CN1262198C - Active peptide feed additive and its preparation method and use - Google Patents

Abstract

The present invention relates to an active peptide feed addictive, as a mixture of one or more plant protein and animal protein, which is the product of the following steps: fermenting and degrading the mixture through bacillus and the like, controlling the degree of hydrolysis at 20-35%, obtaining a fermentation supernatant after a separation post-processing process, concentrating drying the supernatant or directly adsorbing the supernatant onto the carrier, and then low temperature drying it. Said active peptide feed addictive is capable of obviously depressing development of bacillus coli and salmonella, and can prevention and cure diarrhea caused by bacilli or lienteric diarrhea; can exert its functions at a very low concentration, with a very intense effect; can regulate stomach and intestine bacterium group and promote animals digestion functions; can excite animal immune function, reinforce animal disease resistance against a plurality of epidemic diseases and greatly reduce the morbidity and death rate; can promote animal development and improve the growth velocity; can improve the feed use ratio and reduce production costs. Applications of the microorganism fermentation degradation method provided by the present invention have a low fabricating cost, and the obtained peptides have many kinds and high content, without environment pollutions.

Description

A kind of activated peptide forage additive and its production and use

Invention field

The present invention relates to a kind of activated peptide forage additive, specifically relate to a kind of active peptide (or oligopeptides) feed addictive that utilizes the manufacturing of animal and plant albumen and preparation method thereof.

Background technology

Since the 1950's, in animal feeding and feed, be extensive use of growth promotion health-care agents such as antibiotic, chemical synthetic drug, hormone, beta-stimulants, heavy metal, sedative, caused following problem: livestock and poultry and aquatic products product Chinese traditional medicine are residual serious, directly are detrimental to health; The animal products quality descends, and lacks the market competitiveness and trust; Bacterial drug resistance strengthens, antibody-resistant bacterium increases, and threatens human security; Destroy ecological environment etc.Because antibiotic many side effects, nearly twenty or thirty is over year, scientists is being sought antibiotic substitute always, simultaneously, the consumer extremely pays close attention to food-safety problem in recent years, and therefore, countries in the world man is all forbidding or limiting antibiotic use one after another, and accelerate development, popularization new type of safe green feed additive efficiently, to reduce or alternative antibiotic etc additive.

Achievement in research in recent years shows that biologically active peptide is a kind of new type of safe, and animal grow and product formation aspect bringing into play the feed addictive of important effect.But aspect feed industry, can see on the domestic market at present is little peptide or peptide protein feed, rather than activated peptide forage additive truly, and it mostly is imported product, the peptide albumen that is processed by small intestine and the intestinal mucosa of animal; Or contain the peptide protein feed of a certain amount of little peptide.Though they can both improve the production performance of animal, reduce diarrhea rate, mostly have following defective: the addition of (1) product is bigger by 0.1～1.5%; (2) product function is indeterminate, and effect is not obvious; (3) molecular weight of peptide is bigger, is generally about 6000, still has antigenicity, and is unfavorable to animal, easily causes the antigen allergic reaction; (4) preparation cost height or product cost height are unsuitable for animal-breeding and use.These products are mainly used in the part fish meal that replaces in the feed.

On the preparation method of activated peptide forage additive, traditionally, the method for preparing biologically active peptide mainly is an enzyme process, as use plant rennets such as papain, bromelain, or use animal proteases such as trypsase, pepsin, but be easy to generate unacceptable fishy smell or bitter taste, and production cost height; Next is a direct extraction method from raw material, and productive rate is low, cost is high; The 3rd is to be obtained by chemical synthesis, is used for the pharmacology level peptide of the short chain of production high value to long-chain, and the shortcoming of this method is the cost height and may be harmful to healthy and environment in course of reaction; The 4th, DNA recombinant technique method, this method opposes to use the food product of being produced by genetically altered biology because of many consumers, and worries that biological safety and biological pollution are arranged, and is restricted.

Summary of the invention

The addition that the objective of the invention is to overcome the prior art for preparing activated peptide forage additive is bigger, product function is indeterminate, effect is not obvious, peptide molecular weight more still has antigenicity, easily cause the antigen allergic reaction, be not easy to the defective promoted the use of in the animal-breeding industry with the preparation cost height, thereby provide a kind of addition in feed low, definite functions, have improve animal to nutrient (as protein, mineral matter, vitamin etc.) absorption rate, improve milk, meat, egg output and production capacity, improve meat, egg, the quality and the local flavor of milk, stimulate the animal immune allelotaxis and improve premunition and the minimizing incidence of disease, and aquaculture of aquatic animal water quality can be improved obviously, improve aquatic animal to grow speed and survival rate, be widely used in fowl, ox, sheep, pig, aquatic products and economic animal can be used as the activated peptide forage additive of advantages such as antibiotic substitute.

Another object of the present invention is to provide the preparation method of described activated peptide forage additive.

A further object of the present invention is to provide the purposes of described activated peptide forage additive.

The objective of the invention is to be achieved through the following technical solutions:

Activated peptide forage additive provided by the invention, its method that is prepared as follows of serving as reasons is controlled at 20～35% with fermentation substrate through fermentation, hydrolysis and degree of hydrolysis (DH%), and through the protein hydrolyzate that ultra high temperature short time sterilization obtains, described preparation method comprises the steps:

1) preparation seed liquor:

With in bacillus, Aspergillus, mould, the actinomyces one or more, insert in the fluid nutrient medium after the sterilization, cultivated 24～48 hours in 25～35 ℃, obtain seed liquor;

Described fluid nutrient medium is in following ratio liquid mixture prepared: peptone 1.3～2.5g, glucose 3～5g, molasses 3～5g, potato immersion liquid 50～100ml, soluble starch 10～20g, sucrose 10～30g, beef extract 2～3g, yeast soak powder 3.5～5g, dregs of beans 1.5～2g, growth factor 1～2ml, water 1000ml; Growth factor wherein is for containing MgSO ₄.7H ₂O 20.00～30.00mg/ml, CaCl ₂.2H ₂O 2.20～3.50mg/ml, ZnSO ₄.7H ₂O0.81～1.10mg/ml, FeSO ₄.7H ₂O 0.44～0.60mg/ml, MnSO ₄.H ₂O 0.13～0.20mg/ml, CuSO ₄.5H ₂The mixture of O 0.02～0.04mg/ml;

Described bacillus is Bacillus cereus, bacillus subtilis, bacillus licheniformis, natto gemma sample bacterium, bacillus megaterium or bacillus pumilus;

Described Aspergillus is black-koji mould, aspergillus terricola bacterium, aspergillus oryzae, cinnamon Aspergillus or Aspergillus usamii bacterium;

Described actinomyces are green sugared sporangium, grayish green streptomycete or expense formula streptomycete;

Described Penicillium notatum is purple little mould, penicillium chrysogenum, apple mould, yellowish mould, Taiwan mould, Du Pont's mould or blue brown mould;

2) fermentative degradation:

Put into fermentation tank after fermentation substrate is crushed to 60～120 orders, add entry and carbon source successively, add the weight of entry and the weight portion proportioning of the contained protein of fermentation substrate is 100: 5～18, add the nitrogen content of the phosphorus content of carbon source and fermentation substrate weight ratio be 1: 2～35, with zymotic fluid at 0.1～0.13MPa, sterilized 10～30 minutes for 115～125 ℃, be cooled to 20～37 ℃, add the seed liquor that step 1) obtains, the volume ratio of seed liquor and zymotic fluid is 0.3～1: 10, mix the back at 25～42 ℃, speed with 0.5～2.5vvm feeds filtrated air, stir fermentation with 150～300rpm speed, use conventional ninhydrin method determination of color degree of hydrolysis during this time, degree of hydrolysis of detection in per 6 hours before 24 hours, between 24～48 hours, detected a degree of hydrolysis in per 4 hours, degree of hydrolysis of detection in per 2 hours after 48 hours, reach 20～35% up to degree of hydrolysis, then in 140～145 ℃ of sterilizations 2～8 seconds, obtain containing the protein hydrolyzate of active peptide, be activated peptide forage additive;

Described fermentation substrate is one or more the mixture that is selected from vegetable protein or the animal protein;

Described vegetable protein comprises soybean protein, dregs of beans, soya-bean cake, Peas albumen, zein, red bean albumen, wheat gluten, big aleuronat, the cottonseed dregs of rice or rapeseed dregs;

Described animal protein comprises fish meal, blood meal, feather meal, lactoprotein, muscles, casein, the discarded internal organs of animal, earthworm protein powder, squid albumen, fish-skin or other aquatic livestock albumen;

Described carbon source comprises corn flour, brown sugar, edible white sugar, corn syrup, starch, cane molasses, beet molasses.

The method that is used to prepare activated peptide forage additive of the present invention also comprises step 3) and step 4):

Described step 3) is centrifugal slagging-off, the ultrafiltration, concentrated of protein hydrolyzate:

With step 2) protein hydrolyzate through sterilization that obtains separates through 5000～7000rpm continuous centrifugal and removed slag in 10～30 minutes, and the supernatant of telling obtains the filtrate of molecular weight less than 5000Da by ultrafiltration membrance filter; This filtrate is concentrated to 50% of original volume through negative-pressure vacuum, obtains concentrate;

Described step 4) is for adding carrier adsorption dry or spray-drying:

Adding carrier adsorption dry is the concentrate that step 3) is obtained, and is added in the carrier that is pre-mixed, and the weight ratio of concentrate and carrier is 0.5～0.8: 1,45～80 ℃ of dryings, obtains activated peptide forage additive then; Described carrier is made up of 2～5 weight portion organic carriers and 3～5 weight portion inorganic carriers; Described organic carrier comprises bran and puffed soybean organic carrier; Described bran organic carrier is one or more the mixture in wheat bran, maize cob meal, degreasing powdered rice hulls and the defatted rice bran; Described puffed soybean organic carrier is one or more the mixture in bean cake powder, soya-bean cake powder and the corn protein powder; Described inorganic carrier is one or more the mixture in precipitated calcium carbonate, stone flour, medical stone and the zeolite powder;

Spray-drying is the concentrate that step 3) is obtained, press operating condition: volume ratio adds 0.3～1% dextrin and 2～8% precipitated calcium carbonates, after mixing, on spray dryer, press 100 ℃～130 ℃ of air intakes, 60～80 ℃ of air-out, 1.8～2.3kg, flow 1000～1500ml/min are pressed in spray, carry out spray-drying, obtain activated peptide forage additive.

The preparation method of activated peptide forage additive provided by the invention comprises the steps:

1) preparation seed liquor:

With in bacillus, Aspergillus, mould, the actinomyces one or more, insert in the fluid nutrient medium after the sterilization, cultivated 24～48 hours in 25～35 ℃, obtain seed liquor;

Described fluid nutrient medium is in following ratio liquid mixture prepared: peptone 1.3～2.5g, glucose 3～5g, molasses 3～5g, potato immersion liquid 50～100ml, soluble starch 10～20g, sucrose 10～30g, beef extract 2～3g, yeast soak powder 3.5～5g, dregs of beans 1.5～2g, growth factor 1～2ml, water 1000ml; Growth factor wherein is for containing MgSO ₄.7H ₂O 20.00～30.00mg/ml, CaCl ₂.2H ₂O 2.20～3.50mg/ml, ZnSO ₄.7H ₂O0.81～1.10mg/ml, FeSO ₄.7H ₂O 0.44～0.60mg/ml, MnSO ₄.H ₂O 0.13～0.20mg/ml, CuSO ₄.5H ₂The mixture of O 0.02～0.04mg/ml;

Described bacillus is Bacillus cereus, bacillus subtilis, bacillus licheniformis, bafillus natto, bacillus megaterium or bacillus pumilus;

Described Aspergillus is black-koji mould, aspergillus terricola bacterium, aspergillus oryzae, cinnamon Aspergillus or Aspergillus usamii bacterium;

Described actinomyces are green sugared sporangium, grayish green streptomycete or expense formula streptomycete;

Described Penicillium notatum is purple little mould, penicillium chrysogenum, apple mould, yellowish mould, Taiwan mould, Du Pont's mould or blue brown mould;

2) fermentative degradation:

Put into fermentation tank after fermentation substrate is crushed to 60～120 orders, add entry and carbon source successively, add the weight of entry and the weight portion proportioning of the contained protein of fermentation substrate is 100: 5～18, add the nitrogen content of the phosphorus content of carbon source and fermentation substrate weight ratio be 1: 2～35, with zymotic fluid at 0.1～0.13MPa, sterilized 10～30 minutes for 115～125 ℃, be cooled to 20～37 ℃, add the seed liquor that step 1) obtains, the volume ratio of seed liquor and zymotic fluid is 0.3～1: 10, mix the back at 25～42 ℃, the speed feeding filtrated air with 0.5～2.5vvm stirs fermentation with 150～300rpm speed, use conventional ninhydrin method determination of color degree of hydrolysis during this time, detected a degree of hydrolysis in per 6 hours before 24 hours, between 24～48 hours, degree of hydrolysis of detection in per 4 hours, degree of hydrolysis of detection in per 2 hours after 48 hours, reach 20～35% up to degree of hydrolysis, in 140～145 ℃ of sterilizations 2～8 seconds, obtain activated peptide forage additive then;

Described fermentation substrate is one or more the mixture that is selected from vegetable protein or the animal protein;

Described vegetable protein comprises soybean protein, dregs of beans, soya-bean cake, Peas albumen, zein, red bean albumen, wheat gluten, big aleuronat, the cottonseed dregs of rice or rapeseed dregs;

Described animal protein comprises fish meal, blood meal, feather meal, lactoprotein, muscles, casein, the discarded internal organs of animal, earthworm protein powder, squid albumen, fish-skin or other aquatic livestock albumen;

Described carbon source comprises corn flour, brown sugar, edible white sugar, corn syrup, starch, cane molasses, beet molasses.

Its preparation method also comprises step 3) and step 4):

Described step 3) is centrifugal slagging-off, the ultrafiltration, concentrated of protein hydrolyzate:

With step 2) protein hydrolyzate through sterilization that obtains removed slag in centrifugal 10～30 minutes through 5000～7000rpm again, and the supernatant of telling obtains the filtrate of molecular weight less than 5000Da by ultrafiltration membrance filter; This filtrate is concentrated to 50% of original volume through negative-pressure vacuum, obtains concentrate;

Described step 4) is for adding carrier adsorption dry or spray-drying:

Adding carrier adsorption dry is the concentrate that step 3) is obtained, and is added in the carrier that is pre-mixed, and the weight ratio of concentrate and carrier is 0.5～0.8: 1,45～80 ℃ of dryings, obtains activated peptide forage additive then; Described carrier is made up of 2～5 weight portion organic carriers and 3～5 weight portion inorganic carriers; Described organic carrier comprises bran and puffed soybean organic carrier; Described bran organic carrier is one or more the mixture in wheat bran, maize cob meal, degreasing powdered rice hulls and the defatted rice bran; Described puffed soybean organic carrier is one or more the mixture in bean cake powder, soya-bean cake powder and the corn protein powder; Described inorganic carrier is one or more the mixture in precipitated calcium carbonate, stone flour, medical stone and the zeolite powder;

Spray-drying is the concentrate that step 3) is obtained, press operating condition: volume ratio adds 0.3～1% dextrin and 2～8% precipitated calcium carbonates, after mixing, on spray dryer, press 100 ℃～130 ℃ of air intakes, 60～80 ℃ of air-out, 1.8～2.3kg, flow 1000～1500ml/min are pressed in spray, carry out spray-drying, obtain activated peptide forage additive.

Activated peptide forage additive provided by the invention has following effect or purposes:

1. the raising efficiency of feed utilization reduces production costs.Mainly improve animal to nutrient (as protein, mineral matter, vitamin etc.) absorption rate, thereby improve the trans-utilization rate of feed, reduce the generation of bird leg disease and osteoporosis based on active Toplink.

2. the promotion growth of animal can improve the speed of growth.Because of active Toplink promotes Growth and Differentiation, the propagation of cell, and then promote the growth of animal and grow up.Simultaneously can reduce fat deposition under the animal skins, improve lean meat percentage and dressing percentage, and can improve basal metabolic rate, promote the animal microcirculation.

3. can significantly improve the quantity of cellulose-decomposing bacterium in the cud, improve the utilization rate of roughage, thus the speed of growth of the output of milk of raising milk cow and beef cattle, mutton sheep.

4. obviously improve the laying rate of egg fowl, prolong egg-laying peak, optimize nutrient structure and ratio in the birds, beasts and eggs, reduce cholesterol level.

5. improve the quality and the local flavor of meat, egg, milk, make taste brighter, more fragrant.

6. stimulate the animal immune allelotaxis and improve premunition, obviously improve the livestock and poultry survival rate, reduce death rate.

7. have significantly anti-oxidant, anti-aging effects, improve and plant with animal physical efficiency and service time.

8. be used for aquatic livestock, can significantly improve water quality, improve the speed of growth and the survival rate of fry, increase shrimp seedling feed intake and seedling body length.

The using method of this activated peptide forage additive is:

(1) mixes or spice (containing active peptide active ingredient gram/ton feed) piglet, meat chick, cowboy, lamb, 80～120 grams with feed; Sow, growing and fattening pigs, milk cow, fattening sheep and ox, 60～80 grams; Laying hen, duck, goose 60～80 grams; Aquatic livestock 80～120 grams.

(2) also can be directly oral or add in the entry and drink.

Activated peptide forage additive provided by the invention is a kind of green feed additive, compared with the prior art has following superiority:

(1) addition of this activated peptide forage additive in feed is low, and feed per ton only adds about 100 grams, is 1/10 of present peptide protein feed;

(2) definite functions of this activated peptide forage additive has following function:

1. can improve animal to nutrient,, improve efficiency of feed utilization, reduce the sick and osteoporosis generation of bird leg as the absorption rate of important nutrient such as protein, mineral matter, vitamin;

2. can improve milk, meat, egg output and production capacity, improve the quality and the local flavor of meat, egg, milk;

3. can stimulate the animal immune allelotaxis and improve premunition and the minimizing incidence of disease, effectively prevent and treat the generation of livestock and poultry diarrhea, broiler ascites syndrome and watery stools;

4. be used for aquatic livestock, can significantly improve water quality, improve the speed of growth and the survival rate of fry, increase shrimp seedling feed intake and seedling body length, can be widely used in fowl, ox, sheep, pig, aquatic products and economic animal;

(3) molecular weight of this activated peptide forage additive is less;

(4) product cost of the present invention is low, is easy to promote in the breed industry of small profits;

(5) product of the present invention has the animal immune of stimulation allelotaxis, improves functions such as the premunition and the minimizing incidence of disease, can be used as antibiotic substitute, reduces medicament residue;

(6) the method applied in the present invention is a microbe fermentation method, is 1/10 of enzyme process cost, and enzymatic isolation method hydrolysis 1Kg protein raw materials needs 20 yuan of costs, and fermentation method of the present invention only needs 1.8 yuan; And the active peptide of fermentation method production do not have bitter taste, no stink, and the physicochemical property of product is good.

Therefore the exploitation and the development of activated peptide forage additive goods provided by the invention is for saving feed, reducing cost and improve breeding performonce fo animals and premunition is of great immediate significance and social effect.

The specific embodiment

Embodiment 1, preparation activated peptide forage additive L-1 (liquor)

In the fluid nutrient medium after the bacillus subtilis access sterilization, cultivated 48 hours, and obtained seed liquor for 25 ℃.

Described fluid nutrient medium is in following ratio liquid mixture prepared: peptone 1.3g, glucose 3g, molasses 3g, potato immersion liquid 50ml, soluble starch 10g, sucrose 10g, beef extract 2g, yeast soak powder 3.5g, bean cake powder (60 order) 1.5g, growth factor 1ml, water 1000ml; Growth factor wherein is for containing MgSO ₄.7H ₂O 20.00mg/ml, CaCl ₂.2H ₂O 2.20mg/ml, ZnSO ₄.7H ₂O 0.81mg/ml, FeSO ₄.7H ₂O 0.44mg/ml, MnSO ₄.H ₂O 0.13mg/ml, CuSO ₄.5H ₂The mixture of O 0.02mg/ml.

With fermentation substrate---soyabean protein powder is put into fermentation tank after being crushed to 60 orders, add entry and carbon source successively---corn flour and molasses (weight ratio is 1: 1), add the weight of entry and the weight ratio of the contained protein of fermentation substrate is 100: 5, add the nitrogen content of the phosphorus content of carbon source and fermentation substrate weight ratio be 1: 2, with mixture at 0.1MPa, sterilized 30 minutes for 121 ℃, be cooled to 37 ℃, add the seed liquor that obtains as previously mentioned, the volume ratio of seed liquor and zymotic fluid is 0.3: 10, mix the back at 25 ℃, speed with 0.5vvm feeds filtrated air, stir fermentation with 150rpm speed, use conventional ninhydrin method determination of color degree of hydrolysis during this time, degree of hydrolysis of detection in per 6 hours before 24 hours, between 24～48 hours, detected a degree of hydrolysis in per 4 hours, degree of hydrolysis of detection in per 2 hours after 48 hours, degree of hydrolysis (DH%) up to protein hydrolyzate reaches 20%, then through the ultra high temperature short time sterilization machine in 140 ℃ the sterilization 8 seconds, obtain activated peptide forage additive L-1 (liquor).

Degree of hydrolysis (Degree of Hydrolysis, assay method DH) are conventional ninhydrin method development process, now simply are described below:

1. the drafting of calibration curve: at first prepare the standard glycine solution, its concentration is 20ug/ml; Then according to the listed application of sample amount order of table 1 application of sample.

The application of sample amount of table 1 glycine, ninhydrin and ethanol

The test tube numbering	0	1	2	3	4	5	6	7
									Standard glycine solution application of sample amount (ml)	0	0.2	0.4	0.6	0.8	1.0	1.5	2.0
Distilled water application of sample amount (ml)	2.0	1.8	1.6	1.4	1.2	1.0	0.5	0
									Glycine final concentration (ug/ml) during final volume 2ml	0	2	4	6	8	10	15	20
Ninhydrin application of sample amount (ml)	1	1	1	1	1	1	1	1
									40% ethanol application of sample amount (ml)	5	5	5	5	5	5	5	5

Press the listed application of sample amount of table 1, in tool plug test tube, add standard glycine solution, distilled water and ninhydrin developer successively, in boiling water, heat 15min, in cold water, cool off 5min then; Add 40% ethanolic solution cessation reaction again, place 15min; Return to zero in the mensuration A (absorbance) of 570nm place value with the blank pipe.

2. in the hydrolyzed protein liquid-NH ₂Determination on content

Get a certain amount of protein hydrolyzate centrifugal 15min under 4000rpm, get supernatant 100 μ l in the 25ml volumetric flask, constant volume, that is dilute 250 times.Absorption 2ml dilution is in tool plug test tube and add 1ml ninhydrin developer, heats 15min behind the mixing in boiling water; In cold water, cool off 5min then; The ethanolic solution cessation reaction that adds 5ml 40% again, behind the placement 15min, to be measured.At last, utilize calibration curve to calculate in the protein hydrolyzate-NH ₂Content (umol/ml).

3. degree of hydrolysis (DH%) calculates

In following formula:

The relative glycine concentration of protein hydrolyzate: after being meant the absorbance of the protein hydrolyzate of measuring as stated above, the glycine concentration that converses by calibration curve.

The protein concentration of fermentation substrate: the protein concentration that is meant fermentation substrate (as soybean protein etc.).

75.07: the molecular weight that is glycine.

7.8mmol/g: be the peptide bond number (constant) after the soybean protein complete hydrolysis.

0.33mmol/g: be the peptide bond number (constant) in the fermentation substrate (raw material).

Embodiment 2, preparation activated peptide forage additive L-2 (liquor)

In the fluid nutrient medium after the black-koji mould access sterilization, cultivated 24 hours, and obtained seed liquor for 35 ℃.

Described fluid nutrient medium is in following ratio liquid mixture prepared: peptone 2.5g, glucose 5g, molasses 5g, potato immersion liquid 100ml, soluble starch 20g, sucrose 30g, beef extract 3g, yeast soak powder 5g, dregs of beans 2g, growth factor-2 ml, water 1000ml; Growth factor wherein is for containing MgSO ₄.7H ₂O 30.00mg/ml, CaCl ₂.2H ₂O3.50mg/ml, ZnSO ₄.7H ₂O 1.10mg/ml, FeSO ₄.7H ₂O 0.60mg/ml, MnSO ₄.H ₂O 0.20mg/ml, CuSO ₄.5H ₂The mixture of O 0.04mg/ml.

With fermentation substrate---dregs of beans is put into fermentation tank after being crushed to 120 orders, add entry and carbon source successively---corn molasses and white granulated sugar (weight ratio=3: 2), add the weight of entry and the weight ratio of the contained protein of fermentation substrate is 100: 18, add the nitrogen content of the phosphorus content of carbon source and fermentation substrate weight ratio be 1: 35, with mixture at 0.13MPa, sterilized 10 minutes for 125 ℃, be cooled to 20 ℃, add the seed liquor that obtains as previously mentioned, the volume ratio of seed liquor and zymotic fluid is 1: 10, mix the back at 42 ℃, speed with 2.5vvm feeds filtrated air, stir fermentation with 300rpm speed, use conventional ninhydrin method determination of color degree of hydrolysis during this time, degree of hydrolysis of detection in per 6 hours before 24 hours, between 24～48 hours, detected a degree of hydrolysis in per 4 hours, degree of hydrolysis of detection in per 2 hours after 48 hours, degree of hydrolysis (DH%) up to protein hydrolyzate reaches 35%, then through the ultra high temperature short time sterilization machine in 145 ℃ the sterilization 2 seconds, obtain activated peptide forage additive L-2 (liquor).

Embodiment 3, preparation activated peptide forage additive L-3 (liquor)

In the fluid nutrient medium after the sugared sporangium access of the green sterilization, cultivated 36 hours, and obtained seed liquor for 27 ℃.

Described fluid nutrient medium is in following ratio liquid mixture prepared: peptone 1.8g, glucose 4g, molasses 4g, potato immersion liquid 70ml, soluble starch 15g, sucrose 20g, beef extract 2.5g, yeast soak powder 4g, bean cake powder (80 order) 1.8g, growth factor 1.5ml, water 1000ml; Growth factor wherein is for containing MgSO ₄.7H ₂O 25.00mg/ml, CaCl ₂.2H ₂O 3.00mg/ml, ZnSO ₄.7H ₂O 1.00mg/ml, FeSO ₄.7H ₂O 0.50mg/ml, MnSO ₄.H ₂O0.17mg/ml, CuSO ₄.5H ₂The mixed liquor of O 0.03mg/ml.

With fermentation substrate---soya-bean cake is put into fermentation tank after being crushed to 80 orders, add entry and carbon source---starch successively, add the weight of entry and the weight ratio of the contained protein of fermentation substrate is 100: 10, add the nitrogen content of the phosphorus content of carbon source and fermentation substrate weight ratio be 1: 10, with mixture at 0.11MPa, sterilized 20 minutes for 115 ℃, be cooled to 30 ℃, add the seed liquor that obtains as previously mentioned, the volume ratio of seed liquor and zymotic fluid is 0.5: 10, mix the back at 28 ℃, the speed feeding filtrated air with 1.5vvm stirs with 180rpm speed, use conventional ninhydrin method determination of color degree of hydrolysis during this time, detected a degree of hydrolysis in per 6 hours before 24 hours, between 24～48 hours, degree of hydrolysis of detection in per 4 hours, degree of hydrolysis of detection in per 2 hours after 48 hours, degree of hydrolysis (DH%) up to protein hydrolyzate reaches 25%, then through the ultra high temperature short time sterilization machine in 142 ℃ the sterilization 6 seconds, obtain activated peptide forage additive L-3 (liquor).

Embodiment 4～27, preparation activated peptide forage additive L-4～L-27 (liquor)

Listed as table 2, preparation activated peptide forage additive L-4 is to L-27 (liquor).

Table 2,

Peptide feedstuff additive	The preparation method	The used bacterial classification of preparation seed liquor	Fermentation substrate	Carbon source	The protein hydrolyzate degree of hydrolysis
						L-4	With embodiment 1	Bacillus cereus	Fish meal	Corn flour	22
L-5	With embodiment 2	Bacillus licheniformis	Blood meal	Brown sugar	24
						L-6	With embodiment 3	Bafillus natto	Dregs of beans, soya-bean cake	Edible white sugar	34
L-7	With embodiment 1	Bacillus megaterium	Peas albumen	Corn syrup	32
						L-8	With embodiment 2	Bacillus pumilus	Zein	Cane molasses	35
L-9	With embodiment 3	The aspergillus terricola bacterium	Muscles	Beet molasses	26
						L-10	With embodiment 1	Aspergillus oryzae	The earthworm protein powder	Corn flour, brown sugar	28
L-11	With embodiment 2	The cinnamon Aspergillus	Squid albumen	Brown sugar, edible white sugar	27
						L-12	With embodiment 3	The Aspergillus usamii bacterium	Fish-skin	Edible white sugar, corn syrup	31
L-13	With embodiment 1	Grayish green streptomycete	Red bean albumen	Corn syrup, starch	21
						L-14	With embodiment 2	Take the formula streptomycete	Wheat gluten	Starch, cane molasses	29
L-15	With embodiment 3	Purple little mould	Big aleuronat	Starch, beet molasses	25
						L-16	With embodiment 1	Penicillium chrysogenum	The cottonseed dregs of rice	Starch, cane molasses, beet	23

				Molasses
						L-17	With embodiment 2	The apple mould	Rapeseed dregs	Corn flour	30
L-18	With embodiment 3	Yellowish mould	Feather meal	Brown sugar	35
						L-19	With embodiment 1	The Taiwan mould	Lactoprotein	Edible white sugar	33
L-20	With embodiment 2	Du Pont's mould	Casein	Corn syrup	24
						L-21	With embodiment 3	Blue brown mould	Animal discards internal organs	Starch	29
L-22	With embodiment 1	Bacillus cereus, bacillus megaterium	Squid albumen, fish-skin	Cane molasses	33
						L-23	With embodiment 2	Aspergillus oryzae, aspergillus terricola bacterium	Casein, the discarded internal organs of animal	Beet molasses	31
L-24	With embodiment 3	Du Pont's mould, Taiwan mould	Peas albumen, zein	Starch	28
						L-25	With embodiment 1	Grayish green streptomycete, take the formula streptomycete	Blood meal, feather meal	Corn flour	20
L-26	With embodiment 2	Aspergillus oryzae, grayish green streptomycete	Wheat gluten, big aleuronat	Cane molasses	25
						L-27	With embodiment 3	Blue brown mould, bacillus megaterium	Dregs of beans	Starch, beet molasses	28

Embodiment 28, preparation activated peptide forage additive C-1

The degree of hydrolysis (DH%) that embodiment 1 is obtained is that 20% protein hydrolyzate was removed slag in centrifugal 30 minutes through 5000rpm, and the supernatant of telling obtains the filtrate of molecular weight less than 5000Da by ultrafiltration membrance filter; This filtrate is concentrated to 50% of original volume through negative-pressure vacuum, obtains concentrate.

This concentrate is added in the carrier that is pre-mixed, described carrier is 5 weight portion organic carriers (2 weight portion wheat bran, 2 weight portion bean cake powders, 1 weight portion corn protein powder) and 5 weight portion inorganic carriers (precipitated calcium carbonate) form, the weight ratio of concentrate and carrier is 0.5: 1,45 ℃ of dryings, be crushed to 80 orders then, obtain activated peptide forage additive C-1.

Embodiment 29, preparation activated peptide forage additive C-2

The degree of hydrolysis (DH%) that embodiment 2 is obtained is that 35% protein hydrolyzate was removed slag in centrifugal 20 minutes through 6000rpm, and the supernatant of telling obtains the filtrate of molecular weight less than 5000Da by ultrafiltration membrance filter; This filtrate is concentrated to 50% of original volume through negative-pressure vacuum, obtains concentrate.

This concentrate is added in the carrier that is pre-mixed, described carrier is that 2 weight portion organic carriers (maize cob meal) and 3 weight portion inorganic carriers (stone flour) are formed, and the weight ratio of concentrate and carrier is 0.8: 1, then 80 ℃ of dryings, be crushed to 80 orders, obtain activated peptide forage additive C-2.

Embodiment 30, preparation activated peptide forage additive C-3

The degree of hydrolysis (DH%) that embodiment 3 is obtained is that 25% protein hydrolyzate was removed slag in centrifugal 10 minutes through 7000rpm, and the supernatant of telling obtains the filtrate of molecular weight less than 5000Da by ultrafiltration membrance filter; This filtrate is concentrated to 50% of original volume through negative-pressure vacuum, obtains concentrate.

This concentrate is added in the carrier that is pre-mixed, described carrier is 3 weight portion organic carriers (degreasing powdered rice hulls) and 4 weight portion inorganic carriers (medical stone), form, the weight ratio of concentrate and carrier is 0.7: 1, then 60 ℃ of dryings, be crushed to 80 orders, obtain activated peptide forage additive C-3.

Embodiment 31～54, preparation activated peptide forage additive C-4～C-27

The protein hydrolyzate that embodiment 4～27 is obtained is respectively by the method for embodiment 28～30, and is listed as table 3, obtains activated peptide forage additive C-4 to C-27.

Table 3,

Peptide feedstuff additive	Protein hydrolyzate	The preparation method	Organic carrier	Inorganic carrier
					C-4	Embodiment 4	With embodiment 28	Wheat bran 2 weight portions	Precipitated calcium carbonate 3 weight portions
C-5	Embodiment 5	With embodiment 29	Maize cob meal 3 weight portions	Stone flour 4 weight portions
					C-6	Embodiment 6	With embodiment 30	Degreasing powdered rice hulls 4 weight portions	Medical stone 5 weight portions
C-7	Embodiment 7	With embodiment 28	Defatted rice bran 5 weight portions	Zeolite powder 5 weight portions
					C-8	Embodiment 8	With embodiment 29	Bean cake powder 2 weight portions	Precipitated calcium carbonate 2 weight portion stone flours 2 weight portions
C-9	Embodiment 9	With embodiment 30	Soya-bean cake powder 3 weight portions	Stone flour 1 weight portion medical stone 1 weight portion zeolite powder 1 weight portion
					C-10	Embodiment 10	With embodiment 28	Corn protein powder 4 weight portions	Medical stone 2 weight portion zeolite powders 1 weight portion
C-11	Embodiment 11	With embodiment 29	Wheat bran 2 weight portion maize cob meals 3 weight portions	Stone flour 2 weight portion medical stones 2 weight portions
					C-12	Embodiment 12	With embodiment 30	Wheat bran 1 weight portion	Precipitated calcium carbonate 2 weight portions

			Degreasing powdered rice hulls 1 weight portion	Stone flour 1 weight portion medical stone 2 weight portions
					C-13	Embodiment 13	With embodiment 28	Wheat bran 2 weight portion defatted rice brans 1 weight portion	Precipitated calcium carbonate 2 weight portion stone flours 2 weight portions
C-14	Embodiment 14	With embodiment 29	Wheat bran 1 weight portion bean cake powder 3 weight portions	Stone flour 2 weight portion medical stones 1 weight portion
					C-15	Embodiment 15	With embodiment 30	Wheat bran 2 weight portion soya-bean cake powder 3 weight portions	Precipitated calcium carbonate 3 weight portions
C-16	Embodiment 16	With embodiment 28	Maize cob meal 1 weight portion degreasing powdered rice hulls 1 weight portion	Stone flour 4 weight portions
					C-17	Embodiment 17	With embodiment 29	Maize cob meal 1 weight portion bean cake powder 2 weight portions	Medical stone 5 weight portions
C-18	Embodiment 18	With embodiment 30	Degreasing powdered rice hulls 1 weight portion bean cake powder 2 weight portion corn protein powders 1 weight portion	Zeolite powder 5 weight portions
					C-19	Embodiment 19	With embodiment 28	Defatted rice bran 1 weight portion bean cake powder 2 weight portion soya-bean cake powder 2 weight portions	Precipitated calcium carbonate 4 weight portions
C-20	Embodiment 20	With embodiment 29	Maize cob meal 1 weight portion bean cake powder 1 weight portion	Stone flour 3 weight portions
					C-21	Embodiment 21	With embodiment 30	Wheat bran 3 weight portions	Medical stone 3 weight portions
C-22	Embodiment 22	With embodiment 28	Maize cob meal 4 weight portions	Zeolite powder 4 weight portions
					C-23	Embodiment 23	With embodiment 29	Degreasing powdered rice hulls 5 weight portions	Precipitated calcium carbonate 5 weight portions
C-24	Embodiment 24	With embodiment 30	Defatted rice bran 2 weight portions	Stone flour 5 weight portions
					C-25	Embodiment 25	With embodiment 28	Bean cake powder 3 weight portions	Medical stone 4 weight portions
C-26	Embodiment 26	With embodiment 29	Soya-bean cake powder 4 weight portions	Zeolite powder 3 weight portions
					C-27	Embodiment 27	With embodiment 30	Corn protein powder 5 weight portions	Precipitated calcium carbonate 3 weight portions

Embodiment 55, preparation activated peptide forage additive S-1

The degree of hydrolysis (DH%) that embodiment 1 is obtained is that 20% protein hydrolyzate was removed slag in centrifugal 30 minutes through 5000rpm, and the supernatant of telling obtains the filtrate of molecular weight less than 5000Da by ultrafiltration membrance filter; This filtrate is concentrated to 50% of original volume through negative-pressure vacuum, obtains concentrate.

With this concentrate by operating condition: volume ratio adds 0.3% dextrin and 2% precipitated calcium carbonate, after mixing, presses 100 ℃ ℃ of air intakes on spray dryer, 60 ℃ of air-out, 1.8kg, flow 1000ml/min are pressed in spray, carry out spray-drying, obtain activated peptide forage additive S-1.

Embodiment 56, preparation activated peptide forage additive S-2

The degree of hydrolysis (DH%) that embodiment 2 is obtained is that 35% protein hydrolyzate was removed slag in centrifugal 20 minutes through 6000rpm, and the supernatant of telling obtains the filtrate of molecular weight less than 5000Da by ultrafiltration membrance filter; This filtrate is concentrated to 50% of original volume through negative-pressure vacuum, obtains concentrate.

With this concentrate by operating condition: volume ratio adds 1% dextrin and 8% precipitated calcium carbonate, after mixing, presses 130 ℃ of air intakes on spray dryer, 80 ℃ of air-out, 2.3kg, flow 1500ml/min are pressed in spray, carry out spray-drying, obtain activated peptide forage additive S-2.

Embodiment 57, preparation activated peptide forage additive S-3

The degree of hydrolysis (DH%) that embodiment 3 is obtained is that 25% protein hydrolyzate was removed slag in centrifugal 10 minutes through 7000rpm, and the supernatant of telling obtains the filtrate of molecular weight less than 5000Da by ultrafiltration membrance filter; This filtrate is concentrated to 50% of original volume through negative-pressure vacuum, obtains concentrate.

With this concentrate by operating condition: volume ratio adds 0.7% dextrin and 5% precipitated calcium carbonate, after mixing, presses 120 ℃ of air intakes on spray dryer, 70 ℃ of air-out, 2.0kg, flow 1300ml/min are pressed in spray, carry out spray-drying, obtain activated peptide forage additive S-3.

Embodiment 58～81, preparation activated peptide forage additive S-4～S-27

The protein hydrolyzate that embodiment 4～27 is obtained is respectively through the centrifugal slag of removing of 5000rpm, and the supernatant of telling is the ultrafiltration membrance filter of 5nm by membrane aperture; This filtrate is concentrated to 50% of original volume through negative-pressure vacuum, obtains concentrate; This concentrate is pressed operating condition: volume ratio adds 0.3～1% dextrin and 2～8% precipitated calcium carbonates, after mixing, on spray dryer, press 100 ℃～130 ℃ of air intakes, 60～80 ℃ of air-out, 1.8～2.3kg. flow, 1000～1500ml/min is pressed in spray, carry out spray-drying, obtain activated peptide forage additive S-4 to S-27.

According to long-term feeding trial as can be known, no matter activated peptide forage additive provided by the invention is with liquor (L-1～L-27), still to be adsorbed on form on the carrier (C-1～C-27) or the direct pressed powder that obtains of the spray-drying (form of the S-1～S-27) bird of feeding, its effect is only relevant with the degree of hydrolysis and the scale of feeding of contained protein hydrolyzate, and irrelevant with the solid, liquid form of feed.

Peptide ammino acid among embodiment 82, the activated peptide forage additive S-2 (being active peptide) Determination on content

Activated peptide forage additive S-2 according to embodiment 56 methods obtain takes by weighing sample 1.0013g, is dissolved in the 20ml distilled water, with 40ml sulfosalicylic acid precipitating proteins, behind the centrifugal 30min of 3000rpm, gets supernatant 10000rpm secondary centrifuging 40min.Take out 2ml solution from supernatant, 45 ℃ of rotary evaporations dye then and give birth to (with the dissolving of 2ml taurine, add the boric acid of 400 μ L0.05mol/L in test tube, add 100 μ L again and dissolve good sample and 600 μ LFMOC, place 1min).Dye living fully back and extract remaining FMOC with 2000 μ L pentanes.Take out the sample that 100 μ L dye after giving birth to and add 700 μ L eluents, go up in high pressure liquid chromatography (HPLC) and measure, the results are shown in table 4.

Total amino acid (TAA) among table 4, the activated peptide forage additive S-2,

The content of free amino acid (FAA) and peptide ammino acid (PAA)

The amino acid title	FAA(g/g)	TAA(g/g)	PAA(g/g)	PAA accounts for the ratio of TAA
					Asp	0.002166792	0.012578449	0.010411657	82.77％
Ser	0.008550902	0.02781238	0.019261478	69.26％
					Glu	0.00011639	0.037771759	0.037655369	99.69％
Thr	9.3796E-05	0.004508148	0.004414352	97.92％
					Gly	0.004637067	0.011950659	0.007313592	61.20％
Arg	0	0	0	0.00％
					Ala	0.009697688	0.016944848	0.00724716	42.77％
Tyr	8.60159E-05	0.002721595	0.002635579	96.84％
					Pro	0.008556269	0.023238143	0.014681874	63.18％
Val	0	0	0	0.00％
					Phe	0.00100304	0.003769058	0.002766018	73.39％
Ile	0.013641658	0.035264777	0.021623119	61.32％
					leu	0.004459154	0.014044763	0.009585609	68.25％
His	0.001131631	0.138213842	0.137082211	99.18％
					Lys	0.005849348	0.013225191	0.007375843	55.77％
Add up to	0.059989751	0.342043613	0.282053861	82.46％

Annotate: FAA is a free amino acid, and TAA is a total amino acid, and PAA is a peptide ammino acid

As shown in table 4, peptide ammino acid accounts for 82.46% of total amino acid content among the activated peptide forage additive S-2, that is peptide content is 82.46%, and free amino acid only accounts for 17.54% of total amino acid.Wherein the content of histidine is up to 0.138213842g/g, and glutamic acid takes second place, and content is 0.037771759g/g.Alanine content in the free amino acid is higher than alanine content in the peptide ammino acid, and all the other amino acid all are that content is higher in the peptide.Both contained peptide among the activated peptide forage additive S-2 of this explanation fermentation gained, also contain free amino acid, but based on active peptide (accounting for 82.46%), the free amino acid proportion had been very little.

The solubility of embodiment 83, activated peptide forage additive

Table 5 has been listed before the bean pulp fermentation and product---the solubility of activated peptide forage additive S-2 under the different pH values condition that obtains after the fermentation

Solubility change before and after table 5, the bean pulp fermentation

pH	2	3	4	5	6	7	8	9
									S-2(％)	32.68	35.92	33.07	35.04	37.73	37.36	36.86	40.45
Dregs of beans (%)	34.4	1.97	2.36	3.21	13.53	21.71	25.97	27.02

As can be seen from Table 5, the solubility of the dregs of beans before the fermentation is the highest when pH=2 (under the utmost point acid condition), is 34.4%, along with acidity reduces, solubility drops sharply to 1.97%～3.21% when the pH value is 3,4,5, after this, along with acidity further reduces, solubility is gone up again gradually; O'clock (under the strong alkaline condition) reached 27.02% in pH value=9.And the fermentation after obtain product---no matter how Acidity of Aikalinity changes (pH2～9) to activated peptide forage additive S-2, all has higher dissolubility, apparently higher than the preceding dregs of beans of fermentation, and under the condition of different pH values the basic held stationary state of dissolubility, minimum is 32.68%, is up to 40.45%.As seen, activated peptide forage additive provided by the invention has good dissolubility and stability under different soda acid acid-base value conditions.

The heat endurance of embodiment 84, soybean active peptide and soybean protein

After the heat endurance of Soybean Peptide is meant Soybean Peptide solution heated 30min in 100 ℃ of water-bath, measure its absorbance (OD), the percentage of OD value ratio is counted solubility (%) before calculating the heating back and heating, be OD value * 100% before solubility %=heating back OD value/heating, see Table 6, (%) is low for solubility, illustrates that the heating back produces precipitation, sink to the bottom, make solution limpid, thereby the OD value reduce, and indicates poor stability.

The heat endurance method of Soybean Peptide and big dregs of beans is: respectively get 1% Soybean Peptide solution and solution of soybean meal of certain volume, transfer to different pH (2～9) value respectively, 4000rpm, centrifugal 15min.Get supernatant during mensuration and survey its light absorption value in the 420nm place, 30min is heated in the back in 100 ℃ of water-baths, surveys light absorption value as stated above in the 420nm place.By comparing twice OD value, reflect its heat endurance.

Table 6, Soybean Peptide and soybean protein solubility are with the variation of pH

The pH value	2	3	4	5	6	7	8	9
									Soybean Peptide solubility %	83.2	58.17	93.65	89.86	94.28	90.61	96.92	89.92
Soybean protein solubility %	77.40	36.68	69.23	65.08	74.60	61.09	75.76	87.90

As shown in Table 6, the heat endurance of Soybean Peptide is improved largely than its parent protein.When pH=2, the heat endurance of Soybean Peptide and soybean protein is all about 80%, and when pH=3, the solubility of soybean protein has than Soybean Peptide and descends significantly, illustrates that the dissolubility of Soybean Peptide is better than soybean protein.In addition, the heat endurance pH of Soybean Peptide is all comparatively steady in excursion significantly, illustrates that Soybean Peptide has good heat endurance.This is with its female parent---soybean protein is different, and its dissolubility descends significantly after the heat treatment, and poor heat stability promptly is described.

The storage stability of embodiment 85, activated peptide forage additive

Light absorption value before and after table 7, the soybean protein fermentation

	A 0(initial value, normal temperature)	A 1(4℃，12h)	A 2(100 ℃, 30min is cooled to 4 ℃ then)
				S-1	1.0116	1.0245	1.1462
Soybean protein	0.0123	0.0140	0.0185

Table 7 has been listed before the soybean protein fermentation and product---the light absorption value of activated peptide forage additive S-1 that obtains after the fermentation.Hence one can see that, the color and luster of activated peptide forage additive S-1 and turbidity all do not have bigger variation after 4 ℃ of refrigeration, illustrate that refrigeration does not make peptide molecule generation precipitation in the peptide solution, but through 100 ℃, 30min heats and is cooled to 4 ℃ suddenly, and its light absorption value has bigger variation, but compare with the maternal soybean protein before the fermentation, light absorption value changes less, illustrates that under the condition of same concentrations the solution storage stability of activated peptide forage additive of the present invention is obviously more superior than its parent protein.

Embodiment 86, use activated peptide forage additive of the present invention are used in Swine Production

Be the effect of check product of the present invention and the effect on pig industry, and replace antibiotic feasibility.Test is chosen body weight and is divided into 5 processing near 180 healthy of 35 age in days weanling pigs (male and female half and half), 36 pigs of each processing (group), and each handles 6 repetitions, each repeats 6 pigs, be 3 public 3 mothers, in 8 weeks of experimental period (2 months), result of the test is listed in table 8.

Five processing are as follows:

Blank group: do not add any additives, the pig basal diet of only feeding;

The antibiotic control group: pulvis aureomycin 100mg/kg evenly is mixed in the basal diet;

The activated peptide forage additive S-2 of 1 group of active peptide: 40mg/kg embodiment 56 evenly is mixed in the basal diet;

The activated peptide forage additive S-2 of peptide 2 group: 120mg/kg embodiment 56 evenly is mixed in the basal diet;

The activated peptide forage additive S-2 of 3 groups of active peptides: 200mg/kg embodiment 56 evenly is mixed in the basal diet.

Table 8, the result of use in Swine Production

Processing/repetition	29～50 age in days average daily gains (Kg)	29～50 age in days material anharmonic ratioes
			The blank group	0.2707±0.01	2.23±0.09
Antibiotic control group (aureomycin 100mg/kg)	0.3261±0.02	1.68±0.08
			1 group of active peptide (40mg/kg)	0.3049±0.01	1.76±0.07
Peptide 2 group (120mg/kg)	0.3364±0.02	1.68±0.07
			3 groups of active peptides (200mg/kg)	0.3171±0.01	1.84±0.17

As can be seen from Table 8, the average daily gain and the feed efficiency of having added 3 active peptide groups of activated peptide forage additive S-2 all are better than the blank group, can obviously improve pig production performance only, i.e. daily gain and feed efficiency.

Wherein the average daily gain of peptide 2 group (active ingredient 120mg/kg feed) and feed efficiency significantly are better than blank group and antibiotic group, can substitute antibiotic fully.

Embodiment 87, the application of use activated peptide forage additive of the present invention in layer breeding

For checking activated peptide forage additive in result of use on the laying hen and the application prospect in animal husbandry, sea match brown shell layer later stage chicken 3000 plumages are selected in test for use, divide three groups, every group of 1000 laying hens, divide 5 repetitions, each repeats 200 chickens, feed with 3 kinds of different daily rations, be that the A group is the blank group, B organizes the activated peptide forage additive S-3 (i.e. 0.05% activated peptide as additive) that feed per ton adds 20 gram embodiment 57, and C organizes the activated peptide forage additive S-3 (i.e. 0.1% activated peptide as additive) that feed per ton adds 40 gram embodiment 57.2 months experimental periods, the results are shown in table 9.The detection index is rate of broken eggs (%), laying rate (%), egg size (a gram/egg), death rate (%), feed efficiency (feed consumption g/kg egg).

Table 9, the application in layer breeding

Annotate: the right shoulder of colleague's numeral indicates different lowercase persons and is significant difference (P＜0.05).

As can be seen from Table 9, along with the increase of the addition of activated peptide forage additive, laying rate obviously increases, and all has significant difference (p＜0.05) between three groups of A, B, C, wherein, adds active peptide 40/mg and makes laying rate improve 4.6%.Aspect feed efficiency (material/egg ratio), add active Toplink and obviously reduce feedstuff-egg ratio, promptly obviously improved feed efficiency, and significant difference (p＜0.05), but the B group is organized difference not remarkable (p＞0.05) with C; In layer diets, add active peptide 40/mg can obviously increase egg size reach 3.5 grams/piece, increasing degree reaches 5.4%, with control group significant difference (p＜0.05), also obviously can reduce death rate and rate of broken eggs, these all help to reduce aquaculture cost.In a word, this test shows, adds activated peptide forage additive and can obviously improve egg fowl laying rate in egg feedstuff, increases egg size, reduces feedstuff-egg ratio, rate of broken eggs, reduces death rate, improves efficiency of feed utilization, production performance and reduces production costs.

By studies show that of nutrition biochemical test, absorption active or little peptide has the advantages that power consumption is low, be difficult between the saturated and various peptides uncontested property of running and inhibition, peptide itself can improve laying rate, reduces food consumption and increase egg size so add activated peptide forage additive of the present invention in layer diets for the facilitation of the transhipment of amino acid or peptide simultaneously.

Embodiment 88, activated peptide forage additive of the present invention are to the influence of chicken peripheral blood T-lymphopoiesis vigor

For detecting the influence of activated peptide forage additive of the present invention, promptly detect action effect or the influence degree of product of the present invention to meat chick cellular immunity to chicken peripheral blood T-lymphopoiesis vigor.Carried out the micromethod lymphocyte transformation test, promptly add the sterile active peptide liquid of various dose in meat chick LCF, observed the influence of active peptide to meat chick blood medium size lymphocyte proliferation activity, concrete grammar is as follows:

(1) takes a blood sample from the meat chick of raising for 3 ages in week: the heparin tube of sterilization, add aseptic heparin,, shake gently and allow heparin fully mix, to leave standstill with blood 1 hour, obtain aseptic anticoagulated blood from chicken wings passages through which vital energy circulates blood sampling 4ml.

(2) isolated lymphocytes (whole process is carried out sterile working) from aseptic anticoagulated blood: heparin anti-coagulating is done dilution in 1: 1 with no calcium magnesium Hank ' s liquid earlier, add lymphocyte layering liquid 6ml at conical centrifuge tube, then along slowly the superpose heparin anti-coagulating of 3mlHank ' s liquid dilution of tube wall, 3000 rev/mins in horizontal centrifuge is centrifugal 30 minutes, as seen liquid is divided into three layers: the upper strata is a blood plasma, the middle level is a parting liquid, and lower floor is red blood cell and granulocyte.The muddy band of visible skim on the interface between upper strata and the middle level, this is lymphocyte and monocytic mixture.Abandon plasma layer until distance buffy coat 0.5cm place with the suction pipe suction, then with buffy coat and the sucking-off together of a little parting liquid, move in the 10ml centrifuge tube, add 5 times of pH7.2-7.4 and do not have calcium magnesium Hank ' s liquid with upper volume, mixing, with 2000rpm centrifugation 10 minutes, abandoning supernatant was washed secondary again with method.The lymphocyte of precipitation is made into 1 * 10 with RPMI1640 cultivation (not containing FCS) ⁷/ ml lymphocyte suspension.

(3) lymphocyte purity is identified: get above-mentioned lymphocyte suspension smear, after the air dry, Wright's stain dyeing 1min adds equivalent PBS buffer solution and continues dyeing 4min, distilled water flushing airing, microscopic examination, more than 95% of lymphocyte purity.

(4) cell is cultivated: on 96 porocyte culture plates, every hole adds lymphocyte suspension 60 μ l, not adding or add concentration in nutrient solution is the little peptide mother liquor 60 μ l of 640 μ g/ml, making the ultimate density of little peptide in nutrient solution is 0,0.0234375%, 0.046875%, 0.09375%, 0.1875%, 0.375%, 0.75%, 1.5%, repeats three holes.By RPMI1640 nutrient solution (totally four groups, contained FCS is respectively 0,5%, 10%, 15%) total volume of culture is adjusted to 200 μ l, 2 put 5%CO2, cultivate 48h respectively for 37 ℃.Cultivate and finish preceding 4h, supernatant 70 μ l are inhaled in every hole gently, and (haemocyanin is the fundamental component of cell culture fluid, and haemocyanin is sex change formation flocculent deposit easily in organic solvent, influences light transmittance.In order to reduce influence of serum, remove culture supernatant before adding MTT as far as possible), not every on the same group then hole adds 2.5mg/ml, 5.0mg/ml, 7.5mg/ml MTT 10 μ l, and mixing is about 1 minute on oscillator, continues to cultivate 4h.After cultivating end, every hole adds 0.04M acidifying isopropyl alcohol 100 μ l, and the piping and druming mixing dissolves purple crystal fully.

(5) result detects: use enzyme-linked immunosorbent assay instrument, measuring wavelength 570nm, reference wavelength 630nm place measures OD value (OD value), with the lymphocytic proliferation activity of OD value representation.The results are shown in Table 10.

Table 10, to the influence of chicken peripheral blood T-lymphopoiesis vigor

Index	The result
								Active peptide concentration	0.00％	0.03％	0.05％	0.10％	0.20％	0.40％	0.80％
T cell viability (OD value)	0.2078	0.4223	1.7575	2.0822	2.1562	2.1602	2.4192

By the data of table 10 as can be seen, the activated feed additive has the animal immune of stimulation function, strengthen the function of animal T cell viability, the T cell is the main force that participates in nospecific immunity (as antitumor, antiviral) and cellular immunity in the livestock and poultry animal body, and the enhancing explanation of its vigor can improve the premunition of animal.

Embodiment 89, activated peptide forage additive of the present invention are to the influence of bovine serum albumin(BSA) (BSA) antibody titer of chicken

For detecting activated peptide forage additive of the present invention to the influence of fryer to bovine serum albumin(BSA) (BSA) specific antibody titres, the meat chick is divided into 5 groups, each component becomes 5 repetitions, and each repeats 20 chickens, 7 weeks of examination phase.Experimental design sees Table 11, and basal diet proportioning and trophic level see Table 12.Give each injection of bovine serum albumin 0.5ml of fryer left and right sides leg flesh (bovine serum albumin(BSA) concentration is 2.5mg/kg) respectively at 35 ages in days, 42 ages in days

Table 11, feeding of broiler experimental design

Group	Processed group	Finished product addition (/ ton feed)	Active ingredient (g/ ton feed)
				A	Blank	0	0
B	Antibiotic contrast (neomycin+aureomycin)	20+100g	20+100
				C	0.1% active peptide	1kg	40.0
D	0.3% active peptide	3kg	120.0
				E	0.5% active peptide	5kg	200.0

Table 12, meat chick daily ration prescription and trophic level

Feed	Proportioning (%)		Nutrient	Trophic level
						0-3 week	4-7 week	0-3 week	4-7 week
	Corn	55.8		59.8	Metabolizable energy (MJ/kg)					2900.11	3000.55
Dregs of beans			37.6			32.2	Crude protein (%)	21.03	19.00
	Soya-bean oil	2.3		3.36	Calcium (%)					1.01	0.91
Stone flour			1.3			1.2	Total phosphorus (%)	0.70	0.64
	Calcium monohydrogen phosphate	1.92		1.7	Available phosphorus (%)					0.45	0.41
Zeolite powder						0.66	Lysine (%)	1.10	0.96
	Lysine	0.02			Methionine (%)					0.45	0.36
Methionine			0.14			0.07	Egg+Guang (%)	0.79	0.67
	Salt	0.3		0.3	Salt (%)					0.3	0.3
Premix			1			1

Annotate: premix provides to every kilogram of complete diet pellet: Cu 6mg, Fe 75mg, Zn 75mg, Mn 80mg, Se0.2mg, I 0.4mg, zeolite powder 1mg, choline 3mg.Vitamin A 6000IU, cholecalciferol 2000IU, vitamin E 12IU, prokeyvit 2mg, biotin 0.05mg, folic acid 0.4mg, nicotinic acid 32mg, pantothenic acid 7mg, vitamin B1 1mg, vitamin B2 5mg, vitamin B6 1mg, cobalamin 10ug.

Get 12 dirty blood samplings of heart in 28 ages in days, every group of group of 49 ages in days, the centrifugal 10min separation of serum of 3000rpm is frozen in-20 ℃ of refrigerators then, detects the BSA antibody titer with enzyme-linked immunoassay method (ELISA), and the result sees Table 13 with the OD value representation.

Table 13,28 ages in days add the influence of active peptide to meat chick BSA antibody titer

Group	BSA antibody titer (with the OD value representation)
		A (blank group, no medicine)	0.453±0.019
B (antibiotic control group, 120 grams)	0.451±0.022
		C (add peptide 0.1%, contain active peptide 40g/t feed)	0.599±0.021
D (add peptide 0.3%,, contain active peptide 120g/t feed)	0.673±0.032
		E (add peptide 0.5%,, contain peptide 2 00g/t feed)	0.680±0.041

As can be seen from Table 13, the antibody titer of the meat chick of interpolation active peptide group is apparently higher than blank group and antibiotic group, and particularly D group and E group are more remarkable.Illustrate that activated peptide forage additive can obviously improve the antibody titer of meat chick, antibody titer is an important indicator of weighing body opposing cause of disease ability, and the antibody titer height shows that promptly the premunition of body is strong.

Embodiment 90, activated peptide forage additive of the present invention are to the influence of meat chick production performance (speed of weight increment and feed consumption)

Raise table hens by embodiment 89, when 1 age in days is gone into young bird and 4 age ends (28 age in days) in week, 7 age ends in week (49 age in days) be that unit weighs to repeat respectively, and statistics feed consumption rate calculates dead number of elements, weightening finish, feed intake and the feedstuff-meat ratio in 0～4 week and 4～7 all ages.The results are shown in Table 14.

Table 14,49 ages in days add the influence of active peptide to meat chick production performance

Group	Weightening finish (kg)	Feed intake (kg)	Feedstuff-meat ratio
				A (blank group)	2.333±0.076	5.190±0.004Aa	2.227±0.073
B (antibiotic control group)	2.349±0.094	5.184±0.008Aa	2.210±0.092
				C (adding peptide 0.1%)	2.314±0.119	5.124±0.004Bb	2.219±0.121
D (adding peptide 0.3%)	2.380±0.072	5.180±0.005Aa	2.178±0.064
				E (adding peptide 0.5%)	2.377±0.075	5.153±0.003Cc	2.176±0.069

The data of table 14 show that the weight ratio blank component of D group and E group chicken be you can well imagine high 47 grams and 44 grams, you can well imagine high 31 grams and 28 grams than antibiotic component, and simultaneously, the feed consumption rate of D group and E group chicken also is starkly lower than other groups.Illustrate and add the weightening finish that biologically active peptide can obviously improve fryer, also can improve feed efficiency, save the feed spending, increase economic efficiency.

Embodiment 91, activated peptide forage additive of the present invention are to the influence of meat chick product quality

Raising table hens by embodiment 89, when 28 ages in days and 49 ages in days, do Quality of poultry meatan issue of growing importance and measure. each repeats to get two chickens and weighs, butchers, and each handles 10 chickens, gets chest muscle, leg flesh and abdomen fat (only 49 days time get), calculating chest muscle rate, leg flesh rate and abdomen fat rate.The results are shown in Table 15.

Product quality index when table 15, meat chick are delivered (7 age in week) for sale

Group	Chest muscle accounts for the % of trunk	Leg flesh accounts for the % of trunk	Abdomen fat accounts for the % of trunk
				A (blank group)	6.78±0.46	6.79±0.42	1.76±0.31a
B (antibiotic control group)	6.79±0.53	6.81±0.72	1.74±0.27ab
				C (adding peptide 0.1%)	7.22±0.60	6.95±0.63	1.63±0.45c
D (adding peptide 0.3%)	7.35±0.56	7.06±0.42	1.62±0.34cd
				E (adding peptide 0.5%)	7.49±0.97	7.26±0.73	1.62±0.46cd

The ratio that activated peptide forage additive can obviously improve meat chick chest muscle and leg flesh is added in table 15 explanation, can reduce the quantity of stomach fat simultaneously, can obviously improve the quality of broiler carcass (unhairing goes the clean hall chicken of internal organ).

Claims (9)

1, a kind of activated peptide forage additive, its method that is prepared as follows of serving as reasons is controlled at 20～35% with fermentation substrate through fermentation, hydrolysis and degree of hydrolysis, and the protein hydrolyzate that obtains through ultra high temperature short time sterilization, and described preparation method comprises the steps:

1) preparation seed liquor:

With in bacillus, Aspergillus, mould, the actinomyces one or more, insert in the fluid nutrient medium after the sterilization, cultivated 24～48 hours in 25～35 ℃, obtain seed liquor;

Described fluid nutrient medium is in following ratio liquid mixture prepared: peptone 1.3～2.5g, glucose 3～5g, molasses 3～5g, potato immersion liquid 50～100ml, soluble starch 10～20g, sucrose 10～30g, beef extract 2～3g, yeast soak powder 3.5～5g, dregs of beans 1.5～2g, growth factor 1～2ml, water 1000ml; Growth factor wherein is for containing MgSO ₄.7H ₂O 20.00～30.00mg/ml, CaCl ₂.2H ₂O 2.20～3.50mg/ml, ZnSO ₄.7H ₂O0.81～1.10mg/ml, FeSO ₄.7H ₂O 0.44～0.60mg/ml, MnSO ₄.H ₂O 0.13～0.20mg/mg/ml, CuSO ₄.5H ₂The mixture of O 0.02～0.04mg/ml;

2) fermentative degradation:

Put into fermentation tank after fermentation substrate is crushed to 60～120 orders, add entry and carbon source successively, add the weight of entry and the weight portion proportioning of the contained protein of fermentation substrate is 100: 5～18, add the nitrogen content of the phosphorus content of carbon source and fermentation substrate weight ratio be 1: 2～35, with zymotic fluid at 0.1～0.13MPa, sterilized 10～30 minutes for 115～125 ℃, be cooled to 20～37 ℃, add the seed liquor that step 1) obtains, the volume ratio of seed liquor and zymotic fluid is 0.3～1: 10, mix the back at 25～42 ℃, the speed feeding filtrated air with 0.5～2.5vvm stirs fermentation with 150～300rpm speed, use conventional ninhydrin method determination of color degree of hydrolysis during this time, detected a degree of hydrolysis in per 6 hours before 24 hours, between 24～48 hours, degree of hydrolysis of detection in per 4 hours, degree of hydrolysis of detection in per 2 hours after 48 hours, reach 20～35% up to degree of hydrolysis, in 140～145 ℃ of sterilizations 2～8 seconds, obtain activated peptide forage additive then;

Described fermentation substrate is one or more the mixture that is selected from vegetable protein or the animal protein;

Described carbon source comprises corn flour, brown sugar, edible white sugar, corn syrup, starch, cane molasses, beet molasses.

2, activated peptide forage additive as claimed in claim 1 is characterized in that, described bacillus is Bacillus cereus, bacillus subtilis, bacillus licheniformis, bafillus natto, bacillus megaterium or bacillus pumilus; Described Aspergillus is black-koji mould, aspergillus terricola bacterium, aspergillus oryzae, cinnamon Aspergillus or Aspergillus usamii bacterium; Described actinomyces are green sugared sporangium, grayish green streptomycete or expense formula streptomycete; Described Penicillium notatum is purple little mould, penicillium chrysogenum, apple mould, yellowish mould, Taiwan mould, Du Pont's mould or blue brown mould.

3, activated peptide forage additive as claimed in claim 1 is characterized in that, described vegetable protein comprises soybean protein, dregs of beans, soya-bean cake, Peas albumen, zein, red bean albumen, wheat gluten, big aleuronat, the cottonseed dregs of rice and rapeseed dregs; Described animal protein comprises fish meal, blood meal, feather meal, lactoprotein, muscles, casein, the discarded internal organs of animal, earthworm protein powder, squid albumen and fish-skin.

4, activated peptide forage additive as claimed in claim 1 is characterized in that, its preparation method also comprises step 3) and step 4):

Described step 3) is centrifugal slagging-off, the ultrafiltration, concentrated of protein hydrolyzate:

With step 2) protein hydrolyzate through sterilization that obtains removed slag in centrifugal 10～30 minutes through 5000～7000rpm again, and the supernatant of telling obtains the filtrate of molecular weight less than 5000Da by ultrafiltration membrance filter; This filtrate is concentrated to 50% of original volume through negative-pressure vacuum, obtains concentrate;

Described step 4) is for adding carrier adsorption dry or spray-drying:

Adding carrier adsorption dry is the concentrate that step 3) is obtained, and is added in the carrier that is pre-mixed, and the weight ratio of concentrate and carrier is 0.5～0.8: 1,45～80 ℃ of dryings, obtains activated peptide forage additive then; Described carrier is made up of 2～5 weight portion organic carriers and 3～5 weight portion inorganic carriers; Described organic carrier comprises bran and puffed soybean organic carrier; Described bran organic carrier is one or more the mixture in wheat bran, maize cob meal, degreasing powdered rice hulls and the defatted rice bran; Described puffed soybean organic carrier is one or more the mixture in bean cake powder, soya-bean cake powder and the corn protein powder; Described inorganic carrier is one or more the mixture in precipitated calcium carbonate, stone flour, medical stone and the zeolite powder;

Spray-drying is the concentrate that step 3) is obtained, and spray-drying obtains activated peptide forage additive.

5, the preparation method of the described activated peptide forage additive of a kind of claim 1 comprises the steps:

1) preparation seed liquor:

With in bacillus, Aspergillus, mould, the actinomyces one or more, insert in the fluid nutrient medium after the sterilization, cultivated 24～48 hours in 25～35 ℃, obtain seed liquor;

Described fluid nutrient medium is in following ratio liquid mixture prepared: peptone 1.3～2.5g, glucose 3～5g, molasses 3～5g, potato immersion liquid 50～100ml, soluble starch 10～20g, sucrose 10～30g, beef extract 2～3g, yeast soak powder 3.5～5g, dregs of beans 1.5～2g, growth factor 1～2ml, water 1000ml; Growth factor wherein is for containing MgSO ₄.7H ₂O 20.00～30.00mg/ml, CaCl ₂.2H ₂O 2.20～3.50mg/ml, ZnSO ₄.7H ₂O0.81～1.10mg/ml, FeSO ₄.7H ₂O 0.44～0.60mg/ml, MnSO ₄.H ₂O 0.13～0.20mg/ml, CuSO ₄.5H ₂The mixture of O 0.02～0.04mg/ml;

2) fermentative degradation:

Put into fermentation tank after fermentation substrate is crushed to 60～120 orders, add entry and carbon source successively, add the weight of entry and the weight portion proportioning of the contained protein of fermentation substrate is 100: 5～18, add the nitrogen content of the phosphorus content of carbon source and fermentation substrate weight ratio be 1: 2～35, with zymotic fluid at 0.1～0.13MPa, sterilized 10～30 minutes for 115～125 ℃, be cooled to 20～37 ℃, add the seed liquor that step 1) obtains, the volume ratio of seed liquor and zymotic fluid is 0.3～1: 10, mix the back at 25～42 ℃, the speed feeding filtrated air with 0.5～2.5vvm stirs fermentation with 150～300rpm speed, use conventional ninhydrin method determination of color degree of hydrolysis during this time, detected a degree of hydrolysis in per 6 hours before 24 hours, between 24～48 hours, degree of hydrolysis of detection in per 4 hours, degree of hydrolysis of detection in per 2 hours after 48 hours, reach 20～35% up to degree of hydrolysis, in 140～145 ℃ of sterilizations 2～8 seconds, obtain activated peptide forage additive then;

Described fermentation substrate is one or more the mixture that is selected from vegetable protein or the animal protein;

Described carbon source comprises corn flour, brown sugar, edible white sugar, corn syrup, starch, cane molasses, beet molasses.

6, the preparation method of activated peptide forage additive as claimed in claim 5 is characterized in that, described bacillus is Bacillus cereus, bacillus subtilis, bacillus licheniformis, bafillus natto, bacillus megaterium or bacillus pumilus; Described Aspergillus is black-koji mould, aspergillus terricola bacterium, aspergillus oryzae, cinnamon Aspergillus or Aspergillus usamii bacterium; Described actinomyces are green sugared sporangium, grayish green streptomycete or expense formula streptomycete; Described Penicillium notatum is purple little mould, penicillium chrysogenum, apple mould, yellowish mould, Taiwan mould, Du Pont's mould or blue brown mould.

7, the preparation method of activated peptide forage additive as claimed in claim 5, it is characterized in that described vegetable protein comprises soybean protein, dregs of beans, soya-bean cake, Peas albumen, zein, red bean albumen, wheat gluten, big aleuronat, the cottonseed dregs of rice and rapeseed dregs; Described animal protein comprises fish meal, blood meal, feather meal, lactoprotein, muscles, casein, the discarded internal organs of animal, earthworm protein powder, squid albumen and fish-skin.

8, the preparation method of activated peptide forage additive as claimed in claim 5 is characterized in that, also comprises step 3) and step 4):

Described step 3) is centrifugal slagging-off, the ultrafiltration, concentrated of protein hydrolyzate:

With step 2) protein hydrolyzate through sterilization that obtains removed slag in centrifugal 10～30 minutes through 5000～7000rpm again, and the supernatant of telling obtains the filtrate of molecular weight less than 5000Da by ultrafiltration membrance filter; This filtrate is concentrated to 50% of original volume through negative-pressure vacuum, obtains concentrate;

Described step 4) is for adding carrier adsorption dry or spray-drying:

Adding carrier adsorption dry is the concentrate that step 3) is obtained, and is added in the carrier that is pre-mixed, and the weight ratio of concentrate and carrier is 0.5～0.8: 1,45～80 ℃ of dryings, obtains activated peptide forage additive then; Described carrier is made up of 2～5 weight portion organic carriers and 3～5 weight portion inorganic carriers; Described organic carrier comprises bran and puffed soybean organic carrier; Described bran organic carrier is one or more the mixture in wheat bran, maize cob meal, degreasing powdered rice hulls and the defatted rice bran; Described puffed soybean organic carrier is one or more the mixture in bean cake powder, soya-bean cake powder and the corn protein powder; Described inorganic carrier is one or more the mixture in precipitated calcium carbonate, stone flour, medical stone and the zeolite powder;

Spray-drying is the concentrate that step 3) is obtained, and spray-drying obtains activated peptide forage additive.

9, the described activated peptide forage additive of a kind of claim 1 is as the purposes of feed addictive.