CN113826778A - Preparation method of composite probiotic for fermented feed - Google Patents
Preparation method of composite probiotic for fermented feed Download PDFInfo
- Publication number
- CN113826778A CN113826778A CN202111026084.2A CN202111026084A CN113826778A CN 113826778 A CN113826778 A CN 113826778A CN 202111026084 A CN202111026084 A CN 202111026084A CN 113826778 A CN113826778 A CN 113826778A
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- CN
- China
- Prior art keywords
- lactobacillus
- mixing
- bacillus
- fermented feed
- probiotic
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- 239000006041 probiotic Substances 0.000 title claims abstract description 68
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 68
- 238000002360 preparation method Methods 0.000 title claims abstract description 48
- 230000000529 probiotic effect Effects 0.000 title claims abstract description 46
- 239000002131 composite material Substances 0.000 title claims abstract description 37
- 238000002156 mixing Methods 0.000 claims abstract description 57
- 238000000855 fermentation Methods 0.000 claims abstract description 54
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 50
- 230000004151 fermentation Effects 0.000 claims abstract description 48
- 241000894006 Bacteria Species 0.000 claims abstract description 43
- 239000012266 salt solution Substances 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 16
- 239000012452 mother liquor Substances 0.000 claims abstract description 16
- 239000000203 mixture Substances 0.000 claims abstract description 15
- 239000011159 matrix material Substances 0.000 claims abstract description 8
- 235000019764 Soybean Meal Nutrition 0.000 claims abstract description 7
- 240000008042 Zea mays Species 0.000 claims abstract description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 7
- 235000005822 corn Nutrition 0.000 claims abstract description 7
- 235000013312 flour Nutrition 0.000 claims abstract description 7
- 235000013379 molasses Nutrition 0.000 claims abstract description 7
- 239000004455 soybean meal Substances 0.000 claims abstract description 7
- 239000000758 substrate Substances 0.000 claims abstract description 7
- 238000011049 filling Methods 0.000 claims abstract description 5
- 238000003756 stirring Methods 0.000 claims abstract description 5
- 239000000843 powder Substances 0.000 claims description 72
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 43
- 241000186660 Lactobacillus Species 0.000 claims description 40
- 229940039696 lactobacillus Drugs 0.000 claims description 40
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 36
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 36
- 241000194108 Bacillus licheniformis Species 0.000 claims description 35
- 244000063299 Bacillus subtilis Species 0.000 claims description 35
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 35
- 239000012153 distilled water Substances 0.000 claims description 32
- 230000000243 photosynthetic effect Effects 0.000 claims description 30
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 23
- 239000001632 sodium acetate Substances 0.000 claims description 23
- 235000017281 sodium acetate Nutrition 0.000 claims description 23
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 22
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 22
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 20
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 20
- 241000235646 Cyberlindnera jadinii Species 0.000 claims description 17
- 241000190950 Rhodopseudomonas palustris Species 0.000 claims description 17
- 241000235342 Saccharomycetes Species 0.000 claims description 17
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 16
- 241000194031 Enterococcus faecium Species 0.000 claims description 14
- 240000001046 Lactobacillus acidophilus Species 0.000 claims description 14
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 claims description 14
- 244000199866 Lactobacillus casei Species 0.000 claims description 14
- 235000013958 Lactobacillus casei Nutrition 0.000 claims description 14
- 241000186673 Lactobacillus delbrueckii Species 0.000 claims description 14
- 240000006024 Lactobacillus plantarum Species 0.000 claims description 14
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims description 14
- 229940039695 lactobacillus acidophilus Drugs 0.000 claims description 14
- 229940017800 lactobacillus casei Drugs 0.000 claims description 14
- 229940072205 lactobacillus plantarum Drugs 0.000 claims description 14
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 14
- 241000186000 Bifidobacterium Species 0.000 claims description 13
- 238000009360 aquaculture Methods 0.000 claims description 11
- 244000144974 aquaculture Species 0.000 claims description 11
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 11
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 11
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 11
- 229940099596 manganese sulfate Drugs 0.000 claims description 11
- 239000011702 manganese sulphate Substances 0.000 claims description 11
- 235000007079 manganese sulphate Nutrition 0.000 claims description 11
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 9
- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 claims description 8
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000004310 lactic acid Substances 0.000 claims description 2
- 235000014655 lactic acid Nutrition 0.000 claims description 2
- 241000186016 Bifidobacterium bifidum Species 0.000 claims 1
- 229940002008 bifidobacterium bifidum Drugs 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 10
- 230000004083 survival effect Effects 0.000 abstract description 7
- 230000015556 catabolic process Effects 0.000 abstract 1
- 238000006731 degradation reaction Methods 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 description 52
- 238000012258 culturing Methods 0.000 description 37
- 239000001888 Peptone Substances 0.000 description 27
- 108010080698 Peptones Proteins 0.000 description 27
- 229940041514 candida albicans extract Drugs 0.000 description 27
- 235000019319 peptone Nutrition 0.000 description 27
- 239000012138 yeast extract Substances 0.000 description 27
- 238000009630 liquid culture Methods 0.000 description 26
- 239000007787 solid Substances 0.000 description 26
- 239000007788 liquid Substances 0.000 description 22
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 21
- 239000008103 glucose Substances 0.000 description 21
- 230000000052 comparative effect Effects 0.000 description 15
- 238000004108 freeze drying Methods 0.000 description 15
- 239000002609 medium Substances 0.000 description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 13
- 229920001817 Agar Polymers 0.000 description 12
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 12
- 239000008272 agar Substances 0.000 description 12
- 238000005119 centrifugation Methods 0.000 description 12
- 229920000136 polysorbate Polymers 0.000 description 11
- 230000001954 sterilising effect Effects 0.000 description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 241000238553 Litopenaeus vannamei Species 0.000 description 9
- 235000015278 beef Nutrition 0.000 description 9
- 239000000284 extract Substances 0.000 description 9
- 238000011081 inoculation Methods 0.000 description 9
- 239000012137 tryptone Substances 0.000 description 9
- 238000004659 sterilization and disinfection Methods 0.000 description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 6
- 241000238557 Decapoda Species 0.000 description 6
- 230000003321 amplification Effects 0.000 description 6
- 239000001110 calcium chloride Substances 0.000 description 6
- 229910001628 calcium chloride Inorganic materials 0.000 description 6
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 6
- 229910001629 magnesium chloride Inorganic materials 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 6
- 229960001763 zinc sulfate Drugs 0.000 description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 description 6
- 230000000694 effects Effects 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000010564 aerobic fermentation Methods 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 235000021120 animal protein Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Classifications
-
- 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
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- 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
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- 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
-
- 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
- A23K10/33—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from molasses
-
- 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
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/26—Compounds containing phosphorus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/30—Oligoelements
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/113—Acidophilus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/137—Delbrueckii
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Botany (AREA)
- Biochemistry (AREA)
- Inorganic Chemistry (AREA)
- Biomedical Technology (AREA)
- Birds (AREA)
- Sustainable Development (AREA)
- Marine Sciences & Fisheries (AREA)
- Insects & Arthropods (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention relates to the technical field of biological fermented feed, in particular to a preparation method of a composite probiotic for fermented feed. The preparation method comprises the following steps: step 1: preparing a matrix; the method comprises the following steps of (1) mixing dry soybean meal, molasses and corn flour according to the mass ratio of 2-5: 3-8: 6-8, uniformly mixing; step 2: preparing mixed fermentation mother liquor; and (2) mixing the following components in a mass ratio of 8-15: stirring and mixing 0.6-1.1 of fermentation probiotics and a salt solution; and step 3: mixing a substrate and mixed fermentation mother liquor according to a mass ratio of 1: 8-11, and filling the mixture into a sealed bottom for anaerobic fermentation. According to the invention, the composition and the proportion of probiotics are optimized, the concentration of the bacteria of the composite probiotic is increased by adding a salt solution when the composite probiotic is prepared, and the degradation of the excrement and the feed residue of aquatic animals by the composite probiotic fermented feed is promoted by adjusting the composition and the content of the matrix and the composition proportion of the composite probiotic, so that the water quality of a water body is improved, and the survival rate of aquatic products is increased.
Description
Technical Field
The invention relates to the technical field of biological fermented feed, in particular to a preparation method of a composite probiotic for fermented feed.
Background
Aquaculture is a high-investment, high-risk agricultural production process. In the traditional artificial aquaculture industry, because the extensive aquaculture mode and the feed feeding are unscientific, the problems of poor water body environment, insufficient oxygen supply, low immunity of aquatic products, poor development and the like often occur, and even diseases or death of the aquatic products can be caused seriously. In order to reduce the loss, farmers often use a large amount of drugs such as antibiotics and hormones in the cultivation process. However, the antibiotic reduces the concentration of beneficial microorganisms in the body while eliminating the pathogenic microorganisms in the intestinal tract, destroys the balance of a microecosystem in an animal body, reduces the adaptability of the animal to the change of a breeding environment, and is easy to cause the pathogenic microorganisms to generate drug resistance. The overproof antibiotics remained in aquatic products can influence the public health and safety of human beings through a food chain. Therefore, how to breed high-quality and safe aquatic products becomes a hot point of social attention.
The biological fermentation feed is a new industry, and provides a technical solution for upgrading and transforming the development of the aquaculture industry. The biological fermentation feed has been developed in developed countries such as Japan, Europe and America for decades, and practice shows that the biological fermentation feed can promote the healthy growth of cultured animals, reduce the use of antibiotics and other medicaments, improve the feed conversion rate, and is a necessary direction for the development of feed industry. Chinese patent publication No. CN103461647A discloses a probiotic premix using orange seeds as fermentation auxiliary materials and fermentation bacteria as lactic acid bacteria, but the patent uses single probiotic, and has limited effect on improving micro-ecological environment. Chinese patent publication No. CN112205541A discloses a fermented aquatic animal-protective feed, which is prepared by fermenting multiple enzyme preparations, multiple raw materials including marine animal proteins, and composite probiotic strains, but the patent requires secondary anaerobic fermentation after aerobic fermentation, and requires additional addition of multiple raw materials such as decomposition enzymes and vitamins to assist microbial strains in establishing micro-ecological balance, which increases difficulty and cost of the preparation process, and the feed has high price due to high cost, and is not suitable for wide aquaculture users.
Disclosure of Invention
In view of the above, there is a need to provide a method for preparing a complex probiotic for fermented feed. According to the preparation method, the concentration of the thalli of the composite probiotic is improved by adding the salt solution when the composite probiotic is prepared, an enzyme preparation is not required to be added, and the preparation cost is saved; and the composition and content of the matrix and the composition and composition proportion of the composite probiotics are adjusted to promote the composite probiotics fermented feed to degrade the excreta and feed residues of aquatic animals, improve the water quality of water and improve the survival rate of aquatic products.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a preparation method of a composite probiotic for fermented feed, which comprises the following steps:
step 1: preparing a matrix; the method comprises the following steps of (1) mixing dry soybean meal, molasses and corn flour according to the mass ratio of 2-5: 3-8: 6-8, uniformly mixing;
step 2: preparing mixed fermentation mother liquor; and (2) mixing the following components in a mass ratio of 8-15: stirring and mixing 0.6-1.1 of fermentation probiotics and a salt solution;
and step 3: mixing a substrate and mixed fermentation mother liquor according to a mass ratio of 1: 8-11, and filling the mixture into a sealed bottom for anaerobic fermentation.
Further, the mass ratio of the dry soybean meal, the molasses and the corn flour is 3: 5: 6.
further, the mass ratio of the fermentation probiotics to the salt solution is 11: 0.8.
further, the mass ratio of the mixed fermentation mother liquor to the substrate is 10: 1.
further, the fermentation probiotics comprise the following components in a mass ratio of 80-110: 8-12: 2-6: 1-4 of lactobacillus powder, bacillus powder, saccharomycete powder and photosynthetic bacteria powder.
Preferably, the mass ratio of the lactobacillus powder, the bacillus powder, the saccharomycete powder and the photosynthetic bacterium powder is 96: 10: 6: 2.
further, the preparation method of the salt solution comprises the steps of mixing 1.0-3.0 g of monopotassium phosphate, 1.0-3.0 g of diammonium citrate, 0.4-0.8 g of magnesium sulfate, 4.0-8.0 g of sodium acetate, 0.04-0.08 g of manganese sulfate, 0.6-1.1 g of calcium carbonate and 0.6-1.4 mL of Tween-80, and adding distilled water to a constant volume of 1L.
Preferably, the salt solution is prepared by mixing 2.0g of monopotassium phosphate, 2.0g of diammonium citrate, 0.6g of magnesium sulfate, 6.0g of sodium acetate, 0.06g of manganese sulfate, 0.8g of calcium carbonate and 1.0mL of Tween-80, and adding distilled water to a constant volume of 1L.
Further, the lactobacillus powder comprises lactobacillus delbrueckii, bifidobacterium, lactobacillus acidophilus, streptococcus faecium, lactobacillus plantarum and lactobacillus casei; the bacillus powder comprises bacillus subtilis and bacillus licheniformis; the yeast powder comprises beer yeast and candida utilis; the photosynthetic bacteria are rhodopseudomonas palustris.
Further, the bifidobacterium is bifidobacterium CICC21717, the lactobacillus acidophilus is lactobacillus acidophilus CICC 6074, the lactobacillus plantarum is lactobacillus plantarum CICC6009, the lactobacillus delbrueckii is lactobacillus delbrueckii CICC6098, the streptococcus faecium is streptococcus faecium CICC20430, and the lactobacillus casei is lactobacillus casei CICC 6117; the bacillus subtilis is one or more of bacillus subtilis CICC 9011, bacillus subtilis CICC10071 and bacillus subtilis CICC 20872; the bacillus licheniformis is one or more of bacillus licheniformis CICC23584, bacillus licheniformis CICC 10037 and bacillus licheniformis CICC 10181; the beer yeast is beer yeast CICC 1421; the candida utilis is candida utilis CICC 1314; the rhodopseudomonas palustris is rhodopseudomonas palustris CICC 23812; the strains are purchased from China center for culture collection and management of industrial microorganisms.
Further, in the step 3, the fermentation temperature is 25-35 ℃, and the fermentation time is 48-96 hours.
Preferably, the fermentation temperature in step 3 is 30 ℃ and the fermentation time is 72 hours.
Further, the preparation method of the lactobacillus powder comprises the steps of respectively purifying and culturing bifidobacterium CICC21717, lactobacillus acidophilus CICC 6074, lactobacillus plantarum CICC6009, lactobacillus delbrueckii CICC6098, streptococcus faecium CICC20430 and lactobacillus casei CICC 6117 on a lactobacillus solid culture medium, then respectively inoculating the purified and cultured lactobacillus liquid culture medium into the culture medium, culturing the culture medium at 37 ℃ for 8 hours, then mixing the cultured bifidobacterium CICC21717, lactobacillus acidophilus CICC 6074, lactobacillus plantarum CICC6009, lactobacillus delbrueckii CICC6098, streptococcus faecium CICC20430 and lactobacillus casei CICC 6117 in equal volumes to obtain mixed lactobacillus, inoculating the mixed lactobacillus in a liquid fermentation tank into a lactobacillus amplification culture medium according to the volume ratio of the inoculation amount of 1:10, culturing the mixed lactobacillus in the liquid fermentation tank for 12 hours at 37 ℃, performing aseptic centrifugation, and performing low-temperature freeze drying to obtain the lactobacillus powder.
Further, the preparation method of the lactobacillus solid medium comprises the steps of 5.0g of peptone, 5.0g of beef extract powder, 10.0g of tryptone, 5.0g of yeast extract, 8.0g of glucose, 801.0 mL of tween, 2.0g of monopotassium phosphate, 5.0g of sodium acetate, 2.0g of diammonium hydrogen citrate, 0.25g of zinc sulfate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate, 1.0g of calcium carbonate and 15.0g of agar, then adding distilled water to a constant volume of 1L, adjusting the pH to 6.8, and carrying out wet-heat high-pressure sterilization at 118 ℃ for 18 minutes.
Further, the lactobacillus liquid medium is prepared by 5.0g of peptone, 5.0g of beef extract powder, 10.0g of tryptone, 5.0g of yeast extract, 8.0g of glucose, 801.0 mL of tween, 2.0g of monopotassium phosphate, 5.0g of sodium acetate, 2.0g of diammonium hydrogen citrate, 0.25g of zinc sulfate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate and 1.0g of calcium carbonate, then distilled water is added to a constant volume of 1L, the pH value is adjusted to 6.8, and wet-heat autoclaving is carried out at 118 ℃ for 18 minutes.
Further, the preparation method of the lactobacillus amplification medium comprises the steps of 5.0g of peptone, 5.0g of beef extract powder, 10.0g of tryptone, 5.0g of yeast extract, 10.0g of glucose, 801.0 mL of tween, 2.0g of monopotassium phosphate and 5.0g of sodium acetate, then adding distilled water to a constant volume of 1 liter, adjusting the pH value to 6.8, and carrying out damp-heat high-pressure sterilization at 118 ℃ for 18 minutes.
Further, the preparation method of the bacillus powder comprises the steps of respectively purifying and culturing bacillus subtilis CICC 9011, bacillus subtilis CICC10071, bacillus subtilis CICC20872, bacillus licheniformis CICC23584, bacillus licheniformis CICC 10037 and bacillus licheniformis CICC10181 on a bacillus solid culture medium, then respectively inoculating the bacillus solid culture medium into a bacillus liquid culture medium, culturing the bacillus solid culture medium in a shaker at 37 ℃ at 210rpm for 8 hours, then mixing the cultured bacillus subtilis CICC 9011, bacillus subtilis CICC10071 and bacillus subtilis CICC20872 in equal volume to obtain mixed bacillus subtilis, mixing the cultured bacillus licheniformis CICC23584, bacillus licheniformis CICC 10037 and bacillus licheniformis CICC10181 in equal volume to obtain mixed bacillus licheniformis, and mixing the mixed bacillus licheniformis with the mixed bacillus according to the volume ratio of 1: 2, mixing to obtain mixed bacillus, then inoculating the mixed bacillus into a bacillus liquid culture medium in a liquid fermentation tank according to the volume ratio of the inoculation amount of 1:10, culturing for 12 hours at 37 ℃, performing aseptic centrifugation, and performing low-temperature freeze drying to obtain bacillus powder;
further, the bacillus solid medium is prepared by 10.0g of peptone, 5.0g of yeast extract, 10.0g of sodium chloride, 10.0g of glucose and 15.0g of agar, then adding distilled water to a constant volume of 1 liter, adjusting the pH value to 7.0, and carrying out moist-heat autoclaving sterilization at 118 ℃ for 18 minutes.
Further, the bacillus liquid medium is prepared by 10.0g of peptone, 5.0g of yeast extract, 10.0g of sodium chloride and 10.0g of glucose, then adding distilled water to a constant volume of 1 liter, adjusting the pH value to 7.0, and carrying out moist-heat autoclaving at 118 ℃ for 18 minutes.
Further, the preparation method of the saccharomycete powder comprises the steps of respectively purifying and culturing saccharomyces cerevisiae CICC1421 and candida utilis CICC1314 on a saccharomycete solid culture medium, then respectively inoculating the purified and cultured saccharomyces cerevisiae CICC1421 and candida utilis CICC1314 in a saccharomycete liquid culture medium, culturing for 8 hours at 37 ℃ in a shaking table at 210rpm, then mixing the cultured saccharomyces cerevisiae and candida utilis with equal volume to obtain mixed saccharomycete, inoculating the mixed saccharomycete in the saccharomycete liquid culture medium in a liquid fermentation tank according to the volume ratio of the inoculation amount of 1:10, culturing for 12 hours at 37 ℃, performing aseptic centrifugation, and performing low-temperature freeze drying to obtain the saccharomycete powder.
Further, the yeast solid medium is prepared by 5g of yeast extract, 10g of peptone, 20g of glucose, 15.0g of agar, and distilled water to a constant volume of 1L, adjusting pH to 6.4, and autoclaving at 118 ℃ for 20 minutes.
Further, the yeast liquid medium is prepared by adding 5g of yeast extract, 10g of peptone, 20g of glucose and distilled water to a constant volume of 1L, adjusting pH to 6.4, and autoclaving at 118 ℃ for 20 minutes.
Further, the preparation method of the photosynthetic bacteria powder comprises the steps of purifying and culturing rhodopseudomonas palustris CICC 23812 in a photosynthetic bacteria solid culture medium, then inoculating the purified and cultured rhodopseudomonas palustris in a photosynthetic bacteria liquid culture medium, culturing the purified and cultured rhodopseudomonas palustris for 8 hours at 32 ℃ by illumination of a shaking table at 120rpm, inoculating the purified and cultured rhodopseudomonas palustris in a liquid photobioreactor according to the volume ratio of 1:10, culturing the purified and cultured rhodopseudomonas palustris for 96 hours at 32 ℃, performing aseptic centrifugation, and performing low-temperature freeze drying to obtain the photosynthetic bacteria powder.
Further, the preparation method of the photosynthetic bacteria solid medium comprises the steps of 3.5g of sodium acetate, 1.0g of ammonia chloride, 1.2g of monopotassium phosphate, 0.4g of dipotassium phosphate, 0.1g of magnesium chloride, 0.1g of calcium chloride, 0.4g of yeast extract, 0.8g of peptone, 15.0g of agar and fixing the volume to 1 liter of distilled water; the pH was adjusted to 7.0 and autoclaved at 121 ℃ for 20 minutes.
Further, the preparation method of the photosynthetic bacteria liquid medium comprises 3.5g of sodium acetate, 1.0g of ammonia chloride, 1.2g of monopotassium phosphate, 0.4g of dipotassium phosphate, 0.1g of magnesium chloride, 0.1g of calcium chloride, 0.4g of yeast extract, 0.8g of peptone and distilled water to a constant volume of 1 liter; the pH was adjusted to 7.0 and autoclaved at 121 ℃ for 20 minutes.
Further, in the process of preparing the lactobacillus powder, the bacillus powder, the saccharomycete powder and the photosynthetic bacterium powder, the skim milk powder with the mass percent of 16%, the lactose with the mass percent of 1% and the glycerol with the mass percent of 3% are added into the bacterium liquid before low-temperature freeze drying.
Further, the total bacterial count of the composite probiotic inoculum, such as bifidobacterium, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus delbrueckii, streptococcus faecium and lactobacillus casei, is more than or equal to 8 logCFU/g; the total bacterial count of the bacillus subtilis and the bacillus licheniformis is more than or equal to 6 logCFU/g; the total bacterial count of the beer yeast and the candida utilis is more than or equal to 6logCFU/g, and the total bacterial count of the photosynthetic bacteria is more than or equal to 5 logCFU/g.
In a second aspect, the invention provides a composite probiotic for fermented feed, which is prepared by adopting the preparation method.
In a third aspect, the present invention provides a method for preparing a fermented feed for aquaculture, comprising mixing the complex probiotic for fermented feed according to claim 8 or 9 with a complete feed; the mixing ratio of the composite probiotic to the complete feed is 1-2: 9-13 ℃, and the mixing temperature is 25-30 ℃.
Further, the composite probiotic and the complete feed are mixed 8-12 hours before feeding.
The invention has the beneficial effects that:
according to the invention, the composition and the proportion of probiotics are optimized, and a salt solution is added to improve the thallus concentration of the composite probiotic during preparation of the composite probiotic, so that an enzyme preparation is not required to be added, and the preparation cost is saved; and the composition and content of the matrix and the composition and composition proportion of the composite probiotics are adjusted to promote the composite probiotics fermented feed to degrade the excreta and feed residues of aquatic animals, improve the water quality of water and improve the survival rate of aquatic products. The prepared composite probiotic is mixed with complete feed to prepare the fermentation feed for aquaculture, is used for aquaculture of aquatic animals, can effectively improve aquaculture water environment and microbial composition in aquatic intestinal tracts, is beneficial to improvement of animal health, and has the effects of improving water body, improving immune function of aquatic animals, inhibiting growth of harmful bacteria, promoting growth of aquatic animals and increasing water body fertility.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be further clearly and completely described below with reference to the embodiments of the present invention. It should be noted that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The raw materials, reagents or instruments used are not indicated by manufacturers, and are all conventional products which can be obtained by commercial purchase.
Example 1
A preparation method of a composite probiotic for fermented feed comprises the following steps:
step 1: preparing a matrix; mixing dry soybean meal, molasses and corn flour according to the mass ratio of 3: 5: 6, uniformly mixing;
step 2: preparing mixed fermentation mother liquor, mixing fermentation probiotics with a salt solution, and specifically comprising the following steps:
2.1, preparing fermentation probiotics: mixing lactobacillus powder, bacillus powder, saccharomycete powder and photosynthetic bacterium powder according to a ratio of 96: 10: 6: 2, mixing in proportion. The detailed steps are as follows:
2.1.1, preparing lactobacillus powder: respectively purifying and culturing bifidobacterium CICC21717, lactobacillus acidophilus CICC 6074, lactobacillus plantarum CICC6009, lactobacillus delbrueckii CICC6098, streptococcus faecium CICC20430 and lactobacillus casei CICC 6117 on a lactobacillus solid culture medium, then respectively inoculating the purified and cultured lactobacillus liquid culture medium, culturing for 8 hours at 37 ℃, then mixing the cultured bifidobacterium CICC21717, lactobacillus acidophilus CICC 6074, lactobacillus plantarum CICC6009, lactobacillus delbrueckii CICC6098, streptococcus faecium CICC20430 and lactobacillus casei CICC 6117 in equal volume to obtain mixed lactobacillus, inoculating the mixed lactobacillus into a lactobacillus amplification culture medium in a liquid fermentation tank according to the volume ratio of the inoculation amount of 1:10, culturing for 12 hours at 37 ℃, performing aseptic centrifugation, and performing low-temperature freeze drying to obtain lactobacillus powder;
the preparation method of the lactobacillus solid medium comprises the steps of preparing 5.0g of peptone, 5.0g of beef extract powder, 10.0g of tryptone, 5.0g of yeast extract, 8.0g of glucose, 801.0 mL of Tween, 2.0g of monopotassium phosphate, 5.0g of sodium acetate, 2.0g of diammonium hydrogen citrate, 0.25g of zinc sulfate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate, 1.0g of calcium carbonate and 15.0g of agar, fixing the volume to 1L by using distilled water, adjusting the pH to 6.8, and carrying out wet-heat high-pressure sterilization at 118 ℃ for 18 minutes;
the preparation method of the lactobacillus liquid medium comprises the steps of preparing 5.0g of peptone, 5.0g of beef extract powder, 10.0g of tryptone, 5.0g of yeast extract, 8.0g of glucose, 801.0 mL of Tween, 2.0g of monopotassium phosphate, 5.0g of sodium acetate, 2.0g of diammonium hydrogen citrate, 0.25g of zinc sulfate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate and 1.0g of calcium carbonate, fixing the volume to 1L by using distilled water, adjusting the pH value to 6.8, and carrying out wet-heat high-pressure sterilization at 118 ℃ for 18 minutes;
the lactobacillus amplification medium is prepared from peptone 5.0g, beef extract 5.0g, tryptone 10.0g, yeast extract 5.0g, glucose 10.0g, Tween 801.0 mL, potassium dihydrogen phosphate 2.0g, sodium acetate 5.0g, distilled water to constant volume of 1 liter, pH adjusted to 6.8, and wet-heat autoclaving at 118 deg.C for 18 min.
2.1.2, preparing bacillus powder: respectively purifying and culturing bacillus subtilis CICC 9011, bacillus subtilis CICC10071, bacillus subtilis CICC20872, bacillus licheniformis CICC23584, bacillus licheniformis CICC 10037 and bacillus licheniformis CICC10181 on a bacillus solid culture medium, respectively inoculating the bacillus liquid culture medium into a bacillus liquid culture medium, culturing for 8 hours at 37 ℃ by shaking table 210rpm, then mixing the cultured bacillus subtilis CICC 9011, bacillus subtilis CICC10071 and bacillus subtilis CICC20872 in equal volumes to obtain mixed bacillus subtilis, mixing the cultured bacillus licheniformis CICC23584, bacillus licheniformis CICC 10037 and bacillus licheniformis CICC10181 in equal volumes to obtain mixed bacillus licheniformis, and mixing the mixed bacillus subtilis and the mixed bacillus licheniformis according to the volume ratio of 1: 2, mixing to obtain mixed bacillus, then inoculating the mixed bacillus into a bacillus liquid culture medium in a liquid fermentation tank according to the volume ratio of the inoculation amount of 1:10, culturing for 12 hours at 37 ℃, performing aseptic centrifugation, and performing low-temperature freeze drying to obtain bacillus powder;
wherein the Bacillus solid culture medium is prepared from peptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, glucose 10.0g, and agar 15.0g by adding distilled water to a constant volume of 1L, adjusting pH to 7.0, and sterilizing at 118 deg.C under humid heat and high pressure for 18 min.
Wherein the Bacillus liquid culture medium is prepared from peptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, and glucose 10.0g by adding distilled water to a constant volume of 1L, adjusting pH to 7.0, and sterilizing at 118 deg.C under humid heat and high pressure for 18 min.
2.1.3, preparing saccharomycete powder: respectively purifying and culturing saccharomyces cerevisiae CICC1421 and candida utilis CICC1314 on a yeast solid culture medium, then respectively inoculating the purified and cultured saccharomyces cerevisiae CICC1421 and candida utilis CICC1314 in a yeast liquid culture medium, culturing for 8 hours at 37 ℃ by a shaker at 210rpm, then mixing the cultured saccharomyces cerevisiae and candida utilis in equal volume to obtain mixed yeast, inoculating the mixed yeast into the yeast liquid culture medium in a liquid fermentation tank according to the volume ratio of the inoculation amount of 1:10, culturing for 12 hours at 37 ℃, performing aseptic centrifugation, and performing low-temperature freeze drying to obtain yeast powder.
The preparation method of the yeast solid culture medium comprises 5g of yeast extract, 10g of peptone, 20g of glucose, 15.0g of agar, and distilled water with constant volume of 1L, adjusting pH to 6.4, and autoclaving at 118 deg.C for 20 min.
Wherein the yeast liquid culture medium is prepared from yeast extract 5g, peptone 10g, glucose 20g, and distilled water to constant volume of 1L, adjusting pH to 6.4, and autoclaving at 118 deg.C for 20 min.
2.1.4, preparing photosynthetic bacteria powder: purifying and culturing rhodopseudomonas palustris CICC 23812 in a photosynthetic bacteria solid culture medium, then inoculating the rhodopseudomonas palustris CICC 23812 in a photosynthetic bacteria liquid culture medium, and culturing the rhodopseudomonas palustris in a shaking table at 32 ℃ for 8 hours under illumination at 120rpm, wherein the culture medium comprises the following components in percentage by mass of 1: inoculating 10 volume ratio in a liquid photobioreactor, culturing at 32 ℃ for 96 hours, performing aseptic centrifugation, and performing low-temperature freeze drying to obtain photosynthetic bacteria powder.
The preparation method of the photosynthetic bacteria solid culture medium comprises the following steps of 3.5g of sodium acetate, 1.0g of ammonia chloride, 1.2g of potassium dihydrogen phosphate, 0.4g of dipotassium hydrogen phosphate, 0.1g of magnesium chloride, 0.1g of calcium chloride, 0.4g of yeast extract, 0.8g of peptone, 15.0g of agar and constant volume of 1 liter of distilled water; the pH was adjusted to 7.0 and autoclaved at 121 ℃ for 20 minutes.
The preparation method of the photosynthetic bacteria liquid culture medium comprises the following steps of 3.5g of sodium acetate, 1.0g of ammonia chloride, 1.2g of potassium dihydrogen phosphate, 0.4g of dipotassium hydrogen phosphate, 0.1g of magnesium chloride, 0.1g of calcium chloride, 0.4g of yeast extract, 0.8g of peptone and distilled water with the constant volume of 1 liter; the pH was adjusted to 7.0 and autoclaved at 121 ℃ for 20 minutes.
Preferably, in the process of preparing the lactobacillus powder, the bacillus powder, the saccharomycete powder and the photosynthetic bacterium powder, the skim milk powder with the mass percent of 16%, the lactose with the mass percent of 1% and the glycerol with the mass percent of 3% are added into the bacterium liquid before low-temperature freeze drying.
2.2, preparing a salt solution: 2.0g of monopotassium phosphate, 2.0g of diammonium citrate, 0.6g of magnesium sulfate, 6.0g of sodium acetate, 0.06g of manganese sulfate, 0.8g of calcium carbonate and 801.0 mL of Tween, and the volume of distilled water is up to 1L.
2.3, mixing the components in a mass ratio of 11: 0.8 of fermented probiotic bacteria is mixed with the salt solution with stirring.
And step 3: mixing a substrate and mixed fermentation mother liquor according to a mass ratio of 1:10, mixing, filling into a sealed bottom, and performing anaerobic fermentation at the fermentation temperature of 30 ℃ for 72 hours.
Example 2
A preparation method of a composite probiotic for fermented feed comprises the following steps:
step 1: preparing a matrix; the dry soybean meal, the molasses and the corn flour are mixed according to the mass ratio of 4: 6: 8, uniformly mixing;
step 2: preparing mixed fermentation mother liquor, mixing fermentation probiotics with a salt solution, and specifically comprising the following steps:
2.1, preparing fermentation probiotics: mixing lactobacillus powder, bacillus powder, saccharomycete powder and photosynthetic bacterium powder according to a ratio of 96: 10: 6: 2, mixing in proportion. The detailed steps are as follows:
2.1.1, preparing lactobacillus powder: respectively purifying and culturing bifidobacterium CICC21717, lactobacillus acidophilus CICC 6074, lactobacillus plantarum CICC6009, lactobacillus delbrueckii CICC6098, streptococcus faecium CICC20430 and lactobacillus casei CICC 6117 on a lactobacillus solid culture medium, then respectively inoculating the purified and cultured lactobacillus liquid culture medium, culturing for 8 hours at 37 ℃, then mixing the cultured bifidobacterium CICC21717, lactobacillus acidophilus CICC 6074, lactobacillus plantarum CICC6009, lactobacillus delbrueckii CICC6098, streptococcus faecium CICC20430 and lactobacillus casei CICC 6117 in equal volume to obtain mixed lactobacillus, inoculating the mixed lactobacillus into a lactobacillus amplification culture medium in a liquid fermentation tank according to the volume ratio of the inoculation amount of 1:10, culturing for 12 hours at 37 ℃, performing aseptic centrifugation, and performing low-temperature freeze drying to obtain lactobacillus powder;
the preparation method of the lactobacillus solid medium comprises the steps of preparing 5.0g of peptone, 5.0g of beef extract powder, 10.0g of tryptone, 5.0g of yeast extract, 8.0g of glucose, 801.0 mL of Tween, 2.0g of monopotassium phosphate, 5.0g of sodium acetate, 2.0g of diammonium hydrogen citrate, 0.25g of zinc sulfate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate, 1.0g of calcium carbonate and 15.0g of agar, fixing the volume to 1L by using distilled water, adjusting the pH to 6.8, and carrying out wet-heat high-pressure sterilization at 118 ℃ for 18 minutes;
the preparation method of the lactobacillus liquid medium comprises the steps of preparing 5.0g of peptone, 5.0g of beef extract powder, 10.0g of tryptone, 5.0g of yeast extract, 8.0g of glucose, 801.0 mL of Tween, 2.0g of monopotassium phosphate, 5.0g of sodium acetate, 2.0g of diammonium hydrogen citrate, 0.25g of zinc sulfate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate and 1.0g of calcium carbonate, fixing the volume to 1L by using distilled water, adjusting the pH value to 6.8, and carrying out wet-heat high-pressure sterilization at 118 ℃ for 18 minutes;
the lactobacillus amplification medium is prepared from peptone 5.0g, beef extract 5.0g, tryptone 10.0g, yeast extract 5.0g, glucose 10.0g, Tween 801.0 mL, potassium dihydrogen phosphate 2.0g, sodium acetate 5.0g, distilled water to constant volume of 1 liter, pH adjusted to 6.8, and wet-heat autoclaving at 118 deg.C for 18 min.
2.1.2, preparing bacillus powder: respectively purifying and culturing bacillus subtilis CICC 9011, bacillus subtilis CICC10071, bacillus subtilis CICC20872, bacillus licheniformis CICC23584, bacillus licheniformis CICC 10037 and bacillus licheniformis CICC10181 on a bacillus solid culture medium, respectively inoculating the bacillus liquid culture medium into a bacillus liquid culture medium, culturing for 8 hours at 37 ℃ by shaking table 210rpm, mixing the cultured bacillus subtilis CICC 9011, bacillus subtilis CICC10071 and bacillus subtilis CICC20872 in equal volumes to obtain mixed bacillus subtilis, mixing the cultured bacillus CICC23584, the bacillus licheniformis CICC 10037 and the bacillus licheniformis CICC10181 in equal volumes to obtain mixed bacillus licheniformis, and mixing the mixed bacillus subtilis and the mixed bacillus licheniformis according to the volume ratio of 1: 2, mixing to obtain mixed bacillus, then inoculating the mixed bacillus into a bacillus liquid culture medium in a liquid fermentation tank according to the volume ratio of the inoculation amount of 1:10, culturing for 12 hours at 37 ℃, performing aseptic centrifugation, and performing low-temperature freeze drying to obtain bacillus powder;
wherein the Bacillus solid culture medium is prepared from peptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, glucose 10.0g, and agar 15.0g by adding distilled water to a constant volume of 1L, adjusting pH to 7.0, and sterilizing at 118 deg.C under humid heat and high pressure for 18 min.
Wherein the Bacillus liquid culture medium is prepared from peptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g, and glucose 10.0g by adding distilled water to a constant volume of 1L, adjusting pH to 7.0, and sterilizing at 118 deg.C under humid heat and high pressure for 18 min.
2.1.3, preparing saccharomycete powder: respectively purifying and culturing saccharomyces cerevisiae CICC1421 and candida utilis CICC1314 on a yeast solid culture medium, then respectively inoculating the purified and cultured saccharomyces cerevisiae CICC1421 and candida utilis CICC1314 in a yeast liquid culture medium, culturing for 8 hours at 37 ℃ by a shaker at 210rpm, then mixing the cultured saccharomyces cerevisiae and candida utilis in equal volume to obtain mixed yeast, inoculating the mixed yeast into the yeast liquid culture medium in a liquid fermentation tank according to the volume ratio of the inoculation amount of 1:10, culturing for 12 hours at 37 ℃, performing aseptic centrifugation, and performing low-temperature freeze drying to obtain yeast powder.
The preparation method of the yeast solid culture medium comprises 5g of yeast extract, 10g of peptone, 20g of glucose, 15.0g of agar, and distilled water with constant volume of 1L, adjusting pH to 6.4, and autoclaving at 118 deg.C for 20 min.
Wherein the yeast liquid culture medium is prepared from yeast extract 5g, peptone 10g, glucose 20g, and distilled water to constant volume of 1L, adjusting pH to 6.4, and autoclaving at 118 deg.C for 20 min.
2.1.4, preparing photosynthetic bacteria powder: purifying and culturing rhodopseudomonas palustris CICC 23812 in a photosynthetic bacteria solid culture medium, then inoculating the rhodopseudomonas palustris CICC 23812 in a photosynthetic bacteria liquid culture medium, culturing for 8 hours under illumination of 120rpm of a shaking table at 32 ℃, inoculating the rhodopseudomonas palustris CICC 23812 in a liquid photobioreactor according to the volume ratio of the inoculum size of 1:10, culturing for 96 hours at 32 ℃, performing aseptic centrifugation, and performing low-temperature freeze drying to obtain photosynthetic bacteria powder.
The preparation method of the photosynthetic bacteria solid culture medium comprises the following steps of 3.5g of sodium acetate, 1.0g of ammonia chloride, 1.2g of potassium dihydrogen phosphate, 0.4g of dipotassium hydrogen phosphate, 0.1g of magnesium chloride, 0.1g of calcium chloride, 0.4g of yeast extract, 0.8g of peptone, 15.0g of agar and constant volume of 1 liter of distilled water; the pH was adjusted to 7.0 and autoclaved at 121 ℃ for 20 minutes.
The preparation method of the photosynthetic bacteria liquid culture medium comprises the following steps of 3.5g of sodium acetate, 1.0g of ammonia chloride, 1.2g of potassium dihydrogen phosphate, 0.4g of dipotassium hydrogen phosphate, 0.1g of magnesium chloride, 0.1g of calcium chloride, 0.4g of yeast extract, 0.8g of peptone and distilled water with the constant volume of 1 liter; the pH was adjusted to 7.0 and autoclaved at 121 ℃ for 20 minutes.
Preferably, in the process of preparing the lactobacillus powder, the bacillus powder, the saccharomycete powder and the photosynthetic bacterium powder, the skim milk powder with the mass percent of 16%, the lactose with the mass percent of 1% and the glycerol with the mass percent of 3% are added into the bacterium liquid before low-temperature freeze drying.
2.2, preparing a salt solution: 2.0g of monopotassium phosphate, 2.0g of diammonium citrate, 0.6g of magnesium sulfate, 6.0g of sodium acetate, 0.06g of manganese sulfate, 0.8g of calcium carbonate and 801.0 mL of Tween, and the volume of distilled water is up to 1L.
2.3, mixing the components in a mass ratio of 11: 0.8 of fermented probiotic bacteria is mixed with the salt solution with stirring.
And step 3: mixing a substrate and mixed fermentation mother liquor according to a mass ratio of 1:10, mixing, filling into a sealed bottom, and performing anaerobic fermentation at the fermentation temperature of 30 ℃ for 72 hours.
Comparative example 1
A preparation method of a composite probiotic agent for fermented feed is different from that of example 1 in that fermented probiotic bacteria are mixed with water instead of the salt solution in example 1 when a mixed fermentation mother liquor is prepared, and the rest of the preparation method is the same as that of example 1.
Comparative example 2
A preparation method of a composite probiotic for fermented feed is different from that of example 2 in that fermented probiotics are mixed with water instead of the salt solution in example 2 when a mixed fermentation mother liquor is prepared, and the other preparation methods are the same as those of example 2.
Comparative example 3
A preparation method of a composite probiotic for fermented feed is different from that of example 1 in that diammonium citrate and sodium acetate are not added when a saline solution is prepared, and the other preparation methods are the same as those of example 1.
Comparative example 4
A preparation method of a composite probiotic for fermented feed is different from that of example 1 in that 0.5g of diammonium citrate and 12.0g of sodium acetate are added during preparation of a salt solution, and the other preparation methods are the same as those of example 1.
Data comparison
(I) statistics of the number of bacteria
Table 1 shows the statistics of the total bacterial count of each species in the composite probiotics prepared in example 1, example 2 and comparative examples 1-4 (refer to the total bacterial count determined by the food safety national standard food microbiology test GB 4789.2-2016). As can be seen from Table 1, the number of bacteria of each species can be effectively increased in examples 1 to 2 in which the salt solution is added when preparing the mixed fermentation mother liquor, as compared with comparative examples 1 to 2. Compared with the comparative example 3, although the mixed fermentation mother liquor is prepared by adding the salt solution, the diammonium citrate and sodium acetate are lacked in the comparative example 3, the number of strains is reduced, and the salt solution has the advantages that all components are indispensable and have the synergistic effect. Compared with comparative example 4, the salt solution of examples 1-2 has the same composition as comparative example 4, but the different addition amount provides better technical effects for examples 1-2.
TABLE 1
(II) improving the quality of water
In table 2, the water depth was measured by using a sahs disc (a disc spaced in black and white), and the depth of light penetrating into the water surface, i.e., the transparency, was expressed as the water depth. Table 2 shows the water quality improvement of the complex probiotics prepared in example 1, example 2, comparative example 1 and comparative example 2 for different ponds. As can be seen from table 2, the complex probiotics prepared in examples 1 and 2 of the present invention have good water quality improvement effect.
TABLE 2
| Number of loose stool in pond | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Comparative example 1 transparency (cm) | 18.2 | 17.6 | 14.5 | 16.1 | 14.9 | 16.3 | 17.4 | 18.6 | 17.9 | 18.5 |
| Comparative example 2 transparency (cm) | 19.6 | 20.4 | 16.8 | 17.0 | 20.9 | 18.2 | 19.1 | 21.0 | 20.5 | 19.7 |
| Example 1 transparency (cm) | 29.5 | 28.4 | 29.8 | 30.1 | 30.5 | 31.9 | 29.6 | 32.0 | 29.7 | 28.8 |
| Example 2 transparency (cm) | 29.6 | 29.9 | 31.8 | 32.4 | 32.8 | 33.5 | 34.1 | 29.8 | 34.5 | 31.2 |
(III) cultivation conditions
The experimental breeding method comprises the following steps: the experimental pond is selected to be 0.8 mu, the breeding period is 3 months for releasing the seedlings, 7 months for collecting the shrimps, and the seedling releasing density is 6 ten thousand per mu. Feeding for 4 times every day, wherein the feeding time is 7: 00. 12: 00. 18: 00 and 22: 00, feeding the litopenaeus vannamei in a feeding total amount of 3.1-3.5% of the weight of the litopenaeus vannamei every day in the test period, wherein the feeding amount accounts for 40% in the daytime (7: 00 and 12: 00) and 60% in the evening (18: 00 and 22: 00), and the feeding condition is timely observed by taking a feed observation platform as a reference. Weighing 1 jin of litopenaeus vannamei after the shrimp is harvested in 7 months, then obtaining the average weight of the litopenaeus vannamei, weighing the total shrimp weight, and calculating the total tail number of the alive litopenaeus vannamei by dividing the total shrimp weight by the average weight of the litopenaeus vannamei.
Survival (%) × (total mantissa of surviving shrimps/initial mantissa) × 100%.
The composite probiotics prepared in example 1, example 2 and comparative example 1 are mixed with complete feed according to the ratio of 1: 9 to prepare the fermented feed A, B, C. The prepared fermented feed A, B, C is used for culturing the litopenaeus vannamei. In addition, the complete feed without the compound probiotic added is used as a control group.
In the culture method, except for different types of the fed feed, the rest living conditions are the same in the culture process.
The average body weight and the survival rate of the bred litopenaeus vannamei boone are shown in table 3. As can be seen from table 3, the composite probiotics prepared in embodiments 1 and 2 of the present invention can significantly improve the weight and survival rate of litopenaeus vannamei.
| Detecting items | Fermented feed C | Fermented feed A | Fermented feed B | Control group |
| Average weight of prawn (g/one) | 16.1 | 17.9 | 18.5 | 15.3 |
| Survival rate (%) | 70.4±2.19 | 81.3±3.32 | 81.9±1.13 | 58.6±2.98 |
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A preparation method of a composite probiotic for fermented feed is characterized by comprising the following steps:
step 1: preparing a matrix; the method comprises the following steps of (1) mixing dry soybean meal, molasses and corn flour according to the mass ratio of 2-5: 3-8: 6-8, uniformly mixing;
step 2: preparing mixed fermentation mother liquor; and (2) mixing the following components in a mass ratio of 8-15: stirring and mixing 0.6-1.1 of fermentation probiotics and a salt solution;
and step 3: mixing a substrate and mixed fermentation mother liquor according to a mass ratio of 1: 8-11, and filling the mixture into a sealed bottom for anaerobic fermentation.
2. The preparation method of the complex probiotic for fermented feed according to claim 1, wherein the mass ratio of the dry soybean meal, the molasses and the corn flour is 3: 5: 6; the mass ratio of the fermentation probiotics to the salt solution is 11: 0.8; the mass ratio of the mixed fermentation mother liquor to the substrate is 10: 1.
3. the preparation method of the composite probiotic for fermented feed according to claim 1, wherein the fermented probiotic comprises the following components in a mass ratio of 80-110: 8-12: 2-6: 1-4 of lactobacillus powder, bacillus powder, saccharomycete powder and photosynthetic bacteria powder.
4. The method for preparing the complex probiotic for fermented feed according to claim 1, wherein the salt solution is prepared by mixing 1.0-3.0 g of monopotassium phosphate, 1.0-3.0 g of diammonium citrate, 0.4-0.8 g of magnesium sulfate, 4.0-8.0 g of sodium acetate, 0.04-0.08 g of manganese sulfate, 0.6-1.1 g of calcium carbonate and 0.6-1.4 mL of Tween-80, and adding distilled water to a constant volume of 1L.
5. The method for preparing a complex probiotic for fermented feed according to claim 1, wherein the lactic acid bacteria powder comprises lactobacillus delbrueckii, bifidobacterium, lactobacillus acidophilus, streptococcus faecium, lactobacillus plantarum and lactobacillus casei; the bacillus powder comprises bacillus subtilis and bacillus licheniformis; the yeast powder comprises cerevisiae fermentum and Candida utilis; the photosynthetic bacteria are rhodopseudomonas palustris.
6. The method for preparing a complex probiotic for fermented feed according to claim 5, wherein the lactobacillus delbrueckii is lactobacillus delbrueckii cic 6098, the bifidobacterium is bifidobacterium bifidum cic 21717, the lactobacillus acidophilus is lactobacillus acidophilus cic 6074, the streptococcus faecium is streptococcus faecium cic 20430, the lactobacillus plantarum is lactobacillus plantarum cic 6009, and the lactobacillus casei is lactobacillus casei cic 6117; the bacillus subtilis is one or more of bacillus subtilis CICC 9011, bacillus subtilis CICC10071 and bacillus subtilis CICC 20872; the bacillus licheniformis is one or more of bacillus licheniformis CICC23584, bacillus licheniformis CICC 10037 and bacillus licheniformis CICC 10181; the saccharomyces cerevisiae is saccharomyces cerevisiae CICC 1421; the candida utilis is candida utilis CICC 1314; the rhodopseudomonas palustris is rhodopseudomonas palustris CICC 23812.
7. A composite probiotic agent for fermented feed, which is prepared by the preparation method of any one of claims 1 to 6.
8. The complex probiotic for fermented feed according to claim 7, wherein the total number of bifidobacteria, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus delbrueckii, streptococcus faecium and lactobacillus casei in the complex probiotic is not less than 8 logCFU/g; the total bacterial count of the bacillus subtilis and the bacillus licheniformis is more than or equal to 6 logCFU/g; the total bacterial count of the beer yeast and the candida utilis is more than or equal to 6logCFU/g, and the total bacterial count of the photosynthetic bacteria is more than or equal to 5 logCFU/g.
9. A method for preparing a fermented feed for aquaculture, comprising mixing the complex probiotic for fermented feed according to claim 8 or 9 with a complete feed; the mixing ratio of the composite probiotic to the complete feed is 1-2: 9-13 ℃, and the mixing temperature is 25-30 ℃.
10. The method for preparing fermented feed for aquaculture according to claim 9, wherein the composite probiotic and the complete feed are mixed 8-12 hours before feeding.
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| CN106962609A (en) * | 2017-04-01 | 2017-07-21 | 福建龙岩闽雄生物科技股份有限公司 | A kind of direct putting type biological fermentation feed additive and its production and use |
| CN109251874A (en) * | 2018-09-25 | 2019-01-22 | 厦门惠盈动物科技有限公司 | A kind of probiotics preparation and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106962609A (en) * | 2017-04-01 | 2017-07-21 | 福建龙岩闽雄生物科技股份有限公司 | A kind of direct putting type biological fermentation feed additive and its production and use |
| CN109251874A (en) * | 2018-09-25 | 2019-01-22 | 厦门惠盈动物科技有限公司 | A kind of probiotics preparation and its preparation method and application |
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| CN114874949A (en) * | 2022-06-06 | 2022-08-09 | 江苏三仪生物工程有限公司 | Clostridium butyricum, fermentation product thereof, microbial inoculum containing clostridium butyricum and animal feed additive |
| CN114874949B (en) * | 2022-06-06 | 2023-09-12 | 江苏三仪生物工程有限公司 | Clostridium butyricum, fermentation product thereof, microbial inoculum containing clostridium butyricum and animal feed additive |
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