CN101485402B - Composite biological feed additive agent for fattening early weaning mutton sheep - Google Patents
Composite biological feed additive agent for fattening early weaning mutton sheep Download PDFInfo
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- CN101485402B CN101485402B CN2009100777480A CN200910077748A CN101485402B CN 101485402 B CN101485402 B CN 101485402B CN 2009100777480 A CN2009100777480 A CN 2009100777480A CN 200910077748 A CN200910077748 A CN 200910077748A CN 101485402 B CN101485402 B CN 101485402B
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Abstract
The invention relates to an early weaning meat sheep fattening compound biological feed additive, which belongs to the technical field of animals feeding by feed. The feed additive comprises prebiotics consisting of candida utilis, bacillus subtilis and a lactobacillus plantarum microbial agent, non-starch polysaccharide enzyme containing cellulase, xylanase, beta-dextranase, beta-mannase and pectinase and a yeast culture formed by using saccharomyces cerevisiae to completely ferment bean pulp. The additive is specially used for fattening period ration of the early weaning(the weaning lunar age is less than or equal to 2 ages of the moon) meat sheep, and the adding percentage for the full-mixing ration of the fast-fattening meat sheep in 3 to 5 ages of the moon is 1 percent. The additive can provide functional nutrients such as digestive enzyme, B group vitamins and growth promoting factors, and the like which are inadequately produced and insufficient for the early weaning baby sheep and simultaneously contains probiotics for adjusting the micro-ecological environment in intestinal canals, thereby improving the immunity and the healthy level of the baby meat sheep; and after weaning, the daily gaining in weight is more than 10 percent in the prior period and later period of fattening, and the economic benefits are obviously improved.
Description
Technical field
The present invention relates to a kind of composite biological feed additive agent for fattening early weaning mutton sheep, relate in particular in the mutton sheep drylot feeding stable breeding mode of production, be exclusively used in a kind of health care, short long and reduce discharging the green bio feed addictive of pollutant of the fast well-fattened daily ration of early weaning meat lamb.
Background technology
To being the drylot feeding stable breeding of master's TRM with stalk and concentrated feed resource, is that mutton sheep is raised and the dramatic change of the mutton mode of production from the free range husbandry that relies on wild forage grass resource, also is the inevitable direction that mutton sheep is already developed.This change needs multinomial feed and forming a complete and comprehensive system for collecting real estate fees of breeding technology could implement and obtain good result, and one of key technology wherein is the fast well-fattened The Application of Technology of early weaning meat lamb.The fast well-fattened difficult problem of bringing of early weaning lamb has three: one of which; Intestines and stomach health status is poor; Very easily diarrhoea causes production performance to descend even is dead; Usually the method that solves fast well-fattened lamb initial stage diarrhoea problem is to use antibiotics and chemical synthesis additive control disease in a large number, consequently delay cud grow, malnutritive and make production performance performance reduction, and cause environmental pollution and nuisance residual; Its two, lamb fattening phase and cud puberty are synchronous, promote cud normal grow fast improving the lamb general level of the health and roughage digestibility extremely important, but this difficult problem that also is far from the production practices solving; Its three, lamb fattening later stage daily ration contains 30%～40% forage grass or straw in forming, and concentrated feeds such as barley, dregs of beans, causes SNSP content high and reduce digestibility.
Little ecological probio biology feed additive is to promote cud to grow, substitute the environmental protection growth-promoting additive of new generation that antibiotic is used for animal health; The type additive is taken oxygen by force through animal gastrointestinal tract and is kept anaerobic environment, produces a series of effects such as enzyme and product acid; Regulate microecological balance and improve immunity and improve the animal gastrointestinal tract general level of the health, improve the daily ration digestibility; Thereby raising breeding performonce fo animals; Avoid the use of pollution and resistance to the action of a drug side effect that antibiotic brings simultaneously, bacterial classification combination, viable bacteria content and proportioning thereof are the important technology parameters of its function of performance in the probiotics preparation, also are one of emphasis of this field scientific research.
Non-starch polysaccharide enzyme (NSP enzyme) can improve SNSP class ANFs digestibility in children's age ruminant, reduces the viscosity of enteron aisle chyme and improves the whole digestibility of daily ration.Basic element in this enzyme has five types; Be cellulase, zytase, 1,4 beta-glucanase, 'beta '-mannase, pectase; The different combinations ratio should be designed to different animals with raising stage daily ration characteristics and the effect of getting twice the result with half the effort can be produced; In the research at present few in number adopt different single enzyme breed combination prescriptions, complicated technology costs such as liquid fermentation and single enzyme purification are too high to be difficult to practical application to these class methods because of relating to more.
The inventor is engaged in the research work of fattening lamb feed diet for a long time, is probio with bacillus subtilis and saccharomycete once, is applied in the lamb fattening daily ration as enzyme preparation with non-starch polysaccharide enzyme; Find to test and fatten daily gain early stage than control group raising 17.4%; And the later stage raising is merely 5.99%, and the full phase improves 11.04%, does not reach full growth owing to the little cud of age in days in early stage; The secretory volume of digestive ferment is lower; Thereby interpolation probio and non-starch polysaccharide enzyme have significantly improved digestibility and have improved daily gain, and the later stage cud is grown gradually, because the fermentation of cud makes the digestive function of lamb obtain reinforcement; The probio of adding in the feed and the effect of non-starch polysaccharide enzyme reduce, thereby cause above-mentioned phenomenon to take place.
Summary of the invention
To above-mentioned defective; The present invention provides a kind of composite biological feed additive agent for fattening early weaning mutton sheep; Can provide comprehensive nutrition of early weaning lamb and physiology to regulate, promote animal health to grow up, improve immunity; And, significantly improved economic benefit fattening early stage and later stage daily gain raising after the wean all more than 10%.
A kind of composite biological feed additive agent for fattening early weaning mutton sheep; Comprise and account for the probiotic that gross weight is 25-35%, the non-starch polysaccharide enzyme of 40-60% and the yeast culture of 15-25%; Said probiotic is made up of candida utili, bacillus subtilis and Lactobacillus plantarum microbial inoculum, viable bacteria number average>=1 * 10 of its microbial inoculum
10Cfu/g, viable count is than being 0.9-1.1: 0.9-1.1: 0.9-1.1; Crude protein content in the yeast culture>=50%, acid protease activity>=10
6U/kg, non-starch polysaccharide enzyme comprise cellulase, zytase, 1,4 beta-glucanase, 'beta '-mannase and pectase, enzyme activity difference>=4.0 * 10
5U/kg, 5.0 * 10
6U/kg, 5.0 * 10
6U/kg, 3.0 * 10
5U/kg, 5.0 * 10
5U/kg, its enzyme activity portfolio ratio is 1.1-1.3: 12-16: 15-16: 1.0-1.3: 1-2.
Said candida utili is candida utili (Candia utilis Accc20060).
Said bacillus subtilis is bacillus subtilis (Bacillus subtilis CICC 10081).
Said Lactobacillus plantarum be Lactobacillus plantarum (lactobacillus planticola, Accc11118).
Preferred prescription 30% probiotic, 50% SNSP and 20% yeast culture enzyme.
The preparation method of above-mentioned additive: (1) obtains probiotic with the pulvis of candida utili, bacillus subtilis and Lactobacillus plantarum by the viable count mixed; (2) use Wine brewing yeast strain and be that main culture medium carries out solid state fermentation and processes yeast culture with the dregs of beans; (3) be that main culture medium carries out the solid state fermentation mode and makes non-starch polysaccharide enzyme with trichoderma reesei (Trichoderma reesei) Rut C-30 bacterial strain with wheat bran; (4) probiotic, yeast culture, non-starch polysaccharide enzyme are mixed and made into additive of the present invention in proportion.
Said Wine brewing yeast strain be saccharomyces cerevisiae (Saccharomyces cerevisiae, Accc20063).
The fermentation condition of said saccharomyces cerevisiae is that solid medium is a dregs of beans 90%+ wheat bran 10%, and the material-water ratio example is 1: 1.5, inoculum concentration 6%, 30 ℃ of cultivation temperature, incubation time 96 hours.
Said trichoderma reesei (Trichoderma reesei) Rut C-30 bacterial strain solid state fermentation conditions is solid fermentation culture medium wheat bran 95g+ corn flour 5g+K
2HPO
43H
2O 0.1g+MgSO
47H
2O 0.16g, carbon-nitrogen ratio 21; Inoculum concentration 8%, humidity 50% is cultivated 5d in 30 ℃ of ventilation insulating boxs or greenhouse.
The present invention adjusts on the basis of original additive, and the probiotic class is made up of candida utili, bacillus subtilis and Lactobacillus plantarum, through its zymogram product of fermentation condition change of control trichoderma reesei; Make it contain the 'beta '-mannase of definite amount; And the configuration of the vigor of cellulase, zytase, 1,4 beta-glucanase, 'beta '-mannase and pectase is fit to produce feeding non-starch polysaccharide enzyme (NSP enzyme), also adds yeast culture, and the three is auxilliary in proportion to be joined; The lamb fattening feed addictive obtains weaning; Through raising proof, additive of the present invention is grown promoting early weaning lamb cud, and body weight increases; Can put on weight fast well-fattened lamb too for the full grown lamb of later stage cud.
The biologically active probio reaches intestines and stomach and plays a role and receives influence of various factors; Viable count is one of them key factor, and even and the importance of this factor also has a great difference because of the complexity of different domestic animals, poultry ruminant tumor gastric.The present invention is according to the physilogical characteristics of wean lamb, thinks that the probio of feeding adds the order of magnitude and answer>=10 in daily ration
6～10
7Cfu/g, components of additives viable count of the present invention is 10
10～10
11Cfu/g, the adding proportion in daily ration>=0.1% o'clock can reach viable count, promptly>=10
7Cfu/g; So three's viable count ratio is confirmed as about 1: 1: 1, proportion is about 30% in additive simultaneously, when this additive adds 1% in daily ration; Then the adding proportion of every kind of viable bacteria component in daily ration just in time is about 0.1%, just can reach suitable viable count.
1. candida utili
Candida utili is named again and produces protein torula or edible torula, and the content of its protein and Cobastab is all high than saccharomyces cerevisiae.Mutton sheep belongs to ruminant; The lamb fattening phase, its cud was in the growth course from the prematurity to the maturation; The growth that promotes cud to reach maturity as early as possible to help mutton sheep and to the fermentation digestion of SNSP nutrients such as cellulose, hemicellulose improves speed of weight increment and efficiency of feed utilization thereby be beneficial to.Blastocystis is in double oxygen bacterium, but under the condition that aerobic exists, is earlier with the aerobic fermentation.In mutton sheep searches for food process; Brought a large amount of air (oxygen) into; The sweat of viable yeast bacterium can consume the oxygen in the cud rapidly and make cud be in better anaerobic environment; This is the normal microbial body based environment of lumen fermentation, is particularly conducive to beneficial microorganism propagation such as cellulose-decomposing bacterium.Because the increasing substantially of beneficial microbe quantity in the cud strengthened the physiological function of cud, actual result is the quick growth that has promoted the rumen microorganism system, the digestion fiber with utilize the function of lactic acid to be improved, improved the lamb production performance.Yeast cells can pass through absorption noxious material and antagonism pathogen, thereby effectively prevents enterogastric diseases, improves the premunition of mutton sheep.
Saccharomycete itself contains high-quality protein and a large amount of B family vitamins more than 50%, can secrete a large amount of neutral proteinases simultaneously, promotes the raising of mutton sheep small intestine digestibility, thereby improves production performance.
2. bacillus subtilis
Bacillus subtilis is an aerobic bacteria, is characterized in producing a large amount of ectoenzyme (protease, amylase and hydrolysis of hemicellulose enzyme etc.), and these enzymes can promote the digestion and the absorption of feed, improves the utilization rate of feed, thereby promotes growth of animal.Main effect in the meat sheep feed additive is to exhaust gastral oxygen rapidly and produce a large amount of protease.Because this bacterium has high-intensity anti-hydrochloric acid in gastric juice ability; For the acid intestinal environment that Lactobacillus plantarum produces, its activity is unaffected, so in whole gastral environment, all can keep active; Except that lot of consumption cud oxygen content; Simultaneously all play the effect that guarantees anaerobic environment at each section enteron aisle, but and therefore cause the propagation of harmful aerobic bacteria and protect GI health, to regulate the enteric microorganism system in a healthy and balanced way thereby improve the normal anaerobic bacteria breeding of enteron aisle.A large amount of extracellular proteinases in this bacterium reproductive process improve meat sheep feed proteopepsis rate in addition, and this is for children's lamb particular importance in age.
3. Lactobacillus plantarum
Lactobacillus plantarum belongs to lactic acid bacteria class; Lactic acid bacteria breeds in animal intestinal and can produce multiple inhibition compound; Comprise bacteriocin albumen antibiotic such as () lactobacillins, the plain type material of bacterioid and various antagonism material, like hydrogen peroxide and some organic acid etc.The bacteriocin that lactic acid bacteria produces is inhibited to gram-positive bacteria, can suppress the growth of pathogenic microorganism.Lactic acid bacteria can also adhere on the intestinal cell, and occupancy competition and nutrients competition effect are arranged.The organic acid that it produces can reduce the pH value in the enteron aisle; The propagation that suppresses interior pathogen of enteron aisle such as Escherichia coli, Salmonella, clostridium; Reduce the incidence of disease of intestines problem, reduce the adverse effect of bringing because of intestines problem (feed intake reduces, and takes antibiotic etc.).
The adding of above-mentioned probiotic has improved lamb gastrointestinal microorganisms environment, has suppressed growth of pathogenic bacteria, promotes useful anaerobic bacteria growth, improves immunity, has reduced the unfavorable factor that weaning period diarrhoea is brought, and has promoted the growth of cud simultaneously.
Feeding non-starch polysaccharide enzyme (NSP enzyme) is to be widely used in degrading in the feed ANFs SNSP to improve the complex enzyme of feed digestibility, daily gain and economy and environmental benefit; Comprising cellulase, zytase, 1,4 beta-glucanase, 'beta '-mannase and pectase, by various aspergillus, wood bacterial classification production such as mould.Solid state fermentation has better economy and environmental benefit, even also has special advantages in the performance of the activity of enzyme and stability, enzyme and wood is mould and mould produces zytase and cellulase aspects such as technology applicability.Trichoderma reesei Rut C-30 is the mutagenic fungi of wild trichoderma reesei QM6a; It is a kind of saprophytic filamentous fungi; The Primary Study of this bacterial strain solid state fermentation being produced the NSP enzyme shows that this bacterial strain NSP enzyme system is more complete; Wherein xylan and 1,4 beta-glucanase activity are higher, and the various NSP enzyme ratios that the fermentation of different carbon base-material produces are different.The non-starch polysaccharide enzyme that this additive uses this bacterial strain to produce, wherein cellulase activity, zytase and activity of beta-glucanase are high, are more suitable for and fatten the feed diet characteristics of mutton sheep.Increase with the lamb age in days, its alimentary canal changes rapidly, and its main feature is that cud is not initially playing a role basically, after reaching maturity, plays a crucial role.Therefore its fattening period daily ration is formed and generally is divided into two stages; The phase I daily ration contains 80% even higher concentrated feed; Second stage then contains 70%～50% concentrated feed; Be the ratio that second stage has increased considerably forage grass or stalk, so cellulose that should the stage, the content of hemicellulose (being mainly xylan) improve rapidly.In order to adapt to the daily ration of different phase, should have higher cellulase and xylanase activity in the zymogram of non-starch polysaccharide enzyme, take into account the higher activity of dextranase and mannase.And the complex enzyme bases fit that trichoderma reesei Rut C-30 solid fermentation is produced is in the requirement of its daily ration, so confirm to use the enzyme of this bacterial strain production.
The adding of above-mentioned non-starch polysaccharide enzyme has promoted the digestion of the SNSP material in the feed, has reduced the feed viscosity, improves the daily ration digestibility, is beneficial to the growth of intestinal beneficial bacterium simultaneously, keeps the intestinal microecology balance, thereby promotes growth of animal.
Yeast culture is by the formed little ecological goods in yeast process on solid medium abundant fermentation back under specific process conditions control.It is by yeast cells metabolite and the back by fermentation culture medium that makes a variation, and a small amount of yeast cells of non-activity constitutes.In the production process of yeast culture, yeast cells only is a kind of instrument that is used to produce products of cellular metabolism.Yeast culture is a kind of product of complicated component, and it not only contains the nutrients in the yeast cells, also contains the yeast cells metabolite that the fermentation back forms.Known active ingredient such as albumen, amino acid, B group vitamin, living bacterial cells, little peptide, various digestive ferments etc.; Also not research proof is wherein any is main or which kind synergy has been brought into play major function, so there is the people also it to be classified as the unknown function factor.These metabolites can stimulate the microbial bacterial in the animal stomach just, impel the animal health growth, improve efficiency of feed utilization and the purpose of improving the animal level of the productive forces thereby reach.The addition of yeast culture in additive of the present invention is to combine existing result of study; Considered that simultaneously various factors such as space that additive can hold are confirmed; Promptly final ratio in additive of the present invention is about 20%, and the adding proportion in daily ration is 0.2%.Existing feeding experiment shows that yeast culture is safe to ruminant, does not have bad reaction, and the addition that can cross the performance function as additive is between 0.1%～5%.Yeast culture has the cud CELLULOLYTIC BACTERIUM of stimulation and lactic acid bacteria breeding, changes the lumen fermentation mode, reduces cud ammonia concentration, improves effects such as rumen microorganism protein yield and feed digestibility, all plays an important role in lamb wean early stage and later stage.
The purpose of this invention is to provide a kind of weaning period mutton sheep special bio feed addictive.The use of this additive to as if the colony house meat lamb of raising at the daily ration of fast well-fattened phase; Its function is to take oxygen by force at the lamb intestines and stomach, produce enzyme, produce a series of effects such as acid and the breeding of inhibition harmful microorganism through probio; Regulate microecological balance and improve immunity; Improve the lamb general level of the health, improve the daily ration digestibility, avoid the use of pollution and resistance to the action of a drug side effect that antibiotic brings simultaneously; Through SNSP ANFs in non-starch polysaccharide enzyme (NSP enzyme) the hydrolysis lamb daily ration, reduce the viscosity of enteron aisle chyme and improve the daily ration digestibility, and lamb daily gain and feed efficiency; Promote cud to grow through yeast culture; Improve rumen microorganism protein yield and feed digestibility; The present invention joins (reasonable compatibility that comprises component and content) through the rationally auxilliary of three types of materials, offers comprehensive nutrition of early weaning lamb and physiology and regulates, and improves immunity; Promote the lamb healthy growth; Improve feed efficiency, additive of the present invention is exclusively used in early days in the agent for rearing and fattening meat sheep daily ration of (≤2 monthly age) wean, and adding proportion is 1% in fast well-fattened mutton sheep TRM of 3 monthly age～5 monthly ages.Additive of the present invention all can improve daily gain more than 10% in wean lamb fattening early stage and later stage, has significantly improved the economic benefit of lamb.
Description of drawings
Preparation technology's flow chart of Fig. 1 additive of the present invention,
Preparation technology's flow chart of Fig. 2 candida utili microbial inoculum,
Preparation technology's flow chart of Fig. 3 bacillus subtilis microbial agent,
Preparation technology's flow chart of Fig. 4 Lactobacillus plantarum microbial inoculum,
Fig. 5 Lactobacillus plantarum freeze-drying curve figure,
Preparation technology's flow chart of Fig. 6 yeast culture,
Preparation technology's flow chart of Fig. 7 non-starch polysaccharide enzyme.
The specific embodiment
The preparation of embodiment 1 composite biological feed additive agent for fattening early weaning mutton sheep
The 1-1 probiotic is auxilliary joins
The auxilliary commercially available prod that can adopt on the market of joining of probiotic, like pulvis etc., the present invention adopts following method to make.
The preparation of 1-1-1 candida utili microbial inoculum
1-1-1-1 bacterial classification and culture medium
Candida utili (Candia utilis) Accc20060, the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute breeding is preserved, and-80 ℃ of freezing preservation bacterial classifications (5ml dress) can be provided the public.
The potato glucose culture medium: fresh potato, glucose, water, according to the preparation of 10: 1: 50 ratio of weight ratio, natural pH value.
Compound method: peeling potatoes, be cut into fine grained chippings and add water in proportion, boil 30min, two-layer filtered through gauze gets potato and soaks juice, will soak juice and supply moisture to boiling preceding amount.Get this potato and soak juice, glucose, urea, in 5000: 100: 1 ratio preparation mixing, high pressure 0.1MPa sterilization 20 minutes.
The 1-1-1-2 condition of culture
Incubator or greenhouse, the 5000ml blake bottle is cultivated, bottle swingging machine; Every bottle of culture medium liquid measure 3000ml; Inoculum concentration 50ml, 30 ℃ of cultivation temperature, bottle swingging machine rotating speed 150r/min, incubation time 24h.
1-1-1-3 detects
Detect index: viable count; Detect culture medium: the brewer's wort solid medium; Detect incubation time: 24h;
Detection method: adopt dull and stereotyped coating to cultivate counting method.Every sample is got three horizontal dilution factors, three repetitions, and every flat board is got liquid 1 μ L.
Centrifugal and the low temperature drying of 1-1-1-4
Place supercentrifuge centrifugal (4000r/min) with detecting qualified bacterium liquid, gained bacterium mud uses the liquid protectant dilution to be subsequent use bacterium liquid, and mixes low temperature (40 ℃ 4h) dry with carrier protecting agent.The oven dry article are as the prescription raw material for standby of mutton sheep special bio feed addictive.
Liquid protectant prescription: 15% defatted milk.
Carrier protecting agent prescription: peanut hull meal 30%+ wheat bran 70%.
Liquid protectant dosage: 5% of centrifugal preceding bacterium liquid weight
The carrier protecting agent dosage: 1 part of bacterium liquid adds 2 parts of carrier protecting agents.
Subsequent use prescription raw material viable count: the actual living cells content of dry product>=5.0 * 10
10Cfu/g.
Preparation technology sees Fig. 2.
1-1-2. the preparation of bacillus subtilis microbial agent
1-1-2-1 bacterial classification and culture medium
Bacillus subtilis (Bacillus subtilis CICC 10081), there is preservation the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute, can provide the public.
The enlarged culture based formulas: beef extract 2g, peptone 5g, sodium chloride 2.5g, the 500ml sterilized water mixes and packing, the high pressure 0.1Mpa 20min that sterilizes, natural pH.
The 1-1-2-2 condition of culture
The constant temperature shaking table is cultivated, and 32.5 ℃, frequency of oscillation is 250r/min, incubation time 24h.
1-1-2-3 detects
Detect thing: detect culture bacteria liquid; Detect index: every milliliter of bacterium number (cfu/ml); Detect culture medium: the sterilization of the agar powder of adding 2% is processed on enlarged culture based formulas basis; Detection method: counting method is cultivated in dull and stereotyped coating.Every sample is got three horizontal dilution factors, two repetitions, and every flat board is got liquid 1 μ L.
1-1-2-4 centrifugal with mix
Place supercentrifuge centrifugal (4000r/min) with detecting qualified bacterium liquid, gained bacterium mud uses the liquid protectant dilution to be subsequent use bacterium liquid, and mixes low temperature (40 ℃ 4h) dry with carrier protecting agent.The oven dry article are as the prescription raw material for standby of mutton sheep special bio feed addictive.
Liquid protectant prescription: 15% defatted milk.
Carrier protecting agent prescription: the wheat bran of 60 order granularities.
Liquid protectant dosage: 5% of centrifugal preceding bacterium liquid weight
The carrier protecting agent dosage: 1 part of bacterium liquid adds 2 parts of carrier protecting agents.
Subsequent use prescription raw material viable count: the actual living cells content of dry product>=10
11Cfu/g.
Preparation technology sees Fig. 3.
The preparation of 1-1-3 Lactobacillus plantarum microbial inoculum
1-1-3-1 bacterial classification and culture medium
Lactobacillus plantarum (lactobacillus planticola) Accc-1118.There is preservation the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute, can provide the public.
MRS culture medium: peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 20.0g, sodium acetate trihydrate 5.0g, citric acid diamino 2.0g, K
2HFO
42.0g, MgsO4.7H
2O 0.58g, MnSO
4H
2O 0.19g, Tween 80 1.0mL, distilled water 1000mL, pH6.5,121 ℃ of sterilization 20min.
The 1-1-3-2 condition of culture
Liquid seeds is cultivated.Temperature: 37 ℃, incubation time: 24h, (liquid amount 300ml) cultivated in the jolting of 500ml triangular flask; Inoculum concentration 5%.
Expand numerous cultivation.Temperature: 37 ℃, incubation time: 24h, (liquid amount 3000ml) cultivated in the jolting of 5000ml blake bottle; Inoculum concentration 5%.
1-1-3-3 detects index and method
Detection method: counting method, index cfu/ml, cfu/g are cultivated in the dull and stereotyped coating of MRS solid medium.Every sample is got three horizontal dilution factors, three repetitions, and every flat board is got liquid 1 μ L.
Centrifugal and the freeze-drying of 1-1-3-4
Place supercentrifuge centrifugal (4000r/min) with detecting qualified bacterium liquid, isolate thalline bacterium mud, and with freeze drying protectant to mix freeze-drying.Dried frozen aquatic products is as the prescription raw material for standby of mutton sheep special bio feed addictive.
Liquid frozen-dried protective agent prescription: skimmed milk power 15g, sucrose 5g, sterilized water 100ml.
Liquid freeze drying protectant dosage: the bacterium mud after a centrifugal adds the protective agent that is equivalent to centrifugal preceding bacteria liquid long-pending 10%.
The viable count of dried frozen aquatic products:>=10
11Cfu/g.
Preparation technology sees Fig. 4, and freeze-drying curve is seen Fig. 5.
The preparation of 1-1-4 probiotic
The candida utili microbial inoculum, bacillus subtilis microbial agent, the Lactobacillus plantarum microbial inoculum, the part by weight that is converted into dry product raw material of the present invention by viable count content at 1: 1: 1 is that preparation in 2: 1: 1 mixes.
The 1-2 yeast culture
1-2-1 bacterial classification and culture medium
Bacterial classification: saccharomyces cerevisiae (Saccharomyces cervisiae) Accc20063, numerous preservation is expanded in the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute, can provide-80 ℃ of freezing preservation bacterial classifications (5ml dress) to the public
Liquid seed culture medium: the potato glucose culture medium, prescription is following with preparation method.
Fresh potato, glucose, water are according to the preparation of 10: 1: 50 ratio of weight ratio, natural pH value.
Compound method: peeling potatoes, be cut into fine grained chippings and add water in proportion, boil 30min, two-layer filtered through gauze gets potato and soaks juice, will soak juice and supply moisture to boiling preceding amount.Get this potato and soak juice, glucose, urea, in 5000: 100: 1 ratio preparation mixing, high pressure 0.1MPa sterilization 20 minutes.
Yeast culture solid medium: dregs of beans 90%+ wheat bran 10%, material-water ratio example 1: 1.5
The 1-2-2 condition of culture
The liquid seeds condition of culture:
The greenhouse, the 5000ml blake bottle is cultivated; Every bottle of culture medium liquid measure 3000ml; Inoculum concentration 50ml, 30 ℃ of cultivation temperature, bottle swingging machine rotating speed 100r/min, incubation time 24h.
Solid-state condition of culture: 30 ℃ of cultivation temperature, inoculum concentration 6%, incubation time 96h; Uncovered polybag (specification 400 * 800), every bag of 10kg of charge leaves standstill cultivation.
1-2-3 detects index and method
Yeast count: the blood counting chamber counting, measure during fermented and cultured 36h, meet or exceed 10
8Individual/be qualified during g;
Crude protein content: adopt Kjeldahl.
Enzyme: acid protease.The enzyme definition of living: 1g solid state fermentation song is under 40 ℃, pH 3.0 conditions, and per minute hydrolysis concentration is 10gL
-1The casein enzyme amount that produces 1 μ g tyrosine be decided to be an enzyme activity unit.
Preparation technology sees Fig. 6.
The preparation of 1-3 non-starch polysaccharide enzyme
1-3-1 bacterial classification, culture medium and cultural method
Bacterial classification: trichoderma reesei (Trichoderma reesei) Rut C-30, CICC13052, Chinese industrial microorganism fungus kind preservation center (CICC), the public can obtain from this, and there is preservation the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute, also can provide the public.
Slant medium: potato juice, glucose, pH6.8,2% agar;
Fluid nutrient medium: potato juice, glucose, pH6.8;
Solid fermentation culture medium: wheat bran 95g+ corn flour 5g+K
2HPO
43H
2O 0.1g+MgSO
47H
2O 0.16g, carbon-nitrogen ratio 21;
Process bacteria suspension after freeze-drying lactobacillus added sterilized water activation mixing, be inoculated on the slant medium in aseptic.24 ℃, cultivate 7d, see that slant medium is covered with green mycelia, put in 4 ℃ of refrigerators subsequent use.
Be inoculated into respectively in 3 triangular flask fluid nutrient mediums in slant medium bacterial classification picking 3 ring spores and mycelia mixture with oese.24 ℃, shake bottle and cultivate 6d, be covered with hypha body in the culture medium, put in 4 ℃ of refrigerators subsequent use.
The solid-state fermentation culture medium for preparing is put inoculum concentration 8%, add water to humidity 50%, 10kg is loaded on 400 * 800 polybags; Sack is in to be tied up but lax ventilative state is cultivated 5d in 30 ℃ of ventilation insulating boxs or greenhouse, weighs after the fermentation ends; Put 40 ℃ of oven dry, subsequent use.
1-3-2 detects index and method
Detect substrate: sodium carboxymethylcellulose; Glucan (Sigma); Xylan (Sigma); Konjaku powder (Wuhan is gloomy by force), jelly powder (Sigma)
Definition of 1-3-3 enzyme activity and detection method:
Cellulose enzyme vigor assay method adopts national agricultural industry proposed standard NY/T 912-2004.
Xylanase activity enzyme activity determination method is replaced by xylan solution with reference to NY/T 912-2004 with substrate.
1,4 beta-glucanase enzyme activity determination method is replaced by dextran solution with reference to NY/T 912-2004 with substrate.
'beta '-mannase enzyme activity determination method is replaced by konjaku powder solution with reference to NY/T 912-2004 with substrate.Pectase enzyme activity determination method adopts QB 1502-1992.
Preparation technology sees Fig. 7.
The 1-4 additive is auxilliary joins
Auxilliary joining gets final product in following formula rate.
The yeast culture of the probiotic of 25-35%, the non-starch polysaccharide enzyme of 40-60% and 15-25%.
Test Example:
The cold sheep early weaning lamb (2 monthly age) of 48 little tails is divided into control group and test group by the sex principle close with body weight; The control group fed basal diet; Test 1 group and in basal diet, add 0.2% yeast culture; Test and add 1% additive of the present invention in 2 groups of basal diets of feeding, divided for two phases carried out the feeding experiment of totally 8 time-of-weeks.Determination test first phase, test second phase and test the daily gain of full phase respectively.To be 1 group of test adding 0.2% yeast culture fatten the early stage daily gain than control group to the result improves 7.6%, fattens the later stage and improve 4.8%, and the full phase improves 7.6%; Test 2 groups and fatten early stage than the control group daily gain and improve 17.9%, fatten the later stage and improve 12.7%, the full phase improves 15.1%.
Daily gain table of full phase of table 1 feeding experiment
Table 2 feeding experiment is fattened daily gain table in early stage
Table 3 feeding experiment is fattened later stage daily gain table
The feeding experiment conclusion
Make an addition to basal diet with yeast culture as growth-promoting additive and improved early weaning lamb daily gain, but degree is lower; In basal diet, add 1% additive of the present invention, yeast culture is worked in coordination with non-starch polysaccharide enzyme with probio make an addition in the daily ration, make daily gain raising of full phase of early weaning lamb fattening phase 15.1% to have more tangible growth promoting function.This shows that add the effect of probio+non-starch polysaccharide enzyme+yeast culture existence effect stack in the lamb fattening feed, its combination acts synergistically is superior to individual event additive effect wherein.
Claims (9)
1. composite biological feed additive agent for fattening early weaning mutton sheep; Comprise and account for the probiotic that gross weight is 25-35%, the non-starch polysaccharide enzyme of 40-60% and the yeast culture of 15-25%; Said probiotic is made up of candida utili, bacillus subtilis and Lactobacillus plantarum microbial inoculum, and the viable count content of its microbial inoculum all>=1 * 10
10Cfu/g, viable count is than being 0.9-1.1: 0.9-1.1: 0.9-1.1; Crude protein content in the said yeast culture>=50%, acid protease activity>=10
6U/kg; Said non-starch polysaccharide enzyme comprises cellulase, zytase, 1,4 beta-glucanase, 'beta '-mannase and pectase, enzyme activity difference>=4.0 * 10
5U/kg, 5.0 * 10
6U/kg, 5.0 * 10
6U/kg, 3.0 * 10
5U/kg, 5.0 * 10
5U/kg, its enzyme activity portfolio ratio is 1.1-1.3: 12-16: 15-16: 1.0-1.3: 1-2.
2. composite biological feed additive agent for fattening early weaning mutton sheep according to claim 1, said candida utili are candida utili (Candia utilis Accc20060).
3. composite biological feed additive agent for fattening early weaning mutton sheep according to claim 1, said bacillus subtilis are bacillus subtilis (Bacillus subtilis CICC 10081).
4. composite biological feed additive agent for fattening early weaning mutton sheep according to claim 1, said Lactobacillus plantarum be Lactobacillus plantarum (lactobacillus planticola, Accc11118).
5. composite biological feed additive agent for fattening early weaning mutton sheep according to claim 1 is 30% probiotic, 50% non-starch polysaccharide enzyme and 20% yeast culture comprising accounting for weight.
6. the preparation method of the arbitrary composite biological feed additive agent for fattening early weaning mutton sheep of claim 1-5: (1) obtains probiotic with the pulvis of candida utili, bacillus subtilis and Lactobacillus plantarum by the viable count mixed; (2) use Wine brewing yeast strain and be that main culture medium carries out solid state fermentation and processes yeast culture with the dregs of beans; (3) be that main culture medium carries out the solid state fermentation mode and makes non-starch polysaccharide enzyme with trichoderma reesei (Trichoderma reesei) Rut C-30 bacterial strain with wheat bran; (4) probiotic, yeast culture, non-starch polysaccharide enzyme are mixed in proportion.
7. preparation method according to claim 6, said Wine brewing yeast strain be saccharomyces cerevisiae (Saccharomyces cerevisiae, Accc20063).
8. want 7 described preparation methods according to right, the solid state fermentation conditions of said saccharomyces cerevisiae is: solid medium is a dregs of beans 90%+ wheat bran 10%, and the material-water ratio example is 1: 1.5, inoculum concentration 6%, 30 ℃ of cultivation temperature, incubation time 96 hours.
9. preparation method according to claim 6, said trichoderma reesei (Trichoderma reesei) Rut C-30 bacterial strain solid state fermentation conditions is: the solid fermentation culture medium is wheat bran 95g+ corn flour 5g+K
2HPO
43H
2O 0.1g+MgSO
47H
2O0.16g, carbon-nitrogen ratio 21; Inoculum concentration 8%, humidity 50% is cultivated 5d in 30 ℃ of ventilation insulating boxs or greenhouse.
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