CN114246257B - Biological agent for improving fermentation quality of mixed silage and preparation method thereof - Google Patents
Biological agent for improving fermentation quality of mixed silage and preparation method thereof Download PDFInfo
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- CN114246257B CN114246257B CN202210189331.9A CN202210189331A CN114246257B CN 114246257 B CN114246257 B CN 114246257B CN 202210189331 A CN202210189331 A CN 202210189331A CN 114246257 B CN114246257 B CN 114246257B
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- 238000000855 fermentation Methods 0.000 title claims abstract description 81
- 230000004151 fermentation Effects 0.000 title claims abstract description 81
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- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 239000003124 biologic agent Substances 0.000 title claims abstract description 19
- 239000007788 liquid Substances 0.000 claims abstract description 56
- 241000186679 Lactobacillus buchneri Species 0.000 claims abstract description 45
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
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- 230000002401 inhibitory effect Effects 0.000 description 2
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- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
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- 229930013930 alkaloid Natural products 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 230000000433 anti-nutritional effect Effects 0.000 description 1
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- 230000002180 anti-stress Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 1
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- 235000019621 digestibility Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 229930003935 flavonoid Natural products 0.000 description 1
- -1 flavonoid compounds Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
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- 239000008103 glucose Substances 0.000 description 1
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- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
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- 239000011707 mineral Substances 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- 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
-
- 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
Abstract
The invention discloses a biological agent for improving fermentation quality of mixed silage and a preparation method thereof, and belongs to the field of silage additives. The method comprises the following steps: (1) respectively culturing lactobacillus buchneri and lactobacillus plantarum to obtain lactobacillus buchneri seed liquid and lactobacillus plantarum seed liquid; (2) respectively inoculating lactobacillus buchneri seed liquid and lactobacillus plantarum seed liquid into a fermentation culture medium containing a mulberry leaf extract and a jerusalem artichoke extract for culture, filtering to obtain lactobacillus buchneri fermentation liquid and lactobacillus plantarum fermentation liquid, and mixing to obtain the high-activity biological preparation. The invention adds mulberry leaf extract and jerusalem artichoke tuber extract into a culture medium, and cultures fermentation strains to obtain the high-activity biological agent. The fermentation speed is high after the use, the lactic acid can be quickly generated, the growth of putrefying bacteria is effectively inhibited, and the mildew and the rot of the forage are prevented; the palatability of the silage is enhanced, and the contents of crude protein, organic acid, vitamins and amino acid are all improved.
Description
Technical Field
The invention relates to the technical field of silage additives, in particular to a biological agent for improving fermentation quality of mixed silage and a preparation method thereof.
Background
At present, three main methods for treating straws by ensiling at home and abroad are provided, wherein firstly, ammoniation treatment is carried out, chemical fertilizers containing high nitrogen such as ammonium bicarbonate and urea are added into the straws for ammoniation, and the aim of keeping freshness is achieved by inhibiting the activity of microorganisms; secondly, adding organic acid or inorganic acid for antiseptic treatment to inhibit the activity of microorganisms to achieve the aim of fresh-keeping, the method has the advantages that the pH value of the silage can be reduced to a required degree, but the acids easily influence the absorption of the livestock to mineral substances and the activity of the microorganisms in rumen, and simultaneously have high operation requirement and poor palatability; and thirdly, microbial ensiling treatment, which achieves the aim of preservation through the consumption of oxygen and the generation of acid by the activity of functional microbes.
The mixed silage refers to silage prepared by mixing 2 or more than 2 silage raw materials, and has the advantage of rich nutrient content, but the most silage is generally gramineous plants such as pasture grass, sweet sorghum, corn and the like. The woody feed is a novel unconventional feed, is rich in various amino acids, vitamins and organic matters, has rich nutritive value, has high crude protein content in most woody feed plants, can be used as an excellent protein feed resource, and can relieve the serious restriction of the shortage of protein resources on the development of animal husbandry by effective utilization. However, due to the existence of anti-nutritional factors, the untreated woody feed is poor in palatability when being directly fed to livestock and low in digestion utilization rate, so that ensiling is required. However, the mixed silage of woody plants and herbaceous plants is less, and a high-activity biological agent specially aiming at the mixed silage of the woody plants and the herbaceous plants is needed in order to effectively reduce the loss of nutrient substances of the feed, improve the palatability, improve the nutrient components of the feed and improve the feeding effect.
Disclosure of Invention
In view of the prior art, the invention aims to provide a biological agent for improving the fermentation quality of mixed silage and a preparation method thereof. The invention adds mulberry leaf extract and jerusalem artichoke tuber extract into a culture medium, and cultures fermentation strains to obtain the high-activity biological agent. The fermentation speed is high after the use, the lactic acid can be quickly generated, the growth of putrefying bacteria is effectively inhibited, and the mildew and the rot of the forage are prevented; the palatability of the silage is enhanced, and the contents of crude protein, organic acid, vitamins and amino acid are all improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for preparing a biological agent for improving fermentation quality of mixed silage, comprising the steps of:
(1) respectively culturing lactobacillus buchneri and lactobacillus plantarum to obtain lactobacillus buchneri seed liquid and lactobacillus plantarum seed liquid;
(2) respectively inoculating lactobacillus buchneri seed liquid and lactobacillus plantarum seed liquid into a fermentation culture medium containing a mulberry leaf extract and a jerusalem artichoke extract for culture, filtering to obtain lactobacillus buchneri fermentation liquid and lactobacillus plantarum fermentation liquid, and mixing to obtain the biological preparation.
Preferably, in step (1), the Lactobacillus buchneri (B.buchneri) ((B.))Lactobacillus buchneri) The accession number of (A) is CICC 20293, purchased from China center for culture Collection of Industrial microorganisms; said Lactobacillus plantarum: (Lactobacillus plantarum) The accession number of (A) is CICC 20765, and the CICC 20765 is purchased from China center for culture Collection of Industrial microorganisms.
Preferably, in step (2), the Lactobacillus buchneri seed liquid andthe bacterial concentration of the lactobacillus plantarum seed liquid is 1.0 multiplied by 109CFU/mL; the inoculation amount of the lactobacillus buchneri seed liquid and the lactobacillus plantarum seed liquid is 3% -7% of the volume of the fermentation medium.
Preferably, in the step (2), the mulberry leaf extract is prepared by the following method:
a. crushing mulberry leaves, mixing the crushed mulberry leaves with water, steaming the crushed mulberry leaves and the water to obtain mulberry leaf liquid, cooling the mulberry leaf liquid, adding cellulase and pectinase into the mulberry leaf liquid for enzymolysis, and filtering the mixture to obtain enzymatic hydrolysate;
b. concentrating the enzymolysis liquid to obtain an extract, performing polyamide column chromatography on the extract, and performing column chromatography on the extract according to the volume ratio of 4: eluting with ethanol-ethyl acetate as eluent, collecting eluate, concentrating, and spray drying to obtain folium Mori extract.
Preferably, in the step a, the mass ratio of the mulberry leaves to the water is 1: (5-10); the mass ratio of the cellulase to the pectinase is 1: 1, the addition amount of the cellulase accounts for 3-5% of the total mass of the mulberry leaf liquid; the temperature of enzymolysis is 45-55 ℃, and the enzymolysis time is 12-24 h.
Preferably, in the step (2), the jerusalem artichoke extract is prepared by the following method:
a. crushing and drying the jerusalem artichoke tubers to obtain jerusalem artichoke granules;
b. extracting Jerusalem artichoke granules with ethanol under reflux, filtering, concentrating, and drying to obtain Jerusalem artichoke extract.
Preferably, in the step b, the volume concentration of the ethanol is 70-80%, the reflux temperature is 65-75 ℃, and the reflux time is 0.3-1 h; the mass ratio of the jerusalem artichoke granules to the ethanol is 1 (10-15).
Preferably, in step (2), the fermentation medium is an MRS medium; adding 60-10 g of mulberry leaf extract into each liter of MRS culture medium; adding 100-200 g of jerusalem artichoke extract into each liter of MRS culture medium.
Preferably, in the step (2), the mass ratio of the lactobacillus buchneri fermentation liquor to the lactobacillus plantarum fermentation liquor is (1-3): 1; the fermentation temperature is 35-37 ℃, and the fermentation time is 1-2 days.
In a second aspect of the invention, a biological agent prepared by the preparation method for improving the fermentation quality of the mixed silage is provided.
In a third aspect of the invention, there is provided the use of a biological agent for improving the fermentation quality of mixed silage in mixed silage of woody and herbaceous plants.
The invention has the beneficial effects that:
after the woody plant and the herbaceous plant are mixed and ensiled for use, the microbial agent prepared by the invention can quickly generate lactic acid and effectively inhibit the growth of putrefying bacteria; the silage is prevented from generating heat and consuming nutrition, and the loss of nutrient substances is reduced; the fermentation time is shortened, the fermentation efficiency is improved, and the quality of silage is improved; improving digestibility of dry matter and crude fiber; inhibiting the degradation of protein in silage, reducing the ammonia content, improving the quality of crude protein, and increasing the contents of organic acid, amino acid and vitamin; the forage grass flavor is improved, the palatability is enhanced, and the feed intake of livestock is increased; effectively inhibit secondary fermentation and is beneficial to long-term storage; improving the balance of probiotic flora in the intestinal tract of livestock, improving immunity and enhancing disease resistance and anti-stress capability; improve milk yield and quality of meat, egg and milk.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, mixed silage is generally prepared by mixing herbaceous plants or herbaceous and gramineous plants, and becomes a novel unconventional feed due to the rich nutritional value of the woody plants. If herbaceous plants can be mixed with woody plants for silage, the contents of crude protein, amino acids, vitamins and organic matters of the silage can be further increased. The mixed silage has long fermentation time, the fermentation raw materials are easy to go moldy and rot, and even if the silage succeeds, livestock often do not eat due to the problems of palatability and the like.
Based on the above, the invention aims to provide a preparation method of a biological agent for improving the fermentation quality of mixed silage. The mulberry leaves contain flavonoid compounds, alkaloids, phytosterol, gamma-aminobutyric acid, mulberry leaf polysaccharide and the like. The tuber of Jerusalem artichoke is rich in various microelements such as amino acid, sugar, vitamins and the like, and simultaneously, the Jerusalem artichoke also contains rich substances such as synanthrin, pentosan and starch. After the method is adopted for extraction, the mulberry leaf extract and the jerusalem artichoke extract are added into a fermentation culture medium, so that the biological activity of the strain can be effectively improved. The invention firstly takes the mulberry and the alfalfa as the feed to be mixed and ensiled as the woody plant and the herbaceous plant respectively. Through selecting for use different to ferment the misce bene, come the screening and can ferment woody plant and herbaceous plant's zymophyte:
(1) respectively mixing Lactobacillus brevis (Lactobacillus brevis) CICC 20299 (purchased from China center for culture Collection of Industrial microorganisms), Lactobacillus buchneri: (Lactobacillus buchneri) CICC 20293 (purchased from China center for Collection of Industrial microorganisms and strains), Lactobacillus plantarum (C)Lactobacillus plantarum) CICC 20765 (purchased from China center for Collection of Industrial microorganisms and strains), enterococcus faecalis: (CICC 20765)Enterococcus faecalis) CICC 20440 (purchased from China center for Industrial microorganism culture Collection) and Lactobacillus rhamnosus CGMCC No.18233 (purchased from China center for general microorganism culture Collection) are firstly cultured by MRS solid culture medium, colonies are selected and inoculated to MRS liquid culture medium, a medicine bottle is cultured to obtain seed liquid, and the seed liquid is adjusted to the concentration of 1.0 multiplied by 109And (5) performing propagation culture on the CFU/mL to obtain each fermentation microbial inoculum.
Note: except that the culture temperature is set according to the fermentation temperature of each strain, other culture conditions and inoculum sizes are the same.
(2) The fermentation inoculants (the dosage of each sequence group is the same) are respectively used independently or mixed according to the proportion, and the ratio of the fermentation inoculants is 1: 2, fermenting the mixed feed mulberry and alfalfa in a mass ratio, performing sensory evaluation on fermentation raw materials after fermenting for 30 days, and detecting the content of crude protein in the silage (the content of the crude protein refers to ISO5983-1979 'determination of animal feed-nitrogen content and calculation of the content of the crude protein'). The results are shown in Table 1.
TABLE 1
| Serial number | Bacterial agent species | Sensory evaluation | Crude protein content% |
| 1 | Lactobacillus brevis | Slightly sour | 8.0 |
| 2 | Lactobacillus buchneri | Slightly fragrant of fruit | 8.8 |
| 3 | Lactobacillus plantarum | Slightly fragrant of fruit | 8.6 |
| 4 | Enterococcus faecalis | Pungent and sour taste | 7.6 |
| 5 | Lactobacillus rhamnosus | Pungent and sour taste | 7.0 |
| 6 | Lactobacillus buchneri: lactobacillus plantarum = 1: 1 | Fruit fragrance | 10.0 |
| 7 | Lactobacillus brevis: lactobacillus plantarum = 1: 1 | Slightly sour | 8.8 |
| 8 | And (3) lactobacillus brevis: lactobacillus buchneri = 1: 1 | Slightly sour | 9.0 |
Through the research, the lactobacillus buchneri and the lactobacillus plantarum are used as the fermentation microbial inoculum together to ensile the mulberry and alfalfa, and the fermentation effect is optimal.
Furthermore, the inventor adds the mulberry leaf extract and the jerusalem artichoke extract into the fermentation medium, so that the activity of the fermentation microbial inoculum is greatly improved, the mixed silage is more palatable, the contents of crude protein, amino acid, vitamins and organic matters are higher, and the fermentation time is shortened.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available.
Example 1: preparation of mulberry leaf extract
a. Crushing 100g of mulberry leaves, sieving with a 20-mesh sieve, mixing with 750mL of water, cooking for 0.5h to obtain mulberry leaf liquid, cooling 200g of mulberry leaf liquid, adding 4g of cellulase and 4g of pectinase, performing enzymolysis for 18h at 50 ℃, and filtering to obtain enzymatic hydrolysate;
b. concentrating the enzymolysis solution at 65 deg.C under vacuum degree of 80 kpa, and cooling to obtain extract. And (3) performing chromatography on the extract by using a polyamide column (the diameter-height ratio is 1: 10), wherein the volume ratio is 4: eluting with ethanol-ethyl acetate of 1 with 3 times of column volume, collecting eluate, concentrating, and spray drying to obtain folium Mori extract.
Example 2: preparation method of Jerusalem artichoke extract
a. Crushing and drying the jerusalem artichoke tubers to the water content of 8wt% to obtain jerusalem artichoke granules;
b. adding 1.5L ethanol with volume fraction of 80% into 100g Jerusalem artichoke granule, heating to 70 deg.C, reflux extracting for 40min, cooling, filtering to obtain liquid, concentrating, and spray drying to obtain Jerusalem artichoke extract.
Example 3
(1) Respectively scribing Lactobacillus buchneri CICC 20293 and Lactobacillus plantarum CICC 20765 on MRS solid slant, culturing colony of Lactobacillus buchneri at 37 deg.C and Lactobacillus plantarum at 35 deg.C, activating for 2 times, selecting single colony, inoculating into MRS liquid culture medium, shaking overnight (100 rpm/min) to obtain Lactobacillus buchneri seed solution and Lactobacillus plantarum seed solution, adjusting bacterial concentration of Lactobacillus buchneri seed solution and Lactobacillus plantarum seed solution to 1.0 × 109CFU/mL。
(2) Taking 1L of MRS liquid culture medium, adding 80g of mulberry leaf extract prepared in example 1 and 150g of jerusalem artichoke extract prepared in example 2, uniformly mixing to obtain a fermentation culture medium, and uniformly dividing the fermentation culture medium into 2 parts. Respectively inoculating 50mL of lactobacillus buchneri seed liquid and 50mL of lactobacillus plantarum seed liquid into a fermentation culture medium, fermenting for 36h at 36 ℃, and filtering to respectively obtain lactobacillus buchneri fermentation liquid and lactobacillus plantarum fermentation liquid.
And mixing 30mL of lactobacillus buchneri fermentation liquor with 10mL of lactobacillus plantarum fermentation liquor to obtain the biological preparation.
Composition of MRS liquid medium: casein peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, glucose 5.0g, sodium acetate 5.0gDiammonium citrate 2.0g, Twen 801.0 g, K2HPO4 2.0g，MgSO4·7H2O 0.2g，MnSO4·H2O 0.05g，CaCO320.0g of agar, 15.0g of agar and 1.0L of distilled water, and the pH value is 6.8.
Comparative example 1
(1) Lactobacillus buchneri seed fluid and Lactobacillus plantarum seed fluid were prepared according to the method of step (1) of example 3, adjusting the concentration to 1.0X 109CFU/mL。
(2) Taking 1L of MRS liquid culture medium, adding 80g of mulberry leaf extract prepared in example 1, uniformly mixing to obtain a fermentation culture medium, and uniformly dividing the fermentation culture medium into 2 parts. Respectively inoculating 50mL of lactobacillus buchneri seed liquid and 50mL of lactobacillus plantarum seed liquid into a fermentation culture medium, fermenting for 36h at 36 ℃, and filtering to respectively obtain lactobacillus buchneri fermentation liquid and lactobacillus plantarum fermentation liquid. And mixing 30mL of lactobacillus buchneri fermentation liquor with 10mL of lactobacillus plantarum fermentation liquor to obtain the biological preparation.
Comparative example 2
(1) Lactobacillus buchneri seed liquid and Lactobacillus plantarum seed liquid were prepared according to the method of step (1) of example 3, and the concentration of the bacteria was adjusted to 1.0X 109CFU/mL。
(2) Taking 1L of MRS liquid culture medium, adding 150g of the jerusalem artichoke extract prepared in the example 2, uniformly mixing to obtain a fermentation culture medium, and uniformly dividing the fermentation culture medium into 2 parts. Respectively inoculating 50mL of lactobacillus buchneri seed liquid and 50mL of lactobacillus plantarum seed liquid into a fermentation culture medium, fermenting for 36h at 36 ℃, and filtering to respectively obtain lactobacillus buchneri fermentation liquid and lactobacillus plantarum fermentation liquid. And mixing 30mL of lactobacillus buchneri fermentation liquor with 10mL of lactobacillus plantarum fermentation liquor to obtain the biological preparation.
Comparative example 3
The difference from example 3 is that: taking 1L MRS liquid culture medium, adding 80g folium Mori powder (from Fufeng Sinuote Biotechnology Co., Ltd.) and 150g Jerusalem artichoke powder (from Fufeng Sinuote Biotechnology Co., Ltd.), and mixing well to obtain fermentation culture medium.
And mixing 30mL of lactobacillus buchneri fermentation liquor and 10mL of lactobacillus plantarum fermentation liquor to obtain the sexual biological preparation.
Comparative example 4
The difference from example 3 is that: and mixing 10mL of lactobacillus buchneri fermentation liquor and 10mL of lactobacillus plantarum fermentation liquor to obtain the biological preparation.
Comparative example 5
The difference from example 3 is that: MRS liquid culture medium is used as fermentation medium, and other raw materials are not additionally added.
Test examples
Airing branches and leaves of the feed mulberry and the cut alfalfa for 24 hours, cutting the branches and leaves of the feed mulberry and the cut alfalfa into small sections of about 3-5 cm by using a hay cutter, wherein the feed mulberry and the alfalfa are mixed according to the weight ratio of 1: and 2, mixing the materials by mass to obtain the silage. Dividing the biological agents into 7 groups, wherein each group is 10kg, respectively spraying the biological agents prepared in the example 3 and the comparative examples 1-5 onto the silage to be ensiled of each group, and spraying 20mL of the biological agents of each group; and as a control group, no treatment was performed. Fermenting at 35 deg.C, and counting the ensiling time of each group (the control group is not fermented) with green color after ensiling, slight sour taste or fruit fragrance, loose and soft property when taking up and no stickiness as ensiling standard; and the contents of crude protein and organic acid of the silage are detected (the total acid content is determined by titration method and calculated by organic acid). The results are shown in Table 2.
TABLE 2
| Item | Ensiling time of day | Crude protein content% | Organic acid% |
| Control group | — | 6.6 | 5.12 |
| Example 1 | 16 | 14.32 | 7.01 |
| Comparative example 1 | 27 | 10.03 | 6.23 |
| Comparative example 2 | 32 | 9.57 | 6.09 |
| Comparative example 3 | 36 | 10.11 | 6.33 |
| Comparative example 4 | 20 | 12.14 | 6.61 |
| Comparative example 5 | 47 | 8.0 | 5.69 |
As can be seen from Table 2, the ensiling time of example 3 is much shorter than that of comparative examples 1 to 5, and the crude protein content and the organic acid content after ensiling are much higher than those of comparative examples 1 to 5. Therefore, the high-activity biological agent of the invention can shorten the fermentation time and greatly improve the crude protein content and the organic acid content of the silage.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (8)
1. A preparation method of a biological agent for improving fermentation quality of mixed silage is characterized by comprising the following steps:
(1) respectively culturing lactobacillus buchneri and lactobacillus plantarum to obtain lactobacillus buchneri seed liquid and lactobacillus plantarum seed liquid;
the lactobacillus buchneri has a deposit number of CICC 20293, and the lactobacillus plantarum has a deposit number of CICC 20765;
(2) respectively inoculating lactobacillus buchneri seed liquid and lactobacillus plantarum seed liquid into a fermentation culture medium containing a mulberry leaf extract and a jerusalem artichoke extract for culture, filtering to obtain lactobacillus buchneri fermentation liquid and lactobacillus plantarum fermentation liquid, and mixing to obtain a biological preparation of silage mulberry and alfalfa;
the jerusalem artichoke extract is prepared by the following method:
a. crushing and drying the jerusalem artichoke tubers to obtain jerusalem artichoke granules;
b. extracting Jerusalem artichoke granules with ethanol under reflux, filtering, concentrating, and drying to obtain Jerusalem artichoke extract;
the mulberry leaf extract is prepared by the following method:
a. crushing mulberry leaves, mixing the crushed mulberry leaves with water, steaming the crushed mulberry leaves and the water to obtain mulberry leaf liquid, cooling the mulberry leaf liquid, adding cellulase and pectinase into the mulberry leaf liquid for enzymolysis, and filtering the mixture to obtain enzymatic hydrolysate;
b. concentrating the enzymolysis liquid to obtain an extract, performing polyamide column chromatography on the extract, and performing column chromatography on the extract according to the volume ratio of 4: eluting with ethanol-ethyl acetate as eluent, collecting eluate, concentrating, and spray drying to obtain folium Mori extract.
2. The method according to claim 1, wherein the bacterial concentration of the Lactobacillus buchneri seed liquid and the Lactobacillus plantarum seed liquid in step (2) is 1.0 x 109CFU/mL; the inoculation amount of the lactobacillus buchneri seed liquid and the lactobacillus plantarum seed liquid is 3-7% of the volume of the fermentation medium.
3. The method according to claim 1, wherein in the step a of the method for preparing the mulberry leaf extract, the mass ratio of the mulberry leaves to water is 1: (5-10); the mass ratio of the cellulase to the pectinase is 1: 1, the addition amount of the cellulase accounts for 3-5% of the total mass of the mulberry leaf liquid; the temperature of enzymolysis is 45-55 ℃, and the enzymolysis time is 12-24 h.
4. The preparation method according to claim 1, wherein in the step b of the preparation method of the jerusalem artichoke extract, the volume concentration of the ethanol is 70-80%, the reflux temperature is 65-75 ℃, and the reflux time is 0.3-1 h; the mass ratio of the jerusalem artichoke granules to the ethanol is 1 (10-15).
5. The production method according to claim 1, wherein in the step (2), the fermentation medium is an MRS medium; adding 60-10 g of mulberry leaf extract into each liter of MRS culture medium; adding 100-200 g of jerusalem artichoke extract into each liter of MRS culture medium.
6. The preparation method according to claim 1, wherein in the step (2), the mass ratio of the lactobacillus buchneri fermentation liquor to the lactobacillus plantarum fermentation liquor is (1-3): 1; the fermentation temperature is 35-37 ℃, and the fermentation time is 1-2 days.
7. The biological agent for improving the fermentation quality of the mixed silage prepared by the preparation method of any one of claims 1 to 6.
8. Use of the biological agent for improving fermentation quality of mixed silage as claimed in claim 7 in the preparation of mixed silage of woody plants and herbaceous plants, the woody plants being forage mulberry and the herbaceous plants being alfalfa.
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