CN115216426A - Bacillus amyloliquefaciens LL-2 and application thereof - Google Patents
Bacillus amyloliquefaciens LL-2 and application thereof Download PDFInfo
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- CN115216426A CN115216426A CN202210743166.7A CN202210743166A CN115216426A CN 115216426 A CN115216426 A CN 115216426A CN 202210743166 A CN202210743166 A CN 202210743166A CN 115216426 A CN115216426 A CN 115216426A
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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
- C12N1/205—Bacterial isolates
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
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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
- C12N1/14—Fungi; Culture media therefor
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/685—Aspergillus niger
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention relates to the technical field of microorganisms, in particular to bacillus amyloliquefaciens LL-2 and application thereof. The invention separates and purifies soil from a wormwood planting field, and identifies and obtains a probiotic for fermentation, in particular to bacillus amyloliquefaciens LL-2. When the strain and the aspergillus niger are used for fermenting the wormwood, the extraction efficiency of the wormwood polysaccharide and the wormwood flavone is high. The composite microbial agent containing the bacillus amyloliquefaciens LL-2 provided by the invention can be applied to a feed additive after the wormwood is fermented, so that the immune function and the digestion function of broiler chickens can be effectively enhanced, and the production performance of the broiler chickens is improved.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus amyloliquefaciens LL-2 and application thereof.
Background
The mugwort is a plant of the genus Artemisia of the family Compositae, and is a perennial herb or a slightly semi-shrub, and the plant has strong fragrance. The wormwood is widely distributed, the wormwood is generally planted in the prior art, and the total yield is high. The wormwood has the functions of warming channels, removing dampness, dispelling cold, stopping bleeding, diminishing inflammation, relieving asthma, relieving cough, preventing miscarriage, resisting allergy and the like, and is commonly used in the field of traditional Chinese medicine health care, but a large amount of byproducts are not reasonably processed and utilized, so that the waste of resources is caused.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides bacillus amyloliquefaciens LL-2 and application thereof.
In a first aspect, the invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2, wherein the preservation number of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 is CCTCC NO: M2022383.
The invention separates and purifies the soil in the mugwort planting field, identifies and obtains a probiotic which can be used for fermentation, obtains the probiotic as Bacillus amyloliquefaciens by analyzing the physicochemical properties of the probiotic, then names the probiotic as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 and carries out biological preservation on the probiotic, wherein the preservation information is as follows:
the preservation number is: CCTCC NO: m2022383; and (3) classification and naming: bacillus amyloliquefaciens LL-2; the preservation unit: china center for type culture Collection; the preservation address of the mail box of Wuhan university of Wuhan, china: 430027; the preservation date is 2022, 04/06 days.
In a second aspect, the present invention provides a microbial agent comprising the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2;
further, the microbial inoculum also comprises aspergillus niger;
further, the preservation number of the aspergillus niger is CCTCC NO:93027. can be purchased from China center for type culture Collection.
Further, the volume ratio of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 to the aspergillus niger is (1-3): (1-3).
In a third aspect, the invention provides a feed additive, which comprises one or more of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 or the microbial inoculum.
Further, the feed additive is obtained by fermenting wormwood with one or more of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 or the microbial inoculum.
Further, the fermentation conditions are as follows: fermenting for 5-11 days at 26-30 ℃.
The invention further provides application of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2, or aspergillus niger, or the microbial inoculum, or the feed additive in improving the extraction rate of the wormwood content;
the preservation number of the aspergillus niger is CCTCC NO:93027.
further, the wormwood content comprises polysaccharides and/or flavones.
The invention further provides application of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2, or aspergillus niger, or the microbial inoculum, or the feed additive in improving the production performance and the antioxidant capacity of chickens;
the preservation number of the aspergillus niger is CCTCC NO:93027.
further, the production properties include: one or more of slaughter performance, daily gain, parenchymal organ performance, immune organ performance, or digestive organ performance employees.
The invention has the following beneficial effects:
according to the invention, the bacillus amyloliquefaciens LL-2 is obtained through separation, and the composite microbial inoculum is obtained based on the strain and aspergillus niger through compounding, so that the extraction efficiency of polysaccharide and flavone in wormwood can be effectively improved. After the wormwood is fermented, the compound microbial inoculum provided by the invention can be used as a feed additive, so that the immune function and the digestion function of the broiler chicken are enhanced, and the production performance and the oxidation resistance of the broiler chicken are improved.
The invention does not add any carbon source or nitrogen source in the fermentation process (the existing fermentation generally adds the carbon source or the nitrogen source), and has no harm to the natural environment.
The invention utilizes a large amount of wormwood byproducts, not only can relieve the problem of food competition between people and livestock and reduce the feed cost, but also can fully exert the advantages of wormwood such as infection resistance, allergy resistance and oxidation resistance, improve the disease resistance of animals and improve the income of the breeding industry.
According to the invention, the antibacterial and antiviral properties of the wormwood fermented by the composite microbial inoculum are utilized, so that the disease resistance of an animal body can be enhanced, the occurrence of various diseases and the use of antibiotics can be reduced, and the drug residue of the animal body can be reduced; in addition, the fermentation product can effectively improve the micro-ecological balance of animal intestinal tracts, inhibit the growth of pathogenic bacteria, promote the growth of beneficial bacteria and reduce the environmental pollution caused by the emission of harmful pathogenic bacteria in excrement.
The invention provides a compound microbial inoculum and applies the compound microbial inoculum to wormwood fermentation, researches the function and mechanism of the fermented wormwood in promoting the growth of animals and improving the disease resistance of organisms, and provides a theoretical basis for the use of the fermented wormwood as a feed additive to replace antibiotics.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
The probiotic bacteria for fermentation are obtained by initially separating, purifying and identifying the soil in the wormwood planting field, the strain type is identified to be Bacillus amyloliquefaciens by the research on the physiological and biochemical characteristics of the strain and the analysis of a 16S rDNA sequence of the bacteria, and the strain is named as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2, the strain is further subjected to biological preservation, and the preservation information is as follows:
the preservation number is: CCTCC NO: m2022383; and (3) classification and naming: bacillus amyloliquefaciens LL-2; the preservation unit: china center for type culture Collection; the preservation address of the mail box of Wuhan university of Wuhan, china: 430027; the preservation date is 2022, 04 and 06.
Example 2
The invention further aims at the bacillus amyloliquefaciens LL-2 and the aspergillus niger (CCTCC NO:93027 from China center for type culture Collection) provided by the embodiment 1 to carry out experiments, and the experimental method is as follows:
experimental group 1: aspergillus niger (CCTCC NO: 93027) is inoculated with the inoculation amount of 3 percent.
Experimental group 2: inoculating bacillus amyloliquefaciens LL-2 with the inoculation amount of 3 percent.
Control group: no strain was inoculated.
Specifically, the wormwood is fermented by a single strain for 7d in an environment with a feed-liquid ratio of 5, 6 percent and 3 percent of strain inoculation amount and 28 ℃.
The results are shown in the following table:
TABLE 1 extraction of mugwort content of the inoculated strains alone
Compared with a control group, aspergillus niger (CCTCC NO: 93027) can obviously improve the effective extraction rate of wormwood polysaccharide and slightly improve the effective extraction rate of wormwood flavone. While the bacillus amyloliquefaciens LL-2 can reduce the extraction efficiency of the wormwood polysaccharide and the flavonoids.
Example 3
In this example, the same method as that used in example 2 was used to perform a composite inoculation experiment, specifically, an experiment was performed using Aspergillus niger (CCTCC NO: 93027) as the main component and using Bacillus amyloliquefaciens LL-2 and Bacillus amyloliquefaciens LL-2 as the auxiliary components to simultaneously separate two strains LL-8 and LL-16.
Respectively counting viable bacteria of LL-2, LL-8 and LL-16, and then adjusting the concentration of bacterial liquid to be 1 multiplied by 109cfu/mL; culturing the aspergillus niger strains until spores are mature, taking an inoculating loop as a unit, adding 1-loop mature spores into 1mL of PDA liquid culture medium, and uniformly mixing. The two bacteria seed liquids are uniformly mixed according to the corresponding volume ratio (2:2, 2:3 and 3:2), and then fermented by 3% of bacteria inoculation amount (after 100g of wormwood is added with 120mL of distilled water and uniformly mixed, 3mL of mixed bacteria solution is inoculated), wherein the fermentation time is 3d, 5d, 7d, 9d and 11d.
The results are shown in the following table:
TABLE 2 Artemisia argyi content extraction ratio of complex inoculum strains
As can be seen from the table, the effective extraction rate of the wormwood flavone can be obviously improved by the mixed bacteria fermentation.
Example 4
The invention further applies the compound strain in the embodiment 3 in the production of the broiler chicken, and explores the influence of the compound strain on the production performance, the immune function, the digestive function and the antioxidant capacity of the broiler chicken, and the specific steps are as follows:
288 of 1-day-old Kebao broilers with healthy body conditions and approximate body weights are randomly divided into 12 groups, 1 control group (only basic daily ration is fed) and 11 test groups (0.5%, 1% and 2% of aspergillus niger single-strain fermented wormwood powder is respectively added into the basic daily ration, 0.5%, 1% and 2% of LL-2+ aspergillus niger 2:3 mixed-strain fermented wormwood is respectively added into the basic daily ration, 0.5%, 1% and 2% of LL-16+ aspergillus niger 2:2 mixed-strain fermented wormwood is respectively added into the basic daily ration, 1% of wild wormwood powder, 1% of wormwood powder which is treated the same as the fermented wormwood and is not inoculated with strains is respectively added into the basic daily ration), and each group has 3 repetitions, and 8 repetitions are respectively carried out. After the broiler is bought back, the broiler is bred for 3d adaptation by basic daily ration, and an animal test is formally started from the 3-day age of the broiler. The feeding test is divided into three stages, namely a front stage (4-17 days old broiler chickens), a middle stage (18-31 days old broiler chickens) and a later stage (32-45 days old broiler chickens). The basal ration during feeding was formulated according to the broiler nutritional requirements as required by the national research council (NRC, 1994). All broilers were kept under continuous lighting for 24h during the rearing period, with the henhouse temperature being maintained at 32 ℃ 3 days before the rearing process, and then gradually cooled to 26 ℃ by the time of day 21. In the whole feeding process, the broiler chickens are raised in cages, and the feeding management program follows the conventional production and immunization process of the broiler chicken breeding industry in China. The feeding mode ensures that the broiler chicken can eat and drink water freely. Cages for animal feeding were thoroughly cleaned and disinfected prior to the start of the experiment. The feeding trial continued to 45 days of age.
The invention further detects the effects of slaughtering performance, daily gain, parenchymal organs, immune organs and digestive organs of the broiler chicken, and the results are as follows:
TABLE 3 influence of mixed bacteria fermented wormwood on slaughtering performance of broiler chickens
From the results, compared with the basic daily ration control group, the mixed bacteria fermentation group obviously improves the slaughtering rate, the half-bore rate, the full-bore rate and the leg muscle rate of the broiler chicken, and the difference is obvious; the breast muscle rate of the broiler chicken is slightly improved, and the difference is not obvious; there was also a certain improvement compared to the aspergillus niger low dose group. Compared with the control group and the aspergillus niger low-dose group, the mixed bacteria fermentation group obviously reduces the abdominal fat rate of the broiler chicken. Compared with different addition amounts of the mixed bacteria, the effect is optimal when the addition amount of the mixed bacteria is 0.5%.
TABLE 4 influence of mixed bacteria fermented wormwood on average daily gain of broiler chicken
From the average daily gain, the average daily gain of the broilers is not improved by the fermentation products in the early feeding stage, the average daily gain of the broilers is improved by the fermentation product groups in the later feeding stage, the feed-weight ratio of the broilers is reduced, and the feeding efficiency is effectively improved. The average daily gain of the mixed bacteria in the middle feeding stage is higher than that of the Aspergillus niger fermentation group, but the effect of the mixed bacteria fermentation group in the later feeding stage is inferior to that of the Aspergillus niger fermentation group. The higher ratio of the weight of the feed at the early stage is probably caused by waste due to poor palatability, and the increase of the palatability is considered at the later stage.
TABLE 5 Effect of mixed bacteria fermented mugwort on broiler parenchymal organs
From the results shown in Table 5, the fermentation product as an additive can effectively improve the organ indexes of the parenchymal organs of the broilers, the effect of the group added with 2% of mixed bacteria fermentation product in the early stage of feeding is optimal, and the effect of the group added with 0.5% of mixed bacteria fermentation product in the later stage of feeding is better.
TABLE 6 influence of mixed bacteria fermented wormwood on immune organs of broiler chicken
From the results in table 6, the fermentation product as an additive can effectively improve the organ index of the immune organs of the broiler chicken, and the whole result is optimal by adding the 1% mixed bacteria fermentation product group; the effect of adding 0.5% of mixed bacteria fermentation product group in the middle period of feeding is better. The effect of the fermentation product of the mixed bacteria is superior to that of the fermentation product of single aspergillus niger.
TABLE 7 Effect of mixed bacteria fermented mugwort on digestive organs of broiler
From the results shown in Table 7, the fermentation product as an additive can effectively improve the organ index of the digestive organs of the broiler chicken, and different additive combinations can improve the organ index of the digestive organs of the broiler chicken to a certain extent.
The test result shows that the probiotic fermented wormwood can enhance the immune function and the digestive function of the broiler chicken and improve the production performance and the antioxidant capacity of the broiler chicken.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 is characterized in that the preservation number of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 is CCTCC NO: M2022383.
2. A bacterial agent comprising the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 according to claim 1.
3. The microbial inoculum of claim 2, further comprising: aspergillus niger.
4. The microbial inoculum according to claim 3, wherein the Aspergillus niger has a preservation number of CCTCC NO:93027.
5. the microbial inoculum according to claim 3 or 4, wherein the volume ratio of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 to the Aspergillus niger is (1-3): (1-3).
6. A feed additive comprising one or more of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 according to claim 1 or the microbial inoculum according to any one of claims 2 to 5.
7. The feed additive according to claim 6, wherein the feed additive is obtained by fermenting mugwort grass using one or more of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 according to claim 1 or the microbial agents according to any one of claims 2 to 5.
8. The feed additive according to claim 6 or 7, wherein the fermentation conditions are: fermenting for 5-11 days at 26-30 ℃.
9. Use of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 according to claim 1, or aspergillus niger, or the microbial inoculum according to any one of claims 2 to 5, or the feed additive according to any one of claims 6 to 8 for improving the extraction rate of wormwood contents;
the preservation number of the aspergillus niger is CCTCC NO:93027.
10. use of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 according to claim 1, or aspergillus niger, or the bacterial agent according to any one of claims 2 to 5, or the feed additive according to any one of claims 6 to 8 for improving the production performance and the antioxidant capacity of chickens;
the preservation number of the aspergillus niger is CCTCC NO:93027.
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Citations (2)
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| CN112352874A (en) * | 2020-11-11 | 2021-02-12 | 贵州省畜牧兽医研究所 | Anti-piglet diarrhea probiotic preparation and preparation method thereof |
| CN113907192A (en) * | 2021-10-19 | 2022-01-11 | 湖南伟业动物营养集团股份有限公司 | Animal health-care traditional Chinese medicine additive and preparation method and application thereof |
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