CN101186896A - Fermentation material for composite microorganism and preparation method thereof - Google Patents
Fermentation material for composite microorganism and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a composite microorganism ferment material which is characterized in that production strains select production strains of which the growth rate is quick and the enzyme production quantity is large by combining with through ray inducement and production performance detection. The processing technique employs innovative symbiosis compatibility design of probiotics and multi-compound fermentation technology, the number of the live probiotics in the products achieves five billion per gram, and the products simultaneously contain polyoses, polypeptide, organized enzyme, organic acid, phytosterin, triterpennoids saponin and the like active metabolite. Specific enzyme producing inducer is added to growth medium, the latest single creature venting valve is employed on the package, which not only can be added to complete feed of livestock or poultry but also can be directly added into feedstuff, thereby effectively promoting breakdown and absorption of giant molecule nutrient substance in the intestinal canal of livestock or poultry, thereby eliminating the action of anti-nutritional factors in the feedstuff, remarkably increasing utilization ratio of the feedstuff, and reducing the feeding cost.
Description
Technical field
The present invention relates to a kind of fermentation material for composite microorganism and making method, adopt design of probiotic bacterium symbiosis compatibility and multistage composite fermentation technique, every grade of fermentation all has different separately target products, make and be rich in a large amount of useful viable bacterias, polysaccharide, polypeptide, organized enzyme, organic acid, plant sterol, three obedient saponin isoreactivity meta-bolitess in the product, easilier searched for food, digest, absorb, belong to the agro-ecology product technique field by livestock and poultry.
Background technology
Complete feed also claims full-time grain mixed feed.It is meant contains required protein, energy, trace element, the mineral matter and other components of animal certain period growth, can satisfy the nutritional need of the object of feeding comprehensively.These feedstuff raw materials mainly are made up of plant material and animal grease, contain multiple antinutritional factor and macromolecular substance, are difficult for directly being absorbed by animal intestinal mucosa and Digestive tract.So, how fully to absorb and utilize various main nutritive ingredients in the complete feed to become a primary difficult problem that solves in the aquaculture, research about this respect starts to walk very early, except that the new feed resource of exploitation, the utilization ratio that improves existing feed resource also is one of the effective way of dealing with problems.The method of eliminating antinutritional factor and decomposition macromole nutritive substance at present mainly contains following several:
1, physics method, as: heating method, expanded method, mechanical processing method; 2, chemical method; 3, biotechnology method: as zymin method, method of breeding, control consumption method, except that the zymin method, the effect of all the other methods is also not obvious and limited the growth of feed nutrition effect to feeding animals in a way, and because zymin is the very strong catalyzer of specificity, so the numerous antinutritional factor of raw material is difficult to add its effect of eliminating with certain several zymin, and zymin effect simultaneously needs catalytic conditions such as strict temperature, ph.
By at China Intellectual Property Office's patent retrieval query site, a similarly patent application of bulletin, application number is: 03101357.0, be entitled as: " highly effective biological feed ", its main contents are: a biological efficiently chicken feed is provided, be added with fermentative feedstuff of microbe, unsaturated fatty acids in this feed, and the amino-acid chelate of the indispensable metal ion of human body.Behind edible this feed of laying hen, faster to nutritive ingredient absorption in the feed, the nutrient composition content of the more common egg of egg that is produced is greatly improved.Bacterial classification does not pass through any seed selection and induces in this technology, it is very low to produce enzyme effect and total viable count, add simple amino acid prescription simultaneously, take place without any chemistry or bioprocesses, its technological process is simple, can't guarantee the result of use that it " improves food consumption and grazing speed, significantly promote the gross activity of digestive tube endogenous enzyme, reduce irritability diarrhoea ".
Summary of the invention
The purpose of this invention is to provide a kind of raising cultivated animals food consumption and efficiency of feed utilization, improve the fermentation material for composite microorganism and the making method of cultivated animals immunizing power simultaneously.
For realizing above purpose, technical scheme of the present invention provides a kind of fermentation material for composite microorganism, it is characterized in that, form: subtilis 20%-24%, lactobacterium casei 22%-27%, saccharomyces cerevisiae 30%-35%, Rhodopseudomonas palustris 14%-28% by following weight percent raw material.
A kind of preparation method of compound microorganism ferments material, it is characterized in that, producing bacterial classification combines by ray induction and production performance test, screening output height, the production bacterial classification that yield of enzyme is big, production technique adopts design of probiotic bacterium symbiosis compatibility and multistage composite fermentation technique, add in the substratum and produce enzyme inducer, adopt the biological vent valve of individual event on the packaging, its production process and production technique are:
The first step: fermentation strain seed selection
A. the cultivation of going down to posterity: adopt bacillus subtilis strain that Ministry of Agriculture bacterial classification preservation center provides, lactobacterium casei, saccharomyces cerevisiae by the nutrient agar cultivation of going down to posterity, Rhodopseudomonas palustris (photosynthetic bacterium) is cultivated under 18 ℃ of-22 ℃ of illumination conditions by potato culture PDA substratum, seed selection fast growth, the tangible bacterial classification of colony characteristics are preserved bacterial classification under 0 ℃ of-4 ℃ of temperature;
B. induce seed selection: bacillus subtilis strain, lactobacterium casei and saccharomyces cerevisiae are induced respectively, the bacterial classification of preserving is made bacteria suspension, be diluted to 10 under ultraviolet irradiation
-7Is the ultraviolet lamp of 15W at the 30cm place with power, and wavelength is 250nm, handles 18-20s, bacteria suspension after processing temperature in nutrient agar plate is 30 ℃-34 ℃, be inverted and cultivated 45 hours-50 hours, the bacterium colony of selecting fast growth carries out shake flat experiment, and observation growth speed is fast, feature obviously is that total viable count is greater than 5,000,000,000/ml, the lactobacterium casei colony diameter is greater than 3ml, and saccharomyces cerevisiae is greater than 7ml, and subtilis is preserved 0 ℃ of-4 ℃ of temperature greater than the bacterial strain of 5ml;
C. performance test: three kinds of bacterial classifications among the step b are seeded in respectively in the liquid nutritional nutrient agar cultivate, culture temperature is 30 ℃-34 ℃, in being 220 rev/mins-250 rev/mins constant temperature shaking table, rotating speed carries out constant temperature culture, sampling after 24 hours-28 hours, utilize colony counting method to carry out total number of bacterial colony and measure, total number of bacterial colony can be used for the production production of hybrid seeds greater than the bacterial classification of 4,000,000,000/ml;
D. make to produce and use bacterial classification: with the subtilis that filters out, lactobacterium casei, saccharomyces cerevisiae, switching was gone into to be equipped with in the eggplant bottle of nutrient agar under 35 ℃ of-40 ℃ of temperature cultivation 45 hours-50 hours respectively, Rhodopseudomonas palustris inserts and is equipped with in the eggplant bottle of potato culture PDA substratum, cultivated 5-9 days under 20 ℃ of temperature, treat that eggplant bottle surface lawn is covered with, and can take out when band is yellow in white, put into 2-6 ℃ refrigerator then and preserve;
Second step: the liquid at high speed of composite bacteria is cultivated
A. take by weighing each bacterial classification in following ratio: subtilis 20%-24%, lactobacterium casei 22%-27%, saccharomyces cerevisiae 30%-35%, Rhodopseudomonas palustris (photosynthetic bacterium) 14%-28%;
B. the liquid culture of lactobacterium casei and saccharomyces cerevisiae: the lactobacterium casei and the saccharomyces cerevisiae that take by weighing of the ratio of a set by step, wash with stroke-physiological saline solution, make composite bacteria liquid, carry out compound cultivation after the mixing: will mix lawn and be inoculated in 120 ℃-128 ℃, the liquid nutrient medium of 30-45min high-temperature sterilization in 1: 20 ratio together, in fermentor tank, stir 25min-35min, its rotating speed is 200-220r/min, the plastic tank of putting into after stirring after the sterilization left standstill 5-7 days, left standstill the gas of every day in the process measuring the Ph value and discharging output; Sampling is 3 times in the fermenting process, and microscopically detects the thalli growth situation and measures Ph value, stops to ferment when 4,000,000,000/ml concentration and Ph value reach 3.0-4.5 when total viable count reaches;
C. the liquid culture of subtilis, Rhodopseudomonas palustris: subtilis, Rhodopseudomonas palustris lawn are washed with stroke-physiological saline solution, make composite bacteria liquid, in 1: the ratio of 20-25 is inoculated in 120 ℃-128 ℃, the liquid nutrient medium of 30min-45min high-temperature sterilization, air flow maintains 1 during fermentation: 1.0-1.2, fermentation time 24-35 hour;
The 3rd step: the solid complex ferment of bacterial classification:
A. the bacterium liquid of the compound cultivation of step 2 is mixed in the solid enlarged culturing base of back by 1: 20 120 ℃-128 ℃ of ratio accesses, 30min-45min high-temperature sterilization, in substratum, add the diethylamine tetraacethyl of 0.08%-0.1% and make to produce enzyme inducer, stir 30-45min to even, pour sealing and fermenting in the sterilization sealed can into, temperature 32-35 ℃, time 8-10 days;
Sampling is three times in b, the fermenting process, measures moisture and Ph value, takes a sample after the fermentation ends, and mensuration moisture is that 35%-40%, Ph value are 4.5-5.5;
The 4th step: add amino-acid zinc and iron:
A. add according to the purposes difference of fermentation material: if be used for the fermentation of dregs of beans, corn then add amino acid iron and amino-acid zinc, specific practice is: market is bought the amino acid iron and the amino-acid zinc goods that utilize the protein biology synthetic technology to produce and was mixed by 1: 1;
B. with mixed amino-acid zinc and iron in 2-3: 100 ratio is added in the leavened prod in the 3rd step, mixes;
If c. be used for complete feed then do not add;
The 5th step: pulverize and packing:
The product of step 3 fermentation finished thoroughly1 is mixed, and packing into has in the packing bag of individual event vent valve, tightens sack, fastens Sign Board, changes stockyard over to and stacks in order; Sampling detects, significant parameter: moisture content 35%-40%, viable count 〉=5,000,000,000/g, total protein concentration 〉=19%, Methionin 〉=1.2%; Physical behavior: deep yellow pulverulent solids.
The liquid nutrient medium of milk-acid bacteria and saccharomyces cerevisiae composition and weight percent are in the described step 2: corn steep liquor 3%-5%, molasses 12%-15%, yeast extract paste 0.2%-0.4%, sal epsom 0.2%-0.5%, ammonium sulfate 0.5%-0.7%, Sodium phosphate dibasic 0.2%-0.5%, sterilized water 77.8%-83.9%.
Subtilis and Rhodopseudomonas palustris substratum are formed and weight percent in the described step 2: Zulkovsky starch 2%-5%, bean cake powder 0.6%-1.2%, corn steep liquor 1%-6%, potassium primary phosphate 0.2%-0.6%, oxalic acid 1.4%-1.8%, sal epsom 0.4%-1%, defoamer 0.5%-1%, sterilized water 86%-93%.
Solid enlarged culturing base is that at least four kinds of mixtures that sterilized water adds in chaff, bean cake powder, molasses, Semen Maydis powder, potassium primary phosphate, Repone K, sal epsom, urea, the diethylamine tetraacethyl are clearly again formed in the described step 3, and its weight percent proportioning is: clear chaff 12%-14%, wheat bran 30%-33%, soyflour 8%-11%, molasses 8%-10%, Semen Maydis powder 8%-10%, potassium primary phosphate 0.5%-1%, Repone K 1%-1.5%, the diethylamine tetraacethyl of sal epsom 0.4%-0.7%, 0.08%-0.1%, sterilized water 18.7%-32.02%.
The present invention utilizes biotechnology to carry out a plurality of bacterial classifications of screening and optimizing, in complete feed or in the feedstuff raw material, add, nutritive substance can be decomposed or is converted into the small-molecule substance of easier absorption, and eliminate antinutritional factor in common complete feed or the feedstuff raw material, make the feed nutrition nutrient higher, easilier searched for food by livestock and poultry, digestion, absorb, adopt the design of probiotic bacterium symbiosis compatibility and the multistage composite fermentation technique of innovation, every grade of fermentation all has different separately target products, makes and is rich in a large amount of useful viable bacterias in the product, polysaccharide, polypeptide, organized enzyme, organic acid, plant sterol, three obedient saponin isoreactivity meta-bolitess.
Various nutritive ingredients are complete in the complete feed, but main nutrient composition is a macromolecular substance, are difficult to directly be absorbed by the animal digestive tube.Consumption maximum in the protein raw material wherein, just there is protease inhibitor in most important beans of function and grouts thereof, Chinese sorghum and some the piece root tubers, can cause proteinic digestibility decline in the feed, because of its can and trypsinase, stomach en-and Chymotrypsin combination generate the mixture of non-activity, reduce the activity of these enzymes; Can cause the consumption of cultivated animals body internal protein endogenous, antinutritional factor has not only influenced the nutritive value and the palatability of feed, content of cellulose accounts for 15% in the energy feed, wherein Mierocrystalline cellulose, hemicellulose and fruit caving matter are glued together by xylogen, xylogen and silicate are formed the cell walls shell, and is hard smooth.Because xylogen and crystallinity in the cellulose materials, bacterium and enzyme in the livestock digestive tube can not be decomposed and utilize, in addition, it is adipose-derived that animal raw fat can provide, but its molecular structure complexity, cultivated animals decomposes the absorption difficulty, it is wider to utilize the microbial fermentation material to use in straw feed, mainly be to decompose the Mierocrystalline cellulose that utilizes in the stalk, reduce aquaculture cost, but in fact nutritive ingredient is maximum and the most important thing is protein and animal raw fat in the feedstuff raw material, and how decomposing and utilizing these two kinds of materials is a great problems, and the bacterial classification among the present invention promptly is to choose at these three kinds difficult nutritive ingredients of decomposing.Technological line of the present invention is to adopt the probiotics that allows interpolation in the Ministry of Agriculture's 658 bulletins, utilize ray induction technology screening and improvement bacterial classification, make its yield of enzyme and speed of response maximum, a plurality of bacterial classification close fit of while, synergy, adopt liquid at high speed to cultivate and solid enlarged culturing technology, produce and accumulate the thalline of a large amount of nutrition richnesses in the fermenting process, it is lactobacillus, genus bacillus, yeast and useful metabolite, as microbiotic, VITAMIN, organic acid, hormone and Methionin etc., the technology of the present invention has the advantage of zymin method and fermentation method concurrently, can significantly eliminate the antinutritional factor effect in the feedstuff raw material, improve cultivated animals food consumption and efficiency of feed utilization, improve the immunizing power of cultivated animals simultaneously.
Each bacterial classification among the present invention is independently effect of performance separately in raising raw material decomposition and fermentation; cooperatively interact simultaneously; wherein genus bacillus is the beneficial microorganism that a class is high temperature resistant, high pressure reaches low pH value, is at particle or all more stable in liquid feed no matter be used as fodder additives.Genus bacillus enters the animal and bird intestines growth and breeding with the spore state, consume oxygen in the intestines, cause anaerobic environment, milk-acid bacteria, bifidus bacillus are increased, intestinal bacteria reduce, the subtilis of adopting among the present invention can also produce safe endogenous phylactic agent---bacitracin during the fermentation, and bacitracin is the polypeptide antibiotics that is produced by bacillus licheniformis, and leather Lan Shi positive bacteria is had very strong restraining effect.The Zinc-bacitracin fodder additives is applicable to various livestock and poultry, as ox, sheep, pig, chicken, duck etc.Have tangible promotion growth result, improve food conversion ratio and prevent and treat the effect of pig, chicken bacterial diarrhea and chronic respiratory tract disease.Increase the livestock and poultry resistance against diseases, improve health conditions, promote growth, increase trace element, improve efficiency of feed utilization.The cockerel of usefulness such as Kacmmerer is cooked heat stress test on a small scale, thinks and add particle Envelope type Zinc-bacitracin in feed, can eliminate the negative interaction that the continuous high temperature stressors is produced.Bronsch (1984) points out that Zinc-bacitracin may be to be used for increasing weight, lay eggs and making the generation of body internally heated to reduce energy.Manner etc. (1989) are under 34 ℃ of hot conditionss, in feed, add 100mg/kg particle Envelope type Zinc-bacitracin, the test of 33 weeks shows, the test group production performance is much higher than control group, laying hen weightening finish, daily ingestion amount, total egg number and total egg size improve 66.2%, 9.9%, 15.3% and 17% respectively, improve eggshell strength significantly.The generation of bacitracin is that endogenous produces among the present invention, is different from the interpolation of foreign aid's property, has advantages such as low, the active height of cost, drug residue free.
Yeast is a unicellular eukaryote, have growth fast, be easy to genetic manipulation, can translate post-treatment and modification, do not produce characteristics such as toxic products foreign protein, be considered to express the suitable host of foreign protein, the cereuisiae fermentum that adopts in this composite bacteria has fast growth, the characteristics that output is high, wherein protein content Ben Shen reaches more than 50%, in saccharomycetic meta-bolites, contain hormone, VITAMIN, nucleic acid etc. and have the physiologically active substance that promotes the crop growth function.
Milk-acid bacteria all has health care and therapeutic efficiency to humans and animals, and this point all has mass rearing and clinical trial to prove both at home and abroad.Baird (1977) all can increase day weight gain and improve food conversion ratio with Bacterium lacticum feed weanling pig and growing-finishing pig, evidence; Lidbeck etc. (1992) confirm the diarrhoea that lactobacillus can prevent radiotherapy to cause.Cai Huiyi etc. (1993) add up the probiotics result of use, the lactic acid bacteria class probiotics report of pig of feeding wherein, 7 illustrations are bright to improve day weight gain, average improve that 7.67%, 6 illustration is bright improves food conversion ratio, on average improve 5.4%, milk-acid bacteria is a matrix with the carbohydrate that photosynthetic bacterium produces then, secrete lactic acid, lactic acid has extremely strong sterilizing power, and has the movable and corrupt rapidly function of decomposing of organism of inhibition harmful microbe.In addition, the organism that milk-acid bacteria can also be difficult to animals such as xylogen, Mierocrystalline cellulose digestibility and utilization resolves into micromolecular animal and is easy to the nutritive substance that absorbs, utilize, has improved efficiency of feed utilization.
Photosynthetic bacterium is to be the energy with the light and heat, is matrix with lactic acid etc., under anaerobic can carry out photosynthetic bacterium general name.It has the carbohydrate of promotion, amino acids, VITAMIN and the biosynthetic function of physiologically active substance.And can decompose airborne obnoxious flavour, sewage there is very strong detergent power.Between the beneficial microorganisms such as it and vinelandii, actinomycetes, milk-acid bacteria synergy is arranged.In addition, the protein content of photosynthetic bacterium itself is up to more than 60%.
Composite bacteria of the present invention can also produce a large amount of digestive ferments and active metabolite in nutritious complete feed fermenting process, as: as phytase, amylase, proteolytic enzyme,, cellulase, lipase etc.; These enzymes can effectively decompose protein, Mierocrystalline cellulose, the animal raw fat that is difficult in the feedstuff raw material to decompose and is absorbed by animal.Produce the essential nutritive ingredient of animal body simultaneously, as Methionin, methionine(Met), tryptophane, VITAMIN etc.; These materials no longer need to add after using product of the present invention, greatly reduce feed and aquaculture cost.
Because Bacillus subtilus is a spore production bacteria, later stage fermentation liquid is alkalescence, and two kinds of milk-acid bacterias are typical acid culturing bacterium, direct compound cultivation has very big difficulty, so product of the present invention has adopted the liquid single culture, carry out the way that solid is tamed compound cultivation again, both guaranteed the bacterial classification number of viable, with the reservation maximization of meta-bolites, guaranteed the effect of product again.Simultaneously in the finished product, add carrier, guaranteed the survival time of bacterial classification in transportation and preservation process.
The present invention adds some inductor, tensio-active agent and some other product enzyme promotor in the fermentation using enzyme process, can increase the yield of enzyme of thalline greatly.The present invention is by test, selected a kind of tensio-active agent for use: the diethylamine tetraacethyl, inductor as zymin, by the test contrast, production of enzyme 2-5% can be provided, promotor can promote the reason that output increases: mainly be the perviousness of having improved cell, strengthened the transmission speed of oxygen, improved the effective utilization of thalline to oxygen.
After the product fermentation is finished, because a large amount of useful viable bacterias exists and the active metabolism material exists, so can not adopt conventional vacuum-drying packing, this product has adopted the biological vent valve of a kind of novel individual event, the gas of packaged fermentation product can in time be discharged outside the packing, and the extraneous space of containing assorted bacterium can not enter, and this just guarantees that product is in the optimum activity state, can bring into play maximum effect in the quality guaranteed period.
Compare with other methods of eliminating antinutritional factor of present stage, advantage of the present invention is:
1. adopt inductive technology improvement bacterial classification in the strain improvement process,, guaranteed each bacterial classification yield of enzyme and speed of growth efficiently in substratum simultaneously in conjunction with the bacterial classification performance test;
2. adopt design of probiotic bacterium symbiosis compatibility and multistage composite fermentation technique, every grade of fermentation all has different separately target products, simultaneously add special product inductor during the fermentation, make and be rich in a large amount of useful viable bacterias and enzyme, peptide, polysaccharide and amino acid isoreactivity meta-bolites in the product;
3. by producing a large amount of endogenous organized enzymes, antioxidant and microbiotic, improve the feed palatability, improve food consumption, strengthen the anti-stress and the immunizing power of cultivated animals, improve colony's reguarity, and improve the speed of growth and efficiency of feed utilization, reduce aquaculture cost;
4. packing adopts exclusive biological individual event vent valve, ensures that interior viable bacteria of product and meta-bolites guarantee that product is in the optimum activity state, can bring into play maximum effect in the quality guaranteed period;
5. product of the present invention both can be added in the complete feed, and fermentable raw material ferments back soluble protein content raising more than 12% again, and neutral detergent fiber descends more than 15%, and useful total viable count is greater than 5,000,000,000/ml;
6. product of the present invention adopts the Ministry of Agriculture's 658 bulletins to allow the microbial strains of adding, and does not have hazard residue in the cultivated animals body, has reduced the usage quantity of microbiotic in breed simultaneously, is a kind of safe, green microniological proudcts.
Embodiment
The invention will be further described below in conjunction with embodiment.
Embodiment 1
A kind of preparation method of compound microorganism ferments material is:
The present invention adopts bacterial classification and is numbered Lactobacillus casei (ACCC10640) lactobacterium casei, Saccharomyces cerevisiae (IFO0203) saccharomyces cerevisiae, Bacillus licheniformis (ACCC10619) subtilis, Rhodopseudomonas palustris (ACCC10649) Rhodopseudomonas palustris, all bacterial classifications are provided by Ministry of Agriculture bacterial classification preservation center.
The first step: fermentation strain seed selection
A. the cultivation of going down to posterity: the bacillus subtilis strain, lactobacterium casei, saccharomyces cerevisiae, the Rhodopseudomonas palustris (photosynthetic bacterium) that adopt Ministry of Agriculture bacterial classification preservation center to provide, by the nutrient agar cultivation of going down to posterity, Rhodopseudomonas palustris (photosynthetic bacterium) is cultivated under 20 ℃ of illumination conditions by the PDA substratum, seed selection fast growth, the tangible bacterial classification of colony characteristics are preserved bacterial classification under 4 ℃ of temperature;
B. induce seed selection: bacillus subtilis strain, lactobacterium casei and saccharomyces cerevisiae are induced respectively, the bacterial classification of preserving is made bacteria suspension, be diluted to 10 under ultraviolet irradiation
-7Is the ultraviolet lamp of 15W at the 30cm place with power, and wavelength is 250nm, handles 18s, bacteria suspension after processing temperature in nutrient agar plate is 34 ℃, be inverted and cultivated 45 hours, the bacterium colony of selecting fast growth carries out shake flat experiment, and observation growth speed is fast, obviously (total viable count is greater than 5,000,000,000/ml for feature, the lactobacterium casei colony diameter is greater than 3ml, and saccharomyces cerevisiae is greater than 7ml, and subtilis is greater than 5ml) bacterial strain preserved 4 ℃ of temperature;
C. performance test: three kinds of bacterial classifications among the step b are seeded in respectively in the liquid nutritional nutrient agar cultivate, culture temperature is 30 ℃, in being 220 rev/mins constant temperature shaking table, rotating speed carries out constant temperature culture, sampling after 24 hours-28 hours, utilize colony counting method to carry out total number of bacterial colony and measure, total number of bacterial colony can be used for the production production of hybrid seeds greater than the bacterial classification of 4,000,000,000/ml;
D. make to produce and use bacterial classification: with the subtilis that filters out, lactobacterium casei, saccharomyces cerevisiae, switching was gone into to be equipped with in the eggplant bottle of nutrient agar under 35 ℃ of temperature cultivation 45 hours-50 hours respectively, Rhodopseudomonas palustris inserts and is equipped with in the eggplant bottle of PDA substratum, cultivated 7 days under 20 ℃ of temperature, treat that eggplant bottle surface lawn is covered with, and can take out when band is yellow in white, put into 2-6 ℃ refrigerator then and preserve;
Nutrient agar: extractum carnis 3g, peptone 10g, NaCl 5g, agar 15-20g, water 1000ml, pH7.0-7.2,121 ℃ of sterilization 20min;
Potato culture (PDA): potato 200g, sucrose or glucose 20g, agar 15-20g, water 1000ml, pH nature;
Second step: the liquid at high speed of composite bacteria is cultivated
A. take by weighing each bacterial classification in following ratio: subtilis 20%, lactobacterium casei 22%, saccharomyces cerevisiae 30%, Rhodopseudomonas palustris (photosynthetic bacterium) 28%.
B. the liquid culture of lactobacterium casei and saccharomyces cerevisiae: the lactobacterium casei and the saccharomyces cerevisiae that take by weighing of the ratio of a set by step, wash with stroke-physiological saline solution, make composite bacteria liquid, carry out compound cultivation after the mixing: will mix lawn and be inoculated in 121 ℃, the liquid nutrient medium of 30min high-temperature sterilization in 1: 20 ratio together, in fermentor tank, stir 25min-35min, its rotating speed is 200r/min, the plastic tank of putting into after stirring after the sterilization left standstill 5-7 days, left standstill the gas of every day in the process measuring the Ph value and discharging output; Sampling is 3 times in the fermenting process, and microscopically detects the thalli growth situation and measures Ph value, stops to ferment when 4,000,000,000/ml concentration and Ph value reach 3.0-4.5 when total viable count reaches;
The liquid nutrient medium of milk-acid bacteria and saccharomyces cerevisiae is formed and weight percent is: corn steep liquor 3%, molasses 12%, yeast extract paste 0.2%, sal epsom 0.2%, ammonium sulfate 0.5%, Sodium phosphate dibasic 0.2%, sterilized water 83.9%.
C. the liquid culture of subtilis, Rhodopseudomonas palustris: subtilis, Rhodopseudomonas palustris lawn are washed with stroke-physiological saline solution, make composite bacteria liquid, in 1: the ratio of 20-25 is inoculated in 121 ℃, the liquid nutrient medium of 30min high-temperature sterilization, air flow maintains 1: 1 during fermentation, fermentation time 28 hours;
Subtilis and Rhodopseudomonas palustris substratum are formed and weight percent: Zulkovsky starch 3%, bean cake powder 1.2%, corn steep liquor 4%, potassium primary phosphate 0.4%, oxalic acid 1.6%, sal epsom 0.8%, defoamer 0.5%, sterilized water 88.5%.
The 3rd step: the solid complex ferment of bacterial classification:
A. the bacterium liquid of the compound cultivation of step 2 is mixed in the solid enlarged culturing base of back by 1: 20 121 ℃ of ratio accesses, 30min high-temperature sterilization, in substratum, add 0.08% diethylamine tetraacethyl work product enzyme inducer, stir 30-45min to even, pour sealing and fermenting in the sterilization sealed can into, 32 ℃ of temperature, 8 days time;
Sampling is three times in b, the fermenting process, measures moisture and Ph value, takes a sample after the fermentation ends, and mensuration moisture is that 35%-40%, Ph value are 4.5-5.5;
Solid enlarged culturing basic weight amount per distribution ratio is: clear chaff 12%, wheat bran 30%, soyflour 8%, molasses 8%, Semen Maydis powder 8%, potassium primary phosphate 0.5%, Repone K 1%, the diethylamine tetraacethyl of sal epsom 0.4%, 0.08%, sterilized water 31.3%;
The 4th step: add amino-acid zinc and iron:
The amino acid iron and the amino-acid zinc goods that utilize the protein biology synthetic technology to produce are bought in market mixed by 1: 1,
Mixed amino-acid zinc and iron are added in the leavened prod in the 3rd step in 1: 50 ratio, mix;
The 5th step: pulverize and packing:
The product of step 3 fermentation finished thoroughly1 is mixed, and packing into has in the packing bag of individual event vent valve, tightens sack, fastens Sign Board, changes stockyard over to and stacks in order; Sampling detects, significant parameter: moisture content 35%-40%, viable count 〉=5,000,000,000/g, total protein concentration 〉=19%, Methionin 〉=1.2%; Physical behavior: deep yellow pulverulent solids
Embodiment 2:
The first step: fermentation strain seed selection: with embodiment 1
Second step: the liquid at high speed of composite bacteria is cultivated
A. take by weighing each bacterial classification in following ratio: subtilis 24%, lactobacterium casei 27%, saccharomyces cerevisiae 35%, Rhodopseudomonas palustris (photosynthetic bacterium) 14%.
B. the liquid culture of milk-acid bacteria and saccharomyces cerevisiae: the lactobacterium casei and the saccharomyces cerevisiae that take by weighing of the ratio of a set by step, wash with stroke-physiological saline solution, make composite bacteria liquid, carry out compound cultivation after the mixing: will mix lawn and be inoculated in 125 ℃, the liquid nutrient medium of 45min high-temperature sterilization in 1: 25 ratio together, in fermentor tank, stir 35min, its rotating speed is 220r/min, the plastic tank of putting into after stirring after the sterilization left standstill 5-7 days, left standstill the gas of every day in the process measuring the Ph value and discharging output; Sampling is 3 times in the fermenting process, and microscopically detects the thalli growth situation and measures Ph value, stops to ferment when 4,000,000,000/ml concentration and Ph value reach 3.0-4.5 when total viable count reaches;
The liquid nutrient medium of milk-acid bacteria and saccharomyces cerevisiae is formed and weight percent is: corn steep liquor 5%, molasses 15%, yeast extract paste 0.4%, sal epsom 0.5%, ammonium sulfate 0.7%, Sodium phosphate dibasic 0.5%, sterilized water 77.9%.
C. the liquid culture of subtilis, Rhodopseudomonas palustris: subtilis, Rhodopseudomonas palustris lawn are washed with stroke-physiological saline solution, make composite bacteria liquid, be inoculated in 125 ℃, the liquid nutrient medium of 45min high-temperature sterilization in 1: 25 ratio, air flow maintains 1 during fermentation: 1.0-1.2, fermentation time 30 hours;
Liquid nutrient medium weight ratio per-cent is with embodiment 1.
The 3rd step: the solid complex ferment of bacterial classification:
A. the bacterium liquid of the compound cultivation of step 2 is mixed in the solid enlarged culturing base of back by 1: 25 125 ℃ of ratio accesses, 45min high-temperature sterilization, in substratum, add 0.1% diethylamine tetraacethyl work product enzyme inducer, stir 45min to even, pour sealing and fermenting in the sterilization sealed can into, 335 ℃ of temperature, 8 days time;
B, with embodiment 1.
The substratum of solid complex ferment is formed and per distribution ratio is: clear chaff 14%, wheat bran 33%, soyflour 11%, molasses 10%, Semen Maydis powder 10%, potassium primary phosphate 1%, Repone K 1.5%, the diethylamine tetraacethyl of sal epsom 0.7%, 0.1%, sterilized water 18.7%.
The 4th step: add amino-acid zinc and iron:
A. with embodiment 1.
B, mixed amino-acid zinc and iron are added in the leavened prod in the 3rd step in 3: 100 ratio, mix.
C. with embodiment 1.
The 5th step: pulverize and packing: with embodiment 1.
Fully stir with 90% ratio of complete feed or feedstuff raw material in 10% of this preparation during use, can feed after evenly.Attention this product need be deposited in the hermetically drying place, avoid mixing stacking with agricultural chemicals, strong stimulation article etc., break a seal and use as early as possible in back three days, this product shows through the simultaneous test of academy of agricultural sciences, Shanghai City in September, 2006 herding institute in R﹠D process, improve child care piglet day weight gain 9.30% after using this product, improve child care pig starter feed utilization ratio 7.54%, improve growing swine day weight gain 23%, improve growing swine efficiency of feed utilization 3.66%.The application test data that carry out in boar cultivation base, Kingsoft, Shanghai in March, 2007 are:
| The comparison project | Control group | Test group |
| Starting weight (kg) | 58.77±7.56 | 56.92±7.93 |
| Eventually heavy (kg) | 78.54±8.59 | 80.00±8.98 |
| Weightening finish (kg) | 19.77±3.09 | 23.08±4.01 |
| Weightening finish raising rate (%) | 100.00 | 116.71 |
| Feedstuff-meat ratio | 3.60 | 3.24 |
| Price of deed raising rate (%) | 100.00 | 111.11 |
Feed digestibility during the pig 70kg left and right sides
| Digestibility | Test group | Control group | Improve % |
| Crude fiber digestibility (%) | 34.57 | 26.48 | 30.55 |
| Crude protein digestibility (%) | 71.80 | 67.62 | 6.18 |
| Ash digestibility (%) | 47.73 | 58.40 | -18.27 |
| Organic matter digestibility (%) | 79.66 | 78.02 | 2.10 |
Above numeral shows that the relevant data of the art of this patent product shows that the art of this patent product can obviously reduce the influence of multiple antinutritional factor to feed nutrition, significantly improve efficiency of feed utilization, improve palatability and the food consumption of animal, reach volume increase, synergy, the culture efficiency that reduces cost.
Claims (5)
1. a fermentation material for composite microorganism is characterized in that, is made up of following weight percent raw material: subtilis 20%-24%, lactobacterium casei 22%-27%, saccharomyces cerevisiae 30%-35%, Rhodopseudomonas palustris 14%-28%.
2. the preparation method of a kind of compound microorganism ferments material according to claim 1, it is characterized in that, producing bacterial classification combines by ray induction and production performance test, screening output height, the production bacterial classification that yield of enzyme is big, production technique adopts design of probiotic bacterium symbiosis compatibility and multistage composite fermentation technique, add in the substratum and produce enzyme inducer, adopt the biological vent valve of individual event on the packaging, its production process and production technique are:
The first step: fermentation strain seed selection
A. the cultivation of going down to posterity: adopt bacillus subtilis strain that Ministry of Agriculture bacterial classification preservation center provides, lactobacterium casei, saccharomyces cerevisiae by the nutrient agar cultivation of going down to posterity, Rhodopseudomonas palustris is cultivated under 18 ℃ of-22 ℃ of illumination conditions by potato culture PDA substratum, seed selection fast growth, the tangible bacterial classification of colony characteristics are preserved bacterial classification under 0 ℃ of-4 ℃ of temperature;
B. induce seed selection: bacillus subtilis strain, lactobacterium casei and saccharomyces cerevisiae are induced respectively, the bacterial classification of preserving is made bacteria suspension, be diluted to 10 under ultraviolet irradiation
-7Is the ultraviolet lamp of 15W at the 30cm place with power, and wavelength is 250nm, handles 18-20s, bacteria suspension after processing temperature in nutrient agar plate is 30 ℃-34 ℃, be inverted and cultivated 45 hours-50 hours, the bacterium colony of selecting fast growth carries out shake flat experiment, and observation growth speed is fast, feature obviously is that total viable count is greater than 5,000,000,000/ml, the lactobacterium casei colony diameter is greater than 3ml, and saccharomyces cerevisiae is greater than 7ml, and subtilis is preserved 0 ℃ of-4 ℃ of temperature greater than the bacterial strain of 5ml;
C. performance test: three kinds of bacterial classifications among the step b are seeded in respectively in the liquid nutritional nutrient agar cultivate, culture temperature is 30 ℃-34 ℃, in being 220 rev/mins-250 rev/mins constant temperature shaking table, rotating speed carries out constant temperature culture, sampling after 24 hours-28 hours, utilize colony counting method to carry out total number of bacterial colony and measure, total number of bacterial colony can be used for the production production of hybrid seeds greater than the bacterial classification of 4,000,000,000/ml;
D. make to produce and use bacterial classification: with the subtilis that filters out, lactobacterium casei, saccharomyces cerevisiae, switching was gone into to be equipped with in the eggplant bottle of nutrient agar under 35 ℃ of-40 ℃ of temperature cultivation 45 hours-50 hours respectively, Rhodopseudomonas palustris inserts and is equipped with in the eggplant bottle of potato culture PDA substratum, cultivated 5-9 days under 20 ℃ of temperature, treat that eggplant bottle surface lawn is covered with, and can take out when band is yellow in white, put into 2-6 ℃ refrigerator then and preserve;
Second step: the liquid at high speed of composite bacteria is cultivated
A. take by weighing each bacterial classification in following ratio: subtilis 20%-24%, lactobacterium casei 22%-27%, saccharomyces cerevisiae 30%-35%, Rhodopseudomonas palustris 14%-28%;
B. the liquid culture of lactobacterium casei and saccharomyces cerevisiae: the lactobacterium casei and the saccharomyces cerevisiae that take by weighing of the ratio of a set by step, wash with stroke-physiological saline solution, make composite bacteria liquid, carry out compound cultivation after the mixing: will mix lawn and be inoculated in 120 ℃-128 ℃, the liquid nutrient medium of 30-45min high-temperature sterilization in 1: 20 ratio together, in fermentor tank, stir 25min-35min, its rotating speed is 200-220r/min, the plastic tank of putting into after stirring after the sterilization left standstill 5-7 days, left standstill the gas of every day in the process measuring the Ph value and discharging output; Sampling is 3 times in the fermenting process, and microscopically detects the thalli growth situation and measures Ph value, stops to ferment when 4,000,000,000/ml concentration and Ph value reach 3.0-4.5 when total viable count reaches;
C. the liquid culture of subtilis, Rhodopseudomonas palustris: subtilis, Rhodopseudomonas palustris lawn are washed with stroke-physiological saline solution, make composite bacteria liquid, in 1: the ratio of 20-25 is inoculated in 120 ℃-128 ℃, the liquid nutrient medium of 30min-45min high-temperature sterilization, air flow maintains 1 during fermentation: 1.0-1.2, fermentation time 24-35 hour;
The 3rd step: the solid complex ferment of bacterial classification:
A. the bacterium liquid of the compound cultivation of step 2 is mixed in the solid enlarged culturing base of back by 1: 20 120 ℃-128 ℃ of ratio accesses, 30min-45min high-temperature sterilization, in substratum, add the diethylamine tetraacethyl of 0.08%-0.1% and make to produce enzyme inducer, stir 30-45min to even, pour sealing and fermenting in the sterilization sealed can into, temperature 32-35 ℃, time 8-10 days;
B. take a sample three times in the fermenting process, measure moisture and Ph value, take a sample after the fermentation ends, mensuration moisture is that 35%-40%, Ph value are 4.5-5.5;
The 4th step: add amino-acid zinc and iron:
A. add according to the purposes difference of fermentation material: if be used for the fermentation of dregs of beans, corn then add amino acid iron and amino-acid zinc, specific practice is: market is bought the amino acid iron and the amino-acid zinc goods that utilize the protein biology synthetic technology to produce and was mixed by 1: 1;
B. with mixed amino-acid zinc and iron in 2-3: 100 ratio is added in the leavened prod in the 3rd step, mixes;
If c. be used for complete feed then do not add;
The 5th step: pulverize and packing:
The product of step 3 fermentation finished thoroughly1 is mixed, and packing into has in the packing bag of individual event vent valve, tightens sack, fastens Sign Board, changes stockyard over to and stacks in order; Sampling detects, significant parameter: moisture content 35%-40%, viable count 〉=5,000,000,000/g, total protein concentration 〉=19%, Methionin 〉=1.2%; Physical behavior: deep yellow pulverulent solids.
3. the preparation method of a kind of compound microorganism ferments material according to claim 2, it is characterized in that the liquid nutrient medium of milk-acid bacteria and saccharomyces cerevisiae composition and weight percent are in the described step 2: corn steep liquor 3%-5%, molasses 12%-15%, yeast extract paste 0.2%-0.4%, sal epsom 0.2%-0.5%, ammonium sulfate 0.5%-0.7%, Sodium phosphate dibasic 0.2%-0.5%, sterilized water 77.8%-83.9%.
4. the preparation method of a kind of compound microorganism ferments material according to claim 2, it is characterized in that subtilis and Rhodopseudomonas palustris substratum are formed and weight percent in the described step 2: Zulkovsky starch 2%-5%, bean cake powder 0.6%-1.2%, corn steep liquor 1%-6%, potassium primary phosphate 0.2%-0.6%, oxalic acid 1.4%-1.8%, sal epsom 0.4%-1%, defoamer 0.5%-1%, sterilized water 86%-93%.
5. the preparation method of a kind of compound microorganism ferments material according to claim 2, it is characterized in that, solid enlarged culturing base in the described step 3 is that sterilized water adds chaff clearly again, bean cake powder, molasses, Semen Maydis powder, potassium primary phosphate, Repone K, sal epsom, urea, at least four kinds of mixtures in the diethylamine tetraacethyl are formed, and its weight percent proportioning is: clear chaff 12%-14%, wheat bran 30%-33%, soyflour 8%-11%, molasses 8%-10%, Semen Maydis powder 8%-10%, potassium primary phosphate 0.5%-1%, Repone K 1%-1.5%, sal epsom 0.4%-0.7%, the diethylamine tetraacethyl of 0.08%-0.1%, sterilized water 18.7%-32.02%.
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