CN105981915A - Application of active saccharomycetes in improving utilization efficiency of coarse feed - Google Patents
Application of active saccharomycetes in improving utilization efficiency of coarse feed Download PDFInfo
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- CN105981915A CN105981915A CN201510055296.1A CN201510055296A CN105981915A CN 105981915 A CN105981915 A CN 105981915A CN 201510055296 A CN201510055296 A CN 201510055296A CN 105981915 A CN105981915 A CN 105981915A
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Abstract
The invention discloses an application of active saccharomycetes in improving the utilization efficiency of a coarse feed. The application is performed through the following steps of step A: utilizing an artificial rumen culture system in vitro, and adding Candida utilis CICC No.1314 of different concentrations to perform culture in vitro on the coarse feed; B, keeping the different concentration to be 0.1*10<7> cfu to 0.75*10<7> cfu; C, collecting cow rumen fluid through fistulas by the artificial rumen culture system in vitro, and mixing the filtered cow rumen fluid with anaerobic buffer fluid so as to obtain fermentation culture fluid; D, introducing carbon dioxide into the mixed culture fluid rapidly before fermentation, placing the culture fluid with the carbon dioxide on a shaking bath for culturing, adding saccharomycetes culture fluid with different concentration gradients and in-vitro-fermentation culture fluid, introducing carbon dioxide, adding a stopper cover, performing outgassing with a syringe needle so as to adjust air pressure, and then placing the mixed fluid in a thermostatic culture box for in vitro fermentation; and E, adding Candida utilis CICC No.1314 with different addition concentration gradients to the coarse feed. The method is simple, the growth speed is high, and the active saccharomycetes is not liable to mutate. The active saccharomycetes can be used as a mode fungus strain for improving the utilization efficiency of the coarse feed so as to be directly used for improving the utilization efficiency of the coarse feed.
Description
Technical field
The present invention relates to animal nutrition and feed technical field, be more particularly to the application in improving roughage utilization efficiency of a kind of active yeast.
Background technology
Due to antibiotic feed additive life-time service in animal productiong and some irrational use, easily cause bacterial drug resistance, in animal product and environment the problems such as residual.Therefore, research and development green feed additive product has been referred to as global research topic, and the green feed additive kind the most gained public acceptance has plant extract, enzyme preparation, probiotics etc..Yeast is a kind of unicellular fungi quasi-microorganism, has simple unicellular bacteria and grows the features such as fast, easily operated.Its application in Animal Nutrition and Feed Science field at present includes stimulating ruminant tumor gastric microbial growth, strengthening the aspect such as rumen microorganism activity, change rumen zymosis pattern.But, its application in terms of nutritive digestibility and food utilization efficiency is the most very limited, and one of them major reason is a lack of really being effectively improved the yeast strain of food utilization efficiency.
Summary of the invention
It is an object of the invention to the application that there are provided a kind of active yeast in improving roughage utilization efficiency, the yeast strain of the present inventionCandida utilis CICC No.1314 is that a strain has high vigor, roughage utilization efficiency is improved very competent bacterial strain, its cultural method is simple, fast growth, it is difficult to variation, can serve as yeast and improve the type strain of roughage utilization efficiency, it is often more important that be used directly for improving the utilization ratio of roughage.
In order to realize above-mentioned purpose, the present invention uses techniques below measure:
The application in improving roughage utilization efficiency of a kind of active yeast, the steps include:
A, utilize rumen ectogenesis system (FYL-YS-100L), by adding variable concentrationsCandida utilis CICC No.1314 carries out In vitro culture to roughage (rice straw and corn straw).Described roughage (rice straw and corn straw) is pulverized through 80-110 mesh aperture sieve respectively.
B, describedCandida utilis CICC No.1314 variable concentrations is 0.1-0.75 × 107cfu。
C, rumen ectogenesis system gather cow rumen liquid by fistula before feeding in the morning, are sufficiently mixed as fermentation culture with the ratio of 1:9 with anaerobism buffer with the cow rumen liquid after four layers of filtered through gauze.
D, the fermentation culture mixed are passed through rapidly carbon dioxide after 2-4 minute before fermentation, are placed on the shaking bath (ZHSY-50) of 39 DEG C and wait cultivation.Before In Vitro Fermentation, 22-26h weighs the 0.4-0.6 gram of roughage (rice straw and corn straw) pulverized through 80-110 mesh aperture sieve in advance in each anaerobic fermentation bottle, successively add yeast culture fluid and the 40-60ml In Vitro Fermentation culture fluid of variable concentrations gradient immediately, and again it is continually fed into carbon dioxide 2-4min, plus using rapidly syringe needle venting that air pressure adjustment is become 0 after bottle stopper lid, this is the atmospheric pressure value of 0h point.Immediately it is positioned in 39 DEG C of constant incubators beginning In Vitro Fermentation 46-50 hour.
Above-mentioned cud In Vitro Fermentation anaerobism buffer composition includes macroelement solution, trace element solution, reductant solution, resazurin solution and buffer solution.Wherein macroelement solution composition: every 500 milliliters of solution include 7.185 grams of disodium hydrogen phosphate (Na2HPO4·12H2O), 3.1 grams of potassium dihydrogen phosphate (KH2PO4) and 0.3 gram of Magnesium sulfate heptahydrate (MgSO4·7H2O)；Trace element solution forms: every 500 milliliters of solution include 9.97 grams of calcium chloride (CaCl2), 8.0 grams of Iron trichloride hexahydrate (FeCl3·6H2O), 10.0 grams of manganese chloride tetrahydrate (MnCl2·4H2O), 5.9 gram of one water zinc chloride (ZnCl2·H2O), 1.0 grams of cobalt chloride hexahydrate (CoCl2·6H2O) and 1.25 grams of copper chloride dihydrate (CuCl2·2H2O)；Reductant solution forms: every 100 milliliters of solution include 25 gram of nine water cure disodium (Na2S·9H2O)；Resazurin solution forms: every 100 milliliters of solution include 0.1 gram of "diazoresorcinol"；Buffer solution forms: every 1000 milliliters of solution include 4.0 grams of ammonium hydrogen carbonate (NH4HCO3) and 35.0 grams of sodium bicarbonate (NaHCO3).
The preparation of cud In Vitro Fermentation anaerobism buffer: be sequentially added into following solution in 400 milliliters of distilled water: 0.5 milliliter of liquid microelement, 200 milliliters of buffer solution, 200 milliliters of macroelement liquid, 1.0 milliliters of resazurin solution and 1.6 milliliters of reducing solutions.
E, in order to improve roughage utilization efficiency, add described in roughageCandida
utilis CICC
It is 0.1-0.75 × 10 that No.1314 difference adds Concentraton gradient7cfu。
This bacterial strain can improve the utilization ratio of roughage multiple nutritional components, is particularly particularly effective raising dry (DM), nitrogen (N), Organic substance (OM), the utilization ratio of neutral detergent fiber (NDF).
Test proved in 48 hours,Candida utilis CICC No.1314 has been respectively increased 5%, 7%, 6% and 10% to roughage rice straw DM, N, OM, NDF degradation in vitro；Corn straw DM, N, OM, NDF degradation in vitro has been respectively increased 6%, 8%, 9% and 12%.
The yeast strain of raising roughage utilization efficiency provided by the present invention isCandida utilis, this culture presevation is in Chinese industrial Microbiological Culture Collection administrative center, and deposit number is CICC No.1314.This strain can arrive Chinese industrial Microbiological Culture Collection administrative center and buy.
Examining under a microscope, the cell of this bacterial strain is rounded, oval or sausage shape, and size is (3.5～4.5) μ m (7～13) μm.Liquid culture does not produce mould, has bacterial sediment at the bottom of pipe；In wort agar culture medium, bacterium colony milky, smooth, with or without gloss, neat in edge or mycelioid；With in the corn meal agar culture medium of cover plate, form original pseudohypha or underdeveloped pseudohypha, or without pseudohypha；Energy glucose fermentation, sucrose, Raffinose, azymic maltose, galactose, lactose and 6-(.alpha.-D-galactosido)-D-glucose., do not reduce fat, nitrate can be assimilated.
Above-mentioned yeast strain can be cultivated as followsCandida utilis CICC No.1314: receive on inclined-plane with inoculating loop picking thalline, at 25-32 DEG C, slant medium grows 1-3 days, then picking slant strains accesses fluid medium, at 25-32 DEG C, cultivates 1-3 days, can obtain the thalline of this bacterial strain by centrifugation.Described culture medium calculates by weight, comprises following components: glucose 2.0 parts, peptone 2.0 parts, yeast 1.0 parts of powder of leaching, 2.0 parts of agar (fluid medium is not added with agar), natural pH, solvent is distilled water.
The present invention compared with prior art, has the following advantages and effect:
The yeast strain of the present inventionCandida utilis CICC No.1314 is that a strain has high vigor, roughage utilization efficiency is improved very competent bacterial strain, its cultural method is simple, fast growth, it is difficult to variation, can serve as yeast and improve the type strain of roughage utilization efficiency, it is often more important that be used directly for improving the utilization ratio of roughage.This bacterial strain has in the industrial applications prospect improving ruminant roughage utilization efficiency.After In Vitro Fermentation 46-50h,Candida utilis It is 0.25-0.75 × 10 that CICC No.1314 adds concentration7During cfu, roughage rice straw and corn straw degradation rate being promoted effect the most obvious, wherein roughage rice straw DM, N, OM, NDF degradation in vitro is respectively increased 5%, 7%, 6% and 10%；Corn straw DM, N, OM, NDF degradation in vitro is respectively increased 6%, 8%, 9% and 12%.
Detailed description of the invention
Embodiment 1:
The application in roughage utilization efficiency of a kind of active yeast, the steps include:
A、Candida utilis The cultivation of CICC No.1314 thalline:
With inoculating loop picking yeast strainCandida utilis CICC No.1314 receives on slant medium, and slant medium calculates by weight, comprises following components: glucose 2.0 parts, peptone 2.0 parts, yeast soak 1.0 parts of powder, 2.0 parts of agar (fluid medium is not added with agar), natural pH.In temperature is set to the incubator of 30 DEG C, cultivate 1-3 days, the thalline of white occurs.Then, receiving equipped with in the triangular flask of culture fluid with the inoculating loop well-grown thalline of picking from inclined-plane, culture fluid calculates by weight, comprises following components: glucose 2.0 parts, peptone 2.0 parts, yeast soak 1.0 parts of powder.Temperature be set to 30 DEG C, rotating speed be to cultivate 1-3 days on the shaking table of 200rpm, gained thalline centrifugal (9000rpm) 10 minutes, thalline physiological saline solution (0.8%) and sterilized water wash, then are centrifuged, 2-3 time repeatedly, can obtain white free of contaminationCandida utilis CICC No.1314 yeast cells.
B, rumen ectogenesis system:
Before raising morning, gather cow rumen liquid by fistula, be sufficiently mixed as fermentation culture with the ratio of 1:9 with anaerobism buffer with after four layers of filtered through gauze.The culture fluid mixed is passed through rapidly carbon dioxide after 2-3 minute before fermentation, is placed on the shaking bath of 39 DEG C wait and cultivates.In each anaerobic fermentation bottle, weigh 0.4 or 0.5 or 0.6 gram of roughage (rice straw and corn straw) after 80 or 90 or 100 or 110 mesh aperture sieve are pulverized in advance in 24h before In Vitro Fermentation, be successively separately added into 0.1-0.75 × 10 immediately7The yeast culture fluid of cfu Concentraton gradient and 50ml In Vitro Fermentation culture fluid, and again it is continually fed into carbon dioxide 3 minutes, use rapidly syringe needle venting that air pressure adjustment is become 0 after adding bottle stopper lid, this is the atmospheric pressure value of 0h point.Immediately it is positioned in 39 DEG C of constant incubators beginning In Vitro Fermentation 48 hours.
C, addition in roughageCandida utilis CICC No.1314 improves roughage utilization efficiency:
In each anaerobic fermentation bottle, weigh 0.4 or 0.5 or 0.6 gram of roughage (rice straw and corn straw) after 1mm aperture sieve is pulverized according to step 2, be successively separately added into 0,0.25 × 10 immediately7cfu、0.50×107Cfu and 0.75 × 107The yeast strain of cfu Concentraton gradientCandida utilis CICC No.1314 and 50ml external mixing fermentation culture liquid, and again it is continually fed into carbon dioxide 3 minutes, use rapidly syringe needle venting that air pressure adjustment is become 0 after adding bottle stopper lid, this is the atmospheric pressure value of 0h point.Immediately it is positioned in 39 DEG C of constant incubators beginning In Vitro Fermentation 48 hours.In Vitro Fermentation takes out fermentation flask after cultivating 48 hours, residue is fully transferred in silica crucible repeatedly rinse with hot distilled water after crossing 400 mesh nylon cloths by fermentation liquid, it is subsequently placed in 105 DEG C of baking ovens and dries 8 hours with DM, Slag recovering sample sack after measuring DM degradation rate subsequently seals and preserves, N, OM, NDF degradation rate to be measured.
Described rice straw and corn straw carry out pulverizing 80 or 90 or 100 or 110 mesh respectively.
After In Vitro Fermentation 48 hours,Candida utilis It is 0.50 × 10 that CICC No.1314 adds concentration7During cfu, roughage rice straw and corn straw degradation rate being promoted effect the most obvious, wherein roughage rice straw DM, N, OM, NDF degradation in vitro is respectively increased 5%, 7%, 6% and 10%；Corn straw DM, N, OM, NDF degradation in vitro is respectively increased 6%, 8%, 9% and 12%.
Claims (1)
1. an active yeast application in improving roughage utilization efficiency, the steps include:
A, utilize rumen ectogenesis system, by adding variable concentrationsCandida utilis CICC No.1314 carries out In vitro culture to roughage；
B, described variable concentrations are 0.1-0.75 × 107cfu；
C, rumen ectogenesis system gather cow rumen liquid by fistula before feeding in the morning, are sufficiently mixed as fermentation culture with the ratio of 1:9 with anaerobism buffer with the cow rumen liquid after four layers of filtered through gauze；
D, the culture fluid mixed are passed through rapidly carbon dioxide after 2-4 minute before fermentation, it is placed on the shaking bath of 39 DEG C wait to cultivate, before In Vitro Fermentation, 22-26h weighs 0.4-0.6 gram of roughage in advance in each anaerobic fermentation bottle, add yeast culture fluid and the 40-60ml In Vitro Fermentation culture fluid of variable concentrations gradient, again it is continually fed into carbon dioxide 2-4min, plus using rapidly syringe needle venting after bottle stopper lid, air pressure adjustment is become 0, the atmospheric pressure value of 0h point, is subsequently placed in 39 DEG C of constant incubators start In Vitro Fermentation 46-50 hour；
E, addition in roughageCandida utilis CICC
It is 0.1-0.75 × 10 that No.1314 difference adds Concentraton gradient7cfu。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101305769A (en) * | 2008-07-02 | 2008-11-19 | 内蒙古粮食科学研究设计院有限责任公司 | Preparation method of microbial fermentative feedstuff |
| CN103689218A (en) * | 2013-12-05 | 2014-04-02 | 中国农业大学 | Complex fungicide for improving feed fiber degradation rate and positively regulating rumen fermentation propionic acid generation as well as application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101305769A (en) * | 2008-07-02 | 2008-11-19 | 内蒙古粮食科学研究设计院有限责任公司 | Preparation method of microbial fermentative feedstuff |
| CN103689218A (en) * | 2013-12-05 | 2014-04-02 | 中国农业大学 | Complex fungicide for improving feed fiber degradation rate and positively regulating rumen fermentation propionic acid generation as well as application thereof |
Non-Patent Citations (1)
| Title |
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| 庞德公 等: "高精料全混合日粮中产朊假丝酵母添加水平对体外瘤胃发酵特性和纤维降解的影响", 《动物营养学报》 * |
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