CN111685227A - Probiotic feed additive composition, preparation method and application thereof - Google Patents
Probiotic feed additive composition, preparation method and application thereof Download PDFInfo
- Publication number
- CN111685227A CN111685227A CN202010673577.4A CN202010673577A CN111685227A CN 111685227 A CN111685227 A CN 111685227A CN 202010673577 A CN202010673577 A CN 202010673577A CN 111685227 A CN111685227 A CN 111685227A
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- Prior art keywords
- probiotic
- waste liquid
- additive composition
- feed additive
- acid
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- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
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- 125000004122 cyclic group Chemical group 0.000 description 1
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- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 235000019838 diammonium phosphate Nutrition 0.000 description 1
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 1
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- 239000008103 glucose Substances 0.000 description 1
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- 230000000968 intestinal effect Effects 0.000 description 1
- 230000003871 intestinal function Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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- CEIZFXOZIQNICU-UHFFFAOYSA-N tenuazonic acid Chemical compound CCC(C)C1NC(=O)C(C(C)=O)=C1O CEIZFXOZIQNICU-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/14—Pretreatment of feeding-stuffs with enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
- A23K10/38—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material from distillers' or brewers' waste
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention relates to a preparation method of a probiotic feed additive composition, which comprises the following steps: (1) adding an enzyme into the fermentation waste liquid, and carrying out enzymolysis to obtain an enzymolysis liquid, wherein the content of soluble protein in the fermentation waste liquid is not less than 1%, and the content of free amino acid in the fermentation waste liquid is not less than 0.05%; (2) and inoculating probiotics into the prepared enzymolysis liquid and then fermenting to obtain the probiotic feed additive composition. According to the invention, macromolecular substances in the fermentation waste liquid are subjected to enzymolysis to form micromolecular nutrient substances beneficial to growth of probiotics, and the probiotic feed additive composition is prepared through probiotic fermentation, so that resource utilization of the fermentation waste liquid is realized.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a probiotic feed additive composition, and a preparation method and application thereof.
Background
The probiotics are active microorganisms which are beneficial to a host and are formed by changing the flora of a certain part of the host through colonization in a human body or an animal body. The health of the intestinal tract is kept by promoting the absorption of nutrients by regulating the immune function of the host mucous membrane and the system or by regulating the balance of flora in the intestinal tract, so that single microorganisms or mixed microorganisms with definite compositions which are beneficial to the health are generated. The beneficial effect of the probiotics in the animal body is to adjust the physiological balance among animal intestinal flora, and reduce the abnormal proliferation of putrefying bacteria and the invasion and colonization of pathogenic bacteria in the intestinal tract by improving the proportion of the probiotics, thereby reducing or eliminating the generation of vivotoxin. And thus are often used as feed additives. A common preparation method of the probiotic feed additive comprises the steps of inoculating probiotic live bacteria into a culture medium, culturing by using high-quality single or mixed flora in the probiotic to obtain high-activity probiotic fermentation liquor, and preparing the live bacteria agent by using a vacuum freeze-drying machine. But has the defects of large resource consumption, high cost and the like. Therefore, an alternative probiotic feed additive and a preparation method thereof are urgently needed to be found.
The microbial fermentation method mostly uses glucose as a substrate to produce a target product through microbial fermentation, has the advantages of bio-based raw materials, high-efficiency conversion, mild reaction, environmental protection, economy and the like, and is widely applied to the industry. The fermentation liquor is subjected to sterilization, desalting, mixed acid removal, concentration and other treatment, so that a target product is prepared and a large amount of fermentation waste liquor is generated. The fermentation waste liquid has complex components, contains various amino acids, thalli, pigments, broken thalli, amino-mixed acid, incompletely reacted saccharide raw materials, intermediate conversion substances, unused culture media (such as diammonium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, vitamins and the like), and the like, is rich in nutrients such as protein, nucleic acid and the like, and also has high Chemical Oxygen Demand (COD) and ammonia nitrogen properties. In the prior art, after the fermentation waste liquid is concentrated, dried and crushed, the fermentation waste liquid is used as a fertilizer substrate, a yeast fermentation culture medium or an edible fungus cultivation raw material, and the defects of low utilization value, low added value of products and the like exist. Therefore, how to improve the comprehensive utilization efficiency of the fermentation waste liquid, change the waste into valuable, perform three-waste treatment in fermentation and reduce the cost becomes a technical problem which is urgently needed to be solved in the field.
Disclosure of Invention
The invention aims to provide a preparation method of a probiotic feed additive composition, which comprises the following steps: (1) adding an enzyme into the fermentation waste liquid, and carrying out enzymolysis to obtain an enzymolysis liquid, wherein the content of soluble protein in the fermentation waste liquid is not less than 1%, and the content of free amino acid in the fermentation waste liquid is not less than 0.05%; (2) and inoculating probiotics into the prepared enzymolysis liquid and then fermenting to obtain the probiotic feed additive composition.
In the preferable technical scheme of the invention, the content of soluble protein in the fermentation waste liquid is not lower than 2%.
In the preferable technical scheme of the invention, the content of free amino acid in the fermentation waste liquid is not lower than 0.1%.
In the preferable technical scheme of the invention, the fermentation waste liquid is subjected to pretreatment of sterilization and concentration before enzymolysis.
In the preferred technical scheme of the invention, the sterilization and concentration steps of the pretreatment are not in sequence.
In a preferred embodiment of the present invention, the sterilization is selected from any one of chemical sterilization, radiation sterilization, dry heat sterilization, and moist heat sterilization, or a combination thereof.
In the preferred technical scheme of the invention, the sterilization temperature is 60-100 ℃, preferably 70-90 ℃, and more preferably 80-85 ℃.
In a preferred embodiment of the present invention, the sterilization time is 10 to 60 minutes, preferably 15 to 50 minutes, and more preferably 20 to 40 minutes.
In a preferred embodiment of the present invention, the concentration is selected from any one of vacuum concentration, membrane concentration, atmospheric concentration, and membrane concentration, or a combination thereof.
In the preferred technical scheme of the invention, the concentration temperature is 40-90 ℃, preferably 45-80 ℃, more preferably 50-70 ℃, and most preferably 55-65 ℃.
In a preferred embodiment of the present invention, the membrane concentration is selected from any one of ultrafiltration, nanofiltration, reverse osmosis or a combination thereof.
In the preferred technical scheme of the invention, the concentration is 10-40 times, preferably 15-35 times, and more preferably 20-30 times of the fermentation waste liquid collected after the target product is separated.
In a preferred embodiment of the present invention, the fermentation waste liquid is a waste liquid obtained by separating a target product from a microbial fermentation liquid, and is preferably selected from any one or a combination of an amino acid fermentation waste liquid, a corn fermentation waste liquid, and an alcohol fermentation waste liquid.
In a preferred embodiment of the present invention, the amino acid fermentation waste liquid is a waste liquid obtained by separating a target amino acid from a microbial fermentation liquid, and is preferably selected from any one or a combination of an alanine fermentation waste liquid, a valine fermentation waste liquid, a lysine fermentation waste liquid, a glutamic acid fermentation waste liquid, a phenylalanine fermentation waste liquid, a leucine fermentation waste liquid, a threonine fermentation waste liquid, a tryptophan fermentation waste liquid, a methionine fermentation waste liquid, and an isoleucine fermentation waste liquid.
In a preferable technical scheme of the invention, the addition amount of the enzyme agent in the fermentation waste liquid is 0.5-10g/L, preferably 1.5-9g/L, and more preferably 3.5-8 g/L.
In the preferred technical scheme of the invention, the enzyme agent is a complex enzyme, and the preferred complex enzyme consists of lysozyme and hydrolase.
In the preferred technical scheme of the invention, the lysozyme in the compound enzyme is: the mass ratio of the hydrolase is 0.5:1-5:1, preferably 0.8:1-3:1, and more preferably 1:1-2: 1.
In the preferred technical scheme of the invention, when the complex enzyme is added into the fermentation waste liquid, the lysozyme and the hydrolase which form the complex enzyme are added simultaneously or sequentially, and the lysozyme and the hydrolase are preferably added after the lysozyme is added.
In a preferred embodiment of the present invention, the lysozyme is selected from any one or a combination of glucanase, chitinase, cellulase and pectinase, and preferably glucanase.
In a preferred embodiment of the present invention, the hydrolase is selected from any one or a combination of alkaline protease, neutral protease and acidic protease, and is preferably alkaline protease.
In a preferred embodiment of the present invention, the pH of the enzymatic reaction is 6.5-9.0, preferably 7.0-8.5, and more preferably 7.5-8.0.
In the preferred technical scheme of the invention, the enzymolysis reaction temperature is 40-60 ℃, preferably 42-55 ℃, and more preferably 45-50 ℃.
In a preferred technical scheme of the invention, the enzymolysis reaction time is 5-20 hours, preferably 8-18 hours, and more preferably 9-15 hours.
In the preferred technical scheme of the invention, the inoculated probiotics is a solid microbial inoculum with the viable count of the probiotics being 10-100 hundred million/g.
In the preferable technical scheme of the invention, the addition amount of the inoculated probiotics in the enzymolysis liquid is 0.05-1g/L, preferably 0.08-0.5g/L, and more preferably 0.1-0.3 g/L.
In a preferred technical scheme of the invention, the probiotics is any one of lactic acid bacteria, yeast and bacillus or a combination thereof.
In a preferred embodiment of the present invention, the lactic acid bacteria are at least one selected from the group consisting of lactococcus, lactobacillus, and bifidobacterium.
In the preferred technical scheme of the invention, the yeast is candida utilis or saccharomyces cerevisiae.
In a preferred technical scheme of the invention, the bacillus is bacillus subtilis or bacillus licheniformis.
In the preferred technical scheme of the invention, the probiotics is a composite bacterium, preferably the composite bacterium consists of lactococcus lactis, lactobacillus and bifidobacteria, more preferably the lactococcus lactis in the composite bacterium: and (3) lactobacillus: the mass ratio of the bifidobacteria is 1-1: 1-2: 1-5, preferably 1:1:2
In the preferred technical scheme of the invention, the probiotics is composite bacteria, preferably the composite bacteria consists of lactic acid bacteria, saccharomycetes and bacillus, more preferably the lactic acid bacteria in the composite bacteria are: yeast: the mass ratio of the bacillus is 1-4:1-6:1-4, preferably 2:3: 2.
In the preferable technical scheme of the invention, the fermentation temperature is 30-50 ℃, preferably 35-45 ℃, and more preferably 37-44 ℃.
In a preferred embodiment of the present invention, the fermentation time is 12 to 36 hours, preferably 18 to 30 hours, and more preferably 20 to 24 hours.
In a preferred embodiment of the present invention, the fermentation pH is 6.5 to 9.0, preferably 6 to 8, and more preferably 6.5 to 7.0.
In a preferred embodiment of the present invention, the pH adjuster is selected from any one of sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, calcium hydroxide, magnesium hydroxide, quicklime (calcium oxide), hydrochloric acid, sulfuric acid, chloric acid, nitric acid, hydrobromic acid, hydrofluoric acid, phosphoric acid, sulfonic acid, malic acid, fumaric acid, citric acid, carboxylic acid, hydroxy acid, keto acid, acetic acid, oxalic acid, citric acid, succinic acid, formic acid, acetic acid, propionic acid, butyric acid, malonic acid, succinic acid, pyruvic acid, glutamic acid, tartaric acid, lactic acid, itaconic acid, ascorbic acid, fumaric acid, α -ketoglutaric acid, and fruit acid, or a combination thereof.
In a preferred embodiment of the present invention, the pH adjusting agent is a buffering agent, and the buffering agent is preferably selected from any one of or a combination of citric acid, potassium citrate, sodium citrate, malic acid, sodium malate, potassium hydroxide, sodium bicarbonate, sodium hydroxide, calcium carbonate, sodium carbonate, phosphoric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, monoethanolamine, diethanolamine, triethanolamine, lactic acid, sodium lactate, potassium lactate, propionic acid, tartaric acid, sodium tartrate, sodium fumarate, potassium tartrate, potassium fumarate, and fumaric acid.
In a preferred technical scheme of the invention, the number of the live probiotics in the probiotic feed additive composition is not less than 1.0 hundred million/ml, and preferably not less than 5.0 hundred million/ml.
In a preferred technical scheme of the invention, the dry weight percentage content of the crude protein in the probiotic feed additive composition is not less than 10%, preferably not less than 12%.
In a preferred technical scheme of the invention, the probiotic feed additive composition is free from peculiar smell.
In the preferable technical scheme of the invention, the probiotic feed additive composition does not contain aflatoxin.
Another object of the present invention is to provide a probiotic feed additive composition, the preparation method of which comprises the following steps: (1) adding an enzyme into the fermentation waste liquid, and carrying out enzymolysis to obtain an enzymolysis liquid, wherein the content of soluble protein in the fermentation waste liquid is not less than 1%, and the content of free amino acid in the fermentation waste liquid is not less than 0.05%; (2) and inoculating probiotics into the prepared enzymolysis liquid and then fermenting to obtain the probiotic feed additive composition.
In the preferable technical scheme of the invention, the content of soluble protein in the fermentation waste liquid is not lower than 2%.
In the preferable technical scheme of the invention, the content of free amino acid in the fermentation waste liquid is not lower than 0.1%.
In the preferable technical scheme of the invention, the fermentation waste liquid is subjected to pretreatment of sterilization and concentration before enzymolysis.
In the preferred technical scheme of the invention, the sterilization and concentration steps of the pretreatment are not in sequence.
In a preferred embodiment of the present invention, the sterilization is selected from any one of chemical sterilization, radiation sterilization, dry heat sterilization, and moist heat sterilization, or a combination thereof.
In the preferred technical scheme of the invention, the sterilization temperature is 60-100 ℃, preferably 70-90 ℃, and more preferably 80-85 ℃.
In a preferred embodiment of the present invention, the sterilization time is 10 to 60 minutes, preferably 15 to 50 minutes, and more preferably 20 to 40 minutes.
In a preferred embodiment of the present invention, the concentration is selected from any one of vacuum concentration, membrane concentration, atmospheric concentration, and membrane concentration, or a combination thereof.
In the preferred technical scheme of the invention, the concentration temperature is 40-90 ℃, preferably 45-80 ℃, more preferably 50-70 ℃, and most preferably 55-65 ℃.
In a preferred embodiment of the present invention, the membrane concentration is selected from any one of ultrafiltration, nanofiltration, reverse osmosis or a combination thereof.
In the preferred technical scheme of the invention, the concentration is 10-40 times, preferably 15-35 times, and more preferably 20-30 times of the fermentation waste liquid collected after the target product is separated.
In a preferred technical scheme of the invention, the fermentation waste liquid is a waste liquid obtained after a target product is separated from a microbial fermentation liquid, and is preferably any one or a combination of an amino acid fermentation waste liquid, a corn fermentation waste liquid and an alcohol fermentation waste liquid.
In a preferred embodiment of the present invention, the amino acid fermentation waste liquid is a waste liquid obtained by separating a target amino acid from a microbial fermentation liquid, and is preferably any one or a combination of an alanine fermentation waste liquid, a valine fermentation waste liquid, a lysine fermentation waste liquid, a glutamic acid fermentation waste liquid, a phenylalanine fermentation waste liquid, a leucine fermentation waste liquid, a threonine fermentation waste liquid, a tryptophan fermentation waste liquid, a methionine fermentation waste liquid, and an isoleucine fermentation waste liquid.
In a preferable technical scheme of the invention, the addition amount of the enzyme agent in the fermentation waste liquid is 0.5-10g/L, preferably 1.5-9g/L, and more preferably 3.5-8 g/L.
In the preferred technical scheme of the invention, the enzyme agent is a complex enzyme, and the preferred complex enzyme consists of lysozyme and hydrolase.
In the preferred technical scheme of the invention, the lysozyme in the compound enzyme is: the mass ratio of the hydrolase is 0.5:1-5:1, preferably 0.8:1-3:1, and more preferably 1:1-2: 1.
In the preferred technical scheme of the invention, when the complex enzyme is added into the fermentation waste liquid, the lysozyme and the hydrolase which form the complex enzyme are added simultaneously or sequentially, and the lysozyme and the hydrolase are preferably added after the lysozyme is added.
In a preferred embodiment of the present invention, the lysozyme is selected from any one or a combination of glucanase, chitinase, cellulase and pectinase, and preferably glucanase.
In a preferred embodiment of the present invention, the hydrolase is selected from any one or a combination of alkaline protease, neutral protease and acidic protease, and is preferably alkaline protease.
In a preferred embodiment of the present invention, the pH of the enzymatic reaction is 6.5-9.0, preferably 7.0-8.5, and more preferably 7.5-8.0.
In the preferred technical scheme of the invention, the enzymolysis reaction temperature is 40-60 ℃, preferably 42-55 ℃, and more preferably 45-50 ℃.
In a preferred technical scheme of the invention, the enzymolysis reaction time is 5-20 hours, preferably 8-18 hours, and more preferably 9-15 hours.
In the preferred technical scheme of the invention, the inoculated probiotics is a solid microbial inoculum with the viable count of the probiotics being 10-100 hundred million/g.
In the preferable technical scheme of the invention, the addition amount of the inoculated probiotics in the enzymolysis liquid is 0.05-1g/L, preferably 0.08-0.5g/L, and more preferably 0.1-0.3 g/L.
In a preferred technical scheme of the invention, the probiotics is any one of lactic acid bacteria, yeast and bacillus or a combination thereof.
In a preferred embodiment of the present invention, the lactic acid bacteria are selected from any one of or a combination of lactococcus, lactobacillus, and bifidobacterium.
In the preferred technical scheme of the invention, the yeast is candida utilis or saccharomyces cerevisiae.
In a preferred technical scheme of the invention, the bacillus is bacillus subtilis or bacillus licheniformis.
In the preferred technical scheme of the invention, the probiotics is a composite bacterium, preferably the composite bacterium consists of lactococcus lactis, lactobacillus and bifidobacteria, more preferably the lactococcus lactis in the composite bacterium: and (3) lactobacillus: the mass ratio of the bifidobacteria is 1-1: 1-2: 1-5, preferably 1:1:2
In the preferred technical scheme of the invention, the probiotics is composite bacteria, preferably the composite bacteria consists of lactic acid bacteria, saccharomycetes and bacillus, more preferably the lactic acid bacteria in the composite bacteria are: yeast: the mass ratio of the bacillus is 1-4:1-6:1-4, preferably 2:3: 2.
In the preferred technical scheme of the invention, the fermentation temperature is 30-50 ℃, preferably 35-45 ℃, and more preferably 37-44 ℃.
In a preferred embodiment of the present invention, the fermentation time is 12 to 36 hours, preferably 18 to 30 hours, and more preferably 20 to 24 hours.
In a preferred embodiment of the present invention, the fermentation pH is 6.5 to 9.0, preferably 6 to 8, and more preferably 6.5 to 7.0.
In a preferred embodiment of the present invention, the pH adjuster is selected from any one of sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, calcium hydroxide, magnesium hydroxide, quicklime (calcium oxide), hydrochloric acid, sulfuric acid, chloric acid, nitric acid, hydrobromic acid, hydrofluoric acid, phosphoric acid, sulfonic acid, malic acid, fumaric acid, citric acid, carboxylic acid, hydroxy acid, keto acid, acetic acid, oxalic acid, citric acid, succinic acid, formic acid, acetic acid, propionic acid, butyric acid, malonic acid, succinic acid, pyruvic acid, glutamic acid, tartaric acid, lactic acid, itaconic acid, ascorbic acid, fumaric acid, α -ketoglutaric acid, and fruit acid, or a combination thereof.
In a preferred embodiment of the present invention, the pH adjusting agent is a buffering agent, and the buffering agent is preferably selected from any one of or a combination of citric acid, potassium citrate, sodium citrate, malic acid, sodium malate, potassium hydroxide, sodium bicarbonate, sodium hydroxide, calcium carbonate, sodium carbonate, phosphoric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, monoethanolamine, diethanolamine, triethanolamine, lactic acid, sodium lactate, potassium lactate, propionic acid, tartaric acid, sodium tartrate, sodium fumarate, potassium tartrate, potassium fumarate, and fumaric acid.
In a preferred technical scheme of the invention, the number of the live probiotics in the probiotic feed additive composition is not less than 1.0 hundred million/ml, and preferably not less than 5.0 hundred million/ml.
In a preferred technical scheme of the invention, the dry weight percentage content of the crude protein in the probiotic feed additive composition is not less than 10%, preferably not less than 12%.
In a preferred technical scheme of the invention, the probiotic feed additive composition is free from peculiar smell.
In the preferable technical scheme of the invention, the probiotic feed additive composition does not contain aflatoxin.
Another object of the present invention is to provide the use of the probiotic feed additive of the present invention for the preparation of a probiotic fermented feed.
The invention also aims to provide a probiotic fermented feed, which consists of 85-99.95% of basic ration and 0.05-15% of probiotic feed additive composition.
In a preferred technical scheme of the invention, the amount of the probiotic feed additive composition in the probiotic fermented feed is 0.1-10%, and preferably 1-5%.
In a preferred technical scheme of the invention, the basic ration is any one of chicken basic ration, duck basic ration, goose basic ration, pig basic ration, cattle basic ration, horse basic ration, donkey basic ration, cat basic ration, dog basic ration, fish basic ration, rabbit basic ration and sheep basic ration.
In a preferred technical scheme of the invention, the probiotic fermented feed is selected from any one of chicken probiotic fermented feed, duck probiotic fermented feed, goose probiotic fermented feed, pig probiotic fermented feed, cow probiotic fermented feed, horse probiotic fermented feed, donkey probiotic fermented feed, cat probiotic fermented feed, dog probiotic fermented feed, fish probiotic fermented feed, rabbit probiotic fermented feed and sheep probiotic fermented feed.
The invention also aims to provide a preparation method of the probiotic fermented feed, which comprises the following steps: weighing required amounts of the probiotic feed additive composition and the basic ration, and fully and uniformly mixing the probiotic feed additive composition and the basic ration to obtain the probiotic fermented feed, wherein the probiotic fermented feed consists of 85% -99.95% of the basic ration and 0.05% -15% of the probiotic feed additive composition.
In a preferred technical scheme of the invention, the amount of the probiotic feed additive composition in the probiotic fermented feed is 0.1-10%, and preferably 1-5%.
In a preferred technical scheme of the invention, the basic ration is any one of chicken basic ration, duck basic ration, goose basic ration, pig basic ration, cattle basic ration, horse basic ration, donkey basic ration, cat basic ration, dog basic ration, fish basic ration, rabbit basic ration and sheep basic ration.
In a preferred technical scheme of the invention, the probiotic fermented feed is selected from any one of chicken probiotic fermented feed, duck probiotic fermented feed, goose probiotic fermented feed, pig probiotic fermented feed, cow probiotic fermented feed, horse probiotic fermented feed, donkey probiotic fermented feed, cat probiotic fermented feed, dog probiotic fermented feed, fish probiotic fermented feed, rabbit probiotic fermented feed and sheep probiotic fermented feed.
Unless otherwise indicated, when the present invention relates to percentages between liquids, said percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentages between solid and liquid, said percentages being weight/volume percentages; the balance being weight/weight percent.
Unless otherwise indicated, the present invention measures soluble protein content, free amino acid content, lactic acid content, viable probiotic count, dry weight percent crude protein, aflatoxin and odor as follows:
1. and (3) soluble protein content determination: and (4) determining according to a Coomassie brilliant blue method for determining the protein content in the imported and exported milk, egg and bean food of SN/T3926 and 2014 in import and export industry standards.
2. And (3) measuring the content of free amino acid: the content of the free amino acid in the water-soluble fertilizer is determined according to the agricultural standard NY/T1975-2010 determination.
3. And (3) measuring the content of lactic acid: the content of lactic acid in import and export juice is determined according to the import and export industry standard SN/T1511.1-2005 inspection method of lactic acid content in import and export juice.
4. And (3) measuring the viable count of the lactic acid bacteria: measured according to GB4789.35-2016 food safety national standard food microbiology inspection lactobacillus inspection.
5. B, bacillus subtilis quantity determination: the detection is carried out according to GB/T26428 & 2016 detection of Bacillus subtilis in feed biological agents.
6. Measuring the quantity of the yeast: the determination is carried out according to GB 4789.15-2016 food safety national standard food microbiology test mould and yeast count.
7. And (3) determining the dry weight percentage of the crude protein: the determination is carried out according to the national standard GB/T18868-2002 feed moisture, crude protein, crude fiber, crude fat, lysine and methionine rapid determination near infrared spectroscopy.
8. And (3) measuring aflatoxin: according to the determination of the aflatoxin, the zearalenone and the T-2 toxin in the feed of NY/T2071 and 2011 in the agricultural standard, the determination is carried out by a liquid chromatography-tandem mass spectrometry method.
9. And (3) odor detection: and (4) artificial sensory measurement.
Compared with the prior art, the invention has the following beneficial technical effects:
1. the probiotic feed additive composition prepared by the invention is used for preparing the probiotic feed by carrying out enzymolysis on macromolecular substances in the fermentation waste liquid into micromolecular nutrient substances beneficial to growth of probiotics and then fermenting the micromolecular nutrient substances through the probiotics, so that the cyclic utilization of the fermentation waste liquid and resources is realized, the intestinal function of animals is obviously improved, the growth performance of the animals is improved, and the increase of the generation cost caused by three-waste treatment is avoided.
2. The preparation method of the invention has the advantages of simple operation, obviously shortened production period, further obviously reduced production cost and suitability for large-scale industrial production.
Drawings
FIG. 1 comparison of lactic acid content of comparative example 2 (without enzyme) and example 4 (with enzyme)
FIG. 2 comparison of viable count of lactic acid bacteria between comparative example 2 (without enzyme) and example 4 (with enzyme)
FIG. 3 comparison of the percent dry weight of crude protein for comparative example 2 (without enzyme) and example 4 (with enzyme)
FIG. 4 comparison of viable probiotic bacteria count for comparative example 3 (without enzyme) and example 6 (with enzyme)
FIG. 5 comparison of the percent dry weight of crude protein for comparative example 3 (without enzyme) and example 6 (with enzyme)
Detailed Description
The present invention will be described below with reference to examples, but the present invention is not limited to the examples.
The dextranase, alkaline protease, chitinase, cellulase, pectinase, acid protease used in the embodiments are purchased from bioscience, Inc., Yonghua, Tokyo. The used lactococcus lactis, lactobacillus and bifidobacterium are purchased from Jiangsu Yuanshan biotechnology limited.
Comparative example 1 and examples 1-3
Take 0.1m3L-valine fermentation waste liquid was sterilized at 80 ℃ for 20min, concentrated 10-fold with an ultrafiltration membrane, and divided into four equal parts, which were used in comparative example 1 and examples 1 to 3 (see Table 1).
In comparative example 1 no enzyme was added. In the examples 1-3, 3g/L of lysozyme was added, the enzymolysis temperature was controlled at 40 ℃ and the pH was 6.5, and after stirring and reacting for 10 hours, 3g/L of hydrolase was added, the enzymolysis temperature was controlled at 45 ℃ and the pH was 7, and the stirring and reacting were continued for 10 hours to obtain an enzymatic hydrolysate. The lysozyme and hydrolase selections are detailed in table 1.
And detecting the content of soluble protein and free amino acid in the enzymolysis liquid. The results are shown in Table 1.
TABLE 1 comparison of soluble protein and free amino acid content in enzymatic hydrolysate after addition of different enzymatic agents
Comparative example 2Fermentation experiment of lactic acid bacteria
According to the weight ratio of the lactic acid coccus: and (3) lactobacillus: the mass ratio of the bifidobacteria is 1:1:2 preparing the lactobacillus composite bacteria. And inoculating the lactobacillus composite microbial inoculum into the enzymolysis liquid of the comparative example 1 according to 0.1g/L, stirring, adjusting the pH of the enzymolysis liquid to 6.5, and naturally fermenting at 37 ℃ for 24 hours to obtain the lactobacillus feed additive composition.
Example 4Fermentation experiment of lactic acid bacteria
According to the weight ratio of the lactic acid coccus: and (3) lactobacillus: the mass ratio of the bifidobacteria is 1:1:2 preparing the lactobacillus composite bacteria. And inoculating the lactobacillus complex microbial inoculum into the enzymolysis liquid of the embodiment 1 according to 0.1g/L, stirring, adjusting the pH of the enzymolysis liquid to 6.5, and naturally fermenting at 37 ℃ for 24 hours to obtain the lactobacillus feed additive composition.
The lactic acid bacteria feed additive compositions obtained by fermentation in comparative example 2 (without using an enzyme agent) and example 4 (with an enzyme agent) were tested for lactic acid content, viable bacterial colony count of lactic acid bacteria, crude protein content, aflatoxin and odor according to the method of the present invention. The results are shown in FIGS. 1-3. From the 12 th hour of fermentation, the off-flavor of comparative example 2 appeared, the off-flavor became stronger with time, and aflatoxin was detected in the final product. Example 4 there was no off-flavor in the whole fermentation process and no aflatoxin in the final product.
Example 5Preparation of lactobacillus fermented feed
According to the standard of 'T/CFIAS 001-containing 2018 piglet and growing-finishing pig compound feed', the basic ration for the piglets is prepared as follows: 70.24 percent of corn, 20.00 percent of soybean meal, 1.58 percent of corn protein powder, 2.60 percent of fish meal, 1.10 percent of soybean oil, 1.50 percent of monocalcium phosphate, 1.30 percent of stone powder, 0.30 percent of salt, 0.30 percent of L-lysine, 0.18 percent of methionine, 0.15 percent of threonine, 0.25 percent of chromium oxide and 0.50 percent of basic ration premix.
Adding the lactobacillus feed additive prepared in the embodiment 4 into basal ration of piglets according to the addition of 0.5%, and uniformly stirring to obtain lactobacillus fermented feed.
The method of the invention detects that the number of live lactobacillus in the lactobacillus fermented feed is more than 5 hundred million/ml. When the lactobacillus fermented feed is used for feeding piglets, the growth performance of the piglets is obviously improved.
Comparative example 3Probiotic compound bacteria fermentation experiment
According to the lactic acid bacteria: yeast: the mass ratio of the bacillus is 2:3:2, preparing the probiotic compound bacteria. And inoculating the probiotic composite microbial inoculum into the enzymolysis liquid of the comparative example 1 according to 0.1g/L, stirring, adjusting the pH of the enzymolysis liquid to 6.5, and naturally fermenting for 24 hours at 37 ℃ to obtain the probiotic feed additive composition.
Example 6Probiotic compound bacteria fermentation experiment
According to the lactic acid bacteria: yeast: the mass ratio of the bacillus is 2:3:2, preparing the probiotic compound bacteria. And inoculating the probiotic compound bacteria agent into the enzymolysis liquid in the embodiment 1 according to 0.1g/L, stirring, adjusting the pH of the enzymolysis liquid to 6.5, and naturally fermenting at 37 ℃ for 24 hours to obtain the probiotic feed additive composition.
The probiotic bacteria colony count, crude protein content, aflatoxin and odor of the probiotic bacteria feed additive compositions fermented in comparative example 3 (without enzyme agent) and example 6 (with enzyme agent) were tested according to the method described in the present invention. The results are shown in FIGS. 4-5. From the 12 th hour of fermentation, the off-flavor of comparative example 3 appeared, the off-flavor became stronger with time, and aflatoxin was detected in the final product. Example 6 the whole fermentation process has no peculiar smell, and the end product has no aflatoxin.
Example 7Preparation of probiotic fermented feed
According to the standard of 'T/CFIAS 001-containing 2018 piglet and growing-finishing pig compound feed', the basic ration for the piglets is prepared as follows: 74.49% of corn, 16.00% of soybean meal, 0.96% of corn protein powder, 2.30% of fish meal, 2.00% of soybean oil, 1.50% of monocalcium phosphate, 1.10% of stone powder, 0.30% of salt, 0.32% of L-lysine, 0.09% of methionine, 0.17% of threonine, 0.02% of tryptophan, 0.25% of chromium oxide and 0.50% of basic ration premix.
And (3) adding the probiotic feed additive prepared in the embodiment 6 into basal ration of the piglets according to the addition amount of 0.5%, and uniformly stirring to obtain the probiotic fermented feed.
The number of the live probiotics in the probiotic fermented feed detected by the method is more than 5 hundred million/ml. When the probiotic fermented feed is used for feeding piglets, the growth performance of the piglets is remarkably improved.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined in the appended claims.
Claims (10)
1. A preparation method of a probiotic feed additive composition comprises the following steps: (1) adding an enzyme into the fermentation waste liquid, and carrying out enzymolysis to obtain an enzymolysis liquid, wherein the content of soluble protein in the fermentation waste liquid is not less than 1%, and the content of free amino acid in the fermentation waste liquid is not less than 0.05%; (2) and inoculating probiotics into the prepared enzymolysis liquid and then fermenting to obtain the probiotic feed additive composition.
2. The method of claim 1, wherein the amount of the enzyme agent added to the fermentation waste liquid is 0.5-10g/L, preferably 1.5-9g/L, more preferably 3.5-8g/L, the enzyme agent is a complex enzyme, preferably the complex enzyme is composed of lysozyme and hydrolase, and the ratio of lysozyme: the mass ratio of the hydrolase is 0.5:1-5:1, preferably 0.8:1-3:1, and more preferably 1:1-2: 1.
3. The method according to any one of claims 1-2, wherein the enzymatic hydrolysis reaction is at a pH of 6.5-9.0, preferably at a pH of 7.0-8.5, more preferably at a pH of 7.5-8.0.
4. The method according to any one of claims 1 to 3, wherein the enzymatic hydrolysis reaction temperature is 40 to 60 ℃, preferably 42 to 55 ℃, more preferably 45 to 50 ℃.
5. The method according to any one of claims 1 to 4, wherein the probiotic bacteria are any one of lactic acid bacteria, yeast, bacillus or a combination thereof.
6. The method according to any one of claims 1 to 5, wherein the number of live probiotic bacteria in the probiotic feed additive composition is not less than 1.0 million/ml, preferably not less than 5.0 million/ml.
7. The method according to any one of claims 1 to 6, wherein the dry weight percentage content of crude protein in the probiotic feed additive composition is not less than 10%, preferably not less than 12%.
8. A probiotic feed additive composition prepared according to the method of claims 1-7.
9. Use of a probiotic feed additive composition according to any one of claims 1 to 8 for the preparation of a probiotic fermented feed.
10. A probiotic fermented feed, characterized in that it consists of 85% to 99.95% of a basal ration and 0.05% to 15% of a probiotic feed additive composition according to claim 9.
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