CN104004670B - A kind of feeding thermostable microcapsule yeast probiotics preparation and preparation method thereof and application - Google Patents
A kind of feeding thermostable microcapsule yeast probiotics preparation and preparation method thereof and application Download PDFInfo
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- CN104004670B CN104004670B CN201410234358.0A CN201410234358A CN104004670B CN 104004670 B CN104004670 B CN 104004670B CN 201410234358 A CN201410234358 A CN 201410234358A CN 104004670 B CN104004670 B CN 104004670B
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 123
- 238000002360 preparation method Methods 0.000 title claims abstract description 67
- 239000003094 microcapsule Substances 0.000 title claims abstract description 53
- 230000000529 probiotic Effects 0.000 title claims abstract description 53
- 239000006041 probiotic Substances 0.000 title claims abstract description 53
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 53
- 238000001035 drying Methods 0.000 claims abstract description 19
- 238000005469 granulation Methods 0.000 claims abstract description 13
- 230000003179 granulation Effects 0.000 claims abstract description 13
- 239000000969 carrier Substances 0.000 claims abstract description 5
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 84
- 239000007788 liquid Substances 0.000 claims description 25
- 239000002609 media Substances 0.000 claims description 25
- 229910052760 oxygen Inorganic materials 0.000 claims description 16
- 239000001301 oxygen Substances 0.000 claims description 16
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims description 16
- 239000002245 particle Substances 0.000 claims description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 13
- 239000008103 glucose Substances 0.000 claims description 13
- 229940041514 Candida albicans extract Drugs 0.000 claims description 11
- 235000015097 nutrients Nutrition 0.000 claims description 11
- 239000012138 yeast extract Substances 0.000 claims description 11
- 230000001580 bacterial Effects 0.000 claims description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 8
- OAICVXFJPJFONN-UHFFFAOYSA-N phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052698 phosphorus Inorganic materials 0.000 claims description 8
- 239000011574 phosphorus Substances 0.000 claims description 8
- 235000003534 Saccharomyces carlsbergensis Nutrition 0.000 claims description 7
- 229940081969 Saccharomyces cerevisiae Drugs 0.000 claims description 7
- 239000001963 growth media Substances 0.000 claims description 7
- HDTRYLNUVZCQOY-LIZSDCNHSA-N 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 6
- 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 6
- 239000000203 mixture Substances 0.000 claims description 6
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 6
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 6
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims description 5
- 229920002472 Starch Polymers 0.000 claims description 5
- 239000000654 additive Substances 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims description 5
- 239000008108 microcrystalline cellulose Substances 0.000 claims description 5
- 229940016286 microcrystalline cellulose Drugs 0.000 claims description 5
- 235000019698 starch Nutrition 0.000 claims description 5
- 239000008107 starch Substances 0.000 claims description 5
- 210000000481 Breast Anatomy 0.000 claims description 4
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 claims description 4
- 230000000996 additive Effects 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L cacl2 Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- 230000001808 coupling Effects 0.000 claims description 4
- 238000010168 coupling process Methods 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 238000001125 extrusion Methods 0.000 claims description 4
- 239000006052 feed supplement Substances 0.000 claims description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 4
- 229920000053 polysorbate 80 Polymers 0.000 claims description 4
- 239000003223 protective agent Substances 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- 238000005563 spheronization Methods 0.000 claims description 4
- 238000003828 vacuum filtration Methods 0.000 claims description 4
- 239000002002 slurry Substances 0.000 claims description 3
- 229920001353 Dextrin Polymers 0.000 claims description 2
- 239000004375 Dextrin Substances 0.000 claims description 2
- 238000007792 addition Methods 0.000 claims description 2
- 235000019425 dextrin Nutrition 0.000 claims description 2
- 241000048284 Potato virus P Species 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 24
- 238000000034 method Methods 0.000 abstract description 11
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000003860 storage Methods 0.000 abstract description 3
- 230000004083 survival Effects 0.000 abstract description 3
- 239000003674 animal food additive Substances 0.000 abstract description 2
- 238000000855 fermentation Methods 0.000 abstract description 2
- 230000004151 fermentation Effects 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 230000001681 protective Effects 0.000 abstract description 2
- 238000004140 cleaning Methods 0.000 abstract 1
- 241000282898 Sus scrofa Species 0.000 description 8
- 239000000843 powder Substances 0.000 description 6
- 229940045184 Malt extract Drugs 0.000 description 5
- 210000004027 cells Anatomy 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- 230000000968 intestinal Effects 0.000 description 4
- 230000001954 sterilising Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 241000186660 Lactobacillus Species 0.000 description 3
- 229940039696 Lactobacillus Drugs 0.000 description 3
- 210000002966 Serum Anatomy 0.000 description 3
- 230000003115 biocidal Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 210000003736 Gastrointestinal Contents Anatomy 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000001488 breeding Effects 0.000 description 2
- 239000006047 digesta Substances 0.000 description 2
- 230000002183 duodenal Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 230000001603 reducing Effects 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 229940064005 Antibiotic throat preparations Drugs 0.000 description 1
- 229940083879 Antibiotics FOR TREATMENT OF HEMORRHOIDS AND ANAL FISSURES FOR TOPICAL USE Drugs 0.000 description 1
- 229940042052 Antibiotics for systemic use Drugs 0.000 description 1
- 229940042786 Antitubercular Antibiotics Drugs 0.000 description 1
- 210000001198 Duodenum Anatomy 0.000 description 1
- 210000001035 Gastrointestinal Tract Anatomy 0.000 description 1
- 229940093922 Gynecological Antibiotics Drugs 0.000 description 1
- 210000003405 Ileum Anatomy 0.000 description 1
- 210000004347 Intestinal Mucosa Anatomy 0.000 description 1
- 210000001630 Jejunum Anatomy 0.000 description 1
- 210000004379 Membranes Anatomy 0.000 description 1
- 210000004400 Mucous Membrane Anatomy 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L Sodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
- 229940024982 Topical Antifungal Antibiotics Drugs 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 235000019728 animal nutrition Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003078 antioxidant Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 244000052616 bacterial pathogens Species 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002496 gastric Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 244000005709 gut microbiome Species 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 229940079866 intestinal antibiotics Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002906 microbiologic Effects 0.000 description 1
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- 229940005935 ophthalmologic Antibiotics Drugs 0.000 description 1
- 230000003204 osmotic Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 235000021401 pellet diet Nutrition 0.000 description 1
- 235000020245 plant milk Nutrition 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- 235000015921 sodium selenite Nutrition 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 210000004215 spores Anatomy 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- HNBDQABBWNOTRU-UHFFFAOYSA-N thalline Chemical compound C1=CC=[Tl]C=C1 HNBDQABBWNOTRU-UHFFFAOYSA-N 0.000 description 1
Abstract
The present invention discloses a kind of feeding thermostable microcapsule yeast probiotics preparation and preparation method thereof and application, belongs to field of feed additive technology.The present invention first through fermentation, centrifugal, cleaning, add carrier and protective material, the round as a ball granulation of extruding, drying and other steps and obtain feeding thermostable microcapsule yeast probiotics preparation.Prepare feeding thermostable microcapsule yeast probiotics preparation granular size by the method comparatively even, storage stability is good, has better thermotolerance, can tolerate the processing conditions of high temperature, high humidity etc. in feed granulating process, has comparatively high-survival rate; Survival rate remains on more than 90%, and number of live bacteria of probiotics reaches 5 × 10
9more than cfu/g.
Description
Technical field
The invention belongs to feed additive field, relate to a kind of probiotic bacterium, be specifically related to a kind of feeding thermostable microcapsule yeast probiotics preparation and preparation method thereof and application.
Background technology
From the fifties in last century, along with microbiotic is widely used as fodder additives, fodder antibiotics is while promotion growth of animals or poultry, its drawback manifests day by day, lacking of proper care as caused animal gastrointestinal tract normal microflora, producing resistance, drug residue and reduction livestock product quality etc., thus cause potential harm to human health.These problems have caused global extensive concern.Within 2002, the World Health Organization has proposed the global principle reducing antibiotic usage in edible animal, and European Union, Japan and Korea S forbid using microbiotic in feed respectively at 2006 and 2008.
At present, in the feed of generally acknowledging both at home and abroad, Substitutes For Antibiotic comprises the 6 large classes such as probiotics, antibacterial peptide, zymin, herbal medicine, plant milk extract and souring agent.The balance of probiotic bacterium (probiotics) adjustable gastric intestinal microflora, improves breeding performonce fo animals and has become one of Animal nutrition study hotspot.
It is the microorganism lived that FAO/WHO defines probiotic bacterium, when taking in sufficient amount, plays effect good for health to host.The research of domestic and international probiotic bacterium mainly concentrates on lactobacillus, genus bacillus, bifidus bacillus and the kind such as live yeast.The research of China's feed microbe preparation is suitable for the eighties in 20th century, has obvious gap compared with same kind of products at abroad.Main manifestations is: (1) product category is few, and form is single; (2) host specificity is not worked poor by force, with breeding way; (3) production technique falls behind, and strain activity is low, poor heat stability.Probiotics preparation can be divided into liquid preparation and solid preparation.Liquid preparation is by traditional fermentation culture mode, finally obtains probiotics fermention liquid as product.Solid preparation is generally probiotic bacterium after propagation, by means such as freeze-drying, spraying dry or embeddings, liquid preparation is processed into solid-state form further.Compare, solid formulation comparatively liquid preparation is more convenient for transporting, storing and application.Lyophilization is consuming time, apparatus expensive, and energy consumption is high.Although the spray-drying process time is short, viable bacteria rate of loss is high.
Feeding micro-ecological preparation requires to possess better stability in storage, can to tolerate in the feed granulating course of processing particular surroundingss such as high temperature, high pressure, high humidity simultaneously.Current domestic probiotics mostly is freeze-dried type pulvis, after granulated feed and expanded pellet diet add Tiny ecosystem pulvis in the course of processing, the spore inactivation of 10% ~ 30% can be caused under hot and humid condition, faecalis inactivation reaches more than 90%, Bacterium lacticum is almost all killed, and the viable cell loss of yeast also reaches more than 90%.Therefore need to strengthen the research to probiotic bacterium formulation, improve viable bacteria concentration, improve viable bacteria in probiotics preparation and, to the tolerance of poor environment, extend product preservation period.
Summary of the invention
For overcoming the shortcoming and defect of above-mentioned prior art, primary and foremost purpose of the present invention is to provide a strain with pig enteric microorganism for screening object, be separated the yeast saccharomyces cerevisiae (Saccharomycescerevisiae) obtained, apply in feed manufacturing or preparation of granulating for yeast probiotic bacterium and lay the foundation.
Another object of the present invention is to provide the preparation method utilizing above-mentioned yeast saccharomyces cerevisiae to prepare feeding thermostable microcapsule yeast probiotics preparation, hot and humid environment in the feed granulating course of processing can be tolerated, improve probiotics preparation survival volume in feed manufacturing.
Another object of the present invention is to the feeding thermostable microcapsule yeast probiotics preparation providing above-mentioned preparation method to obtain.
Another object of the present invention is the application providing the feeding high-temperature resistant particle preparation of above-mentioned yeast saccharomyces cerevisiae.
Object of the present invention is achieved through the following technical solutions:
One Accharomyces cerevisiae (Saccharomycescerevisiae), name is called SaccharomycescerevisiaeNKY1, China typical culture collection center (CCTCC) is preserved on December 19th, 2013, preservation address: China. Wuhan. Wuhan University, deposit number is CCTCCNO:M2013677.
The culture condition of described yeast saccharomyces cerevisiae for inclined-plane yeast saccharomyces cerevisiae bacterial classification is seeded in yeast culture medium, pH5.0, culture temperature is 30 DEG C, and 150 ~ 250rpm shaking culture, 24 ~ 36h, namely completes the amplification of yeast saccharomyces cerevisiae, obtains yeast saccharomyces cerevisiae nutrient solution;
Described yeast culture medium is preferably malt juice liquid medium or YPD substratum, and is not limited thereto substratum;
Described malt juice liquid medium is prepared preferably by following steps: malt extract powder 130g/L, saves backup after 121 DEG C of autoclaving 20min;
Described YPD substratum is prepared preferably by following steps: dissolve 10g yeast extract paste, 20g peptone, in 900mL water, after 121 DEG C of autoclaving 20min, adds 100mL200g/L glucose; Wherein, glucose needs filtration sterilization or 115 DEG C of sterilizing 15min;
Utilize above-mentioned yeast saccharomyces cerevisiae to prepare a preparation method for feeding thermostable microcapsule yeast probiotics preparation, comprise the steps:
(1) yeast slurry is prepared
Inclined-plane yeast saccharomyces cerevisiae bacterial classification is seeded in yeast culture medium, pH5.0, culture temperature is 30 ± 1 DEG C, shaking culture 24 ~ 36h, obtain liquid seeds nutrient solution, liquid seeds nutrient solution is seeded in fermention medium in the ratio of 5 ~ 10% (v/v), pH5.0 ~ 5.5, temperature 30 ± 1 DEG C, fed-batch mode is adopted to supplement suitable carbon source, nitrogenous source and phosphorus source, cultivate 24 ~ 36h, obtain yeast saccharomyces cerevisiae breast, in yeast-lactic, add 2 ~ 3g/LNaCl, 3 ~ 4g/LMgCl
26H
2after O and 1 ~ 2g/L tween-80, vacuum filtration is adopted to obtain yeast saccharomyces cerevisiae mud;
(2) round as a ball granulation is extruded
The thermal protecting agent of the carrier of 60 ~ 65 mass parts, the Microcrystalline Cellulose of 15 ~ 20 mass parts and 50 ~ 65 mass parts is added in yeast saccharomyces cerevisiae mud 1000 mass parts obtained in step (1), add the antisticking agent of 1 ~ 2 mass parts, fully be uniformly mixed, mixture is adopted the round as a ball granulation mode plasmid of extruding, obtain diameter 1.6 ~ 2mm spherical particle;
(3) dry: the spherical particle obtained in step (2) to be carried out drying, obtains feeding thermostable microcapsule yeast probiotics preparation.
Yeast culture medium described in step (1) is preferably malt juice liquid medium or YPD substratum, and is not limited thereto substratum;
Described malt juice liquid medium is prepared preferably by following steps: malt extract powder 130g/L, saves backup after 121 DEG C of autoclaving 20min;
Described YPD substratum is prepared preferably by following steps: dissolve 10g yeast extract paste, 20g peptone, in 900mL water, after 121 DEG C of autoclaving 20min, adds 100mL200g/L glucose; When preparation is dull and stereotyped, 15 ~ 20g/L agar powder need be added before autoclaving; Wherein, glucose needs filtration sterilization or 115 DEG C of sterilizing 15min;
Fermention medium described in step (1) is prepared preferably by following steps: glucose 10 ~ 20g/L, (NH
4)
2sO
44 ~ 6g/L, yeast extract paste 5 ~ 10g/L, K
2sO
43 ~ 4g/L, KH
2pO
45 ~ 8g/L, MgSO
47H
2o1 ~ 2g/L, CaCl
22H
2o0.2 ~ 0.3g/L, bubble enemy's defoamer (V/V) 0.05 ~ 0.1%; For subsequent use after 121 DEG C of autoclaving 20 ~ 30min;
Fermention medium described in step (1) is prepared more particularly by following steps: glucose 10g/L, (NH
4)
2sO
45g/L, yeast extract paste 5g/L, K
2sO
43.6g/L, KH
2pO
46g/L, MgSO
47H
2o1.5g/L, CaCl
22H
2o0.2g/L, bubble enemy's defoamer (V/V) 0.06%; For subsequent use after 121 DEG C of autoclaving 20 ~ 30min;
Fed-batch mode described in step (1) supplement suitable carbon source, nitrogenous source and phosphorus source step as follows: start to carry out feed supplement with supplemented medium when the dissolved oxygen of fermented liquid rises, setting dissolved oxygen level is 15 ~ 25%, by control of additive raw material and dissolved oxygen level coupling, according to dissolved oxygen state automatic feeding; Feeding volume is original volume 10 ~ 15%;
Described supplemented medium is prepared preferably by following steps: glucose 300 ~ 500g/L, (NH
4)
2sO
45 ~ 10g/L, yeast extract paste 10 ~ 20g/L, KH
2pO
440 ~ 50g/L, MgSO
47H
2o5 ~ 10g/L, bubble enemy's defoamer (V/V) 0.05 ~ 0.1%; For subsequent use after 121 DEG C of autoclaving 20 ~ 30min;
Described supplemented medium is prepared more particularly by following steps: glucose 500g/L, (NH
4)
2sO
47.5g/L, yeast extract paste 15g/L, KH
2pO
445g/L, MgSO
47H
2o7.5g/L, bubble enemy's defoamer (V/V) 0.06%; For subsequent use after 121 DEG C of autoclaving 20 ~ 30min;
Carrier described in step (2) is preferably starch or dextrin;
Thermal protecting agent described in step (2) is preferably trehalose;
Antisticking agent described in step (2) is preferably the one in PEG4000 or PVP;
The preferred granulation device of extruding round as a ball granulation mode described in step (2) is extrusion spheronization machine, and orifice plate diameter is preferably 2mm;
Drying described in step (3), preferably carries out drying with fluidized-bed or ebullated bed; Its drying temperature is not higher than 50 DEG C, is more preferably 45 DEG C, and the whole moisture control of spherical particle, within 8%, obtains feeding thermostable microcapsule yeast probiotics preparation.
Described feeding thermostable microcapsule yeast probiotics preparation is prepared by above-mentioned preparation method.
In described feeding thermostable microcapsule yeast probiotics preparation, every gram is 5 × 10 containing viable count
9more than cfu/g.
The described application of feeding thermostable microcapsule yeast probiotics preparation on feed, addition is that feed per ton adds the above-mentioned feeding thermostable microcapsule yeast probiotics preparation of 2 ~ 4kg.
Mechanism of the present invention is: trehalose can form unique protective membrane at cell surface under the severe environmental conditions such as high temperature, high and cold, high osmotic pressure and dry dehydration; protected protein matter molecule unchangeability inactivation effectively, thus the vital process of the body that sustains life and biological characteristic.Viable yeast embedding is sealed up for safekeeping by microcapsule agglomeration technique becomes a kind of solia particle product in a kind of minigel, can reduce yeast cell and extraneous contact area, reduce yeast cell mortality ratio under the high temperature conditions.
The present invention compared with prior art, has following advantages and effect:
(1) the present invention is separated and obtains a strain yeast belong probiotic bacterium from health pig intestinal contents, through being accredited as yeast saccharomyces cerevisiae (Saccharomycescerevisiae), and this bacterial strain does not possess high temperature resistant activity, more even by adopting micro capsule technology to prepare active yeast preparation granules size, storage stability is good, there is better thermotolerance, hot and humid environment in feed granulating process can be tolerated, there is comparatively high-survival rate.
(2) for yeast class easy in inactivation probiotic bacterium, the approach solving deactivation prob adopts microcapsule embedded technology to embed viable bacteria body exactly, it is made to separate with extraneous poor environment, after special processing (absorption of microballoon capsule, Cotton seeds etc.), active bacteria formulation stability is improved largely, and in tolerance feed granulating process, after hot and humid high pressure, major part is survived.Meanwhile, the probiotic bacterium after microcapsule embedded forms the molecule of approximate sphericity, is easy to add in feed, is convenient to dispersing and mixing even.
Accompanying drawing explanation
Fig. 1 is the growth figure that Saccharomyces Cerevisiae in S accharomycescerevisiaeNKY1 cultivates 36h on YPD culture plate.
Fig. 2 is the particle figure of the feeding thermostable microcapsule yeast probiotics preparation that the present invention prepares.
Fig. 3 is the comparison diagram that in embodiment 2, import reference substance and the thermotolerance of self-control sample detect the thalline survival rate of pure motility rate.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
The experimental technique of unreceipted specific experiment condition in the following example, usually conveniently experiment condition.
Starch, Microcrystalline Cellulose, PEG4000, PVP and trehalose are delicatessen food level product;
Malt juice liquid medium is prepared as follows: malt extract powder 130g/L, saves backup after 121 DEG C of autoclaving 20min;
Fermention medium is prepared as follows: glucose 10g/L, (NH
4)
2sO
45g/L, yeast extract paste 5g/L, K
2sO
43.6g/L, KH
2pO
46g/L, MgSO
47H
2o1.5g/L, CaCl
22H
2o0.2g/L, bubble enemy's defoamer (V/V) 0.06%; For subsequent use after 121 DEG C of autoclaving 20 ~ 30min.
Embodiment 1
Utilize Modern microbiological isolation technique, be separated from health pig intestinal contents and obtain a strain yeast belong probiotic bacterium.Separating step is as follows: collect Digesta samples from just butchering 14 age in days sodium selenite enteron aisles under aseptic condition, being placed in sterile glass vials, under condition of ice bath, be transferred to laboratory for subsequent use, then aseptically getting a little Digesta samples is seeded in YPD substratum, 30 DEG C, 200rpm shaking culture 36h, coating YPD culture plate after dilution, by colony morphological observation and in conjunction with simple microscope microscopy, the line of picking list bacterium colony is separated, screening obtains a collection of yeast cell bacterial strain, by each strain growth performance of shake-flask culture Method compare, screening obtains the excellent yeast strain of a strain proterties, again through 18SrDNA Molecular Identification, this bacterial strain 18SrDNA sequence adopts BLAST instrument and online database comparison through order-checking, this yeast and SaccharomycescerevisiaestrainFJU-YS5 (GenBank:EF153845.1) bacterial strain homology are up to 99%, confirm that this yeast is yeast saccharomyces cerevisiae (Saccharomycescerevisiae) (see Fig. 1).
The culture condition of described yeast saccharomyces cerevisiae is for be seeded in yeast culture medium by inclined-plane yeast saccharomyces cerevisiae bacterial classification, and pH5.0, culture temperature is 30 DEG C, and 150 ~ 250rpm shaking culture, 24 ~ 36h, namely completes the amplification of yeast saccharomyces cerevisiae, obtains yeast saccharomyces cerevisiae nutrient solution.
18SrDNA sequence is as shown in SEQIDNo.1.
This yeast saccharomyces cerevisiae (Saccharomycescerevisiae), name is called SaccharomycescerevisiaeNKY1, China typical culture collection center (CCTCC) is preserved on December 19th, 2013, preservation address: China. Wuhan. Wuhan University, deposit number is CCTCCNO:M2013677.
The preparation of the feeding thermostable microcapsule yeast probiotics preparation of embodiment 2 one kinds
(1) yeast saccharomyces cerevisiae mud is prepared
Inclined-plane yeast saccharomyces cerevisiae bacterial classification is seeded to malt juice liquid medium (malt extract powder 130g/L, save backup after 121 DEG C of autoclaving 20min) in, pH5.0, culture temperature is 30 DEG C, shaking culture 24h, obtain liquid seeds nutrient solution, liquid seeds nutrient solution is seeded in 20L fermention medium in 5% (v/v) ratio, pH5.5, temperature 30 DEG C, adopts fed-batch mode to supplement suitable carbon source, nitrogenous source and phosphorus source, cultivates 36h, acquisition yeast saccharomyces cerevisiae breast, adds 2.5g/LNaCl, 4g/LMgCl in yeast-lactic
26H
2after O and 1.5g/L tween-80, vacuum filtration is adopted to obtain yeast saccharomyces cerevisiae mud;
Described fed-batch mode supplement suitable carbon source, nitrogenous source and phosphorus source step as follows: start to carry out feed supplement with supplemented medium when the dissolved oxygen of fermented liquid rises, setting dissolved oxygen level is 15 ~ 25%, by control of additive raw material and dissolved oxygen level coupling, according to dissolved oxygen state automatic feeding; Feeding volume is original volume 10 ~ 15%.
Described supplemented medium is prepared as follows: glucose 500g/L, (NH
4)
2sO
47.5g/L, yeast extract paste 15g/L, KH
2pO
445g/L, MgSO
47H
2o7.5g/L, bubble enemy's defoamer (V/V) 0.06%; For subsequent use after 121 DEG C of autoclaving 20 ~ 30min.
(2) round as a ball granulation is extruded
Take yeast saccharomyces cerevisiae mud 1kg prepared by step (1), add starch 65g, Microcrystalline Cellulose 20g, PEG4000 (Macrogol 4000) 1g and trehalose 65g, fully be uniformly mixed, extrusion spheronization machine (machine institute of East China University of Science) is adopted to carry out extruding round as a ball granulation, orifice plate diameter selects 2mm, and obtaining diameter is 1.6 ~ 2mm spherical particle.
(3) dry, obtain feeding thermostable microcapsule yeast probiotics preparation.
Drying described in step (3), for carrying out drying with fluidized-bed (Jiangsu Fanqun Drying Equipment Factory Co., Ltd., FG-3 series); Its drying temperature is 45 DEG C, and time of drying is 3h, and the whole moisture control of spherical particle, within 8%, obtains feeding thermostable microcapsule yeast probiotics preparation, as shown in Figure 2.
(4) thermotolerance detects
Simulation feed granulating hot and humid environment, different time sections yeast particle surviving rate is investigated under 95 DEG C of saturated steam conditions, employing dilution plate counts, contrast with external import Thermotolerant yeast of the same type (hundred good fortune bacterium, purchased from this good fortune of method national music (Mingguang City) company limited) simultaneously.The results are shown in Figure 3.The feeding thermostable microcapsule yeast probiotics preparation of above-mentioned preparation is self-control sample, and gained sample viable count is 7.3 × 10
9cfu/g.
As can be seen from Figure 3, during 3min, self-control sample viable count reach 60% of raw sample, and the viable count of reference substance be only former reference substance less than 50%; During 10min, reference substance is complete deactivation then, and makes sample by oneself and still have the bacterium of 10% to survive, therefore, feeding thermostable microcapsule yeast probiotics preparation prepared by the application, has fine thermotolerance, hot and humid environment in feed granulating process can be tolerated, there is comparatively high-survival rate.
The preparation of the feeding high-temperature resistant particle preparation of embodiment 3 one kinds of yeast saccharomyces cerevisiaes
(1) yeast saccharomyces cerevisiae mud is prepared
Inclined-plane yeast saccharomyces cerevisiae bacterial classification is seeded to malt juice liquid medium (malt extract powder 130g/L, save backup after 121 DEG C of autoclaving 20min) in, pH5.0, culture temperature is 30 DEG C, shaking culture 36h, obtain liquid seeds nutrient solution, liquid seeds nutrient solution is seeded in 20L fermention medium in the ratio of 10% (v/v), pH5.0, temperature 30 DEG C, adopts fed-batch mode to supplement suitable carbon source, nitrogenous source and phosphorus source, cultivates 24h, acquisition yeast saccharomyces cerevisiae breast, adds 2.5g/LNaCl, 4g/LMgCl in yeast-lactic
26H
2after O and 1.5g/L tween-80, vacuum filtration is adopted to obtain yeast saccharomyces cerevisiae mud;
Described fed-batch mode supplement suitable carbon source, nitrogenous source and phosphorus source step as follows: start to carry out feed supplement with supplemented medium when the dissolved oxygen of fermented liquid rises, setting dissolved oxygen level is 15 ~ 25%, by control of additive raw material and dissolved oxygen level coupling, according to dissolved oxygen state automatic feeding; Feeding volume is original volume 10 ~ 15%.
Described supplemented medium is prepared as follows: glucose 500g/L, (NH
4)
2sO
47.5g/L, yeast extract paste 15g/L, KH
2pO
445g/L, MgSO
47H
2o7.5g/L, bubble enemy's defoamer (V/V) 0.06%; For subsequent use after 121 DEG C of autoclaving 20 ~ 30min.
(2) round as a ball granulation is extruded
Take yeast slurry 1kg prepared by step (1), add starch 60g, Microcrystalline Cellulose 15g, PVP (Polyvinylpyrrolidone, polyvinylpyrrolidone) 2g and trehalose 50g, fully be uniformly mixed, extrusion spheronization machine is adopted to carry out extruding round as a ball granulation, orifice plate diameter selects 2mm, and obtaining diameter is 1.6 ~ 2mm spherical particle.
(3) dry, obtain feeding thermostable microcapsule yeast probiotics preparation.
Drying described in step (3), for carrying out drying with ebullated bed (Jiangsu Fanqun Drying Equipment Factory Co., Ltd., FG-3 series); Its drying temperature is 45 DEG C, and time of drying is 3h, and the whole moisture control of spherical particle, within 8%, obtains feeding thermostable microcapsule yeast probiotics preparation, as shown in Figure 2.
(4) thermotolerance detects
Simulation feed granulating hot and humid environment, different time sections yeast particle surviving rate is investigated under 95 DEG C of saturated steam conditions, employing dilution plate counts, contrast with external import Thermotolerant yeast of the same type (hundred good fortune bacterium, purchased from this good fortune of method national music (Mingguang City) company limited) simultaneously.The feeding thermostable microcapsule yeast probiotics preparation of above-mentioned preparation is self-control sample, and gained sample viable count is 8.0 × 10
9cfu/g.
Embodiment 4
The ternary that 14 ages in days wean are chosen in test is mixed piglet, is divided into 2 treatment group at random, each process 4 repetitions by body weight and sex, often repetition 6 piglets, and male and female half and half, tests 3 weeks full phases.Control group is the pig feed not adding yeast, and test group is that the pig of adding feeding thermostable microcapsule yeast probiotics preparation by 3g/kg is raised
Material, tests 3 weeks full phases, and research microcapsule live yeast is on the impact of the aspects such as early-weaned piglets growth performance, intestinal growth and mucosa-immune.Test-results shows: in growth performance: after weaning 3 weeks, microcapsule live yeast group feed conversion rate improves 9.29% (P < 0.05) than control group, serum urea nitrogen (SUN) content reduces 18.84% (P < 0.05) than control group, shows that microcapsule live yeast can improve Growth Performance of Weaning Piglets.In Antioxidant Indexes: in piglet serum, SOD is active, microcapsule live yeast group is significantly higher than control group, and MDA content in serum, microcapsule live yeast group, significantly lower than control group (P < 0.05), shows that microcapsule live yeast can improve weanling pig resistance of oxidation.In intestinal mucosa development: the Duodenal villi height of the 3rd week interpolation microcapsule live yeast group that wean is all higher than control group, microcapsule live yeast group improves 8.36% (P < 0.05) than control group, microcapsule live yeast group on the piglet duodenal recess degree of depth to affect difference remarkable, yeast treatment group improves 3.14% (P > 0.05) than contrast group respectively.But duodenum and jejunum villi height/Crypt depth ratio microcapsule live yeast group improve 5.15% (P < 0.05) and 8.57% (P < 0.05) than control group respectively, show that microcapsule live yeast can promote intestinal growth.In mucosa-immune: IgA, IgG content in the 3rd week mucous membrane of small intestine that wean, microcapsule live yeast group is significantly higher than control group (P < 0.05), shows that microcapsule live yeast can improve weanling pig mucosa-immune ability.Add microcapsule live yeast and can improve probiotics genera lactobacillus and yeast quantity in piglet ileum, and reduce intestinal bacteria quantity.Ileal contents yeast count with microcapsule live yeast group comparatively control group improve 11.98% (P < 0.05); Lactobacillus quantity microcapsule live yeast group comparatively control group improves 2.29% (P > 0.05); And intestinal bacteria quantity comparatively control group reduce 22.50% (P < 0.05).
The proportioning of table 1 pig feed and nutrient composition content table
Preblend * is Xinnandu Feed Science & Technology Co., Ltd., Guangdong's " Baobaole ".
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (9)
1. an Accharomyces cerevisiae (Saccharomycescerevisiae), it is characterized in that: this yeast saccharomyces cerevisiae is SaccharomycescerevisiaeNKY1, China typical culture collection center is preserved on December 19th, 2013, be called for short CCTCC, deposit number is CCTCCNO:M2013677.
2. utilize yeast saccharomyces cerevisiae described in claim 1 to prepare a preparation method for feeding thermostable microcapsule yeast probiotics preparation, it is characterized in that comprising the steps:
(1) yeast slurry is prepared
Inclined-plane yeast saccharomyces cerevisiae bacterial classification is seeded in yeast culture medium, pH5.0, culture temperature is 30 ± 1 DEG C, shaking culture 24 ~ 36h, obtain liquid seeds nutrient solution, by liquid seeds nutrient solution by volume 5 ~ 10% ratio be seeded in fermention medium, pH5.0 ~ 5.5, temperature 30 ± 1 DEG C, fed-batch mode is adopted to supplement suitable carbon source, nitrogenous source and phosphorus source, cultivate 24 ~ 36h, obtain yeast saccharomyces cerevisiae breast, in yeast-lactic, add 2 ~ 3g/LNaCl, 3 ~ 4g/LMgCl
26H
2after O and 1 ~ 2g/L tween-80, vacuum filtration is adopted to obtain yeast saccharomyces cerevisiae mud;
(2) round as a ball granulation is extruded
The thermal protecting agent of the carrier of 60 ~ 65 mass parts, the Microcrystalline Cellulose of 15 ~ 20 mass parts and 50 ~ 65 mass parts is added in yeast saccharomyces cerevisiae mud 1000 mass parts obtained in step (1), add the antisticking agent of 1 ~ 2 mass parts, fully be uniformly mixed, mixture is adopted the round as a ball granulation mode plasmid of extruding, obtain diameter 1.6 ~ 2mm spherical particle;
(3) dry: the spherical particle obtained in step (2) to be carried out drying, obtains feeding thermostable microcapsule yeast probiotics preparation;
Yeast culture medium described in step (1) is malt juice liquid medium or YPD substratum;
Fermention medium described in step (1) is prepared as follows: glucose 10 ~ 20g/L, (NH
4)
2sO
44 ~ 6g/L, yeast extract paste 5 ~ 10g/L, K
2sO
43 ~ 4g/L, KH
2pO
45 ~ 8g/L, MgSO
47H
2o1 ~ 2g/L, CaCl
22H
2o0.2 ~ 0.3g/L, bubble enemy defoamer 0.05 ~ 0.1%; For subsequent use after 121 DEG C of autoclaving 20 ~ 30min; Described percentage ratio is percent by volume.
3. preparation method according to claim 2, it is characterized in that: the fed-batch mode described in step (1) supplement suitable carbon source, nitrogenous source and phosphorus source step as follows: start to carry out feed supplement with supplemented medium when the dissolved oxygen of fermented liquid rises, setting dissolved oxygen level is 15 ~ 25%, by control of additive raw material and dissolved oxygen level coupling, according to dissolved oxygen state automatic feeding; Feeding volume is original volume 10 ~ 15%.
4. preparation method according to claim 3, is characterized in that: described supplemented medium is prepared as follows: glucose 300 ~ 500g/L, (NH
4)
2sO
45 ~ 10g/L, yeast extract paste 10 ~ 20g/L, KH
2pO
440 ~ 50g/L, MgSO
47H
2o5 ~ 10g/L, bubble enemy defoamer 0.05 ~ 0.1%; For subsequent use after 121 DEG C of autoclaving 20 ~ 30min; Described percentage ratio is percent by volume.
5. preparation method according to claim 2, is characterized in that: the carrier described in step (2) is starch or dextrin;
Thermal protecting agent described in step (2) is trehalose;
Antisticking agent described in step (2) is the one in PEG4000 or PVP.
6. preparation method according to claim 2, is characterized in that: the granulation device of the round as a ball granulation mode of the extruding described in step (2) is extrusion spheronization machine, and orifice plate diameter is 2mm;
Drying described in step (3), carries out drying with fluidized-bed or ebullated bed; Its drying temperature is not higher than 50 DEG C, and the whole moisture control of spherical particle, within 8%, obtains feeding thermostable microcapsule yeast probiotics preparation.
7. a feeding thermostable microcapsule yeast probiotics preparation, be is characterized in that: prepared by the preparation method described in any one of claim 2 ~ 6.
8. feeding thermostable microcapsule yeast probiotics preparation according to claim 7, is characterized in that: in described feeding thermostable microcapsule yeast probiotics preparation, every gram is 5 × 10 containing viable count
9more than cfu/g.
9. the feeding thermostable microcapsule yeast probiotics preparation described in claim 7 or 8 is preparing the application in feed, it is characterized in that: addition is the feeding thermostable microcapsule yeast probiotics preparation described in feed interpolation per ton 2 ~ 4kg claim 7 or 8.
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| CN112779171B (en) * | 2021-01-12 | 2022-09-30 | 福建省农业科学院农业工程技术研究所 | Saccharomyces cerevisiae direct-vat-set microbial inoculum for improving heat drying survival rate and preparation method thereof |
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Effective date of registration: 20180829 Address after: 512002 Shaoguan, Wengyuan, Guangdong Guandu economic development test area of the Dragon Industrial Park Patentee after: Wengyuan Nandu new feed Technology Co., Ltd. Address before: 510640 1 Tianhe District Dafeng street, Guangzhou, Guangdong Co-patentee before: Xinnandu Feed Science & Technology Co., Ltd., Guangdong Patentee before: Animal science institute of Guangdong Academy of Agricultural Sciences |