CN103315195B - High-moisture alfalfa silage inoculants bacteria - Google Patents
High-moisture alfalfa silage inoculants bacteria Download PDFInfo
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- CN103315195B CN103315195B CN201310266003.5A CN201310266003A CN103315195B CN 103315195 B CN103315195 B CN 103315195B CN 201310266003 A CN201310266003 A CN 201310266003A CN 103315195 B CN103315195 B CN 103315195B
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- 241000894006 Bacteria Species 0.000 title claims abstract description 39
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 title claims abstract description 38
- 241000219823 Medicago Species 0.000 title claims abstract description 31
- 239000004460 silage Substances 0.000 title claims abstract description 21
- 239000002054 inoculum Substances 0.000 title claims abstract description 11
- 241000196324 Embryophyta Species 0.000 claims abstract description 18
- 244000199866 Lactobacillus casei Species 0.000 claims abstract description 12
- 235000013958 Lactobacillus casei Nutrition 0.000 claims abstract description 12
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 12
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 12
- 229940017800 lactobacillus casei Drugs 0.000 claims abstract description 12
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 12
- 241000219793 Trifolium Species 0.000 claims description 41
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 40
- 235000014655 lactic acid Nutrition 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 20
- 239000002068 microbial inoculum Substances 0.000 claims description 16
- 241000186660 Lactobacillus Species 0.000 claims description 15
- 229940039696 lactobacillus Drugs 0.000 claims description 15
- 235000015097 nutrients Nutrition 0.000 claims description 13
- 239000007787 solid Substances 0.000 claims description 12
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- 230000004913 activation Effects 0.000 claims description 7
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- 238000000576 coating method Methods 0.000 claims description 5
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- 238000012545 processing Methods 0.000 claims description 5
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
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- 108010080698 Peptones Proteins 0.000 claims description 3
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention discloses high-moisture alfalfa silage inoculants bacteria. The active ingredients are lactobacillus plantarum (Lactobacillus plantarum) WN5 and lactobacillus casei SN3 (Lactobacillus casei). When the high-moisture alfalfa is ensiled by using the fungicide, the moisture content of the high-moisture alfalfa achieves 81.7%; the silage time achieves 240 days; steam and leaf of the ensiled alfalfa are completely stored on a plant; the moisture content is much different from that before silage; the protein content is also improved; meanwhile, the antibacterial effect of an ensiling agent is improved; the hidden danger that the herbal property enters the food chain to propagate by the ensiled alfalfa is reduced.
Description
Technical field
The present invention relates to a kind of microbe additive, relate in particular to a kind of high-moisture alfalfa ensilage microbial inoculum.
Background technology
Along with the development of aquaculture and riseing of provision price, develop domestic demand best in quality, that the inexpensive forage feed of safety has become the transformation aquaculture style of economic increase.Clover is the forage grass of phytophagous animal high-quality, its main products is hay, but this production is drenched with rain often, fallen leaves, expensive drying plant and the puzzlement that consumes the problems such as mass energy, alfalfa ensilage not only can be avoided above puzzlement, also can reduce nutrient loss to keep the nutritive property of green forage.
Therefore, a lot of technology disclose the method for alfalfa ensilage, but because nutritional character and the colony characteristics of clover self causes the effect of alfalfa ensilage in existing disclosed technology or method undesirable, subject matter is: one, during a part of technical requirements ensiling, need clover to wilt or the water content below airing to 65%, the requirement of this water content will cause the clover in rainy season that faces results cannot carry out ensiling; Two, clover self is with a large amount of yeast and a small amount of milk-acid bacteria, clover needs to use fast, the antibacterial strong lactic bacteria additive of breeding in ensilage, but the alfalfa ensilage product that add agent of lactic acid bacteria will enter food chain when feeding animals, reduce the risk of antibiotics resistance, the antibiotics resistance that needs to evaluate agent of lactic acid bacteria, agent of lactic acid bacteria is to antibiotic sensitivity test.
The feature when problem existing with lactic bacteria additive in view of above-mentioned alfalfa ensilage and alfalfa ensilage, prior art can not complete simultaneously carries out ensiling to high moisture content clover, improves the fungistatic effect of ensiling agent simultaneously and reduces resistance to enter food chain by Alfalfa Silage and the problem of the hidden danger propagated.
Summary of the invention
For solving deficiency of the prior art, the object of the invention is to provide a kind of high-moisture alfalfa ensilage microbial inoculum.
The present invention is for achieving the above object, and the technique means adopting is: a kind of high-moisture alfalfa ensilage microbial inoculum, its activeconstituents be plant lactobacillus (
lactobacillus plantarum) WN5 and lactobacterium casei SN3 (
lactobacillus casei).
Further, this high-moisture alfalfa ensilage microbial inoculum by the MRS liquid culture of plant lactobacillus WN5 and lactobacterium casei SN3 by cfu than 1:(1-2) form.
Further, component and the content of every liter of described MRS liquid nutrient medium are: peptone 10 g/L, yeast powder 5 g/L, glucose 10 g/L, sucrose 15 g/L, extractum carnis 10 g/L, diammonium hydrogen citrate 2 g/L, sodium acetate 5 g/L, Sodium.alpha.-ketopropionate 0.18 g/L, L-light cystinic acid hydrochloride 0.05 g/L, protoheme 0.5 g/L, vitamin K
22 g/L, calcium carbonate 10 g/L, K
2hPO
42 g/L, MgSO
47H
2o 0.58 g/L, MnSO
4h
2o 0.25 g/L, Tween-80 1ml/L; Its pH value is 6.4,121 ° of C sterilizings, within 15 minutes, makes; The component that MRS solid medium is every liter and content are: in above-mentioned MRS liquid nutrient medium, add 1.5% agar powder (mass percent), pH value is 6.4,121 ° of C sterilizings 15 minutes.
Further, under-20 ° of C conditions, respectively get on plant lactobacillus WN5 and lactobacterium casei SN3 5 microlitres injection MRS solid mediums, coating is cultivated 12～14 hours under the condition of 37 ° of C of temperature; With the single bacterium colony on transfering loop picking MRS solid medium, be inoculated in 10 milliliters of test tubes that 5 milliliters of MRS substratum are housed, under the condition of 37 ° of C of temperature, cultivate the milk-acid bacteria that obtains activation for 16～18 hours, now, in every milliliter of MRS liquid nutrient medium, at least contain 0.993 * 10
10cfu milk-acid bacteria.
Further, when above-mentioned silage inoculants bacteria is applied to high-moisture alfalfa ensilage, clover is cut into length 5～8cm, per kilogram high-moisture clover adds this silage inoculants bacteria of 30～35ml, turns evenly; Then, the Alfalfa Silage after processing is packed into compacting in ensiling device, cover lid, under normal temperature, place; Moisture content≤83% of described high-moisture clover, the phase of cradling is that clover stem height was greater than between the seedling stage to ripening stage of 40cm; Ensiling time≤240 day.
Beneficial effect of the present invention is: while using this microbial inoculum to high-moisture alfalfa ensilage, high-moisture clover moisture content is up to 81.7%, the ensiling time reaches 240 days, and the cauline leaf of the clover after ensiling is complete to be stored on plant, differ seldom before water content and ensiling, and protein content also increases; Improve the fungistatic effect of ensiling agent simultaneously and reduce resistance to enter food chain by Alfalfa Silage and the hidden danger propagated.
Embodiment
Below by specific embodiment, the present invention is further elaborated.
embodiment 1
A high-moisture alfalfa ensilage microbial inoculum, its activeconstituents be plant lactobacillus (
lactobacillus plantarum) WN5 and lactobacterium casei SN3 (
lactobacillus casei).
The MRS liquid culture of described plant lactobacillus WN5 and lactobacterium casei SN3 is pressed cfu than 1:(1-2) form.
Component and content that described MRS liquid nutrient medium is every liter are: peptone 10 g/L, yeast powder 5 g/L, glucose 10 g/L, sucrose 15 g/L, extractum carnis 10 g/L, diammonium hydrogen citrate 2 g/L, sodium acetate 5 g/L, Sodium.alpha.-ketopropionate 0.18 g/L, L-light cystinic acid hydrochloride 0.05 g/L, protoheme 0.5 g/L, vitamin K
22 g/L, calcium carbonate 10 g/L, K
2hPO
42 g/L, MgSO
47H
2o 0.58 g/L, MnSO
4h
2o 0.25 g/L, Tween-80 1ml/L; Its pH value is 6.4,121 ° of C sterilizings, within 15 minutes, makes; The component that MRS solid medium is every liter and content are: in above-mentioned MRS liquid nutrient medium, add 1.5% agar powder (mass percent), pH value is 6.4,121 ° of C sterilizings 15 minutes.
Under-20 ° of C conditions, respectively get on plant lactobacillus WN5 and lactobacterium casei SN3 5 microlitres injection MRS solid mediums, coating is cultivated 12～14 hours under the condition of 37 ° of C of temperature; With the single bacterium colony on transfering loop picking MRS solid medium, be inoculated in 10 milliliters of test tubes that 5 milliliters of MRS substratum are housed, under the condition of 37 ° of C of temperature, cultivate the milk-acid bacteria that obtains activation for 16～18 hours, now, in every milliliter of MRS liquid nutrient medium, at least contain 0.993 * 10
10cfu milk-acid bacteria.
Plant lactobacillus (
lactobacillus plantarum) WN5 and lactobacterium casei (
lactobacillus casei) SN3 is all in No. 3 China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 04 12nd, 2013, plant lactobacillus (
lactobacillus plantarum) preservation registration number of WN5 is CGMCC NO. 7469, lactobacterium casei (
lactobacillus casei) preservation registration number of SN3 is CGMCC NO. 7470.
Experimental results show that this microbial inoculum has following characteristic:
(1) can significantly suppress saccharomycetic growth;
(2) to antibiotic sensitive such as Streptomycin sulphate, Ciprofloxacin, gentamicin, amoxycilline Trihydrate bp, erythromycin and Cephradines;
(3) protoheme and multiprenylmenaquinone can significantly improve their rate of propagation and produce acid amount and improve their survival abilities under nutrition scarcity and acidic conditions;
(4) during bacterial strain co-cultivation, can significantly promote rate of propagation, strengthen acid producing ability and improve the tolerance of poor environment.
When above-mentioned silage inoculants bacteria is applied to high-moisture alfalfa ensilage, clover is cut into length 5～8cm, per kilogram high-moisture clover adds this silage inoculants bacteria of 30～35ml, turns evenly; Then, the Alfalfa Silage after processing is packed into compacting in ensiling device, cover lid, under normal temperature, place; Moisture content≤81.7% of described high-moisture clover, the phase of cradling is that clover stem height was greater than between the seedling stage to ripening stage of 40cm; Ensiling time≤240 day.
The present invention is directed in high-moisture clover that air is many, washiness, yeast mainly with and clover chloroplast(id) and plastosome contain protoheme, vitamin K
2forage feature Deng electron transit mediator, simultaneously, in conjunction with reducing the feature of propagating the risk demand of antibiotics resistance after clover product introduction food chain, on selecting, bacterial classification done certain adjustment, and select, growth good to yeast inhibition and fermentation to need protoheme and vitamin K
2, between antibiotic sensitive and bacterial strain, can promote propagation and survival bacterial strain be microoganism additives for ensiling; Do not select to a little less than yeast inhibition, growth and fermentation do not need protoheme and vitamin K
2, growth and fermentation does not need protoheme, to significantly not promoting the bacterial strain of propagation and survival between anti-in microbiotic or high resistance and bacterial strain; Bacterial strain is to protoheme and vitamin K
2dependency, will promote milk-acid bacteria to reduce the injury of oxygen to them to the utilization of oxygen in clover material; Bacterial strain is to protoheme and vitamin K
2application, can promote bacterial strain rapid, high volume propagation and improve strain fermentation and produce sour speed and produce acid amount; Symbiosis cooperation between milk-acid bacteria can consume rapidly oxygen, provides the anaerobic environment that is beneficial to lactobacter growth, makes the breeding of milk-acid bacteria rapid, high volume, suppresses the activity of undesirable microorganism; In the ensiling later stage, the symbiosis cooperation between bacterial strain can make bacterial strain under nutrition scarcity and lower pH condition, keep the survival of long period, has extended the ensiling time. and this additive has effectively utilized oxygen, protoheme and the vitamin K in clover material
2unfavorable factor by these to milk-acid bacteria propagation and fermentation becomes useful factor, promote multiplication capacity, acid producing ability and the tolerance of bacterial strain, this can increase organic acid content in clover, reduces pH value, suppresses harmful bacteria Growth and Reproduction, improves ensiling quality and extend the ensiling time.
embodiment 2
The activation of milk-acid bacteria: take out the plant lactobacillus that above-mentioned example 1 screening of preserving under-20 ° of C conditions obtains (
lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
lactobacillus casei) SN3 CGMCC NO. 7470, to get respectively 5 microlitres and inject on MRS solid medium, coating is cultivated 12～14 hours under the condition of 37 ° of C of temperature; With the single bacterium colony on transfering loop picking MRS solid medium, be inoculated in 10 milliliters of test tubes that 5 milliliters of MRS substratum are housed, under the condition of 37 ° of C of temperature, cultivate the milk-acid bacteria that obtains activation for 16～18 hours, now, in every milliliter of MRS liquid nutrient medium, at least contain 0.993 * 10
10cfu milk-acid bacteria.
High-moisture alfalfa ensilage microbial inoculum is made: by the plant lactobacillus of above-mentioned cultivation (
lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
lactobacillus casei) the bacterium liquid of SN3 CGMCC NO. 7470 in cfu than being mixed to get the seed liquor of carrying out co-cultivation for the ratio of 1:1, inoculum size by 10%, mixed above-mentioned seed liquor is inoculated in MRS liquid nutrient medium not calciferous, 37 ° of static cultivations of C 14 hours～20 hours, make cell density in every milliliters of liquid substratum reach 0.993 * 10
10cfu～0.832 * 10
11cfu, during ensiling, per kilogram adds 30 milliliters～35 milliliters microbe additives.
The application of microbe additive in high-moisture alfalfa ensilage: add the ratio of 30 milliliters～35 milliliters of microbe additives to add mentioned microorganism additive in per kilogram clover in high-moisture clover (water content is 79.7%～80.6%); Add before microbiobacterial agent, high-moisture clover is cut into 5 centimetres～6 cm long; Add after microbiobacterial agent, turn microbial inoculum and clover so that they mix; Then, the clover of adding microbial inoculum is encased in ensiling device; Room temperature is placed; Measure respectively contained moisture (%), pH value, crude protein (%), water-soluble carbohydrate (%), ammonia-state nitrogen (%), total free aminoacids (%) and reducing sugar (%) in Alfalfa Silage material interval 2 days, 5 days, 9 days, 15 days, 30 days and 90 days.3 repetitions are established in test, and the mean value repeating for 3 times is end value.
The silage effect of high-moisture alfalfa ensilage material: open ensiling device in different time sections, evenly accurately take 20 g samples; Add 180 mL distilled water, stir, add preservative film; Lixiviate 24 h at 4 ℃ are vat liquor through four layers of gauze and twice filtration gained liquid of qualitative filter paper.Vat liquor is used for measuring alfalfa ensilage material pH value and ammonia-state nitrogen (NH
3-N).PH value is used thunder magnetic PHS-25 type pH instrument to measure, and ammonia nitrogen content adopts phenol-clorox colorimetric method for determining.Water content is sample weightless weight after dry 48 h in 65 ℃ of baking ovens, water-soluble carbohydrate adopts anthrone-Sulphuric acid Colorimetry, crude protein adopts Kjeldahl nitrogen determination (FOSS company full-automatic Kjeldahl determination device), reducing sugar adopts 3,5-dinitrosalicylic acid (3,5-Dinitrosalicylic acid, DNS) colorimetric method for determining, total free aminoacids adopts ninhydrin colorimetry to measure.It is as shown in the table for measurement result.
Table one, the silage effect of this microbial inoculum to high-moisture clover
From table one, find out, the clover of processing with microbe additive in this example, the different ensiling time period of 2～90 days, the indices of ensiling all reached good effect.PH value remains in the different ensiling time level that is less than 5, the content of protein, water-soluble carbohydrate and reducing sugar does not all have because the prolongation of ensiling time shows obvious minimizing, and the content of water content, ammonia-state nitrogen and total free aminoacids does not all have because the prolongation of ensiling time shows obvious increase.
embodiment 3
The activation of milk-acid bacteria: take out the plant lactobacillus that above-mentioned example 1 screening of preserving under-20 ° of C conditions obtains (
lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
lactobacillus casei) SN3 CGMCC NO. 7470, to get respectively 5 microlitres and inject on MRS solid medium, coating is cultivated 12～14 hours under the condition of 37 ° of C of temperature; With the single bacterium colony on transfering loop picking MRS solid medium, be inoculated in 10 milliliters of test tubes that 5 milliliters of MRS substratum are housed, under the condition of 37 ° of C of temperature, cultivate the milk-acid bacteria that obtains activation for 16～18 hours, now, in every milliliter of MRS liquid nutrient medium, at least contain 0.993 * 10
10cfu milk-acid bacteria.
The microbe additive of high-moisture alfalfa ensilage is made: by the plant lactobacillus of above-mentioned cultivation (
lactobacillus plantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (
lactobacillus casei) the bacterium liquid of SN3 CGMCC NO. 7470 in cfu than being mixed to get the seed liquor of carrying out co-cultivation for the ratio of 1:2, inoculum size by 10%, mixed above-mentioned seed liquor is inoculated in MRS liquid nutrient medium not calciferous, 37 ° of static cultivations of C 14 hours～20 hours, make cell density in every milliliters of liquid substratum reach 0.993 * 10
10cfu～0.832 * 10
11cfu, during ensiling, per kilogram adds 30 milliliters～35 milliliters microbe additives.
The application of microbe additive in high-moisture alfalfa ensilage: add the ratio of 30 milliliters～35 milliliters of microbe additives to add mentioned microorganism additive in per kilogram clover in high-moisture clover (water content is 79.7%～80.6%); Add before microbiobacterial agent, high-moisture clover is cut into 5 centimetres～6 cm long; Add after microbiobacterial agent, turn microbial inoculum and clover so that they mix; Then, the clover of adding microbial inoculum is encased in ensiling device; Room temperature is placed; Measure respectively contained moisture (%), pH value, crude protein (%), water-soluble carbohydrate (%), ammonia-state nitrogen (%), total free aminoacids (%) and reducing sugar (%) in Alfalfa Silage material interval 2 days, 5 days, 9 days, 15 days, 30 days and 90 days.3 repetitions are established in test, and the mean value repeating for 3 times is end value.
The silage effect of high-moisture alfalfa ensilage material: open ensiling device in different time sections, evenly accurately take 20 g samples; Add 180 mL distilled water, stir, add preservative film; Lixiviate 24 h at 4 ℃ are vat liquor through four layers of gauze and twice filtration gained liquid of qualitative filter paper.Vat liquor is used for measuring alfalfa ensilage material pH value and ammonia-state nitrogen (NH
3-N).PH value is used thunder magnetic PHS-25 type pH instrument to measure, and ammonia nitrogen content adopts phenol-clorox colorimetric method for determining.Water content is sample weightless weight after dry 48 h in 65 ℃ of baking ovens, water-soluble carbohydrate adopts anthrone-Sulphuric acid Colorimetry, crude protein adopts Kjeldahl nitrogen determination (FOSS company full-automatic Kjeldahl determination device), reducing sugar adopts 3,5-dinitrosalicylic acid (3,5-Dinitrosalicylic acid, DNS) colorimetric method for determining, total free aminoacids adopts ninhydrin colorimetry to measure.It is as shown in the table for measurement result.
The silage effect of microbial inoculum to high-moisture clover in table two, this example
From table two, find out, the clover of processing with microbe additive in this example, the different ensiling time period of 2 days～90 days, the indices of ensiling all reached good effect.PH value remains in the different ensiling time level that is less than 5, the content of protein, water-soluble carbohydrate and reducing sugar does not all have because the prolongation of ensiling time shows obvious minimizing, and the content of water content, ammonia-state nitrogen and total free aminoacids does not all have because the prolongation of ensiling time shows obvious increase.
Embodiment recited above is described the preferred embodiment of the present invention; not scope of the present invention is limited; design under spiritual prerequisite not departing from the present invention; various distortion and improvement that the common engineering technical personnel in this area make technical solution of the present invention, all should fall in the definite protection domain of claims of the present invention.
Claims (1)
1. a high-moisture alfalfa ensilage microbial inoculum, is characterized in that: activeconstituents is that preserving number is plant lactobacillus (Lactobacillusplantarum) WN5 of CGMCC NO. 7469 and the lactobacterium casei SN3 (Lactobacillus casei) that preserving number is CGMCC NO. 7470;
Under-20 ° of C conditions, respectively get on plant lactobacillus WN5 and lactobacterium casei SN3 5 microlitres injection MRS solid mediums, coating is cultivated 12～14 hours under the condition of 37 ° of C of temperature; With the single bacterium colony on transfering loop picking MRS solid medium, be inoculated in 10 milliliters of test tubes that 5 milliliters of MRS substratum are housed, under the condition of 37 ° of C of temperature, cultivate the milk-acid bacteria that obtains activation for 16～18 hours, now, in every milliliter of MRS liquid nutrient medium, at least contain 0.993 * 10
10cfu milk-acid bacteria;
By the bacterium liquid of the plant lactobacillus of above-mentioned cultivation (Lactobacillusplantarum) WN5 CGMCC NO. 7469 and lactobacterium casei (Lactobacillus casei) SN3 CGMCC NO.7470 in cfu than for 1:(1-2) ratio be mixed to get the seed liquor of carrying out co-cultivation, inoculum size by 10%, mixed above-mentioned seed liquor is inoculated in MRS liquid nutrient medium not calciferous, 37 ° of static cultivations of C 14 hours～20 hours, make cell density in every milliliters of liquid substratum reach 0.993 * 10
10cfu～0.832 * 10
11cfu;
When above-mentioned silage inoculants bacteria is applied to high-moisture alfalfa ensilage, clover is cut into length 5～8cm, per kilogram high-moisture clover adds 30～35ml silage inoculants bacteria, turns evenly; Then, the Alfalfa Silage after processing is packed into compacting in ensiling device, cover lid, under normal temperature, place; Moisture content≤81.7% of described high-moisture clover, the phase of cradling is that clover stem height was greater than between the seedling stage to ripening stage of 40cm; Ensiling time≤240 day;
Component and content that described MRS substratum is every liter are: peptone 10 g/L, yeast powder 5 g/L, glucose 10 g/L, sucrose 15 g/L, extractum carnis 10 g/L, diammonium hydrogen citrate 2 g/L, sodium acetate 5 g/L, Sodium.alpha.-ketopropionate 0.18 g/L, L-aminothiopropionic acid hydrochloride 0.05g/L, protoheme 0.5 g/L, multiprenylmenaquinone 2g/L, calcium carbonate 10 g/L, K
2hPO
42 g/L, MgSO
47H
2o 0.58 g/L, MnSO
4h
2o 0.25 g/L, Tween-80 1ml/L; Its pH value is 6.4,121 ° of C sterilizings, within 15 minutes, makes.
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