CN103039699A - Mixed fermentation feed and preparation method and application method of mixed fermentation feed - Google Patents
Mixed fermentation feed and preparation method and application method of mixed fermentation feed Download PDFInfo
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Abstract
The invention discloses fermentation feed produced by mixed bacteria such as lactobacillus paracasei, and a preparation method and an application method of the fermentation feed. The lactobacillus paracasei (CICC20296), bacillus subtilis (ACCC10619) and saccharomyces cerevisiae (CICC32236) are subjected to independent liquid fermentation and culture, fermentation liquids are mixed at a volume ratio of 1:1:1, inoculated to a solid culture medium for uniform mixing, and placed in a sealed container for anaerobic fermentation and culture for 75-240h at 10-37 DEG C. The viable count of the feed reaches 200-300*10<8>cfu*g<-1>, and the feed is not required to be dried and can be used directly. According to the preparation method of the fermentation feed, the use amount of antibiotics is reduced, the obtained fermentation feed contains a large number of nutrient substances such as mycoprotein, rich amino acid and vitamins, and facilitates promoting balance of intestinal flora, improving immunity of livestock organisms, improving the production performance and lowering raising cost.
Description
Technical field
The invention belongs to the microbial fermentation field, be specifically related to fermented feed and methods for making and using same thereof that the Mixed Microbes such as a kind of lactobacillus paraceasi is produced.
Background technology
In animal diets, add antibiotic and significantly promoted animal production, and major contribution has been made in the development of intensive animal husbandry.Yet As time goes on, add antibiotic harm in the feed and day by day manifest, and be subject to the extensive concern of society.European Union completely forbids from January, 2006 and add antibiotic in animal and fowl fodder.Subsequently, the Asian countries such as Japan and Korea S have also in succession formulated the livestock and poultry antibiotic-free and have raised standard.Must not add antibiotic in the raisings such as live pig has been internationally recognized food security standard at present.
No-antibiotics feed is to utilize the beneficial microbe fermented feed, forms and is rich in a large amount of active beneficial microbes, organic acid and microbial metabolic products, and do not contain any antibiotic fermentative feedstuff of microbe.To provide true albumen and lactic acid bacteria as main, antibiotic-free is residual in the long-term feeding livestock and poultry body.Be rich in a large amount of lactic acid bacterias, saccharomycete, the synthetic needed amino acid of animal, vitamin, digestive ferment and lactic acid etc. make the nutrition of feed more comprehensively under the effect of microorganism, and biological value is higher.Not only can remedy the amino acid that lacks easily in the conventional feed, and other roughage raw material nutrition is transformed rapidly, reach enhancing and digest and absorb effect.
The domestic animals and fowls of using fermentative feedstuff of microbe to feed do not contain residual antibiotic and harmful chemical addition agent in its body, not only make the standard of edible meat reach and surpass the standard of the edible meat of European Union, and the flavor substance in the meat has been improved, and mouthfeel is better.Simultaneously, because the ill probability of drove lowers, growth is stable, and is easy and simple to handle, thereby greatly reduces peasant's feeding cost, improved fanning economics, is particularly suitable for promoting the use of in the vast rural area of China.
The research and development of " no-antibiotics feed " will promote the production of " nonreactive pork ", be common people, improve China's livestock and poultry breeding industry food conversion utilization rate, meat quality, strengthen the international competitiveness of feed industry product.
Summary of the invention
The purpose of this invention is to provide fermented feed and methods for making and using same thereof that the Mixed Microbes such as a kind of lactobacillus paraceasi is produced, the preparation method of the fermented feed that the present invention proposes has reduced antibiotic use, resulting fermented feed contains the nutriments such as a large amount of mycoproteins, abundant amino acid and vitamin, be conducive to promote the gut flora balance, improve fowl poultry immunity of organisms, improve production performance, reduce feeding cost.
A kind of preparation method of mixed fermentation feed: by lactobacillus paraceasi (CICC20296), bacillus subtilis (ACCC10619), saccharomyces cerevisiae (CICC32236) difference liquid fermentation and culture, carry out the solid fermentation cultivation after the by volume 1:1:1 mixing of zymotic fluid and form.
Specifically may further comprise the steps:
(1) liquid fermentation
To get a ring inclined-plane inoculation through the lactobacillus paraceasi of slant activation and cultivate to the seed shaking flask, condition of culture is: the MRS culture medium, and 35 ~ 37 ℃, the pH nature leaves standstill and cultivates 18 ~ 22h; Then carry out liquid fermentation and culture by the inoculation of 3～7% inoculum concentrations, described culture medium is the MRS culture medium, and described fermentation condition is: 35 ~ 37 ℃, and the pH nature, liquid amount 50 ~ 70% leaves standstill and cultivates 36 ~ 60h;
To get a ring inclined-plane inoculation through the bacillus subtilis of slant activation and cultivate to the seed shaking flask, condition of culture is: the LB culture medium, and 28 ~ 32 ℃, pH6.8～7.2, shaking speed 180 ~ 220r/min cultivates 16 ~ 20h; Then carry out liquid fermentation and culture by the inoculation of 2 ~ 8% inoculum concentrations, described culture medium is the LB culture medium, and described fermentation condition is: 28 ~ 32 ℃, and pH6.8～7.2, liquid amount 10 ~ 30%, shaking speed 180 ~ 220r/min cultivates 20 ~ 24h;
To get a ring inclined-plane inoculation through the saccharomyces cerevisiae of slant activation and cultivate to the seed shaking flask, condition of culture is: PDA culture medium, 26 ~ 30 ℃, pH6.2 ~ 6.8, shaking speed 180 ~ 250rpm; Cultivate 16 ~ 20h; Then carry out liquid fermentation and culture by the inoculation of 4 ~ 8% inoculum concentrations, described culture medium is the PDA culture medium, and described fermentation condition is: 26 ~ 30 ℃, and pH6.2 ~ 6.8, liquid amount 10 ~ 30%, shaking speed 180 ~ 250rpm; Cultivate 26 ~ 34h;
(2) solid fermentation
Get respectively the by volume 1:1:1 mixing of three kinds of zymotic fluids that step (1) obtains, the inoculum concentration of 3-10% is fully mixed zymotic fluid with the culture medium major ingredient by mass percentage, and water content is controlled at 35 ~ 55% scopes; The culture medium major ingredient comprises: livestock and poultry complete diet pellet or feedstuff;
(3) mixed material that step (2) is obtained places airtight container, static fermentation under 10 ~ 37 ℃ of yeasting temperature, continuous solid-state anaerobic fermentation 75 ~ 240 hours; The product viable count reaches 200 ~ 300 * 10
8Cfu.g
-1
Above-mentioned feedstuff comprises one or more in wheat bran, corn flour, the beancake powder.
Above-mentioned solid fermentation is finished in fermentation tank, fermentation vat, plastic containers or the vacuum bag of sealing.
A kind of fermented feed is the feed that is prepared from by above-mentioned method.
The application process of above-mentioned fermented feed is: directly, perhaps add to feed in the feed and raise livestock and poultry; Adding mass concentration in feed or feedstuff is 5%-30%.
Product of the present invention need not drying can be directly feeding.
The lactobacillus paraceasi (CICC20296) that the present invention uses and saccharomyces cerevisiae (CICC32236) are available from Chinese industrial microorganism fungus kind preservation administrative center, and bacillus subtilis (ACCC10619) is available from Chinese agriculture microorganism fungus kind preservation administrative center.
The preparation method of the fermented feed that the present invention proposes has reduced antibiotic use, resulting fermented feed contains the nutriments such as a large amount of mycoproteins, abundant amino acid and vitamin, be conducive to promote the gut flora balance, improve fowl poultry immunity of organisms, improve production performance, reduce feeding cost.
The specific embodiment
Be intended to further specify the present invention below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1
(1) liquid fermentation
To get a ring inclined-plane inoculation through the lactobacillus paraceasi (CICC20296) of slant activation and cultivate to the seed shaking flask, the MRS culture medium, 35 ℃, the pH nature leaves standstill and cultivates 18h; Then carry out liquid fermentation and culture by the inoculation of 3% inoculum concentration, described culture medium is the MRS culture medium, and described fermentation condition is: 35 ℃, and the pH nature, liquid amount 50% leaves standstill and cultivates 36h;
To get a ring inclined-plane inoculation through the bacillus subtilis (ACCC10619) of slant activation and cultivate to the seed shaking flask, the LB culture medium, 28 ℃, pH6.8, shaking speed 180r/min cultivates 16h; Then carry out liquid fermentation and culture by the inoculation of 2% inoculum concentration, described culture medium is the LB culture medium, and described fermentation condition is: 28 ℃, and pH6.8, liquid amount 10%, shaking speed 180r/min cultivates 20h;
To get a ring inclined-plane inoculation through the saccharomyces cerevisiae (CICC32236) of slant activation and cultivate PDA culture medium, 26 ℃, pH6.2, shaking speed 180rpm to the seed shaking flask; Cultivate 16h; Then carry out liquid fermentation and culture by the inoculation of 4% inoculum concentration, described culture medium is the PDA culture medium, and described fermentation condition is: 26 ℃, and pH6.2, liquid amount 10%, shaking speed 180rpm; Cultivate 26h;
(2) solid fermentation
Culture medium major ingredient: livestock and poultry complete diet pellet (or the feedstuff such as wheat bran, corn flour, beancake powder);
Inoculation: get respectively by volume 1:1:1 mixing of zymotic fluid, with 3% inoculum concentration zymotic fluid is fully mixed with solid medium, water content is controlled at 35%;
(3) mixed material is placed airtight container (solid fermentation can be finished at fermentation tank, fermentation vat, plastic containers, the vacuum bag of sealing, adopts the two-step method fermentation), under 10 ℃ of yeasting temperature, carried out the continuous solid-state anaerobic fermentation 240 hours; (4) the product viable count can reach 200 * 10
8Cfu.g
-1, it is directly feeding that product need not drying.
Embodiment 2
(1) liquid fermentation
To get a ring inclined-plane inoculation through the lactobacillus paraceasi (CICC20296) of slant activation and cultivate to the seed shaking flask, the MRS culture medium, 36 ℃, the pH nature leaves standstill and cultivates 20h; Then carry out liquid fermentation and culture by the inoculation of 5% inoculum concentration, described culture medium is the MRS culture medium, and described fermentation condition is: 36 ℃, and the pH nature, liquid amount 60% leaves standstill and cultivates 48h;
To get a ring inclined-plane inoculation through the bacillus subtilis (ACCC10619) of slant activation and cultivate to the seed shaking flask, the LB culture medium, 30 ℃, pH7.0, shaking speed 200r/min cultivates 18h; Then carry out liquid fermentation and culture by the inoculation of 5% inoculum concentration, described culture medium is the LB culture medium, and described fermentation condition is: 30 ℃, and pH7.0, liquid amount 20%, shaking speed 200r/min cultivates 22h.
To get a ring inclined-plane inoculation through the saccharomyces cerevisiae (CICC32236) of slant activation and cultivate PDA culture medium, 28 ℃, pH6.5, shaking speed 220rpm to the seed shaking flask; Cultivate 18h; Then carry out liquid fermentation and culture by the inoculation of 6% inoculum concentration, described culture medium is the PDA culture medium, and described fermentation condition is: 28 ℃, and pH6.5, liquid amount 20%, shaking speed 220rpm; Cultivate 30h.
(2) solid fermentation
Culture medium major ingredient: livestock and poultry complete diet pellet (or the feedstuff such as wheat bran, corn flour, beancake powder);
Inoculation: get respectively by volume 1:1:1 mixing of zymotic fluid, with 6% inoculum concentration zymotic fluid is fully mixed with solid medium, water content is controlled at 45%;
(3) (solid fermentation can be finished at fermentation tank, fermentation vat, plastic containers, the vacuum bag of sealing, adopts the two-step method fermentation, carries out the continuous solid-state anaerobic fermentation 120 hours under 25 ℃ of yeasting temperature mixed material to be placed airtight container; (4) the product viable count can reach 300 * 10
8Cfu.g
-1, it is directly feeding that product need not drying.
Embodiment 3
(1) liquid fermentation
To get a ring inclined-plane inoculation through the lactobacillus paraceasi (CICC20296) of slant activation and cultivate to the seed shaking flask, the MRS culture medium, 37 ℃, the pH nature leaves standstill and cultivates 22h; Then carry out liquid fermentation and culture by the inoculation of 7% inoculum concentration, described culture medium is the MRS culture medium, and described fermentation condition is: 37 ℃, and the pH nature, liquid amount 70% leaves standstill and cultivates 60h;
To get a ring inclined-plane inoculation through the bacillus subtilis (ACCC10619) of slant activation and cultivate to the seed shaking flask, the LB culture medium, 32 ℃, pH7.2, shaking speed 220r/min cultivates 20h; Then carry out liquid fermentation and culture by the inoculation of 8% inoculum concentration, described culture medium is the LB culture medium, and described fermentation condition is: 32 ℃, and pH7.2, liquid amount 30%, shaking speed 220r/min cultivates 24h.
To get a ring inclined-plane inoculation through the saccharomyces cerevisiae (CICC32236) of slant activation and cultivate PDA culture medium, 30 ℃, pH6.8, shaking speed 250rpm to the seed shaking flask; Cultivate 20h; Then carry out liquid fermentation and culture by the inoculation of 8% inoculum concentration, described culture medium is the PDA culture medium, and described fermentation condition is: 30 ℃, and pH6.8, liquid amount 30%, shaking speed 250rpm; Cultivate 34h.
(2) solid fermentation
Culture medium major ingredient: livestock and poultry complete diet pellet (or the feedstuff such as wheat bran, corn flour, beancake powder);
Inoculation: get respectively by volume 1:1:1 mixing of zymotic fluid, with 10% inoculum concentration zymotic fluid is fully mixed with solid medium, water content is controlled at 55%;
(3) will mix material and place airtight container (solid fermentation can be finished at fermentation tank, fermentation vat, plastic containers, the vacuum bag of sealing, adopts the two-step method fermentation), under 37 ℃ of yeasting temperature, carry out the continuous solid-state anaerobic fermentation 75 hours;
(4) the product viable count can reach 250 * 10
8Cfu.g
-1, it is directly feeding that product need not drying.
Embodiment 4 different probios are to the bacteriostatic test of pathogen
As can be seen from Table 1, these 4 kinds of test organisms of lactobacillus paraceasi, bacillus subtilis, cured shape bacillus and bacillus coagulans all have certain fungistatic effect to 4 kinds of pathogens.Wherein lactobacillus paraceasi is best to 4 kinds of pathogen fungistatic effects, and bacillus subtilis takes second place.And lactobacillus paraceasi and bacillus subtilis mixed bacteria liquid also all have stronger bacteriostasis to 4 kinds of pathogens, and therefore selecting the lactobacillus paraceasi of good antimicrobial effect and bacillus subtilis is test strain.
Table 1 unit: mm
Embodiment 5 fermentation feedstuff strain characteristic research
(1) acid resistance test
As shown in Table 2, along with the reduction viable bacteria content of pH descends gradually, lactobacillus paraceasi, bacillus subtilis and saccharomyces cerevisiae all can be survived at the pH2.0 environment, certain tolerance is arranged, wherein, bacillus subtilis is slightly poor to the environment tolerance of pH3.0, but still has quite a few survival rate.Be 3.0 o'clock at pH, the upgrowth situation of yeast is better: when pH5.0, each test organisms all has breeds phenomenon preferably; When pH6.0, each test organisms cultivation effect is obvious; Illustrate that three kinds of test organisms all have certain capacity antacid.
Table 2 unit: (CFU/ml)
(2) bile tolerance test
As shown in Table 3, lactobacillus paraceasi, bacillus subtilis and saccharomyces cerevisiae all have obvious propagation phenomenon when gallbladder salinity is 0.3%; And when gallbladder salinity was 0.6% and 0.9%, bacillus subtilis had and breeds preferably phenomenon; Be 0.3 ~ 1.2 o'clock at gallbladder salinity, saccharomyces cerevisiae all has obvious propagation phenomenon.Illustrate that lactobacillus paraceasi, bacillus subtilis and saccharomyces cerevisiae have certain tolerance to cholate.
Table 3 unit: test organisms (CFU/ml)
(3) tolerance test of artificial simulation gastric juices
Result such as the table 4 of manual simulation pig body gastric juice test, along with the prolongation of time is gradual reduction, effect 2h viable bacteria survival rate is all more than 70%, and test organisms can tolerate the SGF environment time and surpasses 2h.Illustrate that test organisms is larger by the possibility of pig body gastric juice environment survival.
Table 4 unit: CFU/ml
(4) tolerance test of artificial simulation intestinal juice
As shown in Table 5, under manual simulation pig body intestinal juice environment, test organisms all has certain survival rate, on average can reach more than 80%, and tolerance time can surpass 4h.Illustrate that test organisms is larger by the possibility of pig body intestinal juice environment survival.
Table 5 unit: CFU/ml
The present invention is by series of experiments such as bacteriostatic test, acid resistance test, bile tolerance test and manual simulation pig body stomach, intestinal environment, test has been carried out the research of biological characteristics with lactobacillus paraceasi, bacillus subtilis and saccharomyces cerevisiae, final result shows: these three kinds of test organisms all show stronger tolerance, has certain tolerance for environmental conditions such as acid, cholate, can resist pig gastro-intestinal digestion road environment to the tolerance requirement of thalline, lactobacillus paraceasi, bacillus subtilis have preferably fungistatic effect.Test shows that test organisms is suitable for the living environment in the pig body enteron aisle, and the viable bacteria that can ensure certain amount of survival arrives pig body intestines and stomach, can pass through smoothly Stomach duodenum thereby ensure enough viable bacterias, arrives small intestine performance prebiotic effect.So lactobacillus paraceasi, bacillus subtilis and saccharomyces cerevisiae can be suitable as the bacterial strain of selecting of fermented feed, be used for the development of fermented feed.
Determining of the different proportioning ratios of embodiment 6 solid fermentation mixed bacterias
The different proportioning ratios of solid fermentation mixed bacteria all have larger impact to zymotechnique and product.In order to determine to select the best proportioning of bacterial classification, this test is with lactobacillus paraceasi: saccharomyces cerevisiae: bacillus subtilis is respectively with the volume ratio of 1:1:1,2:1:1,1:2:1,1:2:2 and 2:2:1, be inoculated in respectively the solid fermentation culture medium, inoculum concentration 5% (in proportion mixed total inoculum concentration), water content is controlled at 40%; Place airtight container under 28 ℃ of yeasting temperature, to carry out the continuous solid-state anaerobic fermentation 120 hours mixed material; Measure the number of viable of tunning.The result is as shown in table 6.
Table 6
As can be seen from Table 6, when lactobacillus paraceasi, saccharomyces cerevisiae and bacillus subtilis were 1:1:1 in volume ratio, tunning viable bacteria content amplification was the highest, can reach 2.32 * 10
10Cfu/g.Therefore, selecting lactobacillus paraceasi, saccharomyces cerevisiae and bacillus subtilis is that the volume ratio of 1:1:1 is carried out the solids mixing fermentation.
Embodiment 6, compound bacteria-fermented feed are on the impact of weaned piglets
This example carries out on Yongzhou City Heng Hui pig farm, experimental design is selected 150 of healthy weanling pigs (28 ages in days are to the rear piglet of 30 ages in days wean), being divided at random 5 by body weight phase approximately principle processes, each processes 3 repetitions, each repeats 10 piglets, be divided into antibiotic control group, nonreactive daily ration+10% fermented feed group, nonreactive daily ration+20% fermented feed group and nonreactive daily ration+30% fermented feed group, (commercially available fermented feed main component is Lactobacillus plantarum, bacillus licheniformis, saccharomyces cerevisiae to the commercially available fermented feed group of nonreactive daily ration+20%, wherein viable count 〉=50 * 10
8Cfu/ml), 45 days experimental periods, result of the test such as following table.
Table 7 compound bacteria-fermented feed is on the impact of weaned piglets
Above result of the test shows, use that the technology of the present invention produces product---no-antibiotics feed can have the incidence that reduces by a relatively large margin Diarrhea after Piglets " Weaning, improve piglet growth speed, improve the food conversion utilization ratio, be a kind of can substitute antibiotics, control diarrhoea and promote the desirable environmental protection microecological fermented forage of growth.
The present invention has worked out production and the using method thereof that is used for the no-antibiotics feed of substitute antibiotics, and result of the test shows: the no-antibiotics feed of this method preparation has the effect of weightening finish and prevention diarrhoea to piglet.
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement or conversion all belong to the protection domain of claims of the present invention.
Claims (7)
1. the preparation method of a mixed fermentation feed, it is characterized in that: by lactobacillus paraceasi (CICC20296), bacillus subtilis (ACCC10619), saccharomyces cerevisiae (CICC32236) difference liquid fermentation and culture, carry out the solid fermentation cultivation after the by volume 1:1:1 mixing of zymotic fluid and form.
2. method according to claim 1 is characterized in that, specifically may further comprise the steps:
(1) liquid fermentation
To get a ring inclined-plane inoculation through the lactobacillus paraceasi of slant activation and cultivate to the seed shaking flask, condition of culture is: the MRS culture medium, and 35 ~ 37 ℃, the pH nature leaves standstill and cultivates 18 ~ 22h; Then carry out liquid fermentation and culture by the inoculation of 3～7% inoculum concentrations, described culture medium is the MRS culture medium, and described fermentation condition is: 35 ~ 37 ℃, and the pH nature, liquid amount 50 ~ 70% leaves standstill and cultivates 36 ~ 60h;
To get a ring inclined-plane inoculation through the bacillus subtilis of slant activation and cultivate to the seed shaking flask, condition of culture is: the LB culture medium, and 28 ~ 32 ℃, pH6.8～7.2, shaking speed 180 ~ 220r/min cultivates 16 ~ 20h; Then carry out liquid fermentation and culture by the inoculation of 2 ~ 8% inoculum concentrations, described culture medium is the LB culture medium, and described fermentation condition is: 28 ~ 32 ℃, and pH6.8～7.2, liquid amount 10 ~ 30%, shaking speed 180 ~ 220r/min cultivates 20 ~ 24h;
To get a ring inclined-plane inoculation through the saccharomyces cerevisiae of slant activation and cultivate to the seed shaking flask, condition of culture is: PDA culture medium, 26 ~ 30 ℃, pH6.2 ~ 6.8, shaking speed 180 ~ 250rpm; Cultivate 16 ~ 20h; Then carry out liquid fermentation and culture by the inoculation of 4 ~ 8% inoculum concentrations, described culture medium is the PDA culture medium, and described fermentation condition is: 26 ~ 30 ℃, and pH6.2 ~ 6.8, liquid amount 10 ~ 30%, shaking speed 180 ~ 250rpm; Cultivate 26 ~ 34h;
(2) solid fermentation
Get respectively the by volume 1:1:1 mixing of three kinds of zymotic fluids that step (1) obtains, the inoculum concentration of 3-10% is fully mixed zymotic fluid with the culture medium major ingredient by mass percentage, and water content is controlled at 35 ~ 55% scopes; The culture medium major ingredient comprises: livestock and poultry complete diet pellet or feedstuff;
(3) mixed material that step (2) is obtained places airtight container, static fermentation under 10 ~ 37 ℃ of yeasting temperature, continuous solid-state anaerobic fermentation 75 ~ 240 hours; The product viable count reaches 200 ~ 300 * 10
8Cfu.g
-1
3. method according to claim 1 is characterized in that, feedstuff comprises one or more in wheat bran, corn flour, the beancake powder.
4. method according to claim 1 is characterized in that, solid fermentation is finished in fermentation tank, fermentation vat, plastic containers or the vacuum bag of sealing.
5. fermented feed is characterized in that: the feed that is prepared from by each described method of claim 1-4.
6. the application process of fermented feed claimed in claim 5 is characterized in that: directly, perhaps add to feed in the feed and raise livestock and poultry.
7. the application process of fermented feed according to claim 6 is characterized in that: adding mass concentration in feed or feedstuff is 5%-30%.
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