CN109609410B - Alfalfa silage starter and alfalfa silage preparation method - Google Patents
Alfalfa silage starter and alfalfa silage preparation method Download PDFInfo
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- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 title claims abstract description 134
- 241000219823 Medicago Species 0.000 title claims abstract description 127
- 239000007858 starting material Substances 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 239000004460 silage Substances 0.000 title abstract description 30
- 240000004658 Medicago sativa Species 0.000 title description 8
- 238000000855 fermentation Methods 0.000 claims abstract description 146
- 230000004151 fermentation Effects 0.000 claims abstract description 146
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 59
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 59
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 59
- 241000193749 Bacillus coagulans Species 0.000 claims abstract description 49
- 229940054340 bacillus coagulans Drugs 0.000 claims abstract description 49
- 238000000034 method Methods 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims description 54
- 230000001580 bacterial effect Effects 0.000 claims description 21
- 239000002609 medium Substances 0.000 claims description 19
- 239000001963 growth medium Substances 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 10
- 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
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 6
- 235000015278 beef Nutrition 0.000 claims description 6
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 229940099596 manganese sulfate Drugs 0.000 claims description 6
- 235000007079 manganese sulphate Nutrition 0.000 claims description 6
- 239000011702 manganese sulphate Substances 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 229920000136 polysorbate Polymers 0.000 claims description 6
- 239000001632 sodium acetate Substances 0.000 claims description 6
- 235000017281 sodium acetate Nutrition 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims 2
- 229920005610 lignin Polymers 0.000 abstract description 13
- 241000191998 Pediococcus acidilactici Species 0.000 description 13
- 230000003213 activating effect Effects 0.000 description 13
- 230000000813 microbial effect Effects 0.000 description 13
- 241000191996 Pediococcus pentosaceus Species 0.000 description 11
- 238000004321 preservation Methods 0.000 description 11
- 230000004913 activation Effects 0.000 description 8
- 238000007789 sealing Methods 0.000 description 8
- 238000005507 spraying Methods 0.000 description 7
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- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 239000004698 Polyethylene Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
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- 229920000573 polyethylene Polymers 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
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- 239000004459 forage Substances 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
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- 241001147416 Ursus maritimus Species 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 102000038379 digestive enzymes Human genes 0.000 description 2
- 108091007734 digestive enzymes Proteins 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 235000010855 food raising agent Nutrition 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
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- 238000012360 testing method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 235000010624 Medicago sativa Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
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- 235000012041 food component Nutrition 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
- A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
- A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
- A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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Abstract
The invention provides an alfalfa silage starter and an alfalfa silage preparation method, and belongs to the technical field of feeds. The alfalfa ensiling leaven comprises lactobacillus plantarum CICC20765 fermentation liquor and bacillus coagulans ACCC10229 fermentation liquor; the mass ratio of the lactobacillus plantarum CICC20765 fermentation liquor to the bacillus coagulans ACCC10229 fermentation liquor is 0.9-1.1: 0.9-1.1. According to the method, the lactobacillus plantarum CICC20765 fermentation liquor and the bacillus coagulans ACCC10229 fermentation liquor are matched according to the mass ratio of 0.9-1.1: 0.9-1.1, so that the content of lignin in the alfalfa silage is reduced. The results of the examples show that: the lactobacillus plantarum CICC20765 and the bacillus coagulans ACCC10229 are matched to reduce the content of lignin in the alfalfa silage by 1.38-11.98%.
Description
Technical Field
The invention belongs to the technical field of feeds, and particularly relates to an alfalfa silage starter and a preparation method of alfalfa silage.
Background
Alfalfa (Medicago sativa) is a high-quality protein forage widely used in dairy cow breeding and is known as the king of forage. China's climate belongs to the same season of rain and heat, and alfalfa harvest is often overlapped with the rainy season in areas with much rainfall in summer, so that the loss is large. Silage is one of effective means for improving the forage value of pasture and is a preferred method for solving the problem of difficult harvesting and storing of alfalfa in rainy season. Alfalfa ensilage (alfalfalfalfa silage) is soft and juicy, has good palatability and high digestibility, and is convenient for long-term storage.
The alfalfa is rich in lignin, and the lignin is a biological macromolecular substance with a complex and stable structure. The animal body is lack of enzymes for degrading lignin, so the lignin cannot be digested and absorbed, thereby reducing the feeding quality and the nutritional value of the feed and influencing the popularization and the application of the alfalfa ensilage to a certain extent.
Disclosure of Invention
In view of the above, the invention provides an alfalfa ensiling starter and an alfalfa ensiling preparation method, which can effectively reduce the content of lignin in alfalfa ensiling and improve the feeding quality and the nutritional value of alfalfa ensiling.
In order to solve the above problems, the present invention provides the following technical solutions:
the invention provides an alfalfa ensiling starter which comprises lactobacillus plantarum CICC20765 fermentation liquor and bacillus coagulans ACCC10229 fermentation liquor;
the mass ratio of the lactobacillus plantarum CICC20765 fermentation liquor to the bacillus coagulans ACCC10229 fermentation liquor is 0.9-1.1: 0.9-1.1.
Preferably, the preparation method of the lactobacillus plantarum CICC20765 fermentation liquor comprises the following steps: inoculating the lactobacillus plantarum CICC20765 bacterial liquid into a liquid fermentation culture medium, and fermenting for 20-28 h at 30-40 ℃ and 80-120 rpm to obtain lactobacillus plantarum CICC20765 fermentation liquid; the liquid fermentation medium comprises the following components in concentration: 12g/L of peptone, 12g/L of beef powder, 6g/L of yeast powder, 24g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate and 5mL/L of Tween, wherein the pH value of the liquid fermentation medium is 5.9.
Preferably, the preparation method of the bacillus coagulans ACCC10229 fermentation liquor comprises the following steps: inoculating the bacillus coagulans ACCC10229 bacterial liquid into a liquid fermentation culture medium, and fermenting for 20-28 h at 30-40 ℃ and 80-120 rpm to obtain a bacillus coagulans ACCC10229 fermentation liquid; the liquid fermentation medium comprises the following components in concentration: 12g/L of peptone, 12g/L of beef powder, 6g/L of yeast powder, 24g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate and 5mL/L of Tween, wherein the pH value of the liquid fermentation medium is 5.9.
Preferably, the viable count of the lactobacillus plantarum CICC20765 bacterial liquid is 1.0 multiplied by 108～×1.0×1010CFU/g; the viable count of the bacillus coagulans ACCC10229 bacterial liquid is 1.0 multiplied by 108～×1.0×1010CFU/g。
Preferably, the inoculation amount is independently 5-15% of the total weight of the liquid fermentation medium.
The invention provides a preparation method of alfalfa ensilage, which comprises the following steps:
1) mixing alfalfa with the alfalfa ensiling starter in the scheme to obtain a total mixture;
2) and (2) carrying out sealed fermentation on the total mixture obtained in the step 1) for 40-50 d to obtain the alfalfa ensilage.
Preferably, the mass volume ratio of the alfalfa and the alfalfa ensiling starter in the step 1) is 1 Kg: 1.4-1.6 mL.
Preferably, the step 1) of mixing further comprises diluting the alfalfa ensiling starter; the dilution multiple is 3-5 times.
Preferably, before the mixing in the step 1), the alfalfa is cut into 2-5 cm alfalfa segments.
Preferably, the temperature of the sealed fermentation in the step 2) is 20-30 ℃.
The invention provides an alfalfa silage starter and an alfalfa silage preparation method. The alfalfa ensiling leaven comprises lactobacillus plantarum CICC20765 fermentation liquor and bacillus coagulans ACCC10229 fermentation liquor; the mass ratio of the lactobacillus plantarum CICC20765 fermentation liquor to the bacillus coagulans ACCC10229 fermentation liquor is 0.9-1.1: 0.9-1.1. In the invention, lactobacillus plantarum CICC20765 fermentation liquor and bacillus coagulans ACCC10229 fermentation liquor are simultaneously added into the silage for fermentation, so that alfalfa cell walls can be expanded, the porosity among fibers is increased, the contact and digestion of lignocellulose digestive enzyme are facilitated, and the content of lignin in alfalfa silage is reduced. The results of the examples show that the combination of lactobacillus plantarum CICC20765 and bacillus coagulans ACCC10229 can obviously reduce the content of lignin in alfalfa silage by 1.38-11.98%.
Detailed Description
The invention provides an alfalfa ensiling starter which comprises lactobacillus plantarum CICC20765 fermentation liquor and bacillus coagulans ACCC10229 fermentation liquor; the mass ratio of the lactobacillus plantarum CICC20765 fermentation liquor to the bacillus coagulans ACCC10229 fermentation liquor is 0.9-1.1: 0.9-1.1. In the invention, the mass ratio of the lactobacillus plantarum CICC20765 fermentation liquor to the bacillus coagulans ACCC10229 fermentation liquor is preferably 1: 1. In the invention, the viable count of the lactobacillus plantarum CICC20765 fermentation liquor is preferably 1.0 multiplied by 108～×1.0×1010CFU/g, more preferably 1.0X 109CFU/g。
In the present invention, the preparation method of the lactobacillus plantarum cic 20765 fermentation broth preferably comprises: inoculating the lactobacillus plantarum CICC20765 bacterial liquid into a liquid fermentation culture medium, and fermenting for 20-28 h at 30-40 ℃ and 80-120 rpm to obtain lactobacillus plantarum CICC20765 fermentation liquid; the liquid fermentation medium comprises the following components in concentration: 12g/L of peptone, 12g/L of beef powder, 6g/L of yeast powder, 24g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate and 5mL/L of Tween, wherein the pH value of the liquid fermentation medium is 5.9. The source of each component in the liquid fermentation medium is not particularly limited in the invention, and the conventional commercial products in the field can be adopted.
In the invention, the inoculation amount of the lactobacillus plantarum CICC20765 bacterial liquid is preferably 5-15% of the total weight of the liquid fermentation medium, and more preferably 10%. In the invention, the preparation method of the lactobacillus plantarum CICC20765 bacterial liquid preferably comprises the steps of sequentially activating and carrying out amplification culture on the lactobacillus plantarum CICC 20765. In the invention, the activating mode is preferably that lactobacillus plantarum CICC20765 is streaked on an MRS solid slant culture medium, and the streaked culture medium is cultured at 35 ℃ until colonies grow out. In the present invention, the number of activation is preferably 2. In the invention, the mode of the enlarged culture is preferably that the activated single colony is inoculated in an MRS liquid culture medium and is kept overnight at 35 ℃ and 120rpm to obtain the lactobacillus plantarum CICC20765 bacterial liquid.
In the invention, the fermentation temperature is 30-40 ℃, preferably 35 ℃; the stirring speed of the fermentation is 80-120 rpm, preferably 100 rpm; the fermentation time is 20-28 h, preferably 24 h.
In the invention, the lactobacillus plantarum CICC20765 is purchased from China center for culture Collection of industrial microorganisms.
In the present invention, the preparation method of the bacillus coagulans ACCC10229 fermentation liquid preferably comprises: inoculating the bacillus coagulans ACCC10229 bacterial liquid into a liquid fermentation culture medium, and fermenting for 20-28 h at 30-40 ℃ and 80-120 rpm to obtain a bacillus coagulans ACCC10229 fermentation liquid; the liquid fermentation medium comprises the following components in concentration: 12g/L of peptone, 12g/L of beef powder, 6g/L of yeast powder, 24g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate and 5mL/L of Tween, wherein the pH value of the liquid fermentation medium is 5.9. The source of each component in the liquid fermentation medium is not particularly limited in the invention, and the conventional commercial products in the field can be adopted.
In the invention, the inoculation amount of the bacillus coagulans ACCC10229 bacterial liquid is preferably 5-10% of the total weight of the liquid fermentation medium, and is preferably 10%. In the invention, the preparation method of the bacillus coagulans ACCC10229 bacterial liquid preferably comprises the steps of sequentially activating and expanding the bacillus coagulans ACCC 10229. In the present invention, the activation is preferably performed by streaking Bacillus coagulans ACCC10229 on MRS solid slant medium, and culturing the streaked medium at 35 ℃ until colonies grow. In the present invention, the number of activation is preferably 2. In the present invention, the amplification culture method is preferably to inoculate the activated single colony in MRS liquid medium, and obtain the bacillus coagulans ACCC10229 bacterial liquid at 35 ℃ and 120rpm overnight.
In the invention, the fermentation temperature is 30-40 ℃, preferably 35 ℃; the stirring speed of the fermentation is 80-120 rpm, preferably 100 rpm; the fermentation time is 20-28 h, preferably 24 h.
In the present invention, the Bacillus coagulans ACCC10229 is purchased from China agricultural microbial culture Collection management center.
In the invention, the lactobacillus plantarum CICC20765 fermentation liquid and the bacillus coagulans ACCC10229 fermentation liquid are added into the silage for fermentation, so that the alfalfa cell walls can be expanded, the porosity among fibers is increased, and the contact and digestion of lignocellulose digestive enzyme are facilitated.
The invention provides a preparation method of alfalfa ensilage, which comprises the following steps:
1) mixing alfalfa with the alfalfa ensiling starter in the scheme to obtain a total mixture;
2) and (2) carrying out sealed fermentation on the total mixture obtained in the step 1) for 40-50 d to obtain the alfalfa ensilage.
The invention mixes alfalfa and alfalfa ensiling leaven to obtain the total mixture. In the invention, before mixing, the alfalfa is preferably cut into small sections of 2-5 cm, and more preferably 4 cm. In the invention, the mass-volume ratio of the alfalfa to the alfalfa ensiling leavening agent is preferably 1 Kg: 1.4-1.6 mL, more preferably 1 Kg: 1.5 mL.
The origin of alfalfa in the present invention is not particularly limited, and alfalfa conventionally used in the art may be used. In the embodiment of the invention, the polar bear variety provided by Dannong seed company is planted in a 'seaside saline-alkali soil greening soil improvement scientific research test base' of Tianjin agricultural resource and environment research institute.
In the present invention, the alfalfa ensilage starter is preferably diluted prior to said mixing; the dilution multiple is preferably 3-5 times, and more preferably 4 times. The diluent for dilution is preferably water. In the present invention, the mixing is preferably performed by spraying the alfalfa ensiling starter on the alfalfa.
After the total mixture is obtained, the invention preferably carries out sealed fermentation on the total mixture for 40-50 days to obtain the alfalfa ensilage. In the present invention, the sealing is preferably performed by using polyethylene plastic. Air is preferably vented prior to sealing. In the invention, the fermentation temperature is preferably 20-30 ℃, and more preferably 25 ℃. The fermentation time is preferably 45 d.
In order to further illustrate the present invention, the following embodiments are described in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Taking out Lactobacillus plantarum CICC20765 and Bacillus coagulans ACCC10229 from a freezing storage tube by using a sterilized inoculating loop, respectively scribing lines on MRS solid slopes, putting the scribed MRS solid culture medium into a 35 ℃ incubator to culture colonies, activating for 2 times, selecting a single colony, inoculating into an MRS liquid culture medium, standing at 35 ℃ at 120rpm/min, and obtaining Lactobacillus plantarum CICC20765 bacterial liquid and Bacillus coagulans ACCC10229 bacterial liquid for later use.
Respectively adjusting the obtained lactobacillus plantarum CICC20765 bacterial liquid and bacillus coagulans ACCC10229 bacterial liquid to the bacterial concentration of 1.0 × 109After CFU/g, respectively inoculating (the inoculation amount is 10 percent of the weight of the liquid culture medium) into the liquid culture medium (12 g/L of peptone, 12g/L of beef powder, 6g/L of yeast powder, 24g/L of glucose, 2g/L of dipotassium hydrogen phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 5mL of Tween and 1L of distilled water at constant volume), preparing the liquid culture medium according to the formula, adjusting the pH value to 5.9, and culturing for 24 hours at 35 ℃ and 100rpm to obtain the lactobacillus plantarum CICC20765 fermentation liquid and the bacillus coagulans ACCC10229 fermentation liquid.
The alfalfa used for the test is a polar bear variety provided by Dannong seed company, the planting area is 'coastal saline-alkali soil greening soil improvement research and experiment base' of Tianjin city agricultural resources and environmental research institute, and the seeding quantity per mu is 1.2 Kg. And (3) cutting the alfalfa in the first initial flowering period of the alfalfa, airing for 24 hours (the dry matter content is 29.13 +/-0.75%), and cutting the alfalfa into small sections of 2-5 cm by using a hay cutter, wherein the small sections are used for preparing alfalfa silage.
Example 2
And mixing the lactobacillus plantarum CICC20765 fermentation liquor obtained in the example 1 and the bacillus coagulans ACCC10229 fermentation liquor according to the mass ratio of 0.9:1.1 to obtain the alfalfa ensiling starter.
Taking the alfalfa segments of example 1, spraying the alfalfa ensiling starter on the alfalfa segments (the mass volume ratio of the alfalfa to the alfalfa ensiling starter is preferably 1 Kg: 1.6mL, in order to ensure the uniformity of the alfalfa ensiling addition, firstly diluting the starter by 3 times, then spraying the diluted starter on the alfalfa segments), putting the alfalfa segments into polyethylene plastic bags (25cm multiplied by 35cm), setting 3 times for each bag to be 300g, performing air suction and sealing simultaneously by using a sealing machine, fermenting at 20 ℃ for 50d, then opening the bags, and analyzing the nutritional ingredients of the alfalfa.
Example 3
And mixing the lactobacillus plantarum CICC20765 fermentation liquor obtained in the example 1 and the bacillus coagulans ACCC10229 fermentation liquor according to the mass ratio of 1.1:0.9 to obtain the alfalfa ensiling starter.
Taking the alfalfa segments of example 1, spraying the alfalfa ensiling starter on the alfalfa segments (the mass volume ratio of the alfalfa to the alfalfa ensiling starter is preferably 1 Kg: 1.4mL, in order to ensure the uniformity of the alfalfa ensiling addition, firstly diluting the starter by 5 times, then spraying the diluted starter on the alfalfa segments), putting the alfalfa segments into polyethylene plastic bags (25cm multiplied by 35cm), setting 3 times for each bag to be 300g, performing air suction and sealing simultaneously by using a sealing machine, fermenting at 30 ℃ for 40d, then opening the bags, and analyzing the nutritional ingredients of the alfalfa.
Example 4
And mixing the lactobacillus plantarum CICC20765 fermentation liquor obtained in the example 1 and the bacillus coagulans ACCC10229 fermentation liquor according to the mass ratio of 1:1 to obtain the alfalfa ensiling starter.
Taking the alfalfa segments of example 1, spraying the alfalfa ensiling starter on the alfalfa segments (the mass volume ratio of the alfalfa to the alfalfa ensiling starter is preferably 1 Kg: 1.5mL, in order to ensure the uniformity of the alfalfa ensiling addition, firstly diluting the starter by 4 times, then spraying the diluted starter on the alfalfa segments), putting the alfalfa segments into polyethylene plastic bags (25cm multiplied by 35cm), setting 3 times for each bag to be 300g, performing air suction and sealing simultaneously by using a sealing machine, fermenting at 25 ℃ for 45 days, then opening the bags, and analyzing the nutritional ingredients of the alfalfa.
Comparative example 1
Alfalfa ensilage was prepared as control 1 in exactly the same procedure as in example 4, except that no starter was added.
Alfalfa silage was prepared as control 2 using the bacillus coagulans ACCC10229 fermentation broth of example 1 as a alfalfa silage starter, with the same procedure as in example 4.
The activating, expanding culturing and fermenting method of the embodiment 1 is adopted to prepare the fermentation liquor of the pediococcus acidilactici CGMCC1.4 (purchased from China general microbiological culture Collection center), the prepared fermentation liquor of the pediococcus acidilactici CGMCC1.4 is used as the fermentation agent of the alfalfa ensilage, other operation steps are completely the same as the embodiment 4, and the alfalfa ensilage is prepared and used as a control group 3.
The activating, expanding culture and fermenting method of the embodiment 1 is adopted to prepare the fermentation liquor of the pediococcus acidilactici CGMCC1.4 (purchased from China general microbiological culture Collection center), the prepared fermentation liquor of the pediococcus acidilactici CGMCC1.4 and the fermentation liquor of the bacillus coagulans ACCC10229 of the embodiment 1 are mixed according to the mass ratio of 1:1 to be used as the fermentation agent of the alfalfa ensiling, other operation steps are completely the same as the embodiment 4, and the alfalfa ensiling is prepared to be used as a control group 4.
The activating, expanding culture and fermentation method of example 1 was used to prepare a pediococcus pentosaceus CICC22737 (purchased from the china industrial microbial strain collection management center) fermentation broth, the prepared pediococcus pentosaceus CICC22737 fermentation broth was used as an alfalfa ensiling starter, and the other operation steps were completely the same as those of example 4 to prepare alfalfa ensils as a control group 5.
The activating, expanding culture and fermentation method of example 1 was used to prepare a pediococcus pentosaceus CICC22737 (purchased from the china industrial microbial strain collection management center) fermentation broth, the prepared pediococcus pentosaceus CICC22737 fermentation broth and the bacillus coagulans ACCC10229 fermentation broth of example 1 were mixed in a mass ratio of 1:1 to prepare an alfalfa silage starter, and other operation steps were completely the same as those of example 4 to prepare alfalfa silage as a control group 6.
Lactobacillus plantarum ACCC11016 (purchased from China agricultural microbial strain preservation management center) fermentation broth is prepared by the activation, amplification culture and fermentation method of example 1, the prepared Lactobacillus plantarum ACCC11016 fermentation broth is used as alfalfa silage starter, other operation steps are completely the same as example 4, and alfalfa silage is prepared as a control group 7.
Lactobacillus plantarum ACCC11016 (purchased from China agricultural microbial strain preservation management center) fermentation broth is prepared by the activation, amplification culture and fermentation method of example 1, and the prepared Lactobacillus plantarum ACCC11016 fermentation broth and Bacillus coagulans ACCC10229 fermentation broth of example 1 are mixed according to the mass ratio of 1:1 to be used as alfalfa ensiling starter, and the other operation steps are completely the same as those of example 4 to prepare alfalfa ensiling to be used as a control group 8.
The activating, expanding culturing and fermenting method of the embodiment 1 is adopted to prepare lactobacillus plantarum ACCC11016 (purchased from China agricultural microbial strain preservation management center) fermentation liquor and pediococcus acidilactici CGMCC1.4 (purchased from China general microbial strain preservation management center) fermentation liquor, the prepared lactobacillus plantarum ACCC11016 fermentation liquor and pediococcus acidilactici CGMCC1.4 fermentation liquor are mixed according to the mass ratio of 1:1 to be used as alfalfa ensiling leavening agent, other operation steps are completely the same as the embodiment 4, and alfalfa ensiling is prepared to be used as a control group 9.
The method comprises the steps of preparing lactobacillus plantarum ACCC11016 (purchased from China agricultural microbial strain preservation management center) fermentation liquor and pediococcus acidilactici CGMCC1.4 (purchased from China general microbial strain preservation management center) fermentation liquor by adopting the activation, amplification culture and fermentation method in the embodiment 1, mixing the prepared lactobacillus plantarum ACCC11016 fermentation liquor, pediococcus acidilactici CGMCC1.4 fermentation liquor and bacillus coagulans ACCC10229 fermentation liquor in the embodiment 1 according to the mass ratio of 1:1:1, and using the mixture as an alfalfa ensiling starter, wherein other operation steps are completely the same as those in the embodiment 4, and preparing alfalfa ensiling to be used as a control group 10.
Lactobacillus plantarum ACCC11016 (chinese agricultural microbial strain preservation management center) fermentation broth and pediococcus pentosaceus CICC22737 (purchased from chinese industrial microbial strain preservation management center) fermentation broth were prepared by the activation, expansion culture and fermentation method of example 1, and the prepared lactobacillus plantarum ACCC11016 fermentation broth and pediococcus acidilactici 22737 fermentation broth were mixed at a mass ratio of 1:1 to be used as alfalfa ensiling starter, and the other operation steps were completely the same as example 4 to prepare alfalfa ensiling as control group 11.
Lactobacillus plantarum ACCC11016 (purchased from the chinese agricultural microbial strain preservation management center) fermentation broth and pediococcus pentosaceus CICC22737 (purchased from the chinese industrial microbial strain preservation management center) fermentation broth were prepared by the activation, expansion culture and fermentation method of example 1, and the prepared lactobacillus plantarum ACCC11016 fermentation broth, pediococcus pentosaceus CICC22737 fermentation broth and bacillus coagulans ACCC10229 fermentation broth of example 1 were mixed in a mass ratio of 1:1:1 to be used as alfalfa silage starter culture, and the other operation steps were completely the same as in example 4 to prepare alfalfa silage as a control group 12.
Alfalfa ensilage was prepared as a control 13 using the lactobacillus plantarum cic 20765 fermentation broth of example 1 as an alfalfa ensilage starter, with the other procedures being exactly the same as in example 4.
The activating, expanding culturing and fermenting method of the embodiment 1 is adopted to prepare the fermentation liquor of the pediococcus acidilactici CGMCC1.4 (purchased from China general microbiological culture Collection center), the prepared fermentation liquor of the pediococcus acidilactici CGMCC1.4 and the fermentation liquor of the lactobacillus plantarum CICC20765 of the embodiment 1 are mixed according to the mass ratio of 1:1 to be used as the fermentation agent of the alfalfa ensiling, other operation steps are completely the same as the embodiment 4, and the alfalfa ensiling is prepared to be used as a control group 14.
The activating, expanding culturing and fermenting method of the embodiment 1 is adopted to prepare the fermentation liquor of the pediococcus acidilactici CGMCC1.4 (purchased from China general microbiological culture Collection center), the prepared fermentation liquor of the pediococcus acidilactici CGMCC1.4, the fermentation liquor of the bacillus coagulans ACCC10229 of the embodiment 1 and the fermentation liquor of the lactobacillus plantarum CICC20765 of the embodiment 1 are mixed according to the mass ratio of 1:1:1 to be used as the fermentation agent of the alfalfa ensiling, other operation steps are completely the same as the embodiment 4, and the alfalfa ensiling is prepared to be used as a control group 15.
The activating, expanding culture and fermentation method of example 1 is adopted to prepare a pediococcus pentosaceus CICC22737 (purchased from China center for culture Collection of Industrial microorganisms) fermentation broth, the prepared pediococcus pentosaceus CICC22737 fermentation broth and the lactobacillus plantarum CICC20765 fermentation broth of example 1 are mixed according to the mass ratio of 1:1 to be used as an alfalfa ensiling starter, and other operation steps are completely the same as those of example 4 to prepare alfalfa ensils to be used as a control group 16.
The activating, expanding culture and fermentation method of example 1 was used to prepare pediococcus pentosaceus CICC22737 fermentation broth (purchased from the china center for industrial microbial strain preservation management), and the prepared pediococcus pentosaceus CICC22737 fermentation broth, bacillus coagulans ACCC10229 of example 1 and lactobacillus plantarum CICC20765 fermentation broth of example 1 were mixed in a mass ratio of 1:1:1 to prepare alfalfa silage starter, and the other operation steps were completely the same as example 4 to prepare alfalfa silage as control group 17.
Example 5
The nutritional components of the alfalfa ensilage prepared in example 4 and comparative example 1 in control groups 1 to 17 were analyzed. The samples were subjected to nutritional value assessment by near infrared spectroscopy (NIR) by CVAS feed analysis chinese service center to determine the content of lignin. The specific measurement results are shown in table 1.
TABLE 1 Effect of different fermenters on lignin content in alfalfa ensilage
As can be seen from Table 1, the combination of Lactobacillus plantarum CICC20765 and Bacillus coagulans ACCC10229 can significantly reduce the content of lignin in alfalfa silage, which is reduced by 1.38% -11.98% compared with the content of lignin in control groups 1-17.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (4)
1. An alfalfa ensiling leaven, which is characterized by consisting of lactobacillus plantarum CICC20765 zymotic fluid and bacillus coagulans ACCC10229 zymotic fluid;
the mass ratio of the lactobacillus plantarum CICC20765 fermentation liquor to the bacillus coagulans ACCC10229 fermentation liquor is 0.9-1.1: 0.9-1.1;
the preparation method of the lactobacillus plantarum CICC20765 fermentation liquor comprises the following steps: inoculating the lactobacillus plantarum CICC20765 bacterial liquid into a liquid fermentation culture medium, and fermenting for 20-28 h at 30-40 ℃ and 80-120 rpm to obtain lactobacillus plantarum CICC20765 fermentation liquid;
the preparation method of the bacillus coagulans ACCC10229 fermentation liquor comprises the following steps: inoculating the bacillus coagulans ACCC10229 bacterial liquid into a liquid fermentation culture medium, and fermenting for 20-28 h at 30-40 ℃ and 80-120 rpm to obtain a bacillus coagulans ACCC10229 fermentation liquid;
the liquid fermentation medium comprises the following components in concentration: 12g/L of peptone, 12g/L of beef powder, 6g/L of yeast powder, 24g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate and 5mL/L of Tween, wherein the pH value of the liquid fermentation medium is 5.9;
the viable count of the lactobacillus plantarum CICC20765 bacterial liquid is 1.0 multiplied by 108~1.0×1010CFU/g; the viable count of the bacillus coagulans ACCC10229 bacterial liquid is 1.0 multiplied by 108~1.0×1010 CFU/g；
The inoculation amount is independently 5% -15% of the total weight of the liquid fermentation medium.
2. A preparation method of alfalfa ensilage comprises the following steps:
1) mixing alfalfa with the alfalfa ensilage starter of claim 1 to obtain a total mixture;
2) carrying out sealed fermentation on the total mixture obtained in the step 1) for 40-50 d to obtain alfalfa ensilage;
the mass volume ratio of the alfalfa and the alfalfa ensiling starter in the step 1) is 1 Kg: 1.4-1.6 mL;
the temperature of the sealed fermentation in the step 2) is 20-30 ℃.
3. The method of claim 2, wherein the step 1) further comprises diluting the alfalfa starter prior to mixing; the dilution multiple is 3-5 times.
4. The method according to claim 2 or 3, wherein the step 1) of mixing further comprises cutting the alfalfa into 2-5 cm pieces.
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