CN115011508B - Enterococcus faecium, microcapsule preparation and preparation method thereof - Google Patents
Enterococcus faecium, microcapsule preparation and preparation method thereof Download PDFInfo
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- CN115011508B CN115011508B CN202210480031.6A CN202210480031A CN115011508B CN 115011508 B CN115011508 B CN 115011508B CN 202210480031 A CN202210480031 A CN 202210480031A CN 115011508 B CN115011508 B CN 115011508B
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- 239000003094 microcapsule Substances 0.000 title claims abstract description 90
- 241000194031 Enterococcus faecium Species 0.000 title claims abstract description 63
- 238000002360 preparation method Methods 0.000 title claims abstract description 58
- 238000000576 coating method Methods 0.000 claims abstract description 41
- 239000011248 coating agent Substances 0.000 claims abstract description 33
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 239000008188 pellet Substances 0.000 claims abstract description 4
- 239000000463 material Substances 0.000 claims description 33
- 230000001580 bacterial effect Effects 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000002245 particle Substances 0.000 claims description 19
- 239000010802 sludge Substances 0.000 claims description 18
- 230000004151 fermentation Effects 0.000 claims description 16
- 239000002702 enteric coating Substances 0.000 claims description 14
- 238000009505 enteric coating Methods 0.000 claims description 14
- 239000003223 protective agent Substances 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 11
- 239000004925 Acrylic resin Substances 0.000 claims description 10
- 239000004816 latex Substances 0.000 claims description 10
- 229920000126 latex Polymers 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 238000004321 preservation Methods 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 8
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 8
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 8
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 238000009472 formulation Methods 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 239000007921 spray Substances 0.000 claims description 5
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 4
- 229920002261 Corn starch Polymers 0.000 claims description 4
- 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 4
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 4
- 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 4
- 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 4
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims description 4
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 claims description 4
- 229930003268 Vitamin C Natural products 0.000 claims description 4
- 229960000583 acetic acid Drugs 0.000 claims description 4
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims description 4
- 238000000889 atomisation Methods 0.000 claims description 4
- 238000005422 blasting Methods 0.000 claims description 4
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 4
- 239000001110 calcium chloride Substances 0.000 claims description 4
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 4
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 4
- 239000008120 corn starch Substances 0.000 claims description 4
- FPAFDBFIGPHWGO-UHFFFAOYSA-N dioxosilane;oxomagnesium;hydrate Chemical compound O.[Mg]=O.[Mg]=O.[Mg]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O FPAFDBFIGPHWGO-UHFFFAOYSA-N 0.000 claims description 4
- 239000012362 glacial acetic acid Substances 0.000 claims description 4
- 235000011187 glycerol Nutrition 0.000 claims description 4
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 claims description 4
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 claims description 4
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 claims description 4
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 claims description 4
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N iron oxide Inorganic materials [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 claims description 4
- 239000008101 lactose Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 229940016286 microcrystalline cellulose Drugs 0.000 claims description 4
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims description 4
- 239000008108 microcrystalline cellulose Substances 0.000 claims description 4
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical compound O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 claims description 4
- 229940093429 polyethylene glycol 6000 Drugs 0.000 claims description 4
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 claims description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 4
- 229940068968 polysorbate 80 Drugs 0.000 claims description 4
- 229920000053 polysorbate 80 Polymers 0.000 claims description 4
- 239000002356 single layer Substances 0.000 claims description 4
- 239000000661 sodium alginate Substances 0.000 claims description 4
- 235000010413 sodium alginate Nutrition 0.000 claims description 4
- 229940005550 sodium alginate Drugs 0.000 claims description 4
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 claims description 4
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 claims description 4
- 229960002920 sorbitol Drugs 0.000 claims description 4
- 238000005507 spraying Methods 0.000 claims description 4
- 230000001502 supplementing effect Effects 0.000 claims description 4
- 239000004408 titanium dioxide Substances 0.000 claims description 4
- 235000019154 vitamin C Nutrition 0.000 claims description 4
- 239000011718 vitamin C Substances 0.000 claims description 4
- 239000003674 animal food additive Substances 0.000 claims description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 2
- 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 2
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- 108010073771 Soybean Proteins Proteins 0.000 claims description 2
- 240000008042 Zea mays Species 0.000 claims description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 2
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 235000005822 corn Nutrition 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- 230000003100 immobilizing effect Effects 0.000 claims description 2
- 229940057995 liquid paraffin Drugs 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- 238000004062 sedimentation Methods 0.000 claims description 2
- 235000019710 soybean protein Nutrition 0.000 claims description 2
- 230000000087 stabilizing effect Effects 0.000 claims description 2
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 2
- 239000001393 triammonium citrate Substances 0.000 claims description 2
- 235000011046 triammonium citrate Nutrition 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 239000012138 yeast extract Substances 0.000 claims description 2
- 238000007873 sieving Methods 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 23
- 241000194033 Enterococcus Species 0.000 abstract description 9
- 238000005469 granulation Methods 0.000 abstract description 7
- 230000003179 granulation Effects 0.000 abstract description 7
- 244000144972 livestock Species 0.000 abstract description 6
- 244000144977 poultry Species 0.000 abstract description 6
- 239000006041 probiotic Substances 0.000 abstract description 3
- 235000018291 probiotics Nutrition 0.000 abstract description 3
- 239000002131 composite material Substances 0.000 abstract 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 14
- 230000000694 effects Effects 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 238000003860 storage Methods 0.000 description 10
- 239000010410 layer Substances 0.000 description 8
- 239000004310 lactic acid Substances 0.000 description 7
- 235000014655 lactic acid Nutrition 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 238000012545 processing Methods 0.000 description 5
- 229920001661 Chitosan Polymers 0.000 description 4
- 241001052560 Thallis Species 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 241000186660 Lactobacillus Species 0.000 description 2
- 241000235342 Saccharomycetes Species 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 229940039696 lactobacillus Drugs 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000000529 probiotic effect Effects 0.000 description 2
- 210000000813 small intestine Anatomy 0.000 description 2
- 229940071440 soy protein isolate Drugs 0.000 description 2
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000006030 antibiotic growth promoter Substances 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000007931 coated granule Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
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- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
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- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/10—Shaping or working-up of animal feeding-stuffs by agglomeration; by granulation, e.g. making powders
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
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Abstract
The invention discloses a Gao Wenshi-resistant enterococcus, a high-stability microcapsule preparation and a preparation method thereof. According to the invention, the enterococcus faecium mutant with obviously improved high temperature resistance is obtained through repeated high temperature stress domestication, and the enterococcus faecium mutant with Gao Wenshi resistance is subjected to pre-microencapsulation coating and post-fermentation coating, so that the multilayer composite coated enterococcus faecium microcapsule preparation is prepared. The high-stability enterococcus faecium microcapsule preparation has obviously improved survival rate after high-temperature granulation of feed, reaching 50% -70%; the survival rate is obviously improved and is stabilized at 60-70% after the feed is stored for 6 months at room temperature, so that the requirements of adding probiotics microecological preparation into pellet feed products of livestock and poultry feed enterprises can be met.
Description
Technical Field
The invention belongs to the technical field of microorganisms. More particularly, relates to a Gao Wenshi-resistant enterococcus, a high-stability microcapsule preparation and a preparation method thereof.
Background
Although lactic acid bacteria have a plurality of advantages and effects in the aspects of livestock and poultry cultivation and replacement of antibiotic growth promoters, due to the limitation of the characteristics of the lactic acid bacteria, the application of the lactic acid bacteria in livestock and poultry feeds can be influenced by a plurality of adverse factors, wherein the low survival rate in the high-temperature granulation processing process and the normal-temperature transportation and storage process of the feeds is a key and difficult problem for limiting the application of the lactic acid bacteria in the livestock and poultry cultivation field, and the improvement of the survival rate in the granulation processing process and the normal-temperature transportation and storage process of the lactic acid bacteria feeds by improving the characteristics of strains and optimizing the post-fermentation treatment and protection process is a prominent problem in the current market. In order to solve the problems, the protection of lactic acid bacteria by using a microcapsule embedding technology has become a hot spot of research at home and abroad at present. However, most of the existing microcapsule coating technologies and products have porous surfaces, poor stability and poor high temperature resistance, and can be released in the small intestine after entering the digestive tract, but still can be subjected to the effects of acidic gastric juice environment, bile acid, small intestine pancreatin and the like; the method is applied to livestock and poultry feed processing, has low survival rate after high-temperature and high-pressure granulation and normal-temperature transportation and storage, and cannot meet the requirements of feed enterprises, so that the stress resistance of the lactic acid bacteria cannot be greatly improved only by single microcapsule coating and simple post-treatment processing technology.
The Chinese patent with the patent number ZL202011208280.7 discloses a preparation method of a multilayer coated probiotic microcapsule, the principle is that chitosan is added after a single microcapsule is coated with thalli to form a secondary coating, then a granulating coating process is adopted to coat double-layer microcapsule thalli, the problem of surface porosity of lactobacillus or saccharomycete microcapsule preparations is solved by the method, the stress resistance of the coated probiotic microcapsule is improved, but the cost of chitosan used by the method is higher, the fluidity and toughness of the prepared coated granule are not ideal, and although the invention is applicable to lactobacillus and saccharomycete, the stable stress resistance cannot be ensured for different strains. According to the invention, based on the existing microcapsule coating technology and granulating and coating technology, the application of combining the Gao Wenshi-resistant enterococcus strain with the heat-resistant protective agent and the special coating premix is achieved through domestication and mutagenesis, so that the survival rate of enterococcus faecium in the high-temperature granulating and normal-temperature storage processes of the feed is greatly improved, and the enterococcus faecium microcapsule preparation with high stability is obtained.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a Gao Wenshi-resistant enterococcus mutant strain and a multi-layer coated high-stability microcapsule preparation prepared by the same.
The invention also aims to provide an application of the Gao Wenshi-resistant enterococcus mutant strain and a microcapsule preparation with high stability thereof in preparing feed additives and pellet feeds.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect, the invention provides a strain of Gao Wenshi resistant enterococcus (Enterococcus faecium), strain named RS047-14 (3), classified as: enterococcus faecium Enterococcus faecium is preserved in China general microbiological culture collection center (CGMCC) (address: north Chen West Lu No.1, 3 of the area of Chachiensis in Beijing, china center for the 11 th month and 30 th year, and is characterized by having a preservation number of CGMCC No. 24004).
Specifically, the Gao Wenshi-resistant enterococcus (Enterococcus faecium) RS047-14 (3) is an enterococcus faecium mutant strain, and the mutation screening method of the mutant strain is as follows:
the method comprises the steps of storing wild enterococcus faecium (preservation number is CGMCC No. 19787) bacterial powder for 210 days at a constant temperature of 37 ℃, inoculating the bacterial powder to MRS broth culture medium, continuously passaging for 14 times at 50 ℃, and screening by a large number of high-temperature-resistant detection.
In a second aspect, the invention also provides a high-stability enterococcus faecium microcapsule preparation, which comprises the Gao Wenshi-resistant enterococcus faecium (Enterococcus faecium) RS047-14 (3). The microcapsule formulations may be prepared by the preparation methods disclosed in the prior art. However, in order to obtain better high temperature resistance and high stability effects, the invention provides a preparation method of the enterococcus faecium microcapsule preparation with high stability, which comprises the following steps:
(1) Preparing sodium alginate solution with the concentration of 10g/L-20g/L in a feed tank of a fermentation system;
(2) Weighing calcium carbonate according to the mass ratio of 1:1-1.5 with the sodium alginate, adding the calcium carbonate into a material supplementing tank, uniformly mixing, sterilizing, cooling to 35-38 ℃, adding Gao Wenshi-resistant enterococcus seed liquid with the preservation number of CGMCC No.24004 in a growth stabilizing period until the viable bacteria concentration is 1 multiplied by 10 6 -5×10 6 CFU/mL, fully mixing to be a water phase;
(3) Adding span 80 as an oil phase into liquid paraffin, wherein the volume percentage of span 80 is 0.1% -0.3%, adding the oil phase into a fermentation tank, and sterilizing;
(4) Preparing 0.1-0.3mol/L calcium chloride solution in another material supplementing tank as a sedimentation agent, and sterilizing;
(5) Slowly transferring the water phase mixed solution into the oil phase of a fermentation tank according to the volume ratio of the water phase to the oil phase of 1:3-5 at the stirring speed of 400-500rpm, stirring for 2-5min, adding glacial acetic acid according to the volume ratio of glacial acetic acid to the oil phase of 1:500-600, immobilizing for 10-20min, stopping stirring, adding a settling agent into the reaction system according to the volume ratio of the water phase to the oil phase of 1:10-20, slowly settling the microcapsule, and sucking away the oil phase;
(6) Adding a fermentation culture medium suitable for the growth of the Gao Wenshi enterococci into a fermentation tank, controlling the pH value of a fermentation liquid to be 6.2-6.9 by adding ammonia water in a flowing way under the conditions of 35-38 ℃ and 50-100rpm of stirring speed, continuously culturing for 8-22 hours until the Gao Wenshi enterococci is filled with more than 90% of microcapsule inner space, and centrifuging to obtain single-layer microcapsule bacterial mud;
(7) Adding a protective agent into the microcapsule bacterial sludge obtained in the step (6); uniformly mixing, adding granulating auxiliary materials, preparing 20-30 mesh microcapsule particles by a swing granulator, and immediately throwing the microcapsule particles into a spherical shot blasting machine; wherein the composition of the protective agent is as follows: adding 5-10 parts of glycerin, 5-10 parts of lactose, 10-15 parts of soybean protein isolate, 1-2 parts of vitamin C, 0.5-1 part of calcium chloride and 0.5-1 part of D-sorbitol into the microcapsule bacterial mud based on 100 parts of the microcapsule bacterial mud; the granulating auxiliary materials comprise the following components: 100-200 parts of corn starch, 5-10 parts of microcrystalline cellulose, 0.06-0.25 part of sodium carboxymethylcellulose and 100-200 parts of water are added into the microcapsule bacterial sludge based on 100 parts of the microcapsule bacterial sludge;
(8) Transferring the microcapsule particles which are coated with the protective agent and polished in the step (7) into a fluidized granulating coating dryer, controlling the air inlet temperature to be 40-55 ℃, and drying until the water content of the sample is below 10%; the enteric coating material is adopted to spray and coat, and when the enteric coating material is completely sprayed, the enteric coating material is dried and sieved to obtain the high-stability enterococcus faecium microcapsule preparation with the particle size of 20-60 meshes.
Further, in step (2), the seed liquid culture medium is MRS broth.
Further, in the step (6), the formula of the fermentation medium is as follows: 15-20g/L glucose, 15-20g/L peptone, 5-10g/L corn steep liquor dry powder, 3-5g/L yeast extract and KH 2 PO 4 0.5-1g/L, and tri-ammonium citrate 0.2-0.5g/L, mgSO 4 ·7H 2 O 0.5-1g/L，MnSO 4 ·H 2 O 0.5-1g/L。
Further, in the step (8), specific spraying and coating conditions are as follows: the flow rate of the enteric coating material is 4-8rpm, the spray atomization pressure is 0.1-0.15Mpa, the air inlet temperature of the fluidized bed is controlled at 50-60 ℃, and the material temperature is controlled at 35-45 ℃.
In the step (8), the enteric coating material is prepared by adding 20-40% of polyacrylic resin latex and matched premix together into water with the total mass of 1.5 times of that of the polyacrylic resin latex and the matched premix; wherein the matched premix comprises hydroxypropyl methylcellulose, polyethylene glycol 6000, talcum powder, polysorbate 80, titanium dioxide and ferric oxide in a mass ratio of 1:1-2:3-5:0.5-1:1-2:0.3-0.5; the mass ratio of the polyacrylic resin latex to the matched premix is 8-12:1.
In a third aspect, the invention also provides application of the Gao Wenshi-resistant enterococcus faecium microcapsule preparation and the high-stability enterococcus faecium microcapsule preparation in preparing feed additives or pellet feeds.
Compared with the prior art, the invention has the following advantages and effects:
the invention not only screens enterococcus faecium (Enterococcus faecium) RS047-14 (3) with better high temperature resistance, but also prepares the high-stability enterococcus faecium (Enterococcus faecium) RS047-14 (3) microcapsule preparation, which solves the problem of porous capsule surface in the prior microcapsule coating technology, and greatly improves the normal temperature storage shelf life of the microcapsule coated enterococcus faecium preparation and the problem of easy inactivation after feed granulation. The survival rate of the high-stability preparation is obviously improved to 50-70% after the high-temperature granulation of the feed, the survival rate is obviously improved after the preparation is stored for 6 months under the condition of room temperature, and the survival rate is also obviously improved to 60-70%, so that the preparation can meet the requirement of adding enterococcus faecium microecological preparation into the granulated feed product of livestock and poultry feed enterprises.
Preservation description
Biological material of reference (strain): RS047-14 (3)
Suggested class naming: enterococcus faecium
Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No.1 and 3
Preservation date: 2021, 11, 30
Accession numbers of the preservation center: CGMCC No.24004.
Drawings
FIG. 1 survival rates of wild type strains and mutant strains treated at different temperatures for different times.
Detailed Description
The invention will be further illustrated with reference to the following specific examples, which are not intended to limit the scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent suppliers.
EXAMPLE 1 acquisition of Gao Wenshi resistant enterococcus mutant
Meanwhile, a wild enterococcus faecium CGMCC No.19787 and a strain activated by enterococcus faecium CGMCC No.19787 bacterial powder stored for 201 days at 37 ℃ are used as initial strains, the initial strains are inoculated into an MRS broth shake flask, the strains are subjected to static culture at 37 ℃, bacterial solutions which are respectively cultivated to 6 hours (late logarithmic phase), 12 hours (late logarithmic phase) and 24 hours (stable phase) are inoculated into the MRS broth shake flask in 1 percent of inoculum amount, and the strains are subjected to continuous passage domestication culture at 55 ℃, and the result shows that the strain activated by enterococcus faecium CGMCC No.19787 bacterial powder stored for 201 days at 37 ℃ is continuously passaged to 15 generations at 55 ℃, 57 strains subjected to high temperature domestication are randomly selected from the 15 generations, and finally a mutant strain with good high temperature resistance and genetic stability is obtained through high temperature-resistant screening, and the mutant strain is delivered to China general microbiological culture collection center (CGMCC No. 24004). For simplicity of explanation, enterococcus faecium (Enterococcus faecium) RS047-14 (3) preserved in the invention is represented by preservation number CGMCCNo.24004 in the following description.
The high temperature resistance of the strain is shown in figure 1. FIG. 1 shows that the survival rates of the mutant strain CGMCC No.24004 at 60 ℃, 65 ℃, 75 ℃ and 85 ℃ for 3min are respectively 98.85%, 86.22%, 67.21% and 45.73%, the survival rates of the mutant strain CGMCC No.24004 at 5min are respectively 95.18%, 75.81%, 45.53% and 18.87%, and the survival rates of the mutant strain CGMCC No.24004 at 10min are respectively 89.62%, 60.77%, 22.38% and 1.39% respectively, which are obviously higher than the survival rates of the wild strain CGMCC No.19787 under the corresponding treatment conditions.
EXAMPLE 2 preparation of highly stable enterococcus faecium microcapsule formulation
Preparing and obtaining single-layer CGMCC No.24004 microcapsule bacterial sludge according to the methods of the steps (1) - (6);
then, adding a protective agent into the collected CGMCC No.24004 microcapsule bacterial sludge; the composition of the protective agent is as follows: adding 10 parts of glycerin, 10 parts of lactose, 15 parts of soy protein isolate, 2 parts of vitamin C, 1 part of calcium chloride and 1 part of D-sorbitol into the microcapsule bacterial sludge based on 100 parts of the weight of the microcapsule bacterial sludge; after mixing evenly, adding granulating auxiliary materials: adding 200 parts of corn starch, 10 parts of microcrystalline cellulose, 0.25 part of sodium carboxymethyl cellulose and 200 parts of water into the microcapsule bacterial sludge in sequence based on 100 parts of the microcapsule bacterial sludge, fully and uniformly mixing, preparing 20-30 mesh microcapsule particles by a swing granulator, and then throwing the microcapsule particles into a spherical shot blasting machine; then transferring the polished microcapsule particles into a fluidized granulating coating dryer, controlling the air inlet temperature to be 40-55 ℃, and drying until the water content of the sample is below 10%; the enteric coating material is adopted to carry out spray coating, the coating material is prepared by adding 40% by mass of polyacrylic resin latex and a matched premix into water with the total mass of 1.5 times of the polyacrylic resin latex and the matched premix according to the mass ratio of 12:1, the premix comprises hydroxypropyl methylcellulose, polyethylene glycol 6000, talcum powder, polysorbate 80, titanium dioxide and ferric oxide, the mass ratio is 1:2:5:1:2:0.5, stirring is carried out while adding until uniform emulsion is prepared, the flow rate of the coating agent is 4-8rpm, the spray atomization pressure is 0.1-0.15Mpa, the air inlet temperature of a fluidized bed is controlled at 50-60 ℃, the material temperature is controlled at 35-45 ℃, when the coating material is completely sprayed, the drying is continued for 50-60min, and a dried sample is sieved, so as to obtain the enterococcus faecium microcapsule preparation with the particle size of 20-60 meshes and high stability.
The preparation method of the enterococcus faecium microcapsule preparation with high stability has two major differences from the preparation method in Chinese patent with the patent number ZL 202011208280.7: 1. coating the second layer without chitosan; 2. the coating material is added with a matched premix. By adding the matched premix, the obtained coated particles have good fluidity, enhanced particle shape and toughness, and more compact pore diameter on the surfaces of the coated particles, so that the product has better heat resistance and storage stability under the condition of no need of adding chitosan with relatively higher cost for two-layer coating, and the release degree of thalli in the product under the intestinal environment is higher.
EXAMPLE 3 preparation of highly stable enterococcus faecium microcapsule formulation
Preparing and obtaining single-layer CGMCC No.24004 microcapsule bacterial sludge according to the methods of the steps (1) - (6);
then, adding a protective agent into the collected CGMCC No.24004 microcapsule bacterial sludge; the composition of the protective agent is as follows: adding 5 parts of glycerin, 5 parts of lactose, 10 parts of soy protein isolate, 1 part of vitamin C, 0.5 part of calcium chloride and 0.5 part of D-sorbitol into the microcapsule bacterial sludge based on 100 parts of the microcapsule bacterial sludge; after mixing evenly, adding granulating auxiliary materials: adding 100 parts of corn starch, 5 parts of microcrystalline cellulose, 0.06 part of sodium carboxymethyl cellulose and 100 parts of water into the microcapsule bacterial sludge in sequence based on 100 parts of the microcapsule bacterial sludge, fully and uniformly mixing, preparing 20-30 mesh microcapsule particles by a swing granulator, and then throwing the microcapsule particles into a spherical shot blasting machine; then transferring the polished microcapsule particles into a fluidized granulating coating dryer, controlling the air inlet temperature to be 40-55 ℃, and drying until the water content of the sample is below 10%; the enteric coating is prepared by adopting an enteric coating material to carry out spray coating, wherein the coating material is prepared by adding 20% by mass of polyacrylic resin latex and a matched premix into water with the total mass of 1.5 times of the polyacrylic resin latex and the matched premix according to the mass ratio of 8:1, wherein the premix comprises hydroxypropyl methylcellulose, polyethylene glycol 6000, talcum powder, polysorbate 80, titanium dioxide and ferric oxide, the mass ratio of 1:1:3:0.5:1:0.3, stirring is carried out while adding until uniform emulsion is prepared, the flow rate of the coating agent is 4-8rpm, the spray atomization pressure is 0.1-0.15Mpa, the air inlet temperature of a fluidized bed is controlled at 50-60 ℃, the material temperature is controlled at 35-45 ℃, when the coating material is completely sprayed, the drying is continued for 50-60min, and the dried sample is sieved, so that the enterococcus faecium microcapsule preparation with the particle size of 20-60 meshes is obtained.
EXAMPLE 4 high stability enterococcus faecium microcapsule preparation preservation experiment results
The multilayer coating high-stability enterococcus faecium microcapsule preparation prepared by using the Gao Wenshi-resistant enterococcus faecium mutant strain CGMCC No.24004 provided by the invention takes a microcapsule preparation of wild enterococcus faecium CGMCC No.19787 which is not coated after fermentation, a multilayer coating microcapsule preparation of wild enterococcus faecium CGMCC No.19787 prepared by using a multilayer coating method provided by Chinese patent with patent number ZL202011208280.7 and a multilayer coating microcapsule preparation of Gao Wenshi-resistant enterococcus faecium mutant strain CGMCC No.24004 prepared by using a multilayer coating method provided by Chinese patent with patent number ZL202011208280.7 as a control, the storage activities of 4 products are detected, and the survival rates of the products are detected under the conditions of room temperature (20 ℃ -37 ℃), 30 ℃ and 37 ℃ for 6 months, respectively, and are shown in table 1. The result shows that the survival rate of the bacterial powder prepared by the high-temperature resistant mutant combined with the multilayer coating method provided by the Chinese patent with the patent number ZL202011208280.7 at room temperature (20-37 ℃) and 30 ℃ and 37 ℃ for 6 months is obviously higher than that of the enterococcus faecium preparation CGMCC No.19787 prepared by the Chinese patent with the patent number ZL202011208280.7, and the storage activity of the bacterial agent prepared by the same method is improved after the high-temperature resistant mutant replaces a wild bacterial strain. After the multi-layer coating method is further changed into the method, the survival rate of the enterococcus faecium preparation is obviously improved compared with the multi-layer coating microcapsule preparation of the wild enterococcus faecium CGMCC No.19787 prepared by the multi-layer coating method provided by Chinese patent No. ZL202011208280.7 at room temperature (20-37 ℃) and 30 ℃ for 6 months, which shows that the shelf life of the enterococcus faecium preparation product obtained by combining the high temperature resistant mutant with the improved multi-layer coating method is obviously prolonged, and the requirement of keeping the use activity in the transportation and storage processes can be met.
TABLE 1 storage Activity of different microbial agent samples at different temperatures
Example 5 results of feed pelletization experiments for highly Stable enterococcus faecium microcapsule formulations
The multilayer coating high-stability enterococcus faecium microcapsule preparation prepared by the enterococcus faecium mutant strain resistant to Gao Wenshi provided by the invention is characterized in that the microcapsule preparation of the wild enterococcus faecium CGMCC No.19787 which is not coated after fermentation, the multilayer coating microcapsule preparation of the wild enterococcus faecium CGMCC No.19787 prepared by the multilayer coating method provided by the Chinese patent with the patent number ZL202011208280.7 and the multilayer coating microcapsule preparation of the enterococcus faecium mutant strain resistant to Gao Wenshi CGMCC No.24004 prepared by the multilayer coating method provided by the Chinese patent with the patent number ZL202011208280.7 are used as a comparison, the survival rate of 4 products in the high-temperature granulating process of feed is detected, the method for detecting the survival rate of the feed is different from the laboratory simulation granulating method provided by the Chinese patent with the patent number ZL202011208280.7, the 4 bacteria powder products are taken to a feed factory, the full-price feed is added according to the additive amount, the full-price feed is fully mixed, the granulating process is carried out completely according to the actual granulating process of the feed factory, the modulating temperature is carried out at the temperature of 85 ℃ for 2min, the time of 2-3min after the full-price mixed bacteria is cooled, the survival rate is detected, the full-price of the powder is obtained after the full-price granulating is obtained, and the full-price granulating is carried out, and the mixed powder is granulated after the full-price is obtained after the full-price granulating is obtained, and the full-price after the whole-price granulating is obtained, and the survival rate is detected. The result shows that after the enterococcus faecium CGMCC No.19787 is subjected to high temperature resistant domestication mutation, the high temperature resistant activity of the microcapsule preparation prepared by the multilayer coating method provided by the Chinese patent with the patent number ZL202011208280.7 is obviously improved, the microcapsule preparation is further matched with a high temperature resistant protective agent, the high temperature resistant activity of thalli is greatly improved after the strain is subjected to multilayer coating by using a special enteric coating material and a matched premix, and the survival rate of the enterococcus faecium preparation prepared by the Chinese patent with the patent number ZL202011208280.7 is respectively improved by 34.7 percent and 32.21 percent after the actual feed granulation processing.
TABLE 2 survival rates of different microbial agent samples before and after feed pelleting
It is to be understood that the above examples of the present invention are provided by way of illustration only and not by way of limitation of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. Not all embodiments are exhaustive. All obvious changes or modifications which come within the spirit of the invention are desired to be protected.
Claims (6)
1. A method for preparing an enterococcus faecium microcapsule preparation, which is characterized by comprising the following steps:
(1) Preparing sodium alginate solution with the concentration of 10g/L-20g/L in a feed tank of a fermentation system;
(2) Weighing calcium carbonate according to the mass ratio of 1:1-1.5 with the sodium alginate, adding the calcium carbonate into a material supplementing tank, uniformly mixing, sterilizing, cooling to 35-38 ℃, and adding enterococcus faecium with the preservation number of CGMCC No.24004 in the growth stabilizing periodEnterococcus faecium) Seed solution with viable bacteria concentration of 1×10 6 -5×10 6 CFU/mL, fully mixing to be a water phase;
(3) Adding span 80 as an oil phase into liquid paraffin, wherein the volume percentage of span 80 is 0.1% -0.3%, adding the oil phase into a fermentation tank, and sterilizing;
(4) Preparing 0.1-0.3mol/L calcium chloride solution in another material supplementing tank as a sedimentation agent, and sterilizing;
(5) Slowly transferring the water phase mixed solution into the oil phase of a fermentation tank according to the volume ratio of the water phase to the oil phase of 1:3-5 at the stirring speed of 400-500rpm, stirring for 2-5min, adding glacial acetic acid according to the volume ratio of glacial acetic acid to the oil phase of 1:500-600, immobilizing for 10-20min, stopping stirring, adding a settling agent into the reaction system according to the volume ratio of the water phase to the oil phase of 1:10-20, slowly settling the microcapsule, and sucking away the oil phase;
(6) Adding a fermentation medium suitable for the enterococcus faecium growth into a fermentation tank, controlling the pH value of a fermentation liquid to be 6.2-6.9 by adding ammonia water under the conditions of 35-38 ℃ and 50-100rpm stirring speed, continuously culturing for 8-22 hours until the enterococcus faecium is filled with more than 90% of microcapsule inner space, and centrifuging to obtain single-layer microcapsule bacterial mud;
(7) Adding a protective agent into the microcapsule bacterial sludge obtained in the step (6); uniformly mixing, adding granulating auxiliary materials, preparing 20-30 mesh microcapsule particles by a swing granulator, and immediately throwing the microcapsule particles into a spherical shot blasting machine; wherein the composition of the protective agent is as follows: adding 5-10 parts of glycerin, 5-10 parts of lactose, 10-15 parts of soybean protein isolate, 1-2 parts of vitamin C, 0.5-1 part of calcium chloride and 0.5-1 part of D-sorbitol into the microcapsule bacterial mud based on 100 parts of the microcapsule bacterial mud; the granulating auxiliary materials comprise the following components: 100-200 parts of corn starch, 5-10 parts of microcrystalline cellulose, 0.06-0.25 part of sodium carboxymethylcellulose and 100-200 parts of water are added into the microcapsule bacterial sludge based on 100 parts of the microcapsule bacterial sludge;
(8) Transferring the microcapsule particles which are coated with the protective agent and polished in the step (7) into a fluidized granulating coating dryer, controlling the air inlet temperature to be 40-55 ℃, and drying until the water content of the sample is below 10%; coating the enterococcus faecium microcapsule preparation with an enteric coating material, and drying and sieving the enteric coating material to obtain the enterococcus faecium microcapsule preparation with the particle size of 20-60 meshes when the enteric coating material is completely coated;
the enteric coating material is prepared by adding 20-40% of polyacrylic resin latex solution and matched premix together into water with the total mass of 1.5 times of that of the polyacrylic resin latex solution and the matched premix; wherein the matched premix comprises hydroxypropyl methylcellulose, polyethylene glycol 6000, talcum powder, polysorbate 80, titanium dioxide and ferric oxide in a mass ratio of 1:1-2:3-5:0.5-1:1-2:0.3-0.5; the mass ratio of the polyacrylic resin latex to the matched premix is 8-12:1.
2. The method of claim 1, wherein in step (2), the seed liquid medium is MRS broth.
3. The method of claim 1, wherein in step (6), the fermentation medium is formulated as follows: 15-20g/L glucose, 15-20g/L peptone, 5-10g/L corn steep liquor dry powder, 3-5g/L yeast extract and KH 2 PO 4 0.5-1g/L, and tri-ammonium citrate 0.2-0.5g/L, mgSO 4 ·7H 2 O 0.5-1g/L，MnSO 4 ·H 2 O 0.5-1g/L。
4. The method of claim 1, wherein in step (8), specific spraying and coating conditions are: the flow rate of the enteric coating material is 4-8rpm, the spray atomization pressure is 0.1-0.15Mpa, the air inlet temperature of the fluidized bed is controlled at 50-60 ℃, and the material temperature is controlled at 35-45 ℃.
5. Use of the enterococcus faecium microcapsule formulation prepared by the preparation method of any one of claims 1-4 in the preparation of a feed additive.
6. Use of an enterococcus faecium microcapsule formulation prepared by the method of any of claims 1-4 in the preparation of a pellet feed.
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| CN115011508A (en) | 2022-09-06 |
| NL2034764A (en) | 2023-11-14 |
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