for protein content in fermentation raw material.Can from Fig. 2 Go out, in embodiment in 4, α-amylase activity highest in 3 final fermented feed of embodiment is 103U/g, embodiment 4 times Height, it is 70U/g, embodiment 2 is minimum, is 24U/g.From figure 3, it can be seen that in 4 kinds of embodiments, embodiment 3 is finally fermented Acid protease activity highest in feed, it is 123U/g, 4 height of embodiment, are 71U/g, and embodiment 2 is minimum, are 47U/g.From Fig. 4 can be seen that live lactobacillus number highest in 3 final fermented feed of embodiment, and its logarithm value is 12.1.
In addition, mould extracellular proteinase and carbohydrase in fermentation process, mould inoculum concentration are too low, it is impossible to provide enough Hydrolase, and then enough hydrolysates can not be obtained, influence the growth of saccharomycete, bacterium;Mould inoculum concentration is excessive, mould life Length is excessively vigorous, on the one hand because competitive relation can influence the growth of saccharomycete and bacterium, on the other hand can cause oxygen supply deficiency.
Lactic acid bacteria, bacillus subtilis and saccharomycete play different effects in fermentation process, and the proportioning of each strain is direct The quality of fermented feed is had influence on, is specifically shown in Table 1.
The fermented feed bacterium of table 1, saccharomycete inoculum concentration result of the test
As shown in Table 1, fermented when bacillus subtilis inoculum concentration 1%, saccharomycete inoculum concentration 6%, lactobacillus inoculum amount 1% Dietary protein level highest.
Aerobic stage can produce substantial amounts of hydrolase, while be also that single cell protein produces the most important stage.It is good The length in oxygen stage is again related to temperature.To be warming up to 37 DEG C of continuation heat-preservation fermentation 24h after 28-30 DEG C of heat-preservation fermentation 36-48h, Obtain the protein content and enzymatic activity highest of fermented feed.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention Enclose being defined of being defined by claims.

Claims (8)

  1. A kind of 1. method of the step combined ferment feed of multi-cultur es two, it is characterised in that mainly include the following steps that:
    (1) activated spawn, and the pure training of head mold, aspergillus oryzae, bacillus subtilis, Lactobacillus plantarum, brewer's yeast is prepared respectively Support thing;
    (2) fermentation raw material is pre-processed, including raw material crushed, mixes after water mixes and sterilizes;
    (3) aerobic fermentation:Inoculation head mold, aspergillus oryzae, bacillus subtilis, Lactobacillus plantarum, the pure culture of brewer's yeast arrive In fermentation raw material, mixed thoroughly after inoculation, be placed in 28~30 DEG C of moisturizings, 36~48h of aerobic culture, 37 DEG C of constant temperature are placed in after temperature rise Incubator aerlbic culture 24h, control relative humidity 85%~95%;
    (4) anaerobic fermentation:After aerobic stage terminates, break up, mix fermented feed thoroughly, load one-way exhaust hermetic bag, be placed in room temperature 4~8d of lower Anaerobic culturel;
    Step (2) the fermentation raw material pretreatment includes:1. the crushing of fermentation raw material:10~15 parts of corn, bran are taken in mass ratio 10~15 parts of skin, 15~25 parts of dregs of beans, 35~45 parts of palm kernel meal, 10~15 parts of wheat-middlings, 1~2 part of urea, glucose 0.5~1 Part, mix, crush, sieving, fineness is 20~40 mesh;2. fermentation raw material mixes water, material-water ratio 1:0.6~1.0, mix thoroughly, fill Disk;3. the sterilizing of fermentation raw material:Carrying out high pressure steam sterilization to fermentation raw material, the condition of sterilizing is 0.1Mpa~0.2Mpa, 15 ~30min;
    Strain example in mass ratio in the step (3) is inoculated with, material after every 100 parts of sterilizings, and inoculation rhizopus wheat bran seed is pure 0.2~0.7 part of culture, 0.2~0.7 part of aspergillus oryzae wheat bran seed pure culture, bacillus subtilis pure culture 0.8~ 1.2 parts, 0.5~1.5 part of Lactobacillus plantarum pure culture, 2~8 parts of brewer's yeast pure culture.
  2. 2. according to the method for claim 1, it is characterised in that material after every 100 parts of sterilizings, be inoculated with rhizopus wheat bran seed 0.2~0.7 part of pure culture, 0.2~0.7 part of aspergillus oryzae wheat bran seed pure culture, bacillus subtilis pure culture connects Kind amount is 1 part, and the inoculum concentration of Lactobacillus plantarum pure culture is 1 part, and the inoculum concentration of brewer's yeast pure culture is 6 parts.
  3. 3. according to the method for claim 1, it is characterised in that the preparation of head mold seed pure culture:After inoculation activation Head mold mycelium is on dregs of beans bran mass solid medium, 28~30 DEG C of incubated 3~5d.
  4. 4. according to the method for claim 1, it is characterised in that the preparation of aspergillus oryzae seed pure culture:After inoculation activation Aspergillus oryzae filament on dregs of beans bran mass solid medium, 28~30 DEG C of incubated 3~5d.
  5. 5. according to the method for claim 1, it is characterised in that the preparation of bacillus subtilis seed pure culture:Inoculation Bacillus subtilis after slant activation is to 50ml LB seed culture fluids, 37 DEG C of 10~18h of constant-temperature shaking culture.
  6. 6. according to the method for claim 1, it is characterised in that the preparation of Lactobacillus plantarum seed pure culture:Switching Lactobacillus plantarum liquid medium after 1ml~2ml activation is to 50ml MRS seed culture fluids, 37 DEG C of 10~18h of quiescent culture.
  7. 7. according to the method for claim 1, it is characterised in that the preparation of brewer's yeast seed pure culture:Transfer 1ml Brewer's yeast liquid nutrient solution after~2ml activation is to 50ml YPD seed culture fluids, 28~30 DEG C of 12~18h of quiescent culture.
  8. 8. the feed being prepared according to any methods describeds of claim 1-7.
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