CN115216426B - Bacillus amyloliquefaciens LL-2 and application thereof - Google Patents
Bacillus amyloliquefaciens LL-2 and application thereof Download PDFInfo
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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 from mugwort planting soil, and identifies and obtains a probiotics which can be used for fermentation, in particular to bacillus amyloliquefaciens LL-2. When the strain and the aspergillus niger are used for fermenting mugwort, the strain and the aspergillus niger have higher extraction efficiency on mugwort polysaccharide and Ai Caohuang ketone. The composite microbial inoculum comprising 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 has perennial herb or semi-shrub shape, and the plant has strong fragrance. The distribution of the mugwort is wide, the mugwort is widely planted in the prior art, and the total yield is high. The wormwood has the effects 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 resource waste 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 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 from the soil of the mugwort planting land, identifies and obtains a probiotics which can be used for fermentation, and obtains the probiotics which is bacillus amyloliquefaciens through the analysis of physicochemical properties of the probiotics, so the probiotics are named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2, and the probiotics are subjected to biological preservation, and the preservation information is as follows:
preservation number: cctccc NO: m2022383; classification naming: bacillus amyloliquefaciens LL-2; preservation unit: china center for type culture Collection; mailbox of the university of martial arts in China of the preservation address: 430027; the date of preservation was 2022, 04 and 06.
In a second aspect, the invention provides a microbial agent comprising the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2;
further, the microbial inoculum also comprises aspergillus niger;
further, the collection number of the aspergillus niger is CCTCC NO:93027. available from the 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 comprising one or more of the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 or the microbial inoculum.
Further, the feed additive is obtained by fermenting mugwort with one or more of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 or the microbial inoculum.
Further, the fermentation conditions are as follows: fermenting at 26-30 deg.c for 5-11 days.
The invention further provides application of the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2, aspergillus niger or the microbial inoculum or the feed additive in improving the extraction rate of the mugwort content;
the preservation number of the aspergillus niger is CCTCC NO:93027.
further, the mugwort content comprises polysaccharides and/or flavones.
The invention further provides application of the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2, aspergillus niger or the microbial inoculum or the feed additive in improving the production performance and the antioxidation capability of chickens;
the preservation number of the aspergillus niger is CCTCC NO:93027.
further, the production performance includes: slaughter performance, daily gain, parenchymal organ performance, immune organ performance or digestive organ performance staff.
The invention has the following beneficial effects:
the bacillus amyloliquefaciens LL-2 is obtained by separation, and the composite microbial inoculum is obtained based on the combination of the strain and the aspergillus niger, so that the extraction efficiency of polysaccharide and flavone in the wormwood can be effectively improved. The composite microbial inoculum provided by the invention can be used as a feed additive after being fermented on mugwort, so that the immune function and the digestion function of broiler chickens are enhanced, and the production performance and the antioxidation capability of the broiler chickens are improved.
The invention does not add any carbon source or nitrogen source in the fermentation process (the carbon source or the nitrogen source is generally added in the fermentation known at present), and has no harm to the natural environment.
The invention utilizes a large amount of mugwort byproducts, not only can relieve the grain competition problem of people and animals and reduce the feed cost, but also can fully exert the advantages of mugwort such as anti-infection, anti-allergy and anti-oxidation, improve the disease resistance of animals and increase the income of the breeding industry.
The invention utilizes the antibacterial and antiviral properties of the fermented mugwort of the compound microbial inoculum, can enhance the disease resistance of animal organisms, reduce the occurrence of various diseases and the use of antibiotics, thereby reducing the drug residues of the animal organisms; in addition, the fermentation product can effectively improve the microecology 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 discharge of harmful pathogenic bacteria in excrement.
The invention provides a composite microbial inoculum and is applied to the fermentation of mugwort, the effect of fermented mugwort on promoting animal growth and improving organism disease resistance and the mechanism thereof are researched, and a theoretical basis is provided for the fermented mugwort serving as a feed additive to replace antibiotics.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1
The experiment initially separates, purifies and identifies probiotics which can be used for fermentation from the soil of the mugwort planting field, and the strain is identified as a bacillus amyloliquefaciens by combining with bacterial 16S rDNA sequence analysis through the physiological and biochemical characteristic study of the strain, and then the strain is named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2, and the invention further carries out biological preservation on the strain, wherein the preservation information is as follows:
preservation number: cctccc NO: m2022383; classification naming: bacillus amyloliquefaciens LL-2; preservation unit: china center for type culture Collection; mailbox of the university of martial arts in China of the preservation address: 430027; the date of preservation was 2022, 04 and 06.
Example 2
The invention further relates to an experiment for bacillus amyloliquefaciens LL-2 and aspergillus niger (CCTCC NO:93027, from China center for type culture collection) provided by the embodiment 1, and the experimental method is as follows:
experiment group 1: aspergillus niger (CCTCC NO: 93027) was inoculated in an amount of 3%.
Experiment group 2: the bacillus amyloliquefaciens LL-2 was inoculated in an amount of 3%.
Control group: no strain was inoculated.
Specifically, the single strain is used for fermenting the wormwood 7d in the environment of 28 ℃ with the feed liquid ratio of 5:6 and 3 percent of strain inoculation amount.
The results are shown in the following table:
TABLE 1 extraction of mugwort content by inoculating strains alone
Compared with the control group, the Aspergillus niger (CCTCC NO: 93027) can obviously improve the effective extraction rate of the mugwort polysaccharide and slightly improve the effective extraction rate of Ai Caohuang ketone. And bacillus amyloliquefaciens LL-2 can reduce the extraction efficiency of mugwort polysaccharide and flavone.
Example 3
In this example, the same method as in example 2 was used to perform a compound inoculation experiment, specifically, an experiment was performed using Aspergillus niger (CCTCC NO: 93027) as the main body, and Bacillus amyloliquefaciens LL-2 and two strains LL-8 and LL-16 obtained by separating Bacillus amyloliquefaciens LL-2 simultaneously.
Respectively counting live bacteria of LL-2, LL-8 and LL-16, and regulating the concentration of bacterial liquid to be 1 multiplied by 109cfu/mL; the Aspergillus niger strain is cultivated until spores are mature, 1 loop of mature spores are added into 1mL of PDA liquid culture medium by taking an inoculating loop as a unit, and the mixture is uniformly mixed. The two strain seed solutions are uniformly mixed according to the corresponding volume ratio (2:2, 2:3 and 3:2), and then fermented with the strain inoculation amount of 3 percent (100 g of mugwort is added into 120mL of distilled water to be uniformly mixed, and then 3mL of mixed strain solution is inoculated), wherein the fermentation time is 3d, 5d, 7d, 9d and 11d.
The results are shown in the following table:
TABLE 2 extraction yield of mugwort content from composite seed Strain
From the table, the mixed bacteria fermentation can obviously improve the effective extraction rate of Ai Caohuang ketone.
Example 4
The invention further applies the compound strain in the embodiment 3 to the production of the broiler chicken, explores the influence of the compound strain on the production performance, the immune function, the digestion function and the antioxidation capability of the broiler chicken, and comprises the following specific steps:
288 broiler chickens of 1 day old and close to body weight are randomly divided into 12 groups, 1 control group (only fed with basic daily ration) and 11 test groups (0.5%, 1% and 2% of Aspergillus niger single-strain fermented mugwort powder are respectively added in the basic daily ration, 0.5%, 1% and 2% of LL-2+Aspergillus niger 2:3 mixed strain fermented mugwort are respectively added in the basic daily ration, 0.5%, 1% and 2% of LL-16+Aspergillus niger 2:2 mixed strain fermented mugwort are respectively added in the basic daily ration, 1% of wild Ai Caofen and 1% of Ai Caofen of non-inoculated strain are treated by the same treatment as the fermented mugwort), and each group is repeated for 3 times, and 8 times are each repeated. The broiler was fed with basal diet for 3d adaptation after purchase, and animal experiments were formally started from 3 days old. The feeding test is divided into three stages of front (4-17 days old of broiler), middle (18-31 days old of broiler) and later (32-45 days old of broiler). The basal diet during feeding was formulated according to the broiler nutrient requirements of the national research council (NRC, 1994). All broilers were kept continuously illuminated for 24h during the feeding process, and the temperature of the chicken house was kept at 32 ℃ for 3d before the feeding process, and then gradually cooled down to 26 ℃ at 21 d. In the whole raising process, the broiler chickens adopt a cage raising mode, and the raising management program follows the conventional production and immunization flow of the broiler raising industry in China. The feeding mode ensures that the broiler chickens can eat and drink water freely. Prior to the start of the test, the cages for animal feeding were thoroughly cleaned and sterilized. The feeding trial was continued until 45 days of age.
The invention further detects the effects of slaughter performance, daily gain, solid organs, immune organs and digestive organs of broilers, and the results are as follows:
TABLE 3 Effect of Mixed Strain fermented mugwort on slaughter Performance of broiler chickens
From the results, compared with a basic ration control group, the mixed bacteria fermentation group obviously improves the slaughter rate, the half-bore rate, the full-bore rate and the leg muscle rate of broilers, and the difference is obvious; the pectoral muscle rate of the broiler chicken is slightly improved, and the difference is not obvious; there was also a certain amount of improvement compared to the A.niger low dose group. The mixed fermentation group significantly reduced the abdominal fat rate of broiler chickens compared to the control group and the Aspergillus niger low dose group. The effect of the mixed bacteria is optimal with the addition amount of 0.5 percent compared with the addition amount of different mixed bacteria.
TABLE 4 influence of Mixed bacteria fermented mugwort on average daily weight gain of broiler chickens
From the average daily gain, the average daily gain of the broiler chickens is not improved by the fermentation products in the early feeding stage, the average daily gain of the broiler chickens is improved by the fermentation product group in the later feeding stage, the feed weight ratio of the broiler chickens is reduced, and the feeding efficiency is effectively improved. The average daily gain of the mixed bacteria in the middle period of feeding is higher than that of the Aspergillus niger fermentation group, but the effect of the mixed bacteria fermentation group in the later period of feeding is inferior to that of the Aspergillus niger fermentation group. The higher feed weight in the early stages may be due to waste caused by poor palatability, and the later stages should be considered to increase its palatability.
TABLE 5 Effect of Mixed Strain fermented mugwort on broiler chicken parenchymal organs
From the results shown in Table 5, the results show that the fermentation product can effectively increase the organ index of the solid organs of broiler chickens, the effect of the fermentation product group added with 2% of mixed bacteria is optimal in the early stage of feeding, and the effect of the fermentation product group added with 0.5% of mixed bacteria is better in the later stage of feeding.
TABLE 6 Effect of Mixed bacteria fermented mugwort on broiler immune organs
From the results in Table 6, the fermentation product can be used as an additive to effectively improve the organ index of immune organs of broiler chickens, and the overall result has optimal effect by adding 1% of mixed bacteria fermentation product group; the effect of adding 0.5% of mixed bacteria fermentation product group in the middle feeding period is better. The effect of the mixed bacteria fermentation product is better than that of the Aspergillus niger single bacteria fermentation product.
TABLE 7 Effect of Mixed bacteria fermented mugwort on broiler digestive organs
From the results shown in Table 7, the fermentation product as an additive can effectively increase the organ index of the digestive organs of the broiler chickens, and the different additive combinations can increase the organ index of the digestive organs of the broiler chickens to a certain extent.
The test result shows that the probiotic fermented mugwort can enhance the immune function and the digestion function of the broiler chicken and improve the production performance and the antioxidation capability of the broiler chicken.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (2)
1. The application of the microbial inoculum in improving the flavone extraction rate of the wormwood is characterized in that the microbial inoculum is utilized to ferment the wormwood, the microbial inoculum consists of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LL-2 and aspergillus niger, and the volume ratio of the bacillus amyloliquefaciens LL-2 to the aspergillus niger is 2:3, the preservation number of the bacillus amyloliquefaciens LL-2 is CCTCC NO: M2022383, and the preservation number of the aspergillus niger is CCTCC NO:93027.
2. a feed additive, characterized in that: the fermented mugwort of claim 1, wherein the fermentation conditions are: fermenting at 26-30 deg.c for 5-11 days.
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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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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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| Optimization of the Artemisia Polysaccharide Fermentation Process by Aspergillus niger，Ali Tao等，Frontiers in Nutrition9;Ali Tao等;Frontiers in Nutrition;第9卷;1-8 * |
| 曹广添 ; 占秀安 ; 张玲玲 ; 曾新福 ; 刘金松 ; 杨彩梅 ; .解淀粉芽孢杆菌对肉鸡生长性能、小肠发育和血清生化指标及免疫功能的影响.中国畜牧杂志.2016,(第03期),摘要. * |
| 杨敏馨 ; 寇涛 ; 李悦 ; 张昊 ; 金顺顺 ; 苏越 ; 张莉莉 ; 王恬 ; .解淀粉芽孢杆菌ES-2对肉鸡屠宰性能和肉品质及肌肉抗氧化能力的影响.南京农业大学学报.2016,(第02期),摘要. * |
| 艾叶粉对肉鸡免疫器官指数及生长的影响;吴有华;刘力;王敬;程文超;;江西畜牧兽医杂志(第06期);全文 * |
| 解淀粉芽孢杆菌ES-2对肉鸡屠宰性能和肉品质及肌肉抗氧化能力的影响;杨敏馨;寇涛;李悦;张昊;金顺顺;苏越;张莉莉;王恬;;南京农业大学学报(第02期);摘要 * |
| 解淀粉芽孢杆菌对肉鸡生长性能、小肠发育和血清生化指标及免疫功能的影响;曹广添;占秀安;张玲玲;曾新福;刘金松;杨彩梅;;中国畜牧杂志(第03期);摘要 * |
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