CN106834167B - Composite microbial inoculum for corn straw silage - Google Patents
Composite microbial inoculum for corn straw silage Download PDFInfo
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Abstract
The invention relates to the field of silage, and in particular relates to a composite microbial inoculum for silage of corn straws. The lactobacillus composite microbial inoculum is mainly applied to silage of corn straws, and active bacteria of the lactobacillus composite microbial inoculum comprise: lactobacillus paracasei, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus pentosus, and Lactobacillus mucosae. The lactobacillus composite microbial inoculum has the advantages of stable composition, strong adaptability, rapid growth and low cost. The inoculation fermentation effect is very obvious, the fermentation quality of the corn straw silage can be effectively improved, the yield of acetic acid is improved, the aerobic stability of the corn straw silage is improved, the secondary fermentation of the corn straw silage is inhibited, the loss of dry matters is reduced, and the corn straw silage has wide market development prospect and application and popularization values in the silage field.
Description
Technical Field
The invention relates to the field of silage, and in particular relates to a composite microbial inoculum for silage of corn straws.
Background
In recent years, with the development of industrial biogas engineering in China, the demand of anaerobic fermentation raw materials is gradually increased. China has a large amount of unused crop straws every year. Crop straws are a good anaerobic fermentation raw material, and the production of biogas by the anaerobic fermentation of the crop straws is an important biomass energy conversion technology. The preservation of the raw materials is very important, since the production of straw is seasonal, and the operation of industrial biogas projects requires a steady and continuous supply of raw materials.
The preservation of the crop straws mainly comprises two modes of air drying and ensiling. The air drying process can cause soluble substance consumption, increased degree of fibrosis, increased lignin content, serious dry matter loss and the like, thereby reducing the degradability and gas production potential of the straw.
Compared with air drying, ensiling is a better way to preserve crop straws, can effectively preserve the nutrient content of the green straws, and inhibits the fiberization process; and the loss of dry matter from ensiling is much lower than from air-dry storage. However, under poor storage conditions or during the discharge phase, secondary fermentation can occur after the silage has been exposed to oxygen, resulting in loss of dry matter.
Additives are often required during silage making to improve success. The lactic acid bacteria additive is a main fermentation promoter. The purpose of using the lactobacillus additive is to ensure that lactobacillus is dominant in fermentation, inhibit the activity of harmful microorganisms, reduce the nutrient loss and obtain high-quality silage.
There are many lactic acid bacteria preparations on the market, most of which are homofermentative lactic acid bacteria preparations for improving the lactic acid yield applied to silage, and few heterofermentative lactic acid bacteria preparations. Homofermentative lactic acid bacteria preparations can improve the yield of lactic acid, reduce the yield of acetic acid and improve the residual amount of soluble sugar, and lactic acid and soluble sugar are substrates which can be utilized by saccharomycetes, so homofermentative usually reduces aerobic stability, and silage is easy to decay. The heterotype fermentation lactic acid bacteria can improve the yield of acetic acid in the ensiling process, and the acetic acid can inhibit the growth of fungi such as yeast mold and the like, so that the aerobic stability of silage can be improved, secondary fermentation is prevented, and dry matter loss in the discharging stage is reduced, so that sufficient attention should be paid to the development of the heterotype lactic acid bacteria.
In addition, the lactic acid bacteria on the market are usually single purified bacteria or simple mixed bacteria of several lactic acid bacteria. The purified single microbial inoculum has the defects of reduced acid production function in the purification process, narrow adaptation range to growth conditions, difficulty in obtaining an advantage in ensiling and unsatisfactory inoculation effect. The simple mixed microbial inoculum has simple composition, and the cooperative relationship or antagonistic relationship among the composition bacteria is yet to be proved.
Disclosure of Invention
In order to solve the technical problems, the invention provides a composite microbial inoculum for ensiling corn straws, which comprises active bacteria: lactobacillus paracasei, Lactobacillus brevis, and Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus pentosus, and Lactobacillus mucosae.
The composite microbial inoculum has high synergistic relationship and high-efficiency stability among Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus pentosus, Lactobacillus mucosae and other Lactobacillus, can become a dominant bacterial group in corn straws after being inoculated, can improve the yield of lactic acid and acetic acid, improve the ratio of acetic acid to lactic acid, reduce the pH value of the corn silage, improve the aerobic stability and inhibit the secondary fermentation of the corn silage, and further effectively reduce the loss of dry substances.
The composite microbial agent comprises the following components in percentage by taking the number of viable bacteria as the total number of viable bacteria, wherein the components comprise Lactobacillus parachuticum, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus pentosus and Lactobacillus mucosae: 28% -35%, 20% -25%, 5% -8%, 4% -6% and 1% -2.5%.
Preferably, the Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus pentosus and Lactobacillus mucosae are respectively as follows by percentage of viable count to total viable count: 31.7%, 31.7% and 23.3%, 6.7%, 5.0%, 1.6%.
The composite microbial agent provided by the invention has the advantages that the ratio of the viable count of Lactobacillus parachuting, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus pentosus and Lactobacillus mucosae is 28-35: 28-35: 20-25: 5-8: 4-6: 1-2.5, preferably 31.7: 31.7: 23.3: 5.7: 5: 1.6.
preferably, the Lactobacillus parachuting can be Lactobacillus parachuting JCM 12493T；
The Lactobacillus brevis can be specifically Lactobacillus brevis JCM 1170;
the Lactobacillus plantarum can be Lactobacillus plantarum JCM 1149T；
The Lactobacillus paracasei can be specifically Lactobacillus paracasei CGMCC No. 1.3112;
the Lactobacillus pentosus can be Lactobacillus pentosus JCM 1158T；
The Lactobacillus mucosae can be specifically Lactobacillus mucosae CGMCC No. 6733.
The active bacteria of the composite microbial inoculum can only consist of the Lactobacillus paracasei, the Lactobacillus brevis, the Lactobacillus plantarum, the Lactobacillus paracasei, the Lactobacillus pentosus and the Lactobacillus mucosae.
The complex microbial inoculum is a liquid preparation.
The liquid formulation comprises glycerin and EDTA; preferably, the ratio of the active ingredient, the glycerol and the EDTA is 1 × 1011CFU 1-5g glycerol: 1-2g EDTA, more preferably 1X 1011CFU 4g glycerol: 1g of EDTA. The liquid preparation can keep the quantity and activity of viable bacteria stable for a long time, and is convenient to transport and use.
The invention also provides application of the compound microbial inoculum in preparation of corn silage straws.
The technical scheme of the invention has the following beneficial effects: the complex microbial inoculum of the invention has the advantages of stable composition, strong adaptability, rapid growth and low cost. The inoculation fermentation effect is very obvious, the fermentation quality and the acetic acid yield of the corn straw silage can be effectively improved, the aerobic stability of the corn straw silage is improved, the secondary fermentation of the corn straw silage is inhibited, the dry matter loss is reduced, and the corn straw silage has wide market development prospect and application and popularization values in the silage field.
Detailed Description
The following examples further describe embodiments of the present invention in detail. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 screening of active bacteria in composite microbial inoculum for corn stalk silage
Preparation of modified SL broth: mixing 10g of tryptone, 10g of yeast powder, 10g of glucose, 10g of sucrose, 6g of monopotassium phosphate, 2g of ammonium citrate, 15g of anhydrous sodium acetate, 1ml of Tween 80, 0.58g of magnesium sulfate heptahydrate, 0.03g of ferrous sulfate heptahydrate, 0.11g of manganese sulfate tetrahydrate and 100ml of boiled corn water, adding water to 1000ml, and adjusting the pH value to about 5.3 by using acetic acid to obtain the compound feed. The preparation of the corn boiling water comprises the following steps: taking 4 fresh corns with skins, weighing about 1.5kg, adding 2.5L of deionized water, and boiling to about 800ml to obtain the corn feed.
The well fermented Chinese cabbage pickle is used as a source for screening floras. 45mL of the improved SL culture medium was put into a 50mL blue-capped bottle, 5g of kimchi was added, and the mixture was subjected to static culture at 30 ℃. After 72h of culture, inoculating the culture according to the inoculum size of 10 percent (volume fraction ratio) for continuous subculture, keeping the culture which has rapid pH drop, more acetogenesis amount and relatively stable change to the external environment, and eliminating the culture opposite to the culture until obtaining the lactobacillus composite system with stable property and strain composition.
The Lactobacillus composite line is researched by a plate separation technology and a clone library technology, and found that the constituent bacteria mainly comprise Lactobacillus parachuting, Lactobacillus brevis and Lactobacillus plantarum, the proportion of the viable bacteria number to the total viable bacteria number is 31.7%, 31.7% and 23.3%, and the proportion of the viable bacteria number to the total viable bacteria number is 6.7%, 5% and 1.6%.
Example 2 preparation of composite microbial inoculum for corn stalk silage
The species used in this example are all species conventional in the art.
Lactobacillus parachuting, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus pentosus and Lactobacillus mucosae are respectively inoculated into an MRS culture medium and are statically cultured for 48 hours at 30 ℃, 31.7ml of fermentation liquor of the Lactobacillus parachuting, 23.3ml of fermentation liquor of the Lactobacillus brevis and 31.7ml of fermentation liquor of the Lactobacillus parachuting are mixed with 6.7ml of the Lactobacillus parachuting, 5ml of the Lactobacillus pentosus and 1.6ml of the Lactobacillus mucosae, so that the percentages of the viable bacteria of the Lactobacillus parachuting, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus parachuting, Lactobacillus pentosus and the Lactobacillus mucosae in the mixed fermentation liquor are respectively 31.7%: 31.7%: 23.3%: 23.7%, 6.7%, 6.5%, 10% and 10% respectively8CFU/ml。
Mixing the above mixed fermentation broth, glycerol and EDTA at 1 × 103ml: 4 g: 1g of the composite microbial inoculum is mixed to obtain the liquid preparation of the composite microbial inoculum for corn straw ensiling.
Example 3 preparation of Complex microbial inoculum for corn stalk silage
In this example, Lactobacillus parachuting JCM 12493TLactobacillus brevis JCM 1170 and Lactobacillus plantarum JCM 1149TAnd Lactobacillus pentosus JCM 1158TAre all preserved in the Japanese microorganism preservation center, and the Lactobacillus paracasei CGMCC No.1.3112 and the Lactobacillus mucosae CGMCC No.6733 are all preserved in the China general microorganism culture preservation management center.
Lactobacillus parachui strain JCM 12493TLactobacillus brevis JCM 1170 and Lactobacillus plantarum JCM 1149TLactobacillus paracasei CGMCC No.1.3112, Lactobacillus pentosus JCM 1158TAnd Lactobacillus mucosae CGMCC No.6733 are respectively inoculated into an MRS culture medium, standing culture is carried out for 48 hours at 30 ℃, 35ml of fermentation liquor of the Lactobacillus paracasei, 20ml of fermentation liquor of the Lactobacillus brevis and 30ml of fermentation liquor of the Lactobacillus plantarum are mixed to 6.7ml of the Lactobacillus paracasei, 5ml of the Lactobacillus pentosus and 2ml of the Lactobacillus mucosae, so that the number percentage of viable bacteria of the Lactobacillus paracasei, the Lactobacillus brevis, the Lactobacillus plantarum, the Lactobacillus paracasei, the Lactobacillus pentosus and the Lactobacillus mucosae in the mixed fermentation liquor is respectively 31 percent, 35 percent, 20 percent, 6 percent, 2 percent, and the total viable bacteria number in the mixed fermentation liquor is 108CFU/ml。
Mixing the above mixed fermentation broth, glycerol and EDTA at 1 × 103ml: 5 g: 2g of the mixture is mixed to obtain the liquid preparation of the composite microbial inoculum for corn straw ensiling.
Test example 1 inoculation fermentation effect of composite microbial inoculum for corn stalk silage
Crushing freshly harvested fresh corn straws into straw particles with the particle size of 1-2 cm, uniformly mixing the liquid lactobacillus composite microbial inoculum prepared in the embodiment 2 according to the proportion of 24ml to 1000g of the corn straw particles, and sealing and ensiling. And (5) performing sealed ensiling. After 50 days of fermentation at room temperature, the yield of lactic acid of the silage obtained is 22.60g/kg, the yield of acetic acid is 14.11g/kg, and the pH value is 3.7.
Control group 1 was also prepared, namely: and (3) hermetically ensiling the corn straw particles, not inoculating the lactobacillus composite microbial inoculum, and fermenting at room temperature for 50 days to obtain ensilage with the lactic acid yield and the acetic acid yield of 18.53g/kg and 4.33g/kg respectively and the pH value of 4.0.
The results show that the lactic acid bacteria composite system can improve the yield of lactic acid and acetic acid and improve the fermentation effect.
Test example 2 measurement of aerobic stability
Immediately after the silage of test example 1 above was completed, 1000g of silage was placed loosely in a polyethylene foam box without compaction and without sealing, and covered to reduce water evaporation. The temperature change was recorded in a 20 ℃ incubator using a YOKOGAWA DX1006 paperless recorder. And (4) placing each thermocouple probe at the central position of the straw, and setting the instrument to record every 30 s. The aerobic stability is determined by the number of hours required for the core temperature of the silage to be 2 ℃ higher than the external temperature after the silage is exposed in the air.
It was observed that the aerobic stability of the non-inoculated treatment (i.e., control group 1) of test example 1 was 185h, and the aerobic stability of the inoculated treatment was 1682h, which proved that the aerobic stability of the lactobacillus composite inoculum was greatly improved. After the silage is exposed for 5 days in the presence of oxygen, compared with a control group 1, the silage inoculated with the lactobacillus composite inoculant has the dry matter loss reduced by 10.9 percent; after 12 days of aerobic exposure, the silage inoculated with the lactobacillus composite inoculant has a dry matter loss reduced by 31 percent compared with that of a control group 1; therefore, the inoculation of the lactobacillus composite microbial inoculum effectively improves the aerobic stability and reduces the dry matter loss.
Control group 2 was also prepared, namely: a liquid Lactobacillus complex inoculum having a single strain was prepared from Lactobacillus parachui according to the method described in example 2, and the following tests were performed:
(1) an inoculation fermentation effect test is carried out according to the test example 1, and the results of observation show that the yield of lactic acid and acetic acid of the silage obtained is 16.26g/kg and 10.95g/kg respectively, and the pH value is 4.1;
(2) then, the aerobic stability was measured in accordance with test example 2, and it was observed that the resulting silage had an aerobic stability of 268 h; silage has a 8.5% reduction in dry matter loss compared to control 1 after 5 days of aerobic exposure; after 12 days of aerobic exposure, the dry matter loss was reduced by 26.4% compared to control 1.
The results show that the effect of the Lactobacillus composite system in the aspects of improving the yield of acetic acid, improving aerobic stability and reducing dry matter loss is better than that of the Lactobacillus parabrucei inoculated alone.
The embodiments of the present invention have been presented for purposes of illustration and description, and are not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.
Claims (7)
1. The composite microbial inoculum for ensiling the corn straws is characterized in that active bacteria comprise: lactobacillus paracasei, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus pentosus and Lactobacillus mucosae;
the ratio of the viable count of Lactobacillus parachuting, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus pentosus and Lactobacillus mucosae is 28-35: 28-35: 20-25: 5-8: 4-6: 1 to 2.5;
the Lactobacillus parachui is Lactobacillus parachui JCM 12493TThe Lactobacillus brevis is Lactobacillus brevis JCM 1170, and the Lactobacillus plantarum JCM is Lactobacillus plantarum JCM 1149TThe Lactobacillus paracasei is Lactobacillus paracasei CGMCC No.1.3112, and the Lactobacillus pentosus is Lactobacillus pentosus JCM 1158TThe Lactobacillus mucosae is Lactobacillus mucosae CGMCC No. 6733.
2. The composite bacterial agent of claim 1, wherein the Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus pentosus and Lactobacillus mucosae are, respectively, as a percentage of the number of viable bacteria to the total number of viable bacteria of the composite bacterial agent: 28% -35%, 20% -25%, 5% -8%, 4% -6% and 1% -2.5%.
3. The complex microbial inoculant according to claim 1 or 2, wherein the complex microbial inoculant is a liquid preparation.
4. The complex microbial inoculant according to claim 3, wherein the liquid formulation comprises glycerol and EDTA.
5. The complex microbial inoculum of claim 4, wherein the ratio of the active ingredient, glycerol and EDTA is 1 x10111-5g of CFU (glycerol): 1-2g EDTA.
6. The complex microbial inoculum of claim 5, wherein the ratio of the active ingredient, glycerol and EDTA is 1 x1011CFU 4g glycerol: 1g of EDTA.
7. Use of the complex microbial inoculum of any one of claims 1 to 6 in preparation of corn silage straw.
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| CN111040969B (en) * | 2019-12-27 | 2021-11-30 | 广西壮族自治区水牛研究所 | Compound lactobacillus agent and application thereof in buffalo silage |
| CN114621900B (en) * | 2022-04-21 | 2023-04-07 | 四川农业大学 | Lactobacillus paracasei and application thereof |
| CN116790423B (en) * | 2023-05-18 | 2023-11-28 | 中国科学院东北地理与农业生态研究所 | Lactobacillus brevis and application thereof in corn or rice straw micro-storage |
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