CN111808788B - Enterococcus faecium powder and preparation method and application thereof - Google Patents
Enterococcus faecium powder and preparation method and application thereof Download PDFInfo
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- CN111808788B CN111808788B CN202010868204.2A CN202010868204A CN111808788B CN 111808788 B CN111808788 B CN 111808788B CN 202010868204 A CN202010868204 A CN 202010868204A CN 111808788 B CN111808788 B CN 111808788B
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- enterococcus faecium
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- 241000194031 Enterococcus faecium Species 0.000 title claims abstract description 124
- 239000000843 powder Substances 0.000 title claims abstract description 68
- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- 239000003223 protective agent Substances 0.000 claims abstract description 30
- 230000001580 bacterial effect Effects 0.000 claims abstract description 28
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims abstract description 26
- 239000011734 sodium Substances 0.000 claims abstract description 26
- 229910052708 sodium Inorganic materials 0.000 claims abstract description 26
- 239000003995 emulsifying agent Substances 0.000 claims abstract description 25
- 229920000858 Cyclodextrin Polymers 0.000 claims abstract description 17
- 239000001116 FEMA 4028 Substances 0.000 claims abstract description 17
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims abstract description 17
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims abstract description 17
- 229960004853 betadex Drugs 0.000 claims abstract description 17
- -1 sulfobutyl Chemical group 0.000 claims abstract description 16
- 239000004354 Hydroxyethyl cellulose Substances 0.000 claims abstract description 14
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 claims abstract description 14
- 238000001035 drying Methods 0.000 claims abstract description 14
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 claims abstract description 14
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 13
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 13
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 11
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 11
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 10
- 239000002562 thickening agent Substances 0.000 claims abstract description 10
- 239000000945 filler Substances 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 239000007957 coemulsifier Substances 0.000 claims abstract description 8
- 238000000855 fermentation Methods 0.000 claims description 92
- 230000004151 fermentation Effects 0.000 claims description 92
- 239000007788 liquid Substances 0.000 claims description 87
- 238000000034 method Methods 0.000 claims description 28
- 239000001963 growth medium Substances 0.000 claims description 24
- 238000011218 seed culture Methods 0.000 claims description 24
- 238000001694 spray drying Methods 0.000 claims description 19
- 239000002609 medium Substances 0.000 claims description 14
- 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 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 239000001888 Peptone Substances 0.000 claims description 12
- 108010080698 Peptones Proteins 0.000 claims description 12
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 12
- 239000008103 glucose Substances 0.000 claims description 12
- 238000011081 inoculation Methods 0.000 claims description 12
- 235000019319 peptone Nutrition 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 11
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical group [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 10
- 229940073490 sodium glutamate Drugs 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 9
- 239000000706 filtrate Substances 0.000 claims description 9
- 229920002261 Corn starch Polymers 0.000 claims description 8
- 229920002774 Maltodextrin Polymers 0.000 claims description 8
- 239000005913 Maltodextrin Substances 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 239000008120 corn starch Substances 0.000 claims description 8
- 229940035034 maltodextrin Drugs 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- 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 6
- 238000001914 filtration Methods 0.000 claims description 6
- 235000013312 flour Nutrition 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 229940099596 manganese sulfate Drugs 0.000 claims description 6
- 235000007079 manganese sulphate Nutrition 0.000 claims description 6
- 239000011702 manganese sulphate Substances 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 229920000136 polysorbate Polymers 0.000 claims description 6
- 239000001632 sodium acetate Substances 0.000 claims description 6
- 235000017281 sodium acetate Nutrition 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 235000013305 food Nutrition 0.000 claims description 5
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 3
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 3
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims 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 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 3
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 3
- 239000008101 lactose Substances 0.000 claims description 3
- 239000000594 mannitol Substances 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 239000000600 sorbitol Substances 0.000 claims description 3
- 230000003068 static effect Effects 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 239000000230 xanthan gum Substances 0.000 claims description 3
- 229920001285 xanthan gum Polymers 0.000 claims description 3
- 235000010493 xanthan gum Nutrition 0.000 claims description 3
- 229940082509 xanthan gum Drugs 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 2
- 230000002829 reductive effect Effects 0.000 abstract description 12
- 230000004083 survival effect Effects 0.000 abstract description 11
- 239000000463 material Substances 0.000 abstract description 5
- 239000003094 microcapsule Substances 0.000 abstract description 4
- 230000005855 radiation Effects 0.000 abstract description 4
- 241001052560 Thallis Species 0.000 abstract description 3
- 239000002775 capsule Substances 0.000 abstract description 3
- 239000002131 composite material Substances 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- 241000894006 Bacteria Species 0.000 description 9
- 238000003860 storage Methods 0.000 description 8
- 230000006872 improvement Effects 0.000 description 7
- 229960004793 sucrose Drugs 0.000 description 7
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 6
- 235000019797 dipotassium phosphate Nutrition 0.000 description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 6
- 239000000919 ceramic Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000008399 tap water Substances 0.000 description 4
- 235000020679 tap water Nutrition 0.000 description 4
- 238000012258 culturing Methods 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 238000005507 spraying Methods 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000000889 atomisation Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 241000194033 Enterococcus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 229920006318 anionic polymer Polymers 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
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- 230000006378 damage Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
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- 244000005700 microbiome Species 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000005518 polymer electrolyte Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000020681 well water Nutrition 0.000 description 1
- 239000002349 well water Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- 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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Polymers & Plastics (AREA)
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Abstract
The invention provides enterococcus faecium powder and a preparation method and application thereof, wherein the protective agent comprises the following components in parts by weight: 4-5 parts of thickening agent, 3-4 parts of filler, 2-3 parts of bovine serum albumin, 0.4-0.5 part of saccharide, 0.2-0.3 part of emulsifier, 0.4-0.5 part of co-emulsifier, 0.3-0.8 part of hydroxyethyl cellulose sodium, 0.3-0.8 part of sulfobutyl beta-cyclodextrin sodium and 0.5-0.6 part of antioxidant. The protective agent forms a microcapsule composite capsule material structure for wrapping thalli, can effectively block heat conduction and heat radiation in the drying process in the preparation and drying process of enterococcus faecium, plays a role in preventing wall sticking, and reduces the occurrence of wall sticking; the survival rate of the dried enterococcus faecium is effectively improved, the production cost is reduced, the heat resistance of the enterococcus faecium powder is improved, and the loss of the bacterial quantity in the later application is reduced.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to enterococcus faecium powder and a preparation method and application thereof.
Background
Enterococcus faecium is classified as enterococcus, gram-positive bacteria, and facultative anaerobic. The thallus is round or elliptical, is arranged in a single, paired or short-chain shape, has no spores and flagella, is part of normal flora in intestinal tracts of human beings and animals, has stronger tolerance and colonization capacity on intestinal mucosa, has stronger stress resistance and can improve the growth performance of the animals.
The enterococcus faecium has good bacteriostatic ability and drug sensitivity resistance, has a regulation function on the intestinal flora, and can well inhibit pathogenic escherichia coli, staphylococcus aureus, salmonella and the like. Enterococcus faecium is widely used in pharmaceutical, food and feed processing industries.
At present, enterococcus faecium fermentation mainly uses liquid fermentation, bacteria liquid needs to be dried and granulated for convenient transportation and storage, the commonly used drying methods at present mainly comprise freeze drying, spray drying and the like, spray drying is widely applied to production due to high efficiency, strong production capacity and less required personnel, and the high death rate of sprayed strains is a key factor for restricting spray drying due to higher temperature in the spray drying process, so that the improvement of the survival rate of the sprayed strains is particularly important.
Disclosure of Invention
In view of the above, the invention aims to provide enterococcus faecium powder and a preparation method and application thereof, wherein a heat-resistant framework is formed by fully utilizing the synergistic effect of a thickening agent, a filling agent, bovine serum albumin, saccharides, an emulsifier, an auxiliary emulsifier, sodium hydroxyethyl cellulose, sulfobutyl beta-cyclodextrin sodium and an antioxidant, so as to form a microcapsule composite capsule wall material structure for wrapping bacteria, so that heat conduction and heat radiation in a drying process can be effectively blocked in the preparation and spraying process of enterococcus faecium, the wall sticking prevention effect is achieved, and the wall sticking situation is reduced; and the survival rate of the dried enterococcus faecium is effectively improved, the unit quantity of enterococcus faecium bacteria is favorably improved, the yield of qualified enterococcus faecium powder is favorably improved, the production cost is reduced, the heat resistance of the enterococcus faecium powder is improved, and the loss of the bacteria quantity in later application is reduced.
In order to achieve the above purpose, the invention provides the following technical scheme:
a protective agent comprises the following components in parts by weight: 4-5 parts of thickening agent, 3-4 parts of filler, 2-3 parts of bovine serum albumin, 0.4-0.5 part of saccharide, 0.2-0.3 part of emulsifier, 0.4-0.5 part of co-emulsifier, 0.3-0.8 part of hydroxyethyl cellulose sodium, 0.3-0.8 part of sulfobutyl beta-cyclodextrin sodium and 0.5-0.6 part of antioxidant.
As a further improvement of the above technical solution, the thickener comprises corn starch;
preferably, the bulking agent comprises at least one of maltodextrin and beta-cyclodextrin;
preferably, the emulsifier comprises xanthan gum;
preferably, the co-emulsifier comprises at least one of mannitol, sorbitol, and glycerol;
preferably, the saccharide includes at least one of sucrose, trehalose, lactose and maltose;
preferably, the antioxidant comprises sodium glutamate.
A preparation method of enterococcus faecium powder comprises the following steps:
mixing raw materials including enterococcus faecium liquid and protective agent, and drying; the protective agent is the protective agent.
As a further improvement of the above technical solution, the preparation method of the enterococcus faecium liquid comprises:
inoculating enterococcus faecium seed liquid into a closed primary fermentation tank for primary fermentation culture to obtain primary fermentation liquid, and then inoculating the primary fermentation liquid into a closed secondary fermentation tank for secondary fermentation culture;
preferably, the enterococcus faecium seed liquid is inoculated in the primary fermentation tank according to the inoculation amount of 0.28-0.4% (V/V);
preferably, the primary fermentation liquid is inoculated in the secondary fermentation tank according to the inoculation amount of 0.28-0.4% (V/V);
preferably, the composition of the fermentation medium in the primary fermentation tank and the fermentation medium in the secondary fermentation tank comprise: 15-18 g/L of glucose, 3-3.5 g/L of corn flour, 3-3.5 g/L of maltose, 8-10 g/L of peptone, 8-10 g/L of yeast extract powder, 800.7-0.8 g/L of tween, 1.5-1.8 g/L of dipotassium hydrogen phosphate, 0.26-0.3 g/L of magnesium sulfate and 0.12-0.15 g/L of manganese sulfate;
preferably, the tank pressure in the primary fermentation tank and the secondary fermentation tank is 0.05-0.06 MPa;
preferably, the temperature of the primary fermentation culture is 35-39 ℃, and the time is 18-20 h;
preferably, the temperature of the secondary fermentation culture is 35-39 ℃, and the time is 16-18 h.
As a further improvement of the technical scheme, the preparation method of the enterococcus faecium seed liquid comprises the following steps:
picking an enterococcus faecium colony, and inoculating the enterococcus faecium colony into a primary seed culture medium for carrying out primary standing culture to obtain a primary seed liquid;
inoculating the primary seed liquid into a secondary seed culture medium for secondary standing culture;
preferably, 1-2 enterococcus faecium colonies are inoculated in each 100ml of the primary seed culture medium;
preferably, the temperature of the first standing culture is 36-38 ℃, and the time is 20-24 h;
preferably, 6-8 ml of the primary seed liquid is inoculated in every 1L of the secondary seed culture medium;
preferably, the temperature of the second standing culture is 36-38 ℃, and the time is 20-24 h;
preferably, the composition of the primary seed culture medium and the secondary seed culture medium comprises: 8-10 g/L of peptone, 4-5 g/L of yeast powder, 16-20 g/L of glucose, 4-5 g/L of sodium acetate, 1.8-2 g/L of diammonium citrate and 1.8-2 g/L of dipotassium hydrogen phosphate.
As a further improvement of the above technical solution, the preparation method of the enterococcus faecium powder further comprises: the method also comprises the following steps before the enterococcus faecium bacterial liquid and the protective agent are mixed: filtering the enterococcus faecium bacterial liquid, adding water into the obtained filtrate, mixing and concentrating to the volume which is the same as that of the enterococcus faecium bacterial liquid;
preferably, the amount of the added water is 1-1.5 times of the volume of the enterococcus faecium bacterial liquid.
As a further improvement of the technical scheme, the dosage of the protective agent is 11.1-15.5% of the total mass of the enterococcus faecium bacterial liquid.
As a further improvement of the above technical solution, the drying is spray drying;
preferably, during the spray drying process: the air inlet temperature is 135-150 ℃, and the air outlet temperature is 62-70 ℃.
An enterococcus faecium powder is prepared by the preparation method of the enterococcus faecium powder.
An application of the enterococcus faecium powder in feed, food or pharmacy.
The invention has the beneficial effects that:
(1) the protective agent of the invention fully utilizes the synergistic effect of the thickening agent, the filling agent, the bovine serum albumin, the saccharides, the emulsifier, the co-emulsifier, the sodium hydroxyethyl cellulose, the sulfobutyl beta-cyclodextrin sodium and the antioxidant to form a heat-resistant microcapsule composite capsule wall material structure for wrapping thalli, can effectively block heat conduction and heat radiation in the drying process in the preparation and spraying process of enterococcus faecium, plays a role in preventing wall sticking, and reduces the occurrence of wall sticking; and the survival rate of the dried enterococcus faecium is effectively improved, the unit quantity of enterococcus faecium bacteria is favorably improved, the yield of qualified enterococcus faecium powder is favorably improved, the production cost is reduced, the heat resistance of the enterococcus faecium powder is improved, and the loss of the bacteria quantity in later application is reduced.
(2) Furthermore, the invention improves the atomization performance by adopting spray drying and finely adjusting the temperature of inlet air and outlet air of the spray drying, ensures no wall sticking and further reduces the death rate of strains in the preparation and drying process of the enterococcus faecium powder.
In order to make the aforementioned and other objects, features and advantages of the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention.
This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
The terms as used herein:
"prepared from … …" is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
The conjunction "consisting of … …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of … …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.
When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when the range "1 ~ 5" is disclosed, the ranges described should be construed to include the ranges "1 ~ 4", "1 ~ 3", "1 ~ 2 and 4 ~ 5", "1 ~ 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.
In these examples, the parts and percentages are by mass unless otherwise indicated.
"part by mass" means a basic unit of measure indicating a mass ratio of a plurality of components, and 1 part may represent any unit mass, for example, 1g or 2.689 g. If we say that the part by mass of the component A is a part by mass and the part by mass of the component B is B part by mass, the ratio of the part by mass of the component A to the part by mass of the component B is a: b. alternatively, the mass of the A component is aK and the mass of the B component is bK (K is an arbitrary number, and represents a multiple factor). It is unmistakable that, unlike the parts by mass, the sum of the parts by mass of all the components is not limited to 100 parts.
"and/or" is used to indicate that one or both of the illustrated conditions may occur, e.g., a and/or B includes (a and B) and (a or B).
The invention provides a protective agent, which comprises the following components in parts by weight:
4-5 parts of thickening agent, such as 4 parts, 4.2 parts, 4.5 parts, 4.8 parts or 5 parts;
3-4 parts of filler, such as 3 parts, 3.2 parts, 3.5 parts, 3.8 parts or 4 parts;
2-3 parts of bovine serum albumin, such as 2 parts, 2.2 parts, 2.5 parts, 2.8 parts or 3 parts;
0.4-0.5 parts of saccharides, such as 0.4 part, 0.42 part, 0.45 part, 0.48 part or 0.5 part;
0.2-0.3 part of emulsifier, such as 0.2 part, 0.22 part, 0.25 part, 0.28 part or 0.3 part;
0.4-0.5 part of auxiliary emulsifier, such as 0.4 part, 0.42 part, 0.45 part, 0.48 part or 0.5 part;
0.3-0.8 parts of hydroxyethyl cellulose, such as 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part or 0.8 part;
0.3-0.8 part of sulfobutyl beta-cyclodextrin sodium, such as 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part or 0.8 part and the like;
0.5-0.6 part of antioxidant, such as 0.5 part, 0.52 part, 0.55 part, 0.58 part or 0.6 part.
Preferably, the thickener comprises corn starch.
Preferably, the filler includes at least one of maltodextrin and β -cyclodextrin.
Preferably, the emulsifier comprises xanthan gum.
Preferably, the coemulsifier comprises at least one of mannitol, sorbitol and glycerol; the co-emulsifier is used to promote the formation of an emulsion with smaller emulsion droplets.
Preferably, the saccharide includes at least one of sucrose, trehalose, lactose and maltose.
Preferably, the antioxidant comprises sodium glutamate.
The hydroxyethyl cellulose sodium is used as an anionic polymer electrolyte, and has high aqueous solution viscosity, good salt resistance and high thermal stability; the anionic high-water-solubility sulfobutyl beta-cyclodextrin sodium has better water solubility, can form a non-covalent compound with other nitrogen-containing components in the protective agent, and has better affinity and inclusion property; antioxidants such as sodium glutamate are effective in inhibiting irreversible cell membrane destruction; the protective agent is prepared by matching saccharides, bovine serum albumin, a thickening agent, a filler, an emulsifier, an auxiliary emulsifier, sodium hydroxyethyl cellulose, sodium sulfobutyl beta-cyclodextrin and an antioxidant to form a heat-resistant microcapsule structure framework for wrapping thalli.
A preparation method of enterococcus faecium powder comprises the following steps:
mixing the raw materials including the enterococcus faecium liquid and the protective agent, and drying.
The protective agent is added, so that heat conduction and heat radiation in the drying process can be effectively blocked in the preparation and spraying process of enterococcus faecium, the wall sticking prevention effect is achieved, and the wall sticking condition is reduced; and the survival rate of the dried enterococcus faecium is effectively improved, the unit quantity of enterococcus faecium bacteria is favorably improved, the yield of qualified enterococcus faecium powder is favorably improved, the production cost is reduced, the heat resistance of the enterococcus faecium powder is improved, and the loss of the bacteria quantity in later application is reduced.
Further, the preparation method of the enterococcus faecium bacterial liquid comprises the following steps:
inoculating enterococcus faecium seed liquid into a closed primary fermentation tank for primary fermentation culture to obtain primary fermentation liquid, and then inoculating the primary fermentation liquid into a closed secondary fermentation tank for secondary fermentation culture.
Preferably, the enterococcus faecium seed liquid is inoculated in the primary fermentation tank according to the inoculation amount of 0.28-0.4% (V/V, namely the volume percentage of the enterococcus faecium seed liquid in the fermentation medium in the primary fermentation tank).
Preferably, the primary fermentation liquid is inoculated into the secondary fermentation tank according to the inoculation amount of 0.28-0.4% (V/V, namely the volume percentage of the primary fermentation liquid in the fermentation medium in the secondary fermentation tank).
Preferably, the composition of the fermentation medium in the primary fermentation tank and the fermentation medium in the secondary fermentation tank both comprise: 15-18 g/L of glucose, 3-3.5 g/L of corn flour, 3-3.5 g/L of maltose, 8-10 g/L of peptone, 8-10 g/L of yeast extract powder, 800.7-0.8 g/L of tween, 1.5-1.8 g/L of dipotassium hydrogen phosphate, 0.26-0.3 g/L of magnesium sulfate and 0.12-0.15 g/L of manganese sulfate.
Preferably, the tank pressure in the primary fermentation tank and the secondary fermentation tank is 0.05-0.06 MPa.
Preferably, the temperature of the primary fermentation culture is 35-39 ℃, and the time is 18-20 h; the temperature of the secondary fermentation culture is 35-39 ℃, and the time is 16-18 h.
Further, the preparation method of the enterococcus faecium seed liquid comprises the following steps:
picking an enterococcus faecium colony, and inoculating the enterococcus faecium colony into a primary seed culture medium for carrying out primary standing culture to obtain a primary seed liquid;
and inoculating the primary seed liquid into a secondary seed culture medium for secondary static culture.
Preferably, 1-2 enterococcus faecium colonies are inoculated in each 100ml of the primary seed culture medium.
Preferably, the temperature of the first static culture is 36-38 ℃, and the time is 20-24 h.
Preferably, 6-8 ml of the primary seed solution is inoculated in every 1L of the secondary seed culture medium.
Preferably, the temperature of the second standing culture is 36-38 ℃, and the time is 20-24 h.
Preferably, the composition of the primary seed culture medium and the secondary seed culture medium comprises: 8-10 g/L of peptone, 4-5 g/L of yeast powder, 16-20 g/L of glucose, 4-5 g/L of sodium acetate, 1.8-2 g/L of diammonium citrate and 1.8-2 g/L of dipotassium hydrogen phosphate.
Further, the preparation method of the enterococcus faecium powder further comprises the following steps: the method also comprises the following steps before the enterococcus faecium bacterial liquid and the protective agent are mixed: filtering the enterococcus faecium bacterial liquid, adding water into the obtained filtrate, mixing and concentrating to the volume which is the same as that of the enterococcus faecium bacterial liquid;
preferably, the amount of the added water is 1-1.5 times of the volume of the enterococcus faecium liquid. The water is preferably common tap water, and well water, pure water or deionized water can also be adopted.
Preferably, the concentration is performed by using a ceramic membrane filter.
Preferably, the dosage of the protective agent is 11.1-15.5% of the total mass of the enterococcus faecium bacterial liquid.
The drying is preferably spray drying; preferably, during the spray drying: the air inlet temperature is 135-150 ℃, the air outlet temperature is 62-70 ℃, the atomization performance is improved, the wall sticking is avoided, and the death rate of strains in the preparation and drying process of the enterococcus faecium powder is further reduced.
An enterococcus faecium powder is prepared by the preparation method of the enterococcus faecium powder.
An application of the enterococcus faecium powder in feed, food or pharmacy.
In the above description, the food refers to various products and materials for human consumption or drinking.
Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
The protective agent for preparing enterococcus faecium powder provided by the embodiment comprises the following components in parts by weight: 4 parts of corn starch, 3 parts of maltodextrin, 3 parts of bovine serum albumin, 0.4 part of cane sugar, 0.2 part of emulsifier, 0.4 part of auxiliary emulsifier, 0.8 part of hydroxyethyl cellulose sodium, 0.3 part of sulfobutyl beta-cyclodextrin sodium and 0.5 part of sodium glutamate.
The preparation method of enterococcus faecium powder provided by the embodiment comprises the following steps:
(1) selecting 1 target enterococcus faecium colony, inoculating into 100ml of first-stage seed culture medium, and standing and culturing at 38 deg.C for 20 hr to obtain first-stage seed solution; then 8ml of first-stage seed liquid is absorbed and inoculated into 1.2L of seed culture medium, and standing culture is carried out for 20h at 38 ℃ to obtain seed liquid; wherein, the composition of the seed culture medium comprises: 10g/L of peptone, 5g/L of yeast powder, 20g/L of glucose, 5g/L of sodium acetate, 2g/L of diammonium citrate and 2g/L of dipotassium phosphate.
(2) Inoculating the obtained enterococcus faecium seed liquid into a closed primary fermentation tank according to the inoculation amount of 0.28% (V/V) for fermentation culture, keeping the temperature in the fermentation tank at 35 ℃, the rotating speed at 60r/min and the tank pressure at 0.05MPa, controlling the pH value to be not lower than 6.8, transferring the liquid into a closed secondary fermentation tank according to the inoculation amount of 0.28% (V/V) for fermentation culture after fermentation culture for 20 hours, similarly keeping the temperature in the fermentation tank at 35 ℃, the rotating speed at 60r/min and the tank pressure at 0.05MPa in the fermentation process, controlling the pH value to be not lower than 6.8, and performing fermentation culture for 18 hours to obtain an enterococcus faecium liquid; wherein, the fermentation medium in the first order fermentation cylinder and above-mentioned second order fermentation cylinder's constitution includes: 15g/L glucose, 3g/L corn flour, 3g/L maltose, 8g/L peptone, 8g/L yeast extract powder, 800.7 g/L tween, 1.5g/L dipotassium phosphate, 0.3g/L magnesium sulfate and 0.15g/L manganese sulfate.
(3) Filtering the obtained enterococcus faecium bacterial liquid by using a vibrating screen, adding tap water with the same volume into the obtained filtrate, concentrating the filtrate to the volume which is the same as that of the original enterococcus faecium bacterial liquid by using a ceramic membrane filter, transferring the obtained concentrated liquid to a liquid storage tank, adding a protective agent with the mass accounting for 11.1 percent of the total mass of the enterococcus faecium bacterial liquid into the liquid storage tank, uniformly mixing, and then, feeding the mixture into a spray tower for spray drying to obtain enterococcus faecium bacterial powder; wherein, in the spray drying process: the inlet air temperature is 135 ℃, and the outlet air temperature is controlled to be 62 ℃ by adjusting the feeding frequency.
Example 2
This example differs from example 1 in that: the protective agent comprises the following components in parts by weight: 4.5 parts of corn starch, 3.5 parts of maltodextrin, 2.5 parts of bovine serum albumin, 0.45 part of cane sugar, 0.25 part of emulsifier, 0.45 part of auxiliary emulsifier, 0.6 part of hydroxyethyl cellulose sodium, 0.6 part of sulfobutyl beta-cyclodextrin sodium and 0.55 part of sodium glutamate.
The preparation method of enterococcus faecium powder is the same as that of example 1.
Example 3
This example differs from example 1 in that: the protective agent comprises the following components in parts by weight: 5 parts of corn starch, 4 parts of maltodextrin, 2 parts of bovine serum albumin, 0.5 part of cane sugar, 0.3 part of emulsifier, 0.5 part of auxiliary emulsifier, 0.3 part of hydroxyethyl cellulose sodium, 0.8 part of sulfobutyl beta-cyclodextrin sodium and 0.6 part of sodium glutamate.
The preparation method of enterococcus faecium powder is the same as that of example 1.
Example 4
The protective agent for preparing enterococcus faecium powder provided by the embodiment comprises the following components in parts by weight: 4.2 parts of corn starch, 3.2 parts of maltodextrin, 2.8 parts of bovine serum albumin, 0.42 part of cane sugar, 0.22 part of emulsifier, 0.42 part of auxiliary emulsifier, 0.7 part of hydroxyethyl cellulose sodium, 0.4 part of sulfobutyl beta-cyclodextrin sodium and 0.52 part of sodium glutamate.
The preparation method of enterococcus faecium powder provided by the embodiment comprises the following steps:
(1) selecting 2 target enterococcus faecium colonies, inoculating into 100ml of first-stage seed culture medium, and standing and culturing at 36 deg.C for 24 hr to obtain first-stage seed liquid; then, 6ml of first-order seed liquid is absorbed and inoculated into 1L of seed culture medium, and standing culture is carried out for 24h at 36 ℃ to obtain seed liquid; wherein, the composition of the seed culture medium comprises: 10g/L of peptone, 5g/L of yeast powder, 20g/L of glucose, 5g/L of sodium acetate, 2g/L of diammonium citrate and 2g/L of dipotassium phosphate.
(2) Inoculating the obtained enterococcus faecium seed liquid into a closed primary fermentation tank according to the inoculation amount of 0.4% (V/V) for fermentation culture, keeping the temperature in the fermentation tank at 35 ℃, the rotating speed at 60r/min and the tank pressure at 0.05MPa, controlling the pH value to be not lower than 6.8, transferring the liquid into a closed secondary fermentation tank according to the inoculation amount of 0.4% (V/V) for fermentation culture after the fermentation culture is carried out for 20 hours, similarly keeping the temperature in the fermentation tank at 35 ℃, the rotating speed at 60r/min and the tank pressure at 0.05MPa in the fermentation process, controlling the pH value to be not lower than 6.8, and carrying out fermentation culture for 18 hours to obtain an enterococcus faecium liquid; wherein, the fermentation medium in the first order fermentation cylinder and above-mentioned second order fermentation cylinder's constitution includes: 15g/L glucose, 3g/L corn flour, 3g/L maltose, 8g/L peptone, 8g/L yeast extract powder, 800.7 g/L tween, 1.5g/L dipotassium phosphate, 0.3g/L magnesium sulfate and 0.15g/L manganese sulfate.
(3) Filtering the obtained enterococcus faecium bacterial liquid by using a vibrating screen, adding tap water with the same volume into the obtained filtrate, concentrating the filtrate to the volume which is the same as that of the original enterococcus faecium bacterial liquid by using a ceramic membrane filter, transferring the obtained concentrated liquid to a liquid storage tank, adding a protective agent with the mass accounting for 15.5 percent of the total mass of the enterococcus faecium bacterial liquid into the liquid storage tank, uniformly mixing, and then feeding the mixture into a spray tower for spray drying to obtain enterococcus faecium bacterial powder; wherein, in the spray drying process: the air inlet temperature is 150 ℃, and the air outlet temperature is controlled to be 70 ℃ by adjusting the feeding frequency.
Example 5
The protective agent for preparing enterococcus faecium powder provided by the embodiment comprises the following components in parts by weight: 4.8 parts of corn starch, 3.8 parts of maltodextrin, 2.2 parts of bovine serum albumin, 0.48 part of cane sugar, 0.28 part of emulsifier, 0.48 part of auxiliary emulsifier, 0.4 part of hydroxyethyl cellulose sodium, 0.7 part of sulfobutyl beta-cyclodextrin sodium and 0.58 part of sodium glutamate.
The preparation method of enterococcus faecium powder provided by the embodiment comprises the following steps:
(1) selecting 2 target enterococcus faecium colonies, inoculating into 100ml of first-stage seed culture medium, and standing and culturing at 36 deg.C for 22 hr to obtain first-stage seed liquid; then, 6ml of first-order seed liquid is absorbed and inoculated into 1L of seed culture medium, and standing culture is carried out for 22h at 36 ℃ to obtain seed liquid; wherein, the composition of the seed culture medium comprises: 10g/L of peptone, 5g/L of yeast powder, 20g/L of glucose, 5g/L of sodium acetate, 2g/L of diammonium citrate and 2g/L of dipotassium phosphate.
(2) Inoculating the obtained enterococcus faecium seed liquid into a closed primary fermentation tank according to the inoculation amount of 0.35% (V/V) for fermentation culture, keeping the temperature in the fermentation tank at 39 ℃, the rotating speed at 60r/min and the tank pressure at 0.05MPa, controlling the pH value to be not lower than 6.8, transferring the liquid into a closed secondary fermentation tank according to the inoculation amount of 0.35% (V/V) for fermentation culture after fermentation culture for 19h, similarly keeping the temperature in the fermentation tank at 39 ℃, the rotating speed at 60r/min and the tank pressure at 0.05MPa in the fermentation process, controlling the pH value to be not lower than 6.8, and performing fermentation culture for 17h to obtain an enterococcus faecium liquid; wherein, the fermentation medium in the first order fermentation cylinder and above-mentioned second order fermentation cylinder's constitution includes: 15g/L glucose, 3g/L corn flour, 3g/L maltose, 8g/L peptone, 8g/L yeast extract powder, 800.7 g/L tween, 1.5g/L dipotassium phosphate, 0.3g/L magnesium sulfate and 0.15g/L manganese sulfate.
(3) Filtering the obtained enterococcus faecium bacterial liquid by using a vibrating screen, adding tap water with the same volume into the obtained filtrate, concentrating the filtrate to the volume which is the same as that of the original enterococcus faecium bacterial liquid by using a ceramic membrane filter, transferring the obtained concentrated liquid to a liquid storage tank, adding a protective agent with the mass accounting for 13.6 percent of the total mass of the enterococcus faecium bacterial liquid into the liquid storage tank, uniformly mixing, and then, feeding the mixture into a spray tower for spray drying to obtain enterococcus faecium bacterial powder; wherein, in the spray drying process: the inlet air temperature is 140 ℃, and the outlet air temperature is controlled to be 66 ℃ by adjusting the feeding frequency.
Comparative example 1
This comparative example differs from example 1 in that: the composition of the protectant was otherwise the same as in example 1 except that bovine serum albumin and carbohydrates were removed.
Comparative example 2
This comparative example differs from example 1 in that: the composition of the protective agent was otherwise the same as in example 1 except that the emulsifier and co-emulsifier were removed.
Comparative example 3
This comparative example differs from example 1 in that: the composition of the protectant was the same as example 1 except that the hydroxyethyl cellulose was removed.
Comparative example 4
This comparative example differs from example 1 in that: the composition of the protectant was the same as that of example 1 except that sodium sulfobutyl β -cyclodextrin and sodium glutamate were removed.
The concentrated solutions and enterococcus faecium powder obtained in the above examples 1 to 5 and comparative examples 1 to 4 were taken, and the viable bacteria amount of the solutions and powder was measured and the survival rate was calculated by a plate dilution coating counting method, and the results are shown in table 1 below.
Note: the survival rate is calculated, wherein the survival rate is% = the viable bacteria amount of the bacteria powder is multiplied by the weight (kg) of the bacteria powder in unit batch, and the survival rate is multiplied by 100 percent.
TABLE 1
| Viable count of bacterial liquid (x 1010 CFU/mL) | Viable count of bacterial powder (x 1010 CFU/mL) | Weight of dry powder | Survival rate (%) | |
| Example 1 | 1.07 | 6.61 | 74 | 91.43 |
| Example 2 | 0.95 | 6.16 | 72 | 93.37 |
| Example 3 | 0.98 | 6.18 | 73 | 92.07 |
| Example 4 | 1.07 | 6.18 | 78 | 90.10 |
| Example 5 | 1.12 | 6.77 | 75 | 90.67 |
| Comparative example 1 | 1.04 | 3.63 | 89 | 62.13 |
| Comparative example 2 | 0.90 | 4.14 | 70 | 64.40 |
| Comparative example 3 | 1.05 | 5.26 | 72 | 72.14 |
| Comparative example 4 | 1.09 | 5.43 | 72 | 71.74 |
The results in table 1 show that the survival rate of the enterococcus faecium after spray drying can reach more than 90% by adopting the protective agent and the preparation process of the enterococcus faecium powder, the advanced level of the industry is reached, the production cost is greatly reduced, and the storage life of the powder is prolonged.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; although the present invention has been described in detail with reference to the foregoing examples 3, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the invention and form different embodiments. For example, in the claims above, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Claims (14)
1. A preparation method of enterococcus faecium powder is characterized by comprising the following steps: mixing raw materials including enterococcus faecium liquid and protective agent, and drying;
the protective agent comprises the following components in parts by weight: 4-5 parts of a thickening agent, 3-4 parts of a filler, 2-3 parts of bovine serum albumin, 0.4-0.5 part of a saccharide, 0.2-0.3 part of an emulsifier, 0.4-0.5 part of a co-emulsifier, 0.3-0.8 part of sodium hydroxyethyl cellulose, 0.3-0.8 part of sulfobutyl beta-cyclodextrin sodium and 0.5-0.6 part of an antioxidant;
the thickening agent is corn starch;
the filler is at least one of maltodextrin and beta-cyclodextrin;
the emulsifier is xanthan gum;
the auxiliary emulsifier is at least one of mannitol, sorbitol and glycerol;
the saccharide is at least one of sucrose, trehalose, lactose and maltose;
the antioxidant is sodium glutamate.
2. The method for preparing enterococcus faecium powder according to claim 1, wherein the method for preparing the enterococcus faecium liquid comprises:
inoculating enterococcus faecium seed liquid into a closed primary fermentation tank for primary fermentation culture to obtain primary fermentation liquid, and then inoculating the primary fermentation liquid into a closed secondary fermentation tank for secondary fermentation culture.
3. The method for preparing enterococcus faecium powder according to claim 2, wherein the enterococcus faecium seed liquid is inoculated in the primary fermentation tank at an inoculation volume ratio of 0.28-0.4%;
and the primary fermentation liquid is inoculated in the secondary fermentation tank according to the inoculation amount volume ratio of 0.28-0.4%.
4. The method of claim 2, wherein the fermentation medium in the primary fermentor and the fermentation medium in the secondary fermentor are independently composed of: 15-18 g/L of glucose, 3-3.5 g/L of corn flour, 3-3.5 g/L of maltose, 8-10 g/L of peptone, 8-10 g/L of yeast extract powder, 800.7-0.8 g/L of tween, 1.5-1.8 g/L of dipotassium hydrogen phosphate, 0.26-0.3 g/L of magnesium sulfate and 0.12-0.15 g/L of manganese sulfate.
5. The method for producing enterococcus faecium powder according to claim 2, wherein the tank pressure in the primary fermentation tank and the secondary fermentation tank is 0.05 to 0.06 MPa;
the temperature of the primary fermentation culture is 35-39 ℃, and the time is 18-20 h;
the temperature of the secondary fermentation culture is 35-39 ℃, and the time is 16-18 h.
6. The method of preparing enterococcus faecium powder according to claim 2, wherein the method of preparing enterococcus faecium seed liquid comprises:
picking an enterococcus faecium colony, and inoculating the enterococcus faecium colony into a primary seed culture medium for carrying out primary standing culture to obtain a primary seed liquid;
and inoculating the primary seed liquid into a secondary seed culture medium for secondary static culture.
7. The method of claim 6, wherein 1-2 enterococcus faecium colonies are inoculated per 100ml of said primary seed medium;
the temperature of the first standing culture is 36-38 ℃, and the time is 20-24 hours.
8. The method for preparing enterococcus faecium powder according to claim 6, wherein 6-8 ml of the primary seed liquid is inoculated into every 1L of the secondary seed culture medium;
the temperature of the second standing culture is 36-38 ℃, and the time is 20-24 hours.
9. The method of claim 6, wherein the first seed medium and the second seed medium are each independently comprised of: 8-10 g/L of peptone, 4-5 g/L of yeast powder, 16-20 g/L of glucose, 4-5 g/L of sodium acetate, 1.8-2 g/L of diammonium citrate and 1.8-2 g/L of dipotassium hydrogen phosphate.
10. The method for preparing enterococcus faecium powder according to claim 1, further comprising, before mixing the enterococcus faecium liquid and the protective agent: filtering the enterococcus faecium bacterial liquid, adding water into the obtained filtrate, mixing and concentrating to the volume which is the same as that of the enterococcus faecium bacterial liquid;
the amount of the added water is 1-1.5 times of the volume of the enterococcus faecium bacterial liquid.
11. The method for preparing enterococcus faecium powder according to claim 1, wherein the amount of the protective agent is 11.1-15.5% of the total mass of the enterococcus faecium liquid.
12. The method for producing enterococcus faecium powder according to claim 1, wherein the drying is spray drying;
in the spray drying process: the air inlet temperature is 135-150 ℃, and the air outlet temperature is 62-70 ℃.
13. An enterococcus faecium powder, characterized in that the enterococcus faecium powder is prepared by the method for preparing enterococcus faecium powder according to any one of claims 1 to 12.
14. The use of enterococcus faecium powder according to claim 13 for the preparation of a feed, a food or a medicament.
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| CN101701200A (en) * | 2009-11-27 | 2010-05-05 | 南京农业大学 | Enterobacter cloacae and application thereof |
| CN109329649A (en) * | 2018-09-10 | 2019-02-15 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of compound micro-ecological preparation and preparation method thereof of antagonism prawn vibrios |
| CN111304111A (en) * | 2019-11-29 | 2020-06-19 | 天津市天合力药物研发有限公司 | Enterococcus faecium, microbial inoculum thereof, preparation method and application of microbial inoculum |
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| CN101701200A (en) * | 2009-11-27 | 2010-05-05 | 南京农业大学 | Enterobacter cloacae and application thereof |
| CN109329649A (en) * | 2018-09-10 | 2019-02-15 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of compound micro-ecological preparation and preparation method thereof of antagonism prawn vibrios |
| CN111304111A (en) * | 2019-11-29 | 2020-06-19 | 天津市天合力药物研发有限公司 | Enterococcus faecium, microbial inoculum thereof, preparation method and application of microbial inoculum |
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