CN115927058B - Brevibacillus laterosporus producing chlorogenic acid and application thereof - Google Patents
Brevibacillus laterosporus producing chlorogenic acid and application thereof Download PDFInfo
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Abstract
The invention relates to microorganisms, in particular to a strain of Brevibacillus laterosporus producing chlorogenic acid and application thereof, and the strain is named Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170. The strain is preserved in China center for type culture collection (CCTCC NO) in university of Wuhan in Wuhan, hubei province of China for 20 days in 2022, wherein the preservation number is CCTCC NO: m2022932. The strain has high chlorogenic acid content, excellent antibacterial performance and good oxidation resistance, has a preventive effect on ulcerative colitis of mice and has a certain effect of intervening and relieving stress states of the bodies of the mice.
Description
Technical Field
The invention relates to microorganisms, in particular to Brevibacillus laterosporus producing chlorogenic acid and application thereof.
Background
The information disclosed in the background of the invention is intended to enhance an understanding of the general background of the invention, and this disclosure should not necessarily be taken as an acknowledgement or any form of suggestion that this information has become known to a person of ordinary skill in the art.
Chlorogenic acid (Chlorogenic acid) is also called as caffeic tannic acid, is depsipeptide composed of caffeic acid and quinic acid, is a phenylpropanoid compound generated by a shikimic acid way in the aerobic respiration process of plants, has the functions of resisting bacteria, viruses, tumors and the like, is internationally recognized plant gold, and is widely applied to the industries of medicine, daily chemical industry, food and the like.
Chlorogenic acid is widely used as a green feed additive in animal feed, mainly by extraction from various medicinal plants. The common extraction methods mainly comprise a stone mercaptan method, a water extraction-alcohol precipitation method, an alcohol extraction-lead salt precipitation method, a polyamide column chromatography method and the like. In the application, the problems of complicated extraction process, high cost and the like exist, and particularly, when the chlorogenic acid extracted by the methods is applied to livestock and poultry cultivation, hidden danger can be brought to animal organism health.
The chlorogenic acid is produced by microbial fermentation. The presently reported strain belonging to the category of feed additives (2013) and being used for fermenting chlorogenic acid is bacillus subtilis, but the yield is lower. Therefore, the probiotics which can produce chlorogenic acid at high yield are separated and screened to be used as feed additives, so that the effect of chlorogenic acid can be exerted, the probiotics of the fermentation strain can be achieved, and the application prospect is wide.
Disclosure of Invention
The invention provides a strain of Brevibacillus laterosporus which can produce chlorogenic acid with high yield, and lays a foundation for expanding chlorogenic acid medicine sources, protecting rare medicinal plant resources, enriching strain resources and promoting development and utilization of endophytes of plants; the strain also has good antibacterial capability, oxidation resistance, acid production performance and prevention and treatment effects on ulcerative colitis of mice, and has certain intervention and relief effects on stress states of the bodies of the mice.
Specifically, the present invention provides the following technical features, and one or more of the following technical features are combined to form the technical scheme of the present invention.
In a first aspect of the present invention, the present invention provides a strain of Brevibacillus laterosporus, named Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170, which has been deposited in China center for type culture Collection (CCTCC for short, address: university of Wuhan, china) at 2022, month 20, with deposit numbers: cctccc NO: m2022932.
The strain is laterally born, and spores are fusiform, can well survive under normal temperature and aerobic conditions, have good growth performance, and have good antibacterial effects on staphylococcus aureus, clostridium perfringens, escherichia coli, aeromonas hydrophila, salmonella gallinarum and the like; the strain also has good antioxidant capacity, can remove 1, 1-diphenyl-2-picrylhydrazine free radical (DPPH) and hydroxyl free radical (HO), and has good acid production performance, for example, when the strain is fermented and cultured for 24 hours in one embodiment of the invention, the content of lactic acid and acetic acid in fermentation supernatant can reach 1.42g/L and 1.84g/L respectively.
The strain has chlorogenic acid producing capacity. In an embodiment of the present invention, when culturing is performed in an LB medium, the composition (mass percent) of the LB medium is: glucose 0.2%, peptone 1.0%, yeast extract 0.5%, naCl 0.5%, pH 7.0, and the strain shows good chlorogenic acid producing ability. And the yield of chlorogenic acid can be further obviously improved through culture medium optimization. For example, in the embodiment of the present invention, when one or more of glucose, corn starch, and corn steep liquor dry powder is used as a carbon source (particularly, glucose is used as a carbon source), and an organic nitrogen source is used as a nitrogen source (particularly, peptone is used as a nitrogen source), a higher chlorogenic acid yield can be obtained. For example, when copper ions (such as copper sulfate) are added to the medium, the yield of chlorogenic acid is improved as compared with the case where no copper ions are added, for example, when the addition amount of copper ions is 0.001 to 0.005% by mass, the yield of chlorogenic acid is improved, and particularly when the addition amount of copper ions is 0.003% by mass, the yield of chlorogenic acid is significantly improved as compared with the other addition amounts or when no chlorogenic acid is added.
On the basis, the invention provides an optimized culture medium, and the culture medium is used for culturing Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170, so that chlorogenic acid can be produced at high yield. The culture medium takes glucose as a carbon source, peptone as a nitrogen source, the addition amount of the peptone is 1-2% (mass percent), and copper sulfate is added, and the addition amount of the copper sulfate is 0.002-0.005% (mass percent). In some embodiments of the invention, the exemplary composition (mass percent) of the medium is: glucose 0.2%, peptone 1.5%, yeast extract 0.5%, naCl 0.5%, copper sulfate 0.003% and pH 7.0.
In the embodiment of the invention, the Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 is fermented and cultured in an LB culture medium and an optimized culture medium respectively, so that when the optimized culture medium is fermented and cultured for 48 hours, the chlorogenic acid content reaches 11.81 mug/mL, compared with the culture in the LB culture medium for 48 hours (the chlorogenic acid yield is 1.61 mug/mL), the chlorogenic acid yield can be improved by 7.3 times, and the chlorogenic acid content generated by fermentation by adopting an optimized formula is obviously increased along with the increase of the fermentation time, and the chlorogenic acid content in the LB culture medium is not obviously changed along with the increase of the fermentation time.
In a second aspect of the invention, the invention provides a microbial inoculant or feed comprising Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 and/or a fermentation product thereof as described in the first aspect above.
In a third aspect of the invention there is provided a range of uses of Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 and/or its ferments, including in the preparation of a pharmaceutical, a microbial agent or a feed additive having one or more of the following functions;
1) The application in preparing antioxidant medicines, bactericides or feed additives;
2) The application in preparing antibacterial medicines, bactericides or feed additives;
3) Application in preparing medicines, bactericides or feed additives for preventing and treating enteritis;
4) The application of the composition in preparing medicines, bactericides or feed additives for improving the antioxidant stress capability of animals.
In the embodiment of the invention, the Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 and/or the fermentation product thereof has good antibacterial capacity and good antibacterial effect on strains such as staphylococcus aureus, clostridium perfringens, escherichia coli, aeromonas hydrophila, salmonella gallinarum and the like.
In an embodiment of the invention, brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 and/or its fermented product have good antioxidant ability, including DPPH free radical and hydroxyl free radical scavenging.
In an embodiment of the invention, brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 is capable of controlling enteritis, particularly ulcerative colitis, including ameliorating ulcerative colitis and its resultant weight loss, colon shortening, and is capable of modulating serum inflammatory factor levels, including elevating anti-inflammatory cytokine IL-10 levels, and lowering pro-inflammatory cytokine TNF- α and IL-6 levels. Meanwhile, in the embodiment of the invention, the Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 can reduce the content of serum Malondialdehyde (MDA) and improve the content of total superoxide dismutase (T-SOD), has good antioxidant capacity, and can interfere and relieve the stress state of an organism.
In a fourth aspect of the present invention, there is provided a method for producing chlorogenic acid by fermentation, comprising culturing Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 by fermentation.
In an embodiment of the present invention, culturing Brevibacillus fermentation (Brevibacillus laterosporus) BLCC1-0170 with LB medium is capable of producing chlorogenic acid, for example, in one embodiment of the present invention, the composition (mass percent) of the LB medium is: glucose 0.2%, peptone 1.0%, yeast extract 0.5%, naCl 0.5%, pH 7.0.
In the embodiment of the invention, the inventor optimizes the fermentation condition, and the inventor finds that when the bacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 is fermented and cultured, one or more of glucose, corn starch and corn steep liquor dry powder are used as carbon sources, and organic nitrogen source peptone is used as a nitrogen source, the higher chlorogenic acid yield can be obtained.
Wherein, when glucose is used as a carbon source, the yield of chlorogenic acid is higher than that of other carbon sources; and when the organic nitrogen source is peptone and the addition amount thereof is 0.5-2.0% (mass percent), the yield of chlorogenic acid can be remarkably improved, and particularly when the addition amount of peptone is 1.5% (mass percent), the effect of improving the yield is more remarkable.
And, when copper ions (such as copper sulfate) are added to the medium, the yield of chlorogenic acid can be improved when the addition amount of copper ions is 0.001 to 0.005% by mass, and particularly, when the addition amount of copper ions is 0.003% by mass, the yield of chlorogenic acid can be significantly improved as compared with other addition amounts or when no addition is performed.
In the embodiment of the invention, the temperature of the fermentation culture Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 is 35-38 ℃, and the fermentation culture is performed aerobically.
In a preferred embodiment of the present invention, in the fermentation culture of Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170, glucose is used as a carbon source, peptone is used as a nitrogen source, the peptone is added in an amount of 1-2% by mass, and copper sulfate is added in an amount of 0.002-0.005% by mass. In a more preferred embodiment, the specific composition (mass percent) of the medium is: glucose 0.2%, peptone 1.5%, yeast extract 0.5%, naCl 0.5%, copper sulfate 0.003% and pH 7.0. Preferably, the fermentation culture time is 24-48h, preferably the fermentation 48h yield is higher.
Compared with the prior art, the invention has the advantages that: the invention provides a bacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170, which can produce chlorogenic acid with high yield, and the discovery of the strain lays a foundation for expanding chlorogenic acid medicine sources, protecting rare medicinal plant resources, enriching strain resources and promoting the development and utilization of endophytes. In addition, the Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 has good antibacterial capacity, antioxidant capacity and acid production performance, has a preventive effect on ulcerative colitis of mice and has certain intervention and relieving effects on stress states of the bodies of the mice.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
Fig. 1: chlorogenic acid concentration standard curve drawn spectrophotometrically as a primary screen in example 1.
Fig. 2: chlorogenic acid concentration standard curve drawn by high performance liquid chromatography double screening in example 1.
Fig. 3: strain BLCC1-0170 microscopic results (magnification 1000 x).
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.
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. The reagents or materials used in the present invention may be purchased in conventional manners, and unless otherwise indicated, they may be used in conventional manners in the art or according to the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
Example 1 screening of Bacillus chlorogenic acid producing Bacillus and optimization of fermentation conditions
1 materials and methods
1.1 test materials
1.1.1 test strains
The plant endophyte deposited by Shandong Baolili biological engineering stock institute of biological engineering, the strain collection is used as a test strain, and the strain sources are eucommia ulmoides, medlar, honeysuckle and the like.
1.1.2 Medium
LB liquid medium: glucose 0.2%, peptone 1.0%, yeast extract 0.5%, naCl 0.5%, pH 7.0, sterilizing at 121deg.C for 30min, wherein the percentages are mass percentages.
LB solid medium: 1.5% agar powder (mass percent) was added to the LB liquid medium.
1.2 screening of Bacillus chlorogenic acid-producing Bacillus
1.2.1 preparation of fermentation broths
Firstly, activating test strains by using LB solid inclined planes, then respectively inoculating the test strains into 20mL of LB liquid culture medium, and fermenting and culturing at 37 ℃ and 180r/min for 24-48 hours to obtain fermentation liquor. And (3) collecting fermentation supernatant after centrifuging the fermentation liquor for 10min at 5000 r/min.
1.2.2 Primary screening
The chlorogenic acid-producing strain is subjected to primary screening by spectrophotometry, and the determination method is carried out according to Zhang Qiufang (2006), and specifically comprises the following steps: accurately sucking 5mL of fermentation supernatant, adding 0.5mL of freshly prepared 0.01mol/L ferric trichloride solution, mixing uniformly upside down, carrying out water bath at 35 ℃ for 60min, and measuring the absorbance at 755 nm.
1.2.3 double sieves
Filtering the collected fermentation supernatant with a 0.22 μm filter, performing ultrasonic treatment, and re-screening chlorogenic acid-producing strain with high performance liquid chromatography, wherein the specific steps are referred to national standard GB/T22250-2008 "determination of chlorogenic acid in health food".
1.3 optimization of fermentation conditions of Bacillus chlorogenic acid-producing Bacillus
And (3) taking the LB liquid culture medium as a basic culture medium, and optimizing fermentation conditions of the screened bacillus for producing chlorogenic acid.
1.3.1 carbon Source optimization
Glucose, sucrose, maltodextrin, corn starch, maltose, soluble starch and corn steep liquor dry powder which are 0.2 percent (mass percent) are respectively used as carbon sources to prepare corresponding fermentation culture mediums, the fermentation culture mediums are fermented and cultivated for 48 hours at 37 ℃ and 180r/min after inoculation, and chlorogenic acid content of fermentation liquid is measured by high performance liquid chromatography after centrifugation, filtration and ultrasound.
1.3.2 Nitrogen Source optimization
Respectively taking peptone and ammonium chloride with different concentrations (mass percent) as nitrogen sources, preparing corresponding fermentation culture media, inoculating the strain, fermenting and culturing at 37 ℃ and 180r/min for 48 hours, centrifuging the fermentation liquor at 4000r/min for 10min, filtering by a 0.22 mu m filter, and measuring chlorogenic acid content by high performance liquid chromatography.
1.3.3 influence of copper ions
According to a single variable principle, taking an LB liquid culture medium as a basic culture medium, respectively inoculating strains into liquid culture media containing copper sulfate (0.001-0.01%) with different concentrations (mass percent), fermenting and culturing at 37 ℃ and 180r/min for 48 hours, centrifuging, filtering and ultrasonic treating fermentation liquor, and measuring chlorogenic acid content by using a high performance liquid chromatography.
1.4 identification of 16S rDNA of Bacillus chlorogenic acid-producing Bacillus
1.4.1 Amplification and sequence analysis of 16S rDNA
The target strain is inoculated in a fresh fermentation medium for 20 hours, and the bacterial DNA is extracted by using a kit of Tiangen company, and is subjected to 16S rDNA sequence amplification. The primers used were bacterial 16S rDNA universal primer 27F/1492R, and the PCR reaction system (50. Mu.L) was: mixture 25. Mu.L (containing TaqDNA polymerase, dNTP, etc., available from Tiangen Biochemical Co., ltd.), 1. Mu.L of each of the upstream and downstream primers, 2. Mu.L of the template DNA, and 21. Mu.L of ultrapure water. The PCR amplification procedure was 94℃for 5min,94℃for 1min,52℃for 1min,72℃for 2min,30 cycles, and 72℃for 10min. The PCR product was sent to Beijing Boshang Biotechnology Co., ltd for sequencing.
1.4.2 phylogenetic analysis
Logging in GenBank, searching a 16S rDNA sequencing result by using Blast, downloading a 16S rDNA sequence of a related genus, carrying out homology analysis by using DNAMAN, DNAclub, MEGA 3.1.3.1 and other software, and constructing a phylogenetic tree.
2 results and analysis
2.1 screening of Bacillus chlorogenic acid-producing Bacillus
2.1.1 preliminary screening by spectrophotometry
Orange FeCl 3 The solution can react with colorless transparent chlorogenic acid standard solution to generate dark green complex substance. And measuring the absorption value of each reactant solution by using an ultraviolet-visible light spectrophotometer, and drawing a standard working curve by taking the concentration of chlorogenic acid standard solution as an abscissa and the absorption value of a reaction product as an ordinate (see figure 1).
And (3) measuring 45 strain fermentation liquor by a spectrophotometry method, and primarily screening 15 candidate strains such as BLCC1-0615, BLCC1-0170, BLCC1-0719, BLCC1-0766 and the like (see tables 1-1 to 1-3).
TABLE 1-1 spectrophotometry determination of chlorogenic acid content in Strain fermentation broths
| Strain numbering | A 755 | ΔA 755 | Chlorogenic acid concentration (μg/mL) |
| BLCC1-0155 | 0.0255 | -0.0415 | - |
| BLCC1-0552 | 0.053 | -0.014 | - |
| BLCC1-0783 | 0.0275 | -0.0395 | - |
| BLCC1-0793 | 0.072 | 0.005 | 13.55 |
| BLCC1-0795 | 0.0225 | -0.0445 | - |
| BLCC1-0129 | 0.041 | -0.026 | - |
| BLCC1-0507 | 0.0385 | -0.0285 | - |
TABLE 1-2 spectrophotometry determination of chlorogenic acid content in Strain fermentation broths
| Strain numbering | A 755 | ΔA 755 | Chlorogenic acid concentration (μg/mL) |
| BLCC1-0615 | 0.096 | 0.029 | 21.83 |
| BLCC1-0792 | 0.028 | -0.039 | - |
| BLCC1-0794 | 0.081 | 0.014 | 16.66 |
| BLCC1-0152 | 0.023 | -0.0655 | - |
| BLCC1-0154 | 0.067 | -0.0215 | - |
| BLCC1-0160 | 0.063 | -0.0255 | - |
| BLCC1-0164 | 0.05 | -0.0385 | - |
| BLCC1-0552 | 0.072 | -0.0165 | - |
| BLCC1-0707 | 0.045 | -0.0435 | - |
| BLCC1-0709 | 0.035 | -0.0535 | - |
| BLCC1-0153 | 0.039 | -0.0495 | - |
| BLCC1-0159 | 0.0595 | -0.029 | - |
| BLCC1-0162 | 0.03 | -0.0585 | - |
| BLCC1-0170 | 0.1525 | 0.048 | 28.38 |
| BLCC1-0505 | 0.074 | -0.0145 | - |
| BLCC1-0706 | 0.078 | -0.0105 | - |
| BLCC1-0708 | 0.032 | -0.0565 | - |
| BLCC1-0710 | 0.0765 | -0.012 | - |
| BLCC1-0712 | 0.0905 | 0.002 | 12.52 |
TABLE 1-3 spectrophotometry determination of chlorogenic acid content in Strain fermentation broths
| Strain numbering | A 755 | ΔA 755 | Chlorogenic acid concentration (μg/mL) |
| BLCC1-0713 | 0.102 | 0.0135 | 16.48 |
| BLCC1-0716 | 0.087 | -0.0015 | - |
| BLCC1-0775 | 0.079 | -0.0095 | - |
| BLCC1-0714 | 0.07 | -0.0185 | - |
| BLCC1-0715 | 0.104 | 0.0155 | 17.17 |
| BLCC1-0719 | 0.1385 | 0.05 | 29.07 |
| BLCC1-0759 | 0.1065 | 0.018 | 18.03 |
| BLCC1-0732 | 0.0535 | -0.039 | - |
| BLCC1-0740 | 0.098 | 0.0055 | 13.72 |
| BLCC1-0752 | 0.0485 | -0.044 | - |
| BLCC1-0756 | 0.11 | 0.0175 | 17.86 |
| BLCC1-0764 | 0.0785 | -0.014 | - |
| BLCC1-0766 | 0.145 | 0.0525 | 29.93 |
| BLCC1-0772 | 0.0745 | -0.018 | - |
| BLCC1-0733 | 0.111 | 0.0185 | 18.21 |
| BLCC1-0751 | 0.1085 | 0.016 | 17.34 |
| BLCC1-0755 | 0.0575 | -0.035 | - |
| BLCC1-0762 | 0.1005 | 0.008 | 14.59 |
| BLCC1-0765 | 0.0385 | -0.054 | - |
Note that: in the table "-" indicates not measured.
2.1.2 high Performance liquid chromatography double screening
The chlorogenic acid standard use solution is respectively absorbed, diluted by a mobile phase and the concentration of the chlorogenic acid standard use solution in a volumetric flask for constant volume is respectively as follows: 2.0 μg/mL, 10.0 μg/mL, 20.0 μg/mL, 40.0 μg/mL, 80.0 μg/mL standard series. Chlorogenic acid concentration standard curve (see FIG. 2) was drawn based on peak area and chlorogenic acid standard concentration.
By measuring 15 candidate strain broths obtained by preliminary screening (Table 2), it was found that chlorogenic acid was detected in only 2 strains of fermentation broths of BLCC1-0170 and BLCC1-0759 at concentrations of 1.61. Mu.g/mL and 0.12. Mu.g/mL, respectively. And then, the strain BLCC1-0170 is used for optimizing the chlorogenic acid production fermentation condition and applying the chlorogenic acid production fermentation condition.
TABLE 2 high performance liquid chromatography determination of chlorogenic acid content in bacterial Strain fermentation broths
Note that: in the table "-" indicates not measured.
2.2 optimization of fermentation conditions of Bacillus chlorogenic acid-producing Bacillus
2.2.1 carbon Source optimization
As shown in Table 3, when the strain BLCC1-0170 uses glucose, corn starch and corn steep liquor dry powder as carbon sources, the strain can produce chlorogenic acid, and the most suitable carbon source is glucose.
TABLE 3 chlorogenic acid content in fermentation broths of strains BLCC1-0170 under different carbon sources
Note that: in the table "-" indicates not measured.
2.2.2 Nitrogen Source optimization
As can be seen from Table 4, when inorganic nitrogen source ammonium chloride is used as the nitrogen source, chlorogenic acid is not detected in the fermentation broth of strain BLCC 1-0170; when organic nitrogen source peptone is used as nitrogen source, chlorogenic acid with a certain concentration can be detected in the fermentation broth of the strain BLCC1-0170, and when the addition amount of the peptone is 1.5%, the content of chlorogenic acid is the highest and is 4.94 mug/mL.
TABLE 4 chlorogenic acid content in bacterial Strain BLCC1-0170 fermentation broths under different Nitrogen sources
Note that: in the table "-" indicates not measured.
2.2.3 influence of copper ions
The effect of copper ions on the chlorogenic acid content of the strain BLCC1-0170 was investigated with 0.2% glucose as a carbon source and 1.5% peptone as a nitrogen source, and the results are shown in Table 5. When the addition amount of copper sulfate is 0.003%, the content of chlorogenic acid produced by the fermentation of the strain BLCC1-0170 is the highest and reaches 11.50 mug/mL.
TABLE 5 influence of copper ions on chlorogenic acid content produced by fermentation of Strain BLCC1-0170
2.2.4 fermentation Medium validation test
And (3) fermenting and culturing the strain BLCC1-0170 by using an LB liquid culture medium and an optimized culture medium respectively, and determining the chlorogenic acid content in the fermentation broth. Wherein, LB liquid medium is composed of: glucose 0.2%, peptone 1.0%, yeast extract 0.5%, naCl 0.5%, pH 7.0. Optimizing the composition of a culture medium (mass percent): glucose 0.2%, peptone 1.5%, yeast extract 0.5%, naCl 0.5%, copper sulfate 0.003% and pH 7.0.
TABLE 6 chlorogenic acid content in fermentation broths at different fermentation times (Unit: μg/mL)
| Culture medium | Fermenting for 24h | Fermenting for 48h |
| LB liquid medium | 1.36 | 1.61 |
| Optimized medium | 6.90 | 11.81 |
As shown in Table 6, when the optimized culture medium is used for fermentation culture for 48 hours, the chlorogenic acid content reaches 11.81 mug/mL, and compared with culture in LB liquid culture medium for 48 hours, the yield of chlorogenic acid can be improved by 7.3 times. The content of chlorogenic acid produced by fermentation with an optimized culture medium is obviously improved within 48 hours of fermentation along with the extension of fermentation time, and the change of the content of the chlorogenic acid is not obvious along with the extension of fermentation time in an LB liquid culture medium.
2.3 identification of Bacillus chlorogenic acid-producing Bacillus
Microscopic observation of the bacterial form revealed that the spores of strain BLCC1-0170 were laterally grown and that the spores were fusiform (FIG. 3). The strain BLCC1-0170 is a strain of Brevibacillus laterosporus (Brevibacillus laterosporus) whose sequence is shown in seq_1, and the 16S rDNA of the strain is amplified by extracting the DNA of the strain BLCC1-0170, and after sequencing, the sequence is registered in Gen Bank database for sequence alignment analysis.
Example 2 determination of in vitro antibacterial, antioxidant and acidogenic Properties of Bacillus chlorogenic acid-producing bacteria
1 materials and methods
1.1 test strains
The strain BLCC1-0170 screened by the invention, namely, the Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170, is preserved in China center for type culture Collection (CCTCC NO) in 2022 and 06 month for 20 days, and has the preservation number of CCTCC NO: m2022932.
1.2 Medium
LB liquid medium: glucose 0.2%, peptone 1.0%, yeast extract 0.5%, naCl 0.5%, pH 7.0.
Fermentation medium: glucose 0.2%, peptone 1.5%, yeast extract 0.5%, naCl 0.5%, copper sulfate 0.003% and pH 7.0.
NA medium: 1.0% of peptone, 0.3% of beef powder, 0.5% of NaCl, 1.5% of agar powder and pH 7.0.
1.3 determination of in vitro antibacterial Properties
Inoculating the strain BLCC1-0170 into LB liquid medium, culturing at 37deg.C and 180r/min for 24 hr, collecting bacterial liquid, centrifuging at 5000r/min for 10min, and collecting supernatant.
The bacteria inhibition test is carried out by using staphylococcus aureus BLCC8-0004, clostridium perfringens BLCC8-0044, salmonella enteritidis chicken BLCC8-0129, aeromonas hydrophila BLCC8-0121 and escherichia coli BLCC8-0102 as indicator bacteria through a punching method, wherein the indicator bacteria are preserved and provided by Shandong Baoli biological engineering institute of biological engineering, inc. strain preservation center.
The specific method for the bacteriostasis test comprises the following steps: the overnight cultured indicator bacteria are diluted 1000 times, 5mL of indicator bacteria diluent is added into 45mL of NA culture medium, and 15mL of indicator bacteria diluent is sucked and placed in a plate after uniform mixing. After solidification, punching by using an oxford cup, then sucking 40 mu L of supernatant after centrifugation, adding the supernatant into the hole, and culturing at 37 ℃ overnight to observe the antibacterial effect.
1.4 measurement of antioxidant Properties
1.4.1 preparation of samples
The strain BLCC1-0170 was inoculated into 10mL of LB liquid medium, and cultured overnight at 37℃at 180r/min to prepare a seed solution. Taking 800 mu L of seed solution, inoculating into 40mL of fermentation medium, fermenting and culturing at 37 ℃ for 48h at 180r/min, and collecting 5mL of fermentation liquor for later use. In addition, taking 5mL of fermentation liquor, centrifuging for 10min at 5000r/min, collecting supernatant for later use, washing the centrifuged thalli with sterile water for 2 times, and re-suspending the thalli with 5mL of sterile physiological saline to prepare bacterial suspension for later use.
1.4.2 determination of in vitro DPPH free radical scavenging ability
To 4mL of DPPH absolute ethanol solution (0.2 mmol/L) was added 0.5mL of the sample, the mixture was reacted in a dark environment at room temperature (25 ℃) for 30min, centrifuged at 5000r/min for 10min, absorbance at 517nm was measured, and deionized water was zeroed.
DPPH radical clearance (%) = [1- (A1-A2)/A3 ] ×100%
Wherein: a1 is the absorbance of 4mL DPPH absolute ethanol solution plus 0.5mL sample; a2 (blank) absorbance of 4mL absolute ethanol+0.5 mL sample; a3 The absorbance was 4mL of DPPH absolute ethanol solution+0.5 mL of sterile water.
1.4.3 determination of the ability to scavenge hydroxyl radicals
Taking 1mL of O-phenanthroline (0.75 mmol/L) in a test tube, sequentially adding 1mL of PBS (0.05 mol/L, pH 7.4), 0.5mL of sample or distilled water, fully mixing, and adding 1mL of FeSO 4 (2.5 mmol/L), mixing, adding H 2 O 2 (20 mmol/L) 1mL, after 1.5h of reaction in a constant temperature water bath at 37℃the absorbance was measured at 536 nm.
Hydroxyl radical clearance (%) = [ (A2-A1)/(A0-A1) ] ×100%
Wherein: a0 is free of sample and H 2 O 2 (1 mL distilled water was used instead of 1mL H) 2 O 2 ) Is a light absorbance value of (2); a1 is sample-free and contains H 2 O 2 Is a light absorbance value of (2); a2 is a sample and H 2 O 2 Is used for the light absorption value of (a).
1.5 analysis of Strain metabolites (organic acids)
The acid production capacity of the strain BLCC1-0170 is measured by high performance liquid chromatography, the method is referred to Zhang Jianmei, hu Shunzhen, mu Xijun, etc. A bacillus strain with acid production capacity is screened and performance detection [ J ]. Livestock ecological newspaper, 2012. The liquid chromatography conditions were: high performance liquid chromatograph (shimadzu LC-20A), mobile phase: 50mmol/L monoammonium phosphate (pH 2.5): acetonitrile=98: 2 (V: V), flow rate: 0.8mL/min, column temperature: 28 ℃, detection wavelength: 212nm, sample injection amount: 10 mu L.
2 results and analysis
2.1 determination of bacterial inhibition performance of bacterial strain BLCC1-0170
As shown in Table 7, the strain BLCC1-0170 has antibacterial effects on 5 pathogenic bacteria of tested staphylococcus aureus, clostridium perfringens, salmonella enteritidis, aeromonas hydrophila and escherichia coli, wherein the antibacterial effect on escherichia coli is the best, and the diameter of a antibacterial ring is 20.25mm.
TABLE 7 antibacterial results of bacterial strain BLCC1-0170
| Sequence number | Pathogen numbering | Indicator bacteria | Diameter of inhibition zone (mm) |
| 1 | BLCC8-0004 | Staphylococcus aureus | 16.25±0.50 |
| 2 | BLCC8-0044 | Clostridium perfringens | 19.00±1.00 |
| 3 | BLCC8-0102 | Coli bacterium | 20.25±0.50 |
| 4 | BLCC8-0121 | Aeromonas hydrophila | 16.50±0.50 |
| 5 | BLCC8-0129 | Salmonella enteritidis of chicken | 13.50±1.00 |
2.2 determination of antioxidant Capacity of Strain BLCC1-0170
As is clear from Table 8, bacterial suspensions, fermentation broths and supernatants of the strain BLCC1-0170 all had a certain DPPH radical and hydroxyl radical scavenging ability, wherein an active substance having a scavenging ability for DPPH radicals was mainly present in the supernatant and an active substance having a scavenging ability for hydroxyl radicals was mainly present in the bacterial suspension.
Table 8 bacterial strain BLCC1-0170 antioxidant capacity index
| Sequence number | Sample ofName of the name | DPPH radical scavenging Rate (%) | Hydroxyl radical scavenging rate (%) |
| 1 | Supernatant fluid | 53.45 | 35.10 |
| 2 | Bacterial suspension | 2.62 | 49.12 |
| 3 | Fermentation liquor | 58.19 | 88.51 |
2.3 determination of acid producing ability of Strain BLCC1-0170
As shown in Table 9, the strain BLCC1-0170 has a certain acid-producing capacity by fermentation, and the lactic acid and acetic acid contents in the fermentation supernatant are 1.42g/L and 1.84g/L, respectively, when the fermentation is carried out for 24 hours.
TABLE 9 BLCC1-0170 acid production by strain (unit: g/L)
| Detecting items | Oxalic acid | Tartaric acid | Malic acid | Lactic acid | Acetic acid | Citric acid |
| Fermenting for 24h | 0.43 | 1.22 | 0.97 | 1.42 | 1.84 | 0.65 |
| Fermenting for 48h | 0.37 | 0.91 | 1.06 | 0.85 | 1.05 | 0.91 |
EXAMPLE 3 application of Bacillus chlorogenic acid-producing Bacillus in animals
1 test materials
Female Kunming mice for the test were purchased from Jinan Pengyue laboratory animal breeding Co., ltd.
Dextran Sodium Sulfate (DSS), purchased from the company of the next san bio-technology (Shanghai), parts, MW: 36000-50000.
The strain BLCC1-0170, namely Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170, is preserved in China center for type culture Collection (CCTCC NO) in 2022 and 20 months: m2022932.
The strain BLCC1-0716 refers to Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0716 in the invention, and the strain has been preserved in China Center for Type Culture Collection (CCTCC) at 11/2021 with the preservation number of CCTCC NO: m20211397, which is described and disclosed in Chinese patent CN 202111582162.7.
2 test design and method
40 female Kunming mice of 6-8 weeks of age were purchased, fed with basal feed, and pre-fed for 3 days. Then randomly dividing into 4 groups according to the principle of insignificant weight difference, namely a blank group (CK group), a UC (ulcerative colitis ) model group, a strain BLCC1-0170 group and a strain BLCC1-0716 group, wherein the grouped days are the 1 st day of the test, and the test design is shown in Table 10. The construction and evaluation method of the UC model is carried out according to the description in Chinese patent CN 202111582162.7.
Table 10 test groupings and designs
On test days 1-7, the CK group and the UC model group are filled with 200 mu L of physiological saline every day; BLCC1-0170 group irrigated with 200. Mu.L 3X 10 per day 8 CFU/mL BLCC1-0170 broth (i.e., 6X 10 strains per daily administration) 7 CFU); BLCC1-0716 group irrigated with 200. Mu.L 3X 10 per day 8 CFU/mL BLCC1-0716 bacterial liquid (i.e., 6X 10 strains per daily administration) 7 CFU), during which each group of mice was free to drink. From day 8, each group was treated as follows for 10 days: the CK group is filled with 200 mu L of physiological saline water and drinks water freely every day; the UC model group is filled with 200 mu L of physiological saline every day and freely drinks 5% DSS water solution; BLCC1-0170 group lavage 200 μL 3×10 8 CFU/mL BLCC1-0170 broth (i.e., 6X 10 strains per daily administration) 7 CFU) and freely drink 5% dss in water; BLCC1-0716 group lavage 200 μL 3×10 8 CFU/mL BLCC1-0716 bacterial liquid (i.e., 6X 10 strains per daily administration) 7 CFU) and freely drink 5% dss in water. Mice were observed daily for general status, body mass, stool traits, and occult blood status during the trial. After the test is finished, the mice are killed by cervical vertebra removal, the eyeballs are subjected to blood collection,serum is collected; at the same time, the colon is separated, the pathological condition is observed visually, and the length of the colon is measured.
3 statistical analysis of data
One-Way ANOVA procedure in SPSS 13.0 statistical analysis software was used for One-factor analysis of variance, with results expressed as mean ± standard deviation. P <0.05 is used as a difference significance judgment standard.
4 results and analysis
4.1 weight changes
As can be seen from table 11, the CK group of mice continued to increase in body weight during the entire test period due to the absence of induction stimulation by DSS. During the period of drinking DSS, the weights of the mice in the model group and the BLCC1-0170 group are increased and then reduced, and after the 4 th day of drinking, the weights of the mice in the model group and the BLCC1-0170 group are reduced. At the end of the trial, the mice in the model group had significantly lower body weight than the CK group (P < 0.05), the BLCC1-0170 mice had significantly lower body weight but not significantly different body weight (P > 0.05) than the CK group, but had higher body weight than the model group. Thus, brevibacillus laterosporus BLCC1-0170 has the effect of inhibiting weight loss of mice caused by free drinking DSS.
Table 11 daily body weight (unit: g) of mice during the test period
Note that: the same row of shoulder marks are identical or are not marked to indicate that the difference is not obvious (P is more than 0.05), and the same row of shoulder marks are different to indicate that the difference is obvious (P is less than 0.05).
4.2 colon Length Change
After the test, the eyes were taken out for blood collection, then the cervical vertebrae were removed to kill the mice, the whole colon was dissected and taken out, the residual feces were removed, and the length of the colon was measured with a ruler, and the results are shown in Table 12.
Table 12 colon length of mice in each group
Note that: the same row of shoulder marks are identical or are not marked to indicate that the difference is not obvious (P is more than 0.05), and the same row of shoulder marks are different to indicate that the difference is obvious (P is less than 0.05).
Except for the complete colon tissue of the CK group, the faeces in the intestinal cavity are formed, and bleeding occurs in the BLCC1-0170 group, the BLCC1-0716 group and the model group, so that the faeces in the intestinal cavity are reduced. As can be seen from Table 12, the CK group had the longest colon and the model group had a colon length 3.31cm shorter than the CK group. The length of the colon of the BLCC1-0170 group is 2.50cm longer than that of the model group, 32.8% is improved, and the difference is obvious (P is less than 0.05). The colon length of the BLCC1-0716 group, although longer than the model group, was significantly shorter than the CK group (P < 0.05) and 0.34cm shorter than the BLCC1-0170 group (P > 0.05). These results indicate that Brevibacillus laterosporus BLCC1-0170 can regulate and control DSS-induced changes in colon length of mice, and the effect is superior to that of BLCC1-0716 groups.
4.3 serum cytokine level Change
Measuring the change of the serum cytokine level of the mice by using an Shanghai ELISA kit, wherein the measured cytokines comprise TNF-alpha, IL-6 and IL-10; the results are shown in Table 13.
As can be seen from Table 13, serum TNF- α levels were highest in the model group, and BLCC1-0170 group was 320.05pg/mL, 17.6% lower than in the model group, and the differences were significant (P < 0.05). Serum IL-6 levels were highest in the model group, BLCC1-0170 group was significantly lower than in the model group, reduced by 17.2%, and did not differ significantly from the CK group. Serum IL-10 levels were 2.8% higher than model with the lowest CK group and the highest BLCC1-0170 group, but none were significant (P > 0.05). In addition, the levels of both serum pro-inflammatory cytokines TNF- α and IL-6 were lower in the BLCC1-0170 group than in the BLCC1-0716 group, and the serum anti-inflammatory cytokine IL-10 was higher than in the BLCC1-0716 group. These results indicate that Brevibacillus laterosporus BLCC1-0170 can regulate and control the changes of serum inflammatory factor level of mice caused by DSS induction, and the effect is superior to that of BLCC1-0716 groups.
TABLE 13 serum inflammatory factor level changes in mice (Unit: pg/mL)
| Group of | TNF-α | IL-6 | IL-10 |
| CK group | 313.03±19.08 a | 50.97±2.70 a | 196.36±13.61 |
| Model group | 388.41±20.12 b | 64.24±1.66 b | 209.28±23.51 |
| BLCC1-0170 group | 320.05±24.70 a | 53.18±1.88 a | 218.04±23.61 |
| BLCC1-0716 group | 345.68±26.32 ab | 54.02±3.02 a | 217.44±15.48 |
Note that: the same or no label indicates that the difference is not significant (P > 0.05) and the same label indicates that the difference is significant (P < 0.05).
4.4 measurement of serum antioxidant index
Malondialdehyde (MDA) is an in vivo lipid peroxidation product, which can indirectly reflect the condition of oxygen radical metabolism and the degree of lipid peroxidation in the body; superoxide dismutase (SOD) plays a vital role in balancing the oxidation and antioxidation of organisms, and can remove superoxide anions to protect cells from damage. The serum antioxidation index of mice is measured by using Nanjing's biological kit, and the measured antioxidation index comprises MDA, T-SOD (total superoxide dismutase) and T-AOC (total antioxidation capability). The results are shown in Table 14.
From Table 14, the serum MDA content of the model group is significantly higher than that of the CK group (P < 0.05), and the serum MDA content is increased by 64.7%, and the possible reasons for analysis are that the mice body generates stress response after drinking DSS. The MDA value of the BLCC1-0170 group is lower than that of the model group, the T-SOD value is obviously higher than that of the model group (P is less than 0.05), and the improvement is 12.8%; BLCC1-0170 group T-AOC values were 3.2% higher than model group. These results show that Brevibacillus laterosporus BLCC1-0170 has certain effects of intervention and alleviation on stress state of the mouse organism. However, the BLCC1-0716 group has no obvious difference from the model group in MDA, T-SOD and T-AOC indexes, which shows that the strain BLCC1-0716 has no obvious effect in relieving the stress state of the mouse organism.
TABLE 14 determination of serum stress indicators in mice
Note that: the same or no label indicates that the difference is not significant (P > 0.05) and the same label indicates that the difference is significant (P < 0.05).
In conclusion, from 45 endophytes separated from eucommia leaves, medlar, honeysuckle and other plants, 1 endophyte BLCC1-0170 with high chlorogenic acid yield is screened by utilizing a spectrophotometry method and a high performance liquid chromatography, and the chlorogenic acid yield after fermentation optimization can reach 11.81 mug/mL, and the chlorogenic acid yield is obviously higher than that of the currently known bacillus subtilis. In addition, the antibacterial performance, the oxidation resistance and the acid production performance of the strain are measured and the in-vivo effect evaluation experiment of mice is carried out, so that the strain is finally determined to have high chlorogenic acid production content, good antibacterial performance and good oxidation resistance, has the effects of preventing and treating ulcerative colitis of mice and has the effects of certain intervention and relieving stress states of the bodies of the mice.
The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A strain of Brevibacillus laterosporus for high yield of chlorogenic acid is characterized in that the strain is named Brevibacillus laterosporus @Brevibacillus laterosporus) BLCC1-0170, the strain has been preserved in China center for type culture collection (CCTCC NO) in university of Wuhan in Wuhan, hubei province, china for 20 months in 2022: m2022932.
2. A microbial inoculant or feed comprising the Brevibacillus laterosporus of claim 1.
3. Use of a Brevibacillus laterosporus according to claim 1 for the preparation of a pharmaceutical or feed additive having one or more of the following functions;
1) The application in preparing antioxidant medicines or feed additives;
2) The application in preparing antibacterial medicines or feed additives;
3) The application of the composition in preparing medicines or feed additives for preventing and treating enteritis.
4. Use according to claim 3, characterized in that the antioxidant is a scavenging of hydroxyl radicals and/or a scavenging of DPPH radicals.
5. The use according to claim 3, wherein the enteritis is ulcerative colitis.
6. A method for producing chlorogenic acid, comprising fermentatively culturing the Brevibacillus laterosporus of claim 1.
7. The method according to claim 6, wherein the fermentation culture is carried out at a temperature of 35-38deg.C using one or more of glucose, corn starch, and corn steep liquor dry powder as a carbon source and an organic nitrogen source as a nitrogen source.
8. The method according to claim 6, wherein copper ions are added to the medium during the fermentation culture in an amount of 0.001 to 0.005% by mass and the organic nitrogen source is added in an amount of 0.5 to 2.0% by mass.
9. The method according to claim 6, wherein the medium is glucose as a carbon source, peptone as a nitrogen source, the peptone is added in an amount of 1-2% by mass, and copper sulfate is added in an amount of 0.002-0.005% by mass, when the fermentation culture is performed.
10. The method according to claim 6, wherein the composition of the medium during fermentation culture is: glucose 0.2%, peptone 1.5%, yeast extract 0.5%, naCl 0.5%, copper sulfate 0.003%, pH 7.0; the fermentation culture time is 24-48h; the fermentation temperature is 35-38 ℃, and the mass percentage of the fermentation is that of aerobic culture.
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| CN114107134A (en) * | 2021-12-22 | 2022-03-01 | 山东宝来利来生物工程股份有限公司 | Brevibacillus laterosporus and application thereof |
| CN114480197A (en) * | 2022-02-09 | 2022-05-13 | 山东农业大学 | Brevibacillus laterosporus, microbial inoculum and application thereof in biological pesticide fertilizer |
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| CN113980828A (en) * | 2021-05-21 | 2022-01-28 | 广西大学 | Bacillus badius mutant strain with high chlorogenic acid yield |
| CN113575758A (en) * | 2021-08-04 | 2021-11-02 | 广西大学 | Composite probiotics and preparation method thereof |
| CN114107134A (en) * | 2021-12-22 | 2022-03-01 | 山东宝来利来生物工程股份有限公司 | Brevibacillus laterosporus and application thereof |
| CN114480197A (en) * | 2022-02-09 | 2022-05-13 | 山东农业大学 | Brevibacillus laterosporus, microbial inoculum and application thereof in biological pesticide fertilizer |
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