CN102077904A - Method for preparing biological feed additive of lactobacillus - Google Patents
Method for preparing biological feed additive of lactobacillus Download PDFInfo
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- CN102077904A CN102077904A CN201010122147XA CN201010122147A CN102077904A CN 102077904 A CN102077904 A CN 102077904A CN 201010122147X A CN201010122147X A CN 201010122147XA CN 201010122147 A CN201010122147 A CN 201010122147A CN 102077904 A CN102077904 A CN 102077904A
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- 241000186660 Lactobacillus Species 0.000 title claims abstract description 22
- 229940039696 lactobacillus Drugs 0.000 title claims abstract description 22
- 239000003674 animal food additive Substances 0.000 title claims abstract description 13
- 238000000034 method Methods 0.000 title abstract 2
- 241000894006 Bacteria Species 0.000 claims abstract description 32
- 238000002360 preparation method Methods 0.000 claims abstract description 16
- 238000000855 fermentation Methods 0.000 claims abstract description 9
- 230000004151 fermentation Effects 0.000 claims abstract description 9
- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 5
- 238000004108 freeze drying Methods 0.000 claims description 24
- 230000004913 activation Effects 0.000 claims description 18
- 239000008367 deionised water Substances 0.000 claims description 18
- 229910021641 deionized water Inorganic materials 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 16
- 241000186717 Lactobacillus acetotolerans Species 0.000 claims description 14
- 240000001929 Lactobacillus brevis Species 0.000 claims description 14
- 235000013957 Lactobacillus brevis Nutrition 0.000 claims description 14
- 240000006024 Lactobacillus plantarum Species 0.000 claims description 14
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims description 14
- 229940072205 lactobacillus plantarum Drugs 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 12
- 239000012530 fluid Substances 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 238000004659 sterilization and disinfection Methods 0.000 claims description 9
- 235000014655 lactic acid Nutrition 0.000 claims description 8
- 239000004310 lactic acid Substances 0.000 claims description 8
- 244000199866 Lactobacillus casei Species 0.000 claims description 7
- 235000013958 Lactobacillus casei Nutrition 0.000 claims description 7
- 241000186673 Lactobacillus delbrueckii Species 0.000 claims description 7
- 241000186684 Lactobacillus pentosus Species 0.000 claims description 7
- 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 7
- 244000057717 Streptococcus lactis Species 0.000 claims description 7
- 235000014897 Streptococcus lactis Nutrition 0.000 claims description 7
- 229940017800 lactobacillus casei Drugs 0.000 claims description 7
- 239000008101 lactose Substances 0.000 claims description 7
- 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 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 230000003068 static effect Effects 0.000 claims description 6
- 235000015193 tomato juice Nutrition 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 4
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- 235000011187 glycerol Nutrition 0.000 claims description 3
- 238000005304 joining Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 241000894007 species Species 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims 1
- 241000193830 Bacillus <bacterium> Species 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 241001465754 Metazoa Species 0.000 abstract description 3
- 239000000654 additive Substances 0.000 abstract description 3
- 230000000968 intestinal effect Effects 0.000 abstract description 3
- 241000589876 Campylobacter Species 0.000 abstract description 2
- 208000037386 Typhoid Diseases 0.000 abstract description 2
- 230000000996 additive effect Effects 0.000 abstract description 2
- 230000008485 antagonism Effects 0.000 abstract description 2
- 244000052616 bacterial pathogen Species 0.000 abstract description 2
- 230000008827 biological function Effects 0.000 abstract description 2
- 238000001035 drying Methods 0.000 abstract description 2
- 239000002158 endotoxin Substances 0.000 abstract description 2
- 230000036039 immunity Effects 0.000 abstract description 2
- 239000003223 protective agent Substances 0.000 abstract description 2
- 201000008297 typhoid fever Diseases 0.000 abstract description 2
- 241000191940 Staphylococcus Species 0.000 abstract 1
- 239000003242 anti bacterial agent Substances 0.000 abstract 1
- 229940088710 antibiotic agent Drugs 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 208000001848 dysentery Diseases 0.000 abstract 1
- 230000008014 freezing Effects 0.000 abstract 1
- 238000007710 freezing Methods 0.000 abstract 1
- 230000002496 gastric effect Effects 0.000 abstract 1
- 239000007788 liquid Substances 0.000 abstract 1
- 244000144977 poultry Species 0.000 description 4
- 235000016709 nutrition Nutrition 0.000 description 3
- 241000233866 Fungi Species 0.000 description 2
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- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 235000013622 meat product Nutrition 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- VEMLQICWTSVKQH-BTVCFUMJSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;propane-1,2,3-triol Chemical compound OCC(O)CO.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O VEMLQICWTSVKQH-BTVCFUMJSA-N 0.000 description 1
- 241000607764 Shigella dysenteriae Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229940007046 shigella dysenteriae Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention relates to a method for preparing a biological feed additive of lactobacillus. The preparation method comprises the following steps of: culturing high-quality bacterium lacticum and galactococcus in lactobacillus to obtain high-activity lactobacillus fermentation liquid; utilizing skimmed milk, and the like as a protective agent; and utilizing a vacuum freeze drier to prepare a viable additive with a viable organism number up to 1011 per gram. The biological feed additive has the advantages that the lactobacillus viable organism obtained through vacuum freezing and drying has high and strong activity, and the fermentation product has stable biological function, is suitable for industrial continuous culture and has the advantages of low cost, convenience of operation, and the like. In addition, the feed fermented by the feed additive can stimulate animal growth, adjust normal flora in gastrointestinal canals, maintain microeubiosis, deteriorate endotoxin, inhibit the growth of spoilage organisms in the intestinal canal, improve the organism immunity and have antagonism on pathogenic bacteria, such as dysentery bacillus, typhoid bacillus, paratyphoid bacillus, campylobacter, staphylococcus bacteria, and the like so that the invention can be applied to the culture industry to decrease the culture cost and effectively terminate the abuse of antibiotics.
Description
Technical field: the preparation method who the present invention relates to a kind of lactic acid bacteria biological feed additive.
Technical background
Entering the 21 century people has requirement more and more higher to the quality of meat product, and its requirement to nutritional quality is also more and more higher, and its source of quality of improving the meat product is quality of the fodder.Therefore the nutritional quality and the nutritional labeling that improve feed are very important for aquaculture.At present, some poultry and livestock feeds have been realized commercialization, standardization, contain amino acid whose feed; Contain vitamin feed and contain antibiotic feed and contain Chinese herbal feed, these additives are low to the utilization rate of fowl poultry, contain side effect, and hazard residue enters biological chain and directly enters human body, promptly can harm humans health can work the mischief to environment again.
Summary of the invention: the purpose of this invention is to provide a kind of promotion animal intestinal digestive function, reduce drug use, reduce feed cost, reduce marketing time, improve meat quality.Technical solution of the present invention is: a kind of preparation method of lactic acid bacteria biological feed additive: 1 from national microorganism fungus kind preservation center purchase Lactobacillus brevis (Lactobacillus brevis), Lactobacillus delbrueckii (ddlbrueckii subsp.Lactis), Lactobacillus casei (paracasei), Lactococcus lactis (Lactobacill), Lactobacillus pentosus (Lactobacilluspentosaceus), Bu Shi lactobacillus (Lactobacillus), Lactobacillus plantarum (Lactobacillusplantarum), the strain inclined plane of lactobacillus acetotolerans (Lactobacillusacetotolerans) is standby;
2) activation culture culture medium of preparation, the prescription of its culture medium is as follows: soy peptone 50g, powdered beef 30g, dusty yeast 30g, glucose 200g, lactose 20g, tomato juice 50ml, deionized water 1000ml, above component is dissolved in the deionized water, in the triangular flask with its 2l that packs into, the refrigerator of behind high pressure steam sterilization 20min under 121 ℃ the condition, putting into-4 ℃ is stand-by;
3) activation culture: with the culture medium 20ml of an activation culture, divide and install in the 100ml blake bottle, picking Lactobacillus brevis (Lactobacillus brevis), Lactobacillus delbrueckii (delbrueckii subsp.Lactis), Lactobacillus casei (paracasei), Lactococcus lactis (Lactobacill), Lactobacillus pentosus (Lactobacilluspentosaceus), Bu Shi lactobacillus (Lactobacillus), Lactobacillus plantarum (Lactobacillusplantarum), a single bacterium colony on lactobacillus acetotolerans (Lactobacillus acetotolerans) strain inclined plane, in the culture medium with each single bacterium transferred species to time activation culture, in temperature is under 36 ± 1 ℃ of conditions, the static cultivation 16～18h of anaerobism, ph value to be determined is: when 3.8-4.5 is, show that an activation culture state reaches requirement.
4) the preparation secondary enlarges cultivation soy peptone 200g, raffinose 20g, tomato juice 100ml, sodium chloride 10 gram, deionized water 2000ml, with above each components dissolved in deionized water, the capacity of joining is in the small-sized fermentation jar of 4l, and high pressure steam sterilization 20min is stand-by under 121 ℃ of conditions;
5) secondary expansion cultivation is inoculated into the zymotic fluid of an activation culture of each bacterial strain in the nutrient solution of secondary expansion cultivation simultaneously, temperature is transferred to 40 ℃ of constant temperature detests foster static cultivation 12-14h, when the ph that treats zymotic fluid reaches 3.8-4.5, show that secondary enlarges cultivation conditions and reaches requirement;
6) preparation freeze drying protectant: skimmed milk 720g, glycerine 30ml, lactose 200g, glucose 200g, deionized water 6000ml, above component is dissolved in the deionized water, installs to after stirring in the triangular flask of 10l, the refrigerator of putting into-4 ℃ behind high pressure steam sterilization 15min under 118 ℃ of conditions is stand-by;
7) collect zymophyte mud: secondary is enlarged cultivate the zymotic fluid that obtains and under aseptic condition, pack in the centrifuge tube of centrifuge, under the 10000rpm/min condition, behind the centrifugal 5min, in aseptic superclean bench, outwell supernatant, resulting dry is a bacterium mud, collects in the triangular flask that joins the 10l that dynamic protection note is housed behind the bacterium mud immediately stand-by;
8) with bacterium mud and the freeze drying protectant mixing collected, wherein fermentation back bacterium mud and protectant ratio are 1: 3;
9) freeze-drying: the bacterium mud that mixes and freeze drying protectant are packed in the freeze-drying bottle, and the freeze-drying bottle changes pre-freeze 4h in-40 ℃ the refrigerator over to, changes the freeze-drying bottle over to vacuum freeze drier then, and freeze-drying 24h promptly can obtain viable count and reach 10
11The lactobacillus feed additive of/gram.
The invention has the advantages that:
Utilize the lactic acid bacteria strains of studying comparative maturity both at home and abroad to make the fermentation strain of additive for microbe feedstuff, its stable physical-chemical indexes, the biological function stability and safety, effect in fowl poultry enteron aisle is thorough, be easy in enteron aisle, adhere to and bring into play work, and stronger fertility and biologically active are arranged, be convenient to the quality strains that batch production is cultivated.And simple to operate aspect production technology, the low amount of cost can continued operation, and uses this feed addictive can promote the animal intestinal digestive function, reduces drug use, reduces feed cost, reduces marketing time, improves meat quality.The preparation method of above-mentioned biology feed additive, its characteristics are three aspects, the one, and the optimization that bacterial classification is selected; The 2nd, the simple low cost of zymotechnique; The 3rd, freeze-dry process efficient
Characteristics of the present invention are to utilize the quality strains in the lactic acid bacteria: Lactobacillus brevis (Lactobacillus brevis); Lactobacillus delbrueckii (delbrueckii subsp.Lactis); Lactobacillus casei (paracasei); cultivate through once enlarging behind Lactococcus lactis (Lactobacill) Lactobacillus pentosus (Lactobacilluspentosaceus) Bu Shi lactobacillus (Lactobacillus) Lactobacillus plantarum (Lactobacillusplantarum) lactobacillus acetotolerans (Lactobacillus acetotolerans) purifying, secondary enlarges cultivates the viable count 10 that obtains through vacuum freeze drying as protective agent with skimmed milk glycerol-glucose lactose sucrose
9The feed addictive of/gram.
And lactic acid bacteria is the confidential relation of having digested and assimilated of the normal beneficial microbe of fowl poultry enteron aisle and feed.Keep that beneficial flora just often promptly can improve food digestion rate and biological value in the super island; The control endotoxin; Suppress corrupt bacteria growing in the enteron aisle.The biological rhzomorph that probio metabolism simultaneously produces has antagonism to do for pathogenic bacteria such as shigella dysenteriae, typhoid bacillus, paratyphosum Bacterium, campylobacter, grape ball Lin etc., improves immunity of organisms etc.
The specific embodiment:
A kind of preparation method of lactic acid bacteria biological feed additive:
1 from national microorganism fungus kind preservation center purchase Lactobacillus brevis (Lactobacillus brevis), Lactobacillus delbrueckii (delbrueckii subsp.Lactis), Lactobacillus casei (paracasei), Lactococcus lactis (Lactobacill), Lactobacillus pentosus (Lactobacilluspentosaceus), Bu Shi lactobacillus (Lactobacillus), Lactobacillus plantarum (Lactobacillusplantarum), the strain inclined plane of lactobacillus acetotolerans (Lactobacillusacetotolerans) is standby;
2) activation culture culture medium of preparation, the prescription of its culture medium is as follows: soy peptone 50g, powdered beef 30g, dusty yeast 30g, glucose 200g, lactose 20g, tomato juice 50ml, deionized water 1000ml, above component is dissolved in the deionized water, in the triangular flask with its 2l that packs into, the refrigerator of behind high pressure steam sterilization 20min under 121 ℃ the condition, putting into-4 ℃ is stand-by;
3) activation culture: with the culture medium 20ml of an activation culture, divide and install in the 100ml blake bottle, picking Lactobacillus brevis (Lactobacillus brevis), Lactobacillus delbrueckii (delbrueckii subsp.Lactis), Lactobacillus casei (paracasei), Lactococcus lactis (Lactobacill), Lactobacillus pentosus (Lactobacilluspentosaceus), Bu Shi lactobacillus (Lactobacillus), Lactobacillus plantarum (Lactobacillusplantarum), a single bacterium colony on lactobacillus acetotolerans (Lactobacillus acetotolerans) strain inclined plane, in the culture medium with each single bacterium transferred species to time activation culture, in temperature is under 36 ± 1 ℃ of conditions, the static cultivation 16～18h of anaerobism, ph value to be determined is: when 3.8-4.5 is, show that an activation culture state reaches requirement.
4) the preparation secondary enlarges cultivation soy peptone 200g, raffinose 20g, tomato juice 100ml, sodium chloride 10 gram, deionized water 2000ml, with above each components dissolved in deionized water, the capacity of joining is in the small-sized fermentation jar of 4l, and high pressure steam sterilization 20min is stand-by under 121 ℃ of conditions;
5) secondary expansion cultivation is inoculated into the zymotic fluid of an activation culture of each bacterial strain in the nutrient solution of secondary expansion cultivation simultaneously, temperature is transferred to 40 ℃ of constant temperature detests foster static cultivation 12-14h, when the ph that treats zymotic fluid reaches 3.8-4.5, show that secondary enlarges cultivation conditions and reaches requirement;
6) preparation freeze drying protectant: skimmed milk 720g, glycerine 30ml, lactose 200g, glucose 200g, deionized water 6000ml, above component is dissolved in the deionized water, installs to after stirring in the triangular flask of 10l, the refrigerator of putting into-4 ℃ behind high pressure steam sterilization 15min under 118 ℃ of conditions is stand-by;
7) collect zymophyte mud: secondary is enlarged cultivate the zymotic fluid that obtains and under aseptic condition, pack in the centrifuge tube of centrifuge, under the 10000rpm/min condition, behind the centrifugal 5min, in aseptic superclean bench, outwell supernatant, resulting dry is a bacterium mud, collects in the triangular flask that joins the 10l that dynamic protection note is housed behind the bacterium mud immediately stand-by;
8) with bacterium mud and the freeze drying protectant mixing collected, wherein fermentation back bacterium mud and protectant ratio are 1: 3;
9) freeze-drying: the bacterium mud that mixes and freeze drying protectant are packed in the freeze-drying bottle, and the freeze-drying bottle changes pre-freeze 4h in-40 ℃ the refrigerator over to, changes the freeze-drying bottle over to vacuum freeze drier then, and freeze-drying 24h promptly can obtain viable count and reach 10
11The lactobacillus feed additive of/gram.
Claims (1)
1. the preparation method of a lactic acid bacteria biological feed additive is characterized in that:
The strain inclined plane of 1 Lactobacillus brevis (Lactobacillus brevis), Lactobacillus delbrueckii (delbrueckiisubsp.Lactis), Lactobacillus casei (paracasei), Lactococcus lactis (Lactobacill), Lactobacillus pentosus (Lactobacilluspentosaceus), Bu Shi lactobacillus (Lactobacillus), Lactobacillus plantarum (Lactobacillusplantarum), lactobacillus acetotolerans (Lactobacillusacetotol erans) is standby;
2) activation culture culture medium of preparation, the prescription of its culture medium is as follows: soy peptone 50g, powdered beef 30g, dusty yeast 30g, glucose 200g, lactose 20g, tomato juice 50ml, deionized water 1000ml, above component is dissolved in the deionized water, in its triangular flask of 21 of packing into, the refrigerator of behind high pressure steam sterilization 20min under 121 ℃ the condition, putting into-4 ℃ is stand-by;
3) activation culture: with the culture medium 20ml of an activation culture, divide and install in the 100ml blake bottle, picking Lactobacillus brevis (Lactobacillus brevis), Lactobacillus delbrueckii (delbrueckii subsp.Lactis), Lactobacillus casei (paracasei), Lactococcus lactis (Lactobacill), Lactobacillus pentosus (Lactobacilluspentosaceus), Bu Shi lactobacillus (Lactobacillus), Lactobacillus plantarum (Lactobacillusplantarum), a single bacterium colony on lactobacillus acetotolerans (Lactobacillus acetotolerans) strain inclined plane, in the culture medium with each single bacterium transferred species to time activation culture, in temperature is under 36 ± 1 ℃ of conditions, the static cultivation 16～18h of anaerobism, ph value to be determined is: when 3.8-4.5 is, show that an activation culture state reaches requirement.
4) the preparation secondary enlarges cultivation soy peptone 200g, raffinose 20g, tomato juice 100ml, sodium chloride 10 gram, deionized water 2000ml, with above each components dissolved in deionized water, the capacity of joining is that high pressure steam sterilization 20min is stand-by under 121 ℃ of conditions in 41 the small-sized fermentation jar;
5) secondary expansion cultivation is inoculated into the zymotic fluid of an activation culture of each bacterial strain in the nutrient solution of secondary expansion cultivation simultaneously, temperature is transferred to 40 ℃ of constant temperature detests foster static cultivation 12-14h, when the ph that treats zymotic fluid reaches 3.8-4.5, show that secondary enlarges cultivation conditions and reaches requirement;
6) preparation freeze drying protectant: skimmed milk 720g, glycerine 30ml, lactose 200g, glucose 200g, deionized water 6000ml, above component is dissolved in the deionized water, installs to after stirring in 101 the triangular flask, the refrigerator of putting into-4 ℃ behind high pressure steam sterilization 15min under 118 ℃ of conditions is stand-by;
7) collect zymophyte mud: secondary is enlarged cultivate the zymotic fluid that obtains and under aseptic condition, pack in the centrifuge tube of centrifuge, under the 10000rpm/min condition, behind the centrifugal 5min, in aseptic superclean bench, outwell supernatant, resulting dry is a bacterium mud, collect join immediately behind the bacterium mud in 101 the triangular flask that dynamic protection note is housed stand-by;
8) with bacterium mud and the freeze drying protectant mixing collected, wherein fermentation back bacterium mud and protectant ratio are 1: 3;
9) freeze-drying: the bacterium mud that mixes and freeze drying protectant are packed in the freeze-drying bottle, and the freeze-drying bottle changes pre-freeze 4h in-40 ℃ the refrigerator over to, changes the freeze-drying bottle over to vacuum freeze drier then, and freeze-drying 24h promptly can obtain viable count and reach 10
11The lactobacillus feed additive of/gram.
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| CN102250800A (en) * | 2011-06-22 | 2011-11-23 | 南京农业大学 | Oxalic acid degrading bacterium NJODL1 and application thereof |
| CN102370058A (en) * | 2011-09-29 | 2012-03-14 | 孙义 | Mixed fermentation strain combination of high-yield polyunsaturated fatty acid and polysaccharide and composite microbial ecological agent thereof |
| CN102987435A (en) * | 2012-11-13 | 2013-03-27 | 西华大学 | Preparation method for multifunctional complex-fermented powder for tenderization and fermentation and method for preparing fermented yak meat particles |
| CN103320351A (en) * | 2013-05-16 | 2013-09-25 | 沈阳药科大学 | Lactococcus garvieae and biological feed-additive prepared therefrom |
| CN106578450A (en) * | 2016-11-25 | 2017-04-26 | 南宁学院 | Micro-ecologic preparation for improving immunity of calves, and preparation method and application thereof |
| CN106754482A (en) * | 2016-11-28 | 2017-05-31 | 江苏梁丰食品集团有限公司 | A kind of preparation method of the probiotics leaven for producing functional sugar |
| CN106854626A (en) * | 2015-12-08 | 2017-06-16 | 内蒙古沃德生物质科技有限公司 | A kind of maize straw is blue or green（It is yellow）Store the preparation method of enzyme bacterium composite ferment |
| CN107495057A (en) * | 2017-09-27 | 2017-12-22 | 广州市澳米环保科技有限公司 | A kind of compound lactobacillus solid beverage and preparation method thereof |
| CN107981033A (en) * | 2017-11-13 | 2018-05-04 | 佛山科学技术学院 | A kind of feed addictive for preventing chicken paratyphoid disease and preparation method thereof |
| CN109234186A (en) * | 2018-06-24 | 2019-01-18 | 山东省科学院生物研究所 | A kind of novel thermophilic lactic acid bacteria and its application in wet process corn soaking |
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| CN113973997A (en) * | 2021-09-24 | 2022-01-28 | 北京三元种业科技股份有限公司饲料分公司 | Lactobacillus composition and calf starter feed |
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| CN102370058A (en) * | 2011-09-29 | 2012-03-14 | 孙义 | Mixed fermentation strain combination of high-yield polyunsaturated fatty acid and polysaccharide and composite microbial ecological agent thereof |
| CN102987435A (en) * | 2012-11-13 | 2013-03-27 | 西华大学 | Preparation method for multifunctional complex-fermented powder for tenderization and fermentation and method for preparing fermented yak meat particles |
| CN103320351A (en) * | 2013-05-16 | 2013-09-25 | 沈阳药科大学 | Lactococcus garvieae and biological feed-additive prepared therefrom |
| CN106854626A (en) * | 2015-12-08 | 2017-06-16 | 内蒙古沃德生物质科技有限公司 | A kind of maize straw is blue or green（It is yellow）Store the preparation method of enzyme bacterium composite ferment |
| CN106578450A (en) * | 2016-11-25 | 2017-04-26 | 南宁学院 | Micro-ecologic preparation for improving immunity of calves, and preparation method and application thereof |
| CN106754482A (en) * | 2016-11-28 | 2017-05-31 | 江苏梁丰食品集团有限公司 | A kind of preparation method of the probiotics leaven for producing functional sugar |
| CN107495057A (en) * | 2017-09-27 | 2017-12-22 | 广州市澳米环保科技有限公司 | A kind of compound lactobacillus solid beverage and preparation method thereof |
| CN107981033A (en) * | 2017-11-13 | 2018-05-04 | 佛山科学技术学院 | A kind of feed addictive for preventing chicken paratyphoid disease and preparation method thereof |
| CN109234186A (en) * | 2018-06-24 | 2019-01-18 | 山东省科学院生物研究所 | A kind of novel thermophilic lactic acid bacteria and its application in wet process corn soaking |
| CN109234186B (en) * | 2018-06-24 | 2019-12-06 | 山东省科学院生物研究所 | novel thermophilic lactic acid bacteria and application thereof in wet corn soaking |
| CN109259210A (en) * | 2018-09-07 | 2019-01-25 | 浙江九田环保科技有限公司 | The preparation method of probiotic microorganisms fermented product |
| CN113973997A (en) * | 2021-09-24 | 2022-01-28 | 北京三元种业科技股份有限公司饲料分公司 | Lactobacillus composition and calf starter feed |
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