CN110982739A - Lactobacillus plantarum for high-moisture oat silage and application thereof - Google Patents
Lactobacillus plantarum for high-moisture oat silage and application thereof Download PDFInfo
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- 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
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
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- 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
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Abstract
The invention discloses Lactobacillus plantarum for high-moisture oat ensiling, which is preserved in the China general microbiological culture Collection center in 2019, 5, 15 and is Lactobacillus plantarum with the preservation number of CGMCC No. 17740. The invention also provides an application of the plant lactobacillus in preparing oat silage and a preparation method of the oat silage. The strain disclosed by the invention is strong in comprehensive acid production capacity, can quickly generate lactic acid, reduce pH, reduce the content of ammoniacal nitrogen, well improve the palatability of the silage, inhibit the growth of fungi, and is beneficial to improving the aerobic stability of the silage, prolonging the storage life of the silage, and retaining the nutritional quality of oat grass silage raw materials to the greatest extent, and the prepared silage is free of any additive, good in biological safety and low in preparation cost.
Description
Technical Field
The invention belongs to the technical field of silage, and particularly relates to lactobacillus plantarum for high-moisture oat silage.
Background
Silage has become an important component of ruminant diets worldwide. For a long time, the oat is a main food crop and (silage) feed source in semiarid farming and pasturing areas and high-altitude mountain areas in China due to high growth speed, high yield and simple and flexible cultivation mode. However, when the oats are harvested, due to the fact that the oats are not wilted properly in rainy seasons (april and september) and imperfect harvest management measures, the oats cannot be wilted properly under natural conditions and have high water content, so that the activity of butyric acid bacteria in the silage storage process is high, and the oats are not beneficial to the preservation of forage. Studies have shown that environmental factors including rainfall, ultraviolet radiation, air temperature, transport distance, storage and planting area, etc., change the fermentation characteristics of silage, and among these factors, the ambient temperature and humidity at the storage site are the determining factors affecting the fermentation process and aerobic stability of silage. In some cases, suitable storage temperatures facilitate the preservation of functional forage components such as alpha-topherol and beta-carotene, and the microbial community composition and structure in silage is also affected by ambient temperature. Therefore, how to improve the fermentation quality of high-moisture silage is a problem to be solved urgently at present.
Chemical additives such as formic acid, propionic acid, potassium sorbate and the like are added, so that the fermentation quality of high-moisture silage can be improved to a certain extent, however, the production cost of the additives is higher, more microbial preparations are selected by farmers and herdsmen, but the number of lactobacillus preparations aiming at high-moisture oat silage is less at present.
Disclosure of Invention
The invention mainly aims to provide lactobacillus plantarum for high-moisture oat silage aiming at the improved appeal of the high-moisture oat silage fermentation process, and also provides an application of the lactobacillus plantarum, mainly a preparation method of oat silage, wherein the lactobacillus plantarum is subjected to silage fermentation at different temperatures for about 4 months.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
the Lactobacillus plantarum is preserved in the common microorganism center of China general microbiological culture Collection center in 2019, 5 months and 15 days, and the Lactobacillus plantarum strain is Lactobacillus plantarum with the preservation number of CGMCC No.17740 and the preservation address of Beijing north-oriented south district northwest road.
The strain is obtained by separating from samples such as Qinghai-Tibet plateau pickle, silage, livestock manure, gastric juice, yoghourt and the like, and has good biological safety.
The application of the plant lactobacillus suitable for high-moisture oat silage in preparation of oat silage.
The preparation method of the oat silage comprises the following steps:
1) preparing raw materials: manually cutting oats in the 4-month flowering period and oats in the 9-month flowering period, and then cutting the oats to 5-8 cm;
2) strain activation: inoculating Lactobacillus plantarum stored in Ampere bottle in MRS liquid culture medium, culturing at 30 deg.C for 12-14 hr, subculturing for 2 times to obtain activated strain, inoculating activated strain in MRS liquid culture medium, performing amplification culture, coating with cyclodextrin, freeze drying, making into solid preparation, and counting viable bacteria>109cfu/g；
3) Preparing ensiling raw materials and fermenting: diluting the solid-state bacteria preparation with distilled water, inoculating the solid-state bacteria preparation into fresh grass raw materials according to the inoculation amount of the viable count of 100-.
The anaerobic environment is formed by bundling and wrapping, the lactic acid fermentation is easy, and the wrapping is convenient for transportation.
Preferably, the step 3) is to dilute the solid-state bacterium preparation with distilled water, inoculate the diluted solid-state bacterium preparation into fresh grass raw materials according to the inoculation amount of the viable count of 105cfu/g, bundle and wrap the mixture after mixing, and perform ensiling and fermentation for 120 days to obtain the silage.
Preferably, the ensiling fermentation in the step 3) is respectively fermentation in low altitude areas or high altitude areas, wherein the temperature in the low altitude areas is 20-30 ℃ in 5-8 months, the temperature in the 9-12 months is 5-20 ℃, the temperature in the high altitude areas is 4-14 ℃ in 5-8 months, and the temperature in the 9-12 months is-10-10 ℃.
Further, the low-altitude longitude and latitude is N30 degrees 05-31 degrees 26 ', E102 degrees 54-104 degrees 53', the altitude is 450-3600 m, the high-altitude longitude and latitude is N31 degrees 51-33 degrees 33 ', E101 degrees 51-103 degrees 22', and the altitude is 3400-3600 m.
Preferably, the wrapping size in the step 3) is
Density 55kg/m3FM。
And (4) after ensiling and fermentation, carrying out chemical composition, fermentation product, microbial composition and bacterial community analysis on the obtained product. Analysis shows that after the lactobacillus is added, the oat silage has higher soluble carbohydrate and crude protein content; the enrichment degree of the butyric acid bacteria is reduced to a certain extent, the bacterial community structure is remodeled, and the fermentation quality of the silage is improved.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides lactobacillus plantarum for high-moisture oat silage, and also provides an application of the lactobacillus plantarum, mainly a preparation method of oat silage, wherein silage fermentation is carried out at different temperatures for about 4 months.
Drawings
FIG. 1 is a schematic diagram of the fermentation environment temperature of high altitude, low altitude ensiling in an embodiment of the present invention;
FIG. 2 is a schematic diagram of the relative humidity of the high altitude, low altitude ensiling fermentation environment in an embodiment of the present invention;
FIG. 3 is a schematic diagram showing the length of sunshine in a high-altitude, low-altitude ensiling fermentation environment in the example of the present invention;
FIG. 4 shows the relative abundance of bacterial colonies at the phylum level for fresh and silage in the test examples of the present invention;
FIG. 5 shows the relative abundance of bacterial communities at the genus level of fresh and silage in the test examples of the present invention;
FIG. 6 shows the relative abundance of bacterial communities at the species level in fresh and silage samples of the present invention;
FIG. 7 is a Pearson correlation coefficient of butyric acid concentration in the test example of the present invention;
FIG. 8 is a Pearson correlation coefficient of the relative abundance of Clostridia in the detection example of the present invention;
FIG. 9 is a diagram showing PCoA analysis and ANOSIM detection of sample 1 in the detection example of the present invention;
FIG. 10 is a diagram showing PCoA analysis and ANOSIM detection of sample 2 in the detection example of the present invention.
Wherein L represents low altitude, H represents high altitude, C represents blank, and I represents inoculation with the lactic acid bacteria of the present invention.
Detailed Description
The foregoing summary of the invention is described in further detail below with reference to specific embodiments.
It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples. Various substitutions and alterations can be made without departing from the technical idea of the invention as described above, according to the common technical knowledge and conventional means in the field, and the scope of the invention is covered.
Example 1
This example is a strain screening, determination and preservation example.
The lactobacillus plantarum for high-moisture oat ensiling is obtained by separating and screening samples such as Qinghai-Tibet plateau pickle, ensiling feed, livestock excrement, gastric juice and yoghourt for multiple times, is preserved in the common microorganism center of China general microbiological culture Collection center in 2019, 5, 15 and the like, and is L.plantarum with the preservation number of CGMCC No. 17740.
Example 2
The embodiment is an application example of the lactobacillus plantarum obtained in the embodiment 1 in silage fermentation of high-moisture oats, and the specific method steps are as follows:
1) preparing raw materials: 4, manually cutting oats in the flowering period of Xinjin county in Chengdu for 4 months, and then cutting the oats to 5-8 cm;
2) strain activation: inoculating lactobacillus plantarum stored in an ampoule bottle into an MRS liquid culture medium, culturing for 12-14h at the temperature of 30 ℃, subculturing for 2 times to obtain an activated strain, inoculating the activated strain into the MRS liquid culture medium, carrying out amplification culture, wrapping the strain by cyclodextrin, freeze-drying, and making into a solid-state bacterium preparation, wherein the viable count is more than 109 cfu/g;
3) preparing ensiling raw materials and fermenting: diluting the solid-state bacterium preparation with distilled water, inoculating the diluted solid-state bacterium preparation into fresh grass raw materials according to the inoculation amount of viable count of 105cfu/g, uniformly mixing, bundling and wrapping, wherein the wrapping size is as follows:
the density of the silage is 55kg/m3FM (fresh materials), one part of the produced bales is stored in Chengdujin county, the other part of the bales is stored in Hongyuan county of Abao, and silage sample 1 (sample 1) is obtained by performing silage fermentation for 120 d.
Longitude and latitude N30 degrees to 05 degrees to 31 degrees to 26 degrees, E102 degrees to 54 degrees to 104 degrees to 53 degrees, altitude 450 and 500m, temperature 20-30 ℃ in 5-8 months and temperature 5-20 ℃ in 9-12 months in Chengjin county; the longitude and latitude of the region of Hongyuan county of Abaga are N31 degrees, 51 degrees to 33 degrees, E101 degrees, 51 degrees to 103 degrees, 22 degrees, the altitude is 3400-3600m, the temperature is 4-14 ℃ in 5-8 months, and the temperature is-10-10 ℃ in 9-12 months.
The ambient temperature is shown in fig. 1, the ambient relative humidity is shown in fig. 2, and the sunshine length is shown in fig. 3.
Example 3
The embodiment is an application example of the lactobacillus plantarum obtained in the embodiment 1 in silage fermentation of high-moisture oats, and the specific method steps are as follows:
1) preparing raw materials: cutting oat in 9-month Arba Hongyuan county in flowering period to 5-8cm after artificial mowing;
2) strain activation: inoculating lactobacillus plantarum stored in an ampoule bottle into an MRS liquid culture medium, culturing for 12-14h at the temperature of 30 ℃, subculturing for 2 times to obtain an activated strain, inoculating the activated strain into the MRS liquid culture medium, carrying out amplification culture, wrapping the strain by cyclodextrin, freeze-drying, and making into a solid-state bacterium preparation, wherein the viable count is more than 109 cfu/g;
3) preparing ensiling raw materials and fermenting: diluting the solid-state bacterium preparation with distilled water, inoculating the diluted solid-state bacterium preparation into fresh grass raw materials according to the inoculation amount of viable count of 105cfu/g, uniformly mixing, bundling and wrapping, wherein the wrapping size is as follows:
the density of the fresh food is 55kg/m3FM, and the prepared wrapping is partially stored in ChengduxinAnd storing the other part of the Jinxian county in the Hongyuan county of the Abaga, and performing silage fermentation for 120 days to obtain a silage sample 2 (sample 2).
Longitude and latitude N30 degrees to 05 degrees to 31 degrees to 26 degrees, E102 degrees to 54 degrees to 104 degrees to 53 degrees, altitude 450 and 500m, temperature 20-30 ℃ in 5-8 months and temperature 5-20 ℃ in 9-12 months in Chengjin county; the longitude and latitude of the region of Hongyuan county of Abaga are N31 degrees, 51 degrees to 33 degrees, E101 degrees, 51 degrees to 103 degrees, 22 degrees, the altitude is 3400-3600m, the temperature is 4-14 ℃ in 5-8 months, and the temperature is-10-10 ℃ in 9-12 months.
The ambient temperature is shown in fig. 1, the ambient relative humidity is shown in fig. 2, and the sunshine length is shown in fig. 3.
Example 4
The embodiment is an application example of the lactobacillus plantarum obtained in the embodiment 1 in silage fermentation of high-moisture oats, and the specific method steps are as follows:
1) preparing raw materials: 4, manually cutting oats in the flowering period of Xinjin county in Chengdu for 4 months, and then cutting the oats to 5-8 cm;
2) strain activation: inoculating lactobacillus plantarum stored in an ampoule bottle into an MRS liquid culture medium, culturing for 12-14h at the temperature of 30 ℃, subculturing for 2 times to obtain an activated strain, inoculating the activated strain into the MRS liquid culture medium, carrying out amplification culture, wrapping the strain by cyclodextrin, freeze-drying, and making into a solid-state bacterium preparation, wherein the viable count is more than 109 cfu/g;
3) preparing ensiling raw materials and fermenting: diluting the solid-state bacterium preparation with distilled water, inoculating the diluted solid-state bacterium preparation into fresh grass raw materials according to the inoculation amount of viable count of 100 cfu/g, uniformly mixing, bundling and wrapping, wherein the wrapping size is as follows:
the density is 55kg/m3FM (fresh materials), one part of the prepared package is stored in Chengdujin county, the other part of the package is stored in Hongyuan county of Abao, and silage is obtained by ensiling and fermentation for 130 d.
Longitude and latitude N30 degrees to 05 degrees to 31 degrees to 26 degrees, E102 degrees to 54 degrees to 104 degrees to 53 degrees, altitude 450 and 500m, temperature 20-30 ℃ in 5-8 months and temperature 5-20 ℃ in 9-12 months in Chengjin county; the longitude and latitude of the region of Hongyuan county of Abaga are N31 degrees, 51 degrees to 33 degrees, E101 degrees, 51 degrees to 103 degrees, 22 degrees, the altitude is 3400-3600m, the temperature is 4-14 ℃ in 5-8 months, and the temperature is-10-10 ℃ in 9-12 months.
The ambient temperature is shown in fig. 1, the ambient relative humidity is shown in fig. 2, and the sunshine length is shown in fig. 3.
Example 5
The embodiment is an application example of the lactobacillus plantarum obtained in the embodiment 1 in silage fermentation of high-moisture oats, and the specific method steps are as follows:
1) preparing raw materials: cutting oat in 9-month Arba Hongyuan county in flowering period to 5-8cm after artificial mowing;
2) strain activation: inoculating lactobacillus plantarum stored in an ampoule bottle into an MRS liquid culture medium, culturing for 12-14h at the temperature of 30 ℃, subculturing for 2 times to obtain an activated strain, inoculating the activated strain into the MRS liquid culture medium, carrying out amplification culture, wrapping the strain by cyclodextrin, freeze-drying, and making into a solid-state bacterium preparation, wherein the viable count is more than 109 cfu/g;
3) preparing ensiling raw materials and fermenting: diluting the solid-state bacterium preparation with distilled water, inoculating the diluted solid-state bacterium preparation into a fresh grass raw material according to the inoculation amount of viable count of 110cfu/g, uniformly mixing, bundling and wrapping, wherein the wrapping size is as follows:
the density is 55kg/m3FM (fresh materials), one part of the prepared package is stored in Chengdujin county, the other part of the package is stored in Akaba Hongyuan county, and silage is obtained by ensiling and fermentation for 100 d.
Longitude and latitude N30 degrees to 05 degrees to 31 degrees to 26 degrees, E102 degrees to 54 degrees to 104 degrees to 53 degrees, altitude 450 and 500m, temperature 20-30 ℃ in 5-8 months and temperature 5-20 ℃ in 9-12 months in Chengjin county; the longitude and latitude of the region of Hongyuan county of Abaga are N31 degrees, 51 degrees to 33 degrees, E101 degrees, 51 degrees to 103 degrees, 22 degrees, the altitude is 3400-3600m, the temperature is 4-14 ℃ in 5-8 months, and the temperature is-10-10 ℃ in 9-12 months.
The ambient temperature is shown in fig. 1, the ambient relative humidity is shown in fig. 2, and the sunshine length is shown in fig. 3.
Example of detection
In this test example, the chemical composition, fermentation product, microbial composition and bacterial community analysis were performed on the products obtained in examples 2 and 3.
① chemical composition and microbial community of silage raw material (fresh material FM) and silage samples 1, 2:
as shown in tables 1 and 2 below, less lactic acid bacteria (4.33-3.36 log10 cfu/gFM) and more yeast (5.99-6.89log10 cfu/g FM) were present in the silage (fresh FM). After the regulation by inoculating exogenous lactic acid bacteria, the number of the lactic acid bacteria is increased by 52.66-72.75% in a sample 1 and increased by 70.17-133.43% in a sample 2; the number of yeasts in sample 1 was reduced by 43.98-58.20%, and the number of yeasts in sample 2 was reduced by 59.73-66.11%. The lactic acid bacteria preparation increased the soluble carbohydrate content (sample 1, L:1.46vs. 0.30% DM, H:2.85vs. 1.51% DM; sample 2, L:3.77 vs. 2.94% DM, H:2.05vs.1.99) and the Crude Protein (CP) content (sample 1, L:8.46vs. 7.07% DM, H:8.32vs. 7.92% DM; sample 2, L:7.75vs. 7.31% DM, H:6.90vs. 6.26% DM) in the silage. In addition, the lactic acid bacteria preparation also increased the neutral detergent fiber content (L, 57.21vs. 50.91% DM; H, 55.06vs. 51.67% DM) of sample 1.
TABLE 1
TABLE 2
Different letters in the same column represent significance at a P <0.05 level.
② fermentation quality of silage samples
As shown in Table 3 below, the lactic acid bacteria preparation increased the lactic acid content (L,3.84vs. 1.48% DM; H,4.38vs. 2.79% DM) and acetic acid content (L,1.21vs. 0.81% DM; H,1.41vs. 0.82% DM) and decreased the pH (L,4.46vs. 4.56% DM; H,4.36vs. 4.47% DM) of sample 1.
TABLE 3
As shown in table 4 below, the lactic acid bacteria preparation increased the lactic acid content (2.06vs. 1.39% DM), acetic acid content (1.12vs. 0.72% DM) and propionic acid content (0.47vs. 0.32% DM) in sample 2 stored in the red sources, and decreased the silage pH (4.45vs. 4.67% DM) and ammoniacal nitrogen content (16.39vs. 19.65% DM), but had no significant effect on the lactic acid content and acetic acid content of the samples stored in the same way.
TABLE 4
③ bacterial flora composition and structure of silage
As shown in FIGS. 4-6, the richness of firmicutes after ensiling was increased 92.37-145.50%, and the richness of Proteobacteria was decreased 47.90-96.80%, with Lactobacillus being the main bacterium. The genus Lactobacillus (Lactobacillus) is the predominant bacterium. The lactic acid bacteria preparation increases the relative abundance of lactobacillus and decreases the relative abundance of Clostridium tyrobutyricum.
As shown in table 5 below, the lactobacillus preparation increased the number of bacteria OUT stored in the pooled sample 1 (68 vs.152), the abundance index (Chao 1, 112.68vs.251.52) and the diversity index (Shannon, 2.67vs.3.27), and also decreased the number of bacteria OUT stored in the erythrogen sample 1 (89 vs.126).
TABLE 5
As shown in table 6 below, the lactobacillus preparation reduced the number of bacteria OUT (62vs. 142), the abundance index (Chao 1, 116.08vs.220.53) and the diversity index (Shannon, 2.25 vs.3.29) of sample 2 with all present.
TABLE 6
The analysis is shown in the Pearson correlation coefficient for butyric acid concentration of FIG. 7, and the Pearson correlation coefficient for the relative abundance of Clostridium of FIG. 8.
As shown in fig. 9 and 10, the bacterial community structure of the silage significantly changes after inoculation of the lactic acid bacteria preparation.
In conclusion, the invention provides lactobacillus plantarum for high-moisture oat silage, and also provides an application of the lactobacillus plantarum, mainly a preparation method of high-moisture oat silage, wherein silage fermentation is carried out for about 4 months at different temperatures.
Claims (7)
1. A lactobacillus plantarum for high-moisture oat ensiling is characterized in that: the Lactobacillus plantarum is preserved in the general microbiological center of China Committee for Culture Collection of Microorganisms (CCM) in 2019, 5 and 15, and the Lactobacillus plantarum strain is Lactobacillus plantarum with the preservation number of CGMCC No. 17740.
2. Use of the Lactobacillus plantarum used in accordance with claim 1 for high-moisture oat silage preparation.
3. The use according to claim 2, wherein the oat silage is prepared by the following steps:
1) preparing raw materials: manually cutting oats in the 4-month flowering period and oats in the 9-month flowering period, and then cutting the oats to 5-8 cm;
2) strain activation: inoculating Lactobacillus plantarum stored in Ampere bottle into MRS liquid culture medium, culturing at 30 deg.C for 12-14 hr, and culturingPerforming generation culture for 2 times to obtain activated strain, inoculating activated strain, performing amplification culture in MRS liquid culture medium, wrapping with cyclodextrin, freeze drying, making into solid preparation, and counting viable bacteria>109cfu/g；
3) Preparing ensiling raw materials and fermenting: diluting the solid-state bacteria preparation with distilled water, inoculating the solid-state bacteria preparation into fresh grass raw materials according to the inoculation amount of the viable count of 100-.
4. The method for preparing oat silage according to claim 3, characterized in that: and 3) diluting the solid-state bacterium preparation with distilled water, inoculating the diluted solid-state bacterium preparation into the fresh grass raw material according to the inoculation amount of the viable count of 105cfu/g, bundling and wrapping the mixture after uniformly mixing, and performing silage fermentation for 120d to obtain the silage.
5. The method for preparing oat silage according to claim 4, characterized in that: the ensiling fermentation in the step 3) is respectively carried out in low altitude areas or high altitude areas, wherein the temperature in the low altitude areas is 20-30 ℃ in 5-8 months, the temperature in the 9-12 months is 5-20 ℃, the temperature in the high altitude areas is 4-14 ℃ in 5-8 months, and the temperature in the 9-12 months is-10-10 ℃.
6. The method for preparing oat silage according to claim 5, characterized in that: the low-altitude longitude and latitude is N30 degrees 05 '-31 degrees 26', E102 degrees 54 '-104 degrees 53', the altitude is 450-3600 m, the high-altitude longitude and latitude is N31 degrees 51 '-33 degrees 33', E101 degrees 51 '-103 degrees 22', and the altitude is 3400-3600 m.
7. The method for preparing oat silage according to claim 4, characterized in that: the wrapping size in the step 3) is
Density 55kg/m3FM。
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| CN114437991B (en) * | 2022-03-02 | 2024-03-19 | 青海省畜牧兽医科学院 | Lactobacillus plantarum 160 and application thereof |
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