CN115927058A - Brevibacillus laterosporus for producing chlorogenic acid and application thereof - Google Patents
Brevibacillus laterosporus for producing chlorogenic acid and application thereof Download PDFInfo
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Abstract
The invention relates to a microorganism, in particular to a Brevibacillus laterosporus strain for producing chlorogenic acid and application thereof, wherein the strain is named Brevibacillus laterosporus BLCC1-0170. The strain is preserved in China Center for Type Culture Collection (CCTCC) in Wuhan university in Wuhan City, hubei province, china at 20/06/2022, with the preservation number of CCTCC NO: m2022932. The strain has high chlorogenic acid content, excellent antibacterial property and good oxidation resistance, has a prevention effect on mouse ulcerative colitis, and has certain intervention and relief effects on the stress state of a mouse organism.
Description
Technical Field
The invention relates to a microorganism, in particular to a brevibacillus laterosporus for producing chlorogenic acid and application thereof.
Background
The information disclosed in this background of the invention is intended to enhance an understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information has become known prior art to a person skilled in the art.
Chlorogenic acid (also called caffeogenic acid) is depside composed of caffeic acid and quinic acid, is a phenylpropanoid compound generated by plants in the aerobic respiration process through the shikimic acid pathway, has the functions of antibiosis, antivirus, antitumor 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 applied to animal feeds as a green feed additive, and the chlorogenic acid is mainly obtained by extracting from various medicinal plants. The commonly used extraction method mainly comprises a mercaptan method, a water extraction-alcohol precipitation method, an alcohol extraction-lead salt precipitation method, a polyamide column chromatography method and the like. The problems of complicated extraction process, high cost and the like exist in the application, and particularly when the chlorogenic acid extracted by the methods is applied to livestock and poultry breeding by using the feed additive, hidden dangers can be brought to the health of animal organisms.
The production of chlorogenic acid by microbial fermentation is a new preparation method. The currently reported microorganisms belong to feed additive variety catalog (2013), and the strain capable of being used for producing chlorogenic acid by fermentation is bacillus subtilis, but the yield is low. Therefore, the probiotics capable of producing chlorogenic acid at high yield is separated and screened to be used as the feed additive, 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 brevibacillus laterosporus strain, which can produce chlorogenic acid with high yield, and lays a foundation for enlarging the drug source of the chlorogenic acid, protecting rare medicinal plant resources, enriching the strain resources and promoting the development and utilization of endophytes of plants; the strain also has good bacteriostatic ability, oxidation resistance, acid production performance and prevention and treatment effect on mouse ulcerative colitis, and has certain intervention and alleviation effect on the stress state of a mouse organism.
Specifically, the present invention provides the following technical features, and one or a combination of the following technical features constitutes the technical solution of the present invention.
In the first aspect of the invention, the invention provides a Brevibacillus laterosporus with high chlorogenic acid yield, which is named Brevibacillus laterosporus BLCC1-0170 and is preserved in China center for type culture Collection (CCTCC for short, with the address: wuhan, university of Wuhan, china) in 20/06/2022, with the preservation number: CCTCC NO: m2022932.
The strain has lateral spores which are fusiform, can well survive under the aerobic condition at normal temperature, has good growth performance, and has good bacteriostatic effect on staphylococcus aureus, clostridium perfringens, escherichia coli, aeromonas hydrophila, salmonella enteritidis and the like; the strain also has good oxidation resistance, can remove 1,1-diphenyl-2-picrylhydrazyl radical (DPPH. Cndot.) and hydroxyl radical (HO. Cndot.), and has good acid production performance, for example, in one embodiment of the invention, when the strain is cultured for 24 hours in a fermentation mode, the content of lactic acid and acetic acid in a fermentation supernatant can reach 1.42g/L and 1.84g/L respectively.
The strain has chlorogenic acid producing ability. In the embodiment of the invention, when the LB culture medium is used for culture, the composition (mass percentage) of the LB culture medium is as follows: 0.2% of glucose, 1.0% of peptone, 0.5% of yeast extract, 0.5% of NaCl and 7.0% of pH value, and the strain shows good chlorogenic acid production capacity. And the yield of chlorogenic acid can be further remarkably 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), higher yield of chlorogenic acid can be obtained. For example, when copper ions (e.g., copper sulfate) are added to the medium, the yield of chlorogenic acid is improved as compared to the case without copper ions, for example, when the amount of copper ions is 0.001-0.005% (by mass), the yield of chlorogenic acid is improved, and particularly, when the amount of copper ions is 0.003% (by mass), the yield of chlorogenic acid is significantly improved as compared to other amounts or no addition.
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 a high rate. The culture medium takes glucose as a carbon source and peptone as a nitrogen source, the addition amount of the peptone is 1-2% (mass percent), 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, an exemplary composition (in mass percent) of the medium is: glucose 0.2%, peptone 1.5%, yeast extract 0.5%, naCl 0.5%, copper sulfate 0.003%, pH 7.0.
In the embodiment of the invention, brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 is respectively fermented and cultured in LB culture medium and the optimized culture medium of the invention, so that the content of chlorogenic acid in the optimized culture medium reaches 11.81 mu g/mL when the fermentation culture is carried out for 48 hours, compared with the culture medium with 48 hours (the yield of the chlorogenic acid is 1.61 mu g/mL), the yield of the chlorogenic acid can be improved by 7.3 times, the content of the chlorogenic acid produced by fermentation with the optimized formula is obviously increased along with the increase of the fermentation time, and the content of the chlorogenic acid in the LB culture medium is not obviously changed along with the increase of the fermentation time.
In a second aspect of the present invention, there is provided a microbial preparation or feed comprising Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 described in the first aspect above and/or a fermented product thereof.
In a third aspect, the invention provides a series of uses of Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 and/or its fermentation product, including the use in the preparation of a medicament, microbial agent or feed additive with one or more of the following functions;
1) The application in preparing antioxidant medicines, microbial inoculum or feed additives;
2) The application in preparing bacteriostatic drugs, bactericides or feed additives;
3) The application in preparing medicines, microbial inoculum or feed additives for preventing and treating enteritis;
4) The application in preparing medicines, microbial agents or feed additives for improving the antioxidant stress capability of animals.
In the embodiment of the invention, the Brevibacillus laterosporus BLCC1-0170 and/or the fermentation product thereof have good antibacterial capacity and have good antibacterial effect on strains such as staphylococcus aureus, clostridium perfringens, escherichia coli, aeromonas hydrophila, salmonella enteritidis and the like.
In an embodiment of the present invention, brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 and/or its fermentation product has good antioxidant ability, including scavenging DPPH free radical and hydroxyl free radical.
In an embodiment of the invention, brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 can prevent and treat enteritis, especially ulcerative colitis, and can improve ulcerative colitis and weight reduction caused by ulcerative colitis and shorten colon, and can regulate serum inflammatory factor levels, including improving the level of anti-inflammatory cytokines IL-10 and reducing the levels of proinflammatory cytokines TNF-alpha and IL-6. Meanwhile, in the embodiment of the invention, the Brevibacillus laterosporus (BLCC 1-0170) can reduce the content of Malondialdehyde (MDA) in serum, increase the content of total superoxide dismutase (T-SOD), show good antioxidant capacity and can intervene and relieve the stress state of the organism.
In a fourth aspect of the invention, the invention provides a method for the fermentative production of chlorogenic acid, which comprises the fermentative culture of Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170.
In an embodiment of the present invention, culturing Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 in LB medium can produce chlorogenic acid, for example, in an embodiment of the present invention, the composition (mass percent) of 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 conditions, and the inventor finds that when Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 is subjected to fermentation culture, higher chlorogenic acid yield can be obtained when one or more of glucose, corn starch and corn steep liquor dry powder is used as a carbon source and peptone serving as an organic nitrogen source is used as a nitrogen source.
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 of the organic nitrogen source is 0.5-2.0% (mass percentage), the yield of chlorogenic acid can be obviously improved, and particularly when the addition amount of the peptone is 1.5% (mass percentage), the yield improvement effect is more obvious.
And, when copper ions (such as copper sulfate) are added to the medium, the yield of chlorogenic acid can be improved when the amount of copper ions added is 0.001-0.005% (mass%), and particularly, when the amount of copper ions added is 0.003% (mass%), the yield of chlorogenic acid can be significantly increased as compared to other amounts or no addition.
In an embodiment of the present invention, brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 is cultured aerobically at a temperature of 35-38 ℃.
In a preferred embodiment of the present invention, when Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170 is fermentatively cultured, glucose is used as a carbon source, peptone is used as a nitrogen source, the addition amount of the peptone is 1-2% (mass percentage), and copper sulfate is added, and the addition amount of the copper sulfate is 0.002-0.005% (mass percentage). In a more preferred embodiment, the specific composition (mass percentage) of the culture medium is: glucose 0.2%, peptone 1.5%, yeast extract 0.5%, naCl 0.5%, copper sulfate 0.003%, pH 7.0. Preferably, the fermentation culture time is 24-48h, and the fermentation 48h is preferred to have higher yield.
Compared with the prior art, the invention has the advantages that: the invention provides a Brevibacillus laterosporus strain BLCC1-0170 which can produce chlorogenic acid with high yield, and the discovery of the strain lays a foundation for enlarging the drug source of the chlorogenic acid, protecting rare medicinal plant resources, enriching the strain resources and promoting the development and utilization of endophytes of plants. In addition, the Brevibacillus laterosporus BLCC1-0170 has good bacteriostatic ability, oxidation resistance and acid production performance, has a prevention effect on mouse ulcerative colitis, and has certain intervention and relief effects on the stress state of a mouse body.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention. Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
FIG. 1: a standard curve of chlorogenic acid concentration was initially screened by spectrophotometry in example 1.
FIG. 2: chlorogenic acid concentration standard curve prepared by high performance liquid chromatography rescreening in example 1.
FIG. 3: the result of microscopic examination of the strain BLCC1-0170 (magnification of 1000 x).
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
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 starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specification. 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 embodiments and materials described herein are exemplary only.
Example 1 selection of Bacillus chlorogenic acid production and optimization of fermentation conditions
1 materials and methods
1.1 test materials
1.1.1 test strains
The endophyte of the plant preserved by the strain preservation center of the biological engineering research institute of the corporation of Shandong Baolaili is taken as a test strain, and the strain is derived from eucommia bark, medlar, honeysuckle and the like.
1.1.2 culture Medium
LB liquid medium: 0.2% of glucose, 1.0% of peptone, 0.5% of yeast extract, 0.5% of NaCl, 7.0% of pH and 30min of sterilization at 121 ℃, wherein the percentages are mass percentages.
LB solid medium: to the LB liquid medium was added 1.5% agar powder (mass%).
1.2 screening of Bacillus chlorogenic acid
1.2.1 preparation of fermentation broth
Firstly, LB solid slant is used for activating test strains, then the test strains are respectively inoculated into 20mL LB liquid culture medium, and fermentation culture is carried out at 37 ℃ and 180r/min for 24-48h, thus obtaining fermentation liquor. Centrifuging the fermentation liquor at 5000r/min for 10min, and collecting the fermentation supernatant.
1.2.2 prescreening
Primarily screening the chlorogenic acid producing strain by a spectrophotometric method, and performing the determination method by referring to Zhang Qiufang (2006), wherein the specific steps are as follows: accurately absorbing 5mL of fermentation supernatant, adding 0.5mL of freshly prepared 0.01mol/L ferric trichloride solution, turning upside down and uniformly mixing, carrying out water bath at 35 ℃ for 60min, and then measuring the light absorption value at the wavelength of 755 nm.
1.2.3 double sifting
Filtering the collected fermentation supernatant with 0.22 μm filter, performing ultrasonic treatment, and re-screening the chlorogenic acid producing strain with high performance liquid chromatography, wherein the specific steps refer to GB/T22250-2008 "determination of chlorogenic acid in health food".
1.3 optimization of fermentation conditions of Bacillus chlorogenic acid
And (3) optimizing the fermentation conditions of the screened chlorogenic acid producing bacillus by taking an LB liquid culture medium as a basic culture medium.
1.3.1 carbon Source optimization
Respectively taking 0.2 percent (mass percentage) of glucose, sucrose, maltodextrin, corn starch, maltose, soluble starch and corn steep liquor dry powder as carbon sources, preparing corresponding fermentation culture media, inoculating, fermenting and culturing at 37 ℃ and 180r/min for 48 hours, centrifuging, filtering and ultrasonically treating fermentation liquor, and then measuring the content of chlorogenic acid by a high performance liquid chromatography.
1.3.2 Nitrogen Source optimization
Separately using peptone and ammonium chloride with different concentrations (mass percentage) as nitrogen sources, preparing corresponding fermentation culture medium, inoculating strain at 37 deg.C, fermenting and culturing at 180r/min for 48h, centrifuging the fermentation liquor at 4000r/min for 10min, filtering with 0.22 μm filter, and ultrasonically treating with high performance liquid chromatography to determine chlorogenic acid content.
1.3.3 Effect 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%) (mass percent) with different concentrations, performing fermentation culture at 37 ℃ at 180r/min for 48h, centrifuging, filtering and ultrasonically treating fermentation liquor, and then measuring the content of chlorogenic acid by using a high performance liquid chromatography.
1.4 identification of 16S rDNA of Bacillus chlorogenic acid
1.4.1 Amplification and sequence analysis of 16S rDNA
The target strain is inoculated in a fresh fermentation medium for culturing for 20h, and the DNA of the strain is extracted by adopting a kit of Tiangen company and subjected to 16S rDNA sequence amplification. The primers used were bacterial 16S rDNA universal primers 27F/1492R, and the PCR reaction system (50. Mu.L) was: mix 25. Mu.L (containing TaqDNA polymerase and dNTP, purchased from Tiangen Biochemical technology 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 program comprises pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 1min, annealing at 52 ℃ for 1min, extension at 72 ℃ for 2min,30 cycles, and extension at 72 ℃ for 10min. The PCR product was sent to Beijing Boshang Biotechnology Co., ltd for sequence determination.
1.4.2 phylogenetic analysis
And (3) logging in GenBank, retrieving the sequencing result of 16S rDNA by using Blast, downloading the 16S rDNA sequence of related species, performing homology analysis by using DNAMAN, DNAclub, MEGA3.1 and other software, and constructing a phylogenetic tree.
2 results and analysis
2.1 selection of Bacillus chlorogenic acid
2.1.1 prescreening by spectrophotometry
Orange-yellow FeCl 3 The solution can react with colorless and transparent chlorogenic acid standard solution to generate dark green complex substance. Measuring the absorption value of each reactant solution with ultraviolet-visible spectrophotometer, and drawing a standard working curve (see fig. 1) with the concentration of chlorogenic acid standard solution as abscissa and the absorption value of reaction product as ordinate.
45 strain fermentation liquor is measured by a spectrophotometry method, and 15 candidate strains such as BLCC1-0615, BLCC1-0170, BLCC1-0719, BLCC1-0766 and the like are primarily screened (see tables 1-1 to 1-3).
TABLE 1-1 spectrophotometric method for determining chlorogenic acid content in strain fermentation liquor
| Strain numbering | A 755 | ΔA 755 | Chlorogenic acid concentration (μ g/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 spectrophotometric method for determining chlorogenic acid content in strain fermentation liquid
| Strain numbering | A 755 | ΔA 755 | Chlorogenic acid concentration (μ g/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 determination of chlorogenic acid content in fermentation broth of strain by spectrophotometry
| Strain numbering | A 755 | ΔA 755 | Chlorogenic acid concentration (μ g/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: in the table, "-" indicates that no measurement was made.
2.1.2 double screening by high performance liquid chromatography
Respectively sucking standard chlorogenic acid use solution, diluting with mobile phase, and fixing the volume in a volumetric flask to obtain the following concentrations: 2.0. Mu.g/mL, 10.0. Mu.g/mL, 20.0. Mu.g/mL, 40.0. Mu.g/mL, 80.0. Mu.g/mL of the standard series. And (4) drawing a standard curve of the concentration of the chlorogenic acid according to the peak area and the concentration of the chlorogenic acid standard (see figure 2).
By measuring the 15 candidate strain fermentation broths obtained by primary screening (Table 2), it was found that chlorogenic acid was detected in only 2 strains of the fermentation broths of BLCC1-0170 and BLCC1-0759, at concentrations of 1.61. Mu.g/mL and 0.12. Mu.g/mL, respectively. Subsequently, strain BLCC1-0170 is used for optimizing and applying the fermentation condition for producing chlorogenic acid.
TABLE 2 determination of chlorogenic acid content in fermentation broth by high performance liquid chromatography
Note: in the table, "-" indicates that no measurement was made.
2.2 optimization of fermentation conditions for 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 dry corn steep liquor 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 broth of strain BLCC1-0170 under different carbon sources
Note: in the table, "-" indicates no measurement.
2.2.2 Nitrogen Source optimization
As can be seen from Table 4, when ammonium chloride, which is an inorganic nitrogen source, was used as the nitrogen source, chlorogenic acid was not detected in the fermentation broth of the strain BLCC 1-0170; when organic nitrogen source peptone is used as a nitrogen source, chlorogenic acid with a certain concentration can be detected in the strain BLCC1-0170 fermentation broth, and when the addition amount of the peptone is 1.5%, the content of the chlorogenic acid is the highest and is 4.94 mu g/mL.
TABLE 4 chlorogenic acid content in fermentation broth of strain BLCC1-0170 under different nitrogen sources
Note: in the table, "-" indicates no measurement.
2.2.3 Effect of copper ions
The influence of copper ions on the content of chlorogenic acid produced by strain BLCC1-0170 was studied by using 0.2% glucose as carbon source and 1.5% peptone as nitrogen source, with different contents of copper sulfate added, and the results are shown in Table 5. When the addition amount of copper sulfate is 0.003 percent, the chlorogenic acid produced by the strain BLCC1-0170 through fermentation has the highest content which reaches 11.50 mu g/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
Respectively fermenting and culturing strain BLCC1-0170 with LB liquid culture medium and optimized culture medium, and determining chlorogenic acid content in the fermentation broth. Wherein, the LB liquid culture medium comprises: glucose 0.2%, peptone 1.0%, yeast extract 0.5%, naCl 0.5%, pH 7.0. The optimized culture medium (mass percent) comprises the following components: 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 (unit: ug/mL) in fermentation broths at different fermentation times
| Culture medium | Fermenting for 24h | Fermenting for 48h |
| LB liquid culture Medium | 1.36 | 1.61 |
| Optimized cultureBase of | 6.90 | 11.81 |
As can be seen from Table 6, the content of chlorogenic acid reached 11.81. Mu.g/mL when the culture medium was optimized for 48 hours of fermentation culture, and the yield of chlorogenic acid was 7.3 times higher than that obtained by 48 hours of culture in LB liquid medium. Along with the extension of the fermentation time within 48h, the content of the chlorogenic acid generated by adopting the optimized culture medium for fermentation is obviously improved, and along with the extension of the fermentation time in the LB liquid culture medium, the content of the chlorogenic acid is not obviously changed.
2.3 identification of Bacillus chlorogenic acid
The morphology of the strain BLCC1-0170 was observed by microscopic examination, and spores were lateral and spindle (FIG. 3). DNA of the strain BLCC1-0170 is extracted, 16S rDNA of the strain is amplified by PCR, and after sequencing, the strain is logged in a GenBank database for sequence comparison analysis, and the strain BLCC1-0170 is a strain of Brevibacillus laterosporus (Brevibacillus laterosporus) with the sequence shown in Seq _ 1.
Example 2 determination of in vitro bacteriostatic, antioxidant and acid-producing Properties of Bacillus chlorogenic acid
1 materials and methods
1.1 test strains
The strain BLCC1-0170 screened by the invention, namely Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170, is preserved in China center for type culture Collection in 20 months 06 and 2022, and the preservation number is CCTCC NO: m2022932.
1.2 culture 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%, pH 7.0.
NA culture medium: peptone 1.0%, beef powder 0.3%, naCl 0.5%, agar powder 1.5%, pH 7.0.
1.3 determination of in vitro bacteriostatic Properties
Inoculating the strain BLCC1-0170 into LB liquid culture medium, culturing at 37 deg.C and 180r/min for 24h, collecting bacterial liquid, centrifuging at 5000r/min for 10min, and collecting supernatant.
Staphylococcus aureus BLCC8-0004, clostridium perfringens BLCC8-0044, salmonella gallinarum BLCC8-0129, aeromonas hydrophila BLCC8-0121 and escherichia coli BLCC8-0102 are used as indicator bacteria, and a perforation method is used for carrying out bacteriostatic test, wherein the indicator bacteria are preserved and provided by a strain preservation center of bioengineering research institute of biologicalprocess GmbH, shandong Baolaili.
The specific method of the bacteriostatic test comprises the following steps: the indicator bacteria cultured overnight are diluted 1000 times, 5mL of indicator bacteria dilution is added into 45mL of NA culture medium, mixed evenly, 15mL of indicator bacteria dilution is sucked and placed in a plate. After solidification, the wells are punched by an Oxford cup, then 40 mul of centrifuged supernatant is sucked and added into the wells, and the culture is carried out overnight at 37 ℃, and the bacteriostatic effect is observed.
1.4 determination of Oxidation resistance
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. Inoculating 800 μ L of the seed liquid into 40mL of fermentation medium, performing fermentation culture at 37 deg.C for 48h at 180r/min, and collecting 5mL of fermentation liquid for use. In addition, 5mL of fermentation liquor is taken, centrifuged for 10min at 5000r/min, the supernatant is collected for standby, the centrifuged thalli are washed for 2 times by using sterile water, and the thalli are resuspended by using 5mL of sterile physiological saline to prepare a bacterial suspension for standby.
1.4.2 measurement of in vitro DPPH radical scavenging Capacity
0.5mL of the sample was added to 4mL of a DPPH absolute ethanol solution (0.2 mmol/L), reacted at room temperature (25 ℃) in the dark for 30min, centrifuged at 5000r/min for 10min, the absorbance at 517nm was measured, and the sample was zeroed with deionized water.
DPPH radical clearance (%) = [1- (A1-A2)/A3 ] x 100%
Wherein: a1 is the light absorption value of 4mL of DPPH absolute ethyl alcohol solution and 0.5mL of sample; a2 (blank) is the light absorption value of 4mL of absolute ethyl alcohol plus 0.5mL of sample; a3 (control) is absorbance of 4mL of DPPH absolute ethanol solution +0.5mL of sterile water.
1.4.3 measurement of the ability to scavenge hydroxyl radicals
Adding 1mL of O-phenanthroline (0.75 mmol/L) into a test tube, sequentially adding 1mL of PBS (0.05 mol/L, pH 7.4) and 0.5mL of sample or distilled water, mixing well, adding 1mL of FeSO 4 (2.5 mmol/L), mixing well and adding H 2 O 2 (20 mmol/L) was added to 1mL of the reaction mixture, and after 1.5 hours 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%
In the formula: a0 is sample and H 2 O 2 (1 mL of distilled water was used instead of 1mL of H 2 O 2 ) The light absorption value of (a); a1 is sample-free, H 2 O 2 The light absorption value of (a); a2 is a sample and H 2 O 2 The absorbance of (a).
1.5 analysis of metabolites (organic acids) of strains
The acid production capacity of the strain BLCC1-0170 is determined by high performance liquid chromatography, and the method refers to Zhang Jianmei, hu Shunzhen, mu Xijun and the like. The liquid chromatography conditions were: high performance liquid chromatograph (shimadzu LC-20A, japan), mobile phase: 50mmol/L ammonium dihydrogen phosphate (phosphoric acid pH 2.5): acetonitrile =98:2 (V: V), flow rate: 0.8mL/min, column temperature: 28 ℃, detection wavelength: 212nm, sample size: 10 μ L.
2 results and analysis
2.1 determination of bacteriostatic Property of Strain BLCC1-0170
As can be seen from Table 7, the strain BLCC1-0170 has bacteriostatic effects on 5 pathogenic bacteria, namely staphylococcus aureus, clostridium perfringens, salmonella enteritidis, aeromonas hydrophila and escherichia coli to be tested, wherein the bacteriostatic effect on the escherichia coli is the best, and the diameter of a bacteriostatic zone is 20.25mm.
TABLE 7 bacteriostatic results of strain BLCC1-0170
| Serial number | Pathogenic bacteria numbering | Indicator bacterium | Diameter of bacteriostatic circle (mm) |
| 1 | BLCC8-0004 | Staphylococcus aureus (Staphylococcus aureus) | 16.25±0.50 |
| 2 | BLCC8-0044 | Clostridium perfringens | 19.00±1.00 |
| 3 | BLCC8-0102 | Escherichia coli | 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 can be seen from table 8, the bacterial suspensions, fermentation broths and supernatants of the strains BLCC1-0170 all have certain DPPH radicals and hydroxyl radical scavenging abilities, wherein the active substances capable of scavenging DPPH radicals mainly exist in the supernatants, and the active substances capable of scavenging hydroxyl radicals mainly exist in the bacterial suspensions.
TABLE 8 antioxidant capacity index of strain BLCC1-0170
| Serial number | Sample name | DPPH radical scavenging ratio (%) | Hydroxyl radical clearance (%) |
| 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 can be seen from Table 9, the strain BLCC1-0170 has certain fermentation acid production capacity, and the lactic acid content and the acetic acid content in the fermentation supernatant are respectively 1.42g/L and 1.84g/L when the fermentation is carried out for 24 hours.
TABLE 9 acid production amount of BLCC1-0170 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 in animals
1 test Material
Female Kunming mice were used for the experiments, purchased from Jinanpunyue laboratory animal Breeding Co., ltd.
Dextran Sodium Sulfate (DSS), available from saint next biotechnology (shanghai) gmbh, MW:36000 to 50000.
The strain BLCC1-0170, namely Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0170, is preserved in China center for type culture Collection (CCTCC NO) at 20 months and 06 months in 2022, and the preservation number is CCTCC NO: m2022932.
The strains BLCC1-0716 in the invention refer to Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0716, which have been deposited in China center for type culture Collection at 11 months and 11 days in 2021, with the deposition number being 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 old are purchased and fed with basal feed for 3 days. Then, the test samples were randomly divided into 4 groups according to the principle that the body weight difference was not significant, which were respectively a blank group (CK group), a UC (ulcerative colitis) model group, a strain BLCC1-0170 group and a strain BLCC1-0716 group, and the divided day was recorded as the 1 st day of the test, and the test design is shown in Table 10. The UC model is constructed and evaluated according to the record in Chinese patent CN 202111582162.7.
TABLE 10 test grouping and design
On the 1 st to 7 th days of the test, the CK group and the UC model group are perfused with 200 mu L of normal saline every day; BLCC1-0170 groups perGastrodia elata 200. Mu.L 3X 10 8 CFU/mL BLCC1-0170 bacterial liquid (i.e. 6X 10 strains per daily administration) 7 CFU); the BLCC1-0716 groups administered with 200 μ L of 3 × 10 per day 8 CFU/mL BLCC1-0716 bacterial suspension (i.e. 6X 10 strains per daily administration) 7 CFU) during which the mice in each group had free access to water. Starting on day 8, each group was treated as follows for a period of 10 days: CK group drenches 200 μ L normal saline every day, freely drinks water; gavage 200 μ L of physiological saline daily for the UC model group, and freely drink 5% DSS aqueous solution; gavage 200 uL 3X 10 for BLCC1-0170 group 8 CFU/mL BLCC1-0170 bacterial liquid (i.e. 6X 10 strains per daily administration) 7 CFU), and 5% dss aqueous solution was freely drunk; intragastric administration of 200. Mu.L 3X 10 for BLCC1-0716 group 8 CFU/mL BLCC1-0716 bacterial suspension (i.e. 6X 10 strains per daily administration) 7 CFU), and 5% DSS aqueous solution was freely drunk. The general condition, body mass, stool characteristics and occult blood of the mice were observed daily during the test period. After the test is finished, taking off the cervical vertebra, killing the mouse, taking blood from the eyeball, and collecting serum; meanwhile, the colon is isolated, and the lesion condition is visually observed to measure the length of the colon.
Statistical analysis of data
One-Way ANOVA program in SPSS 13.0 statistical analysis software was used for One-Way ANOVA with results expressed as mean ± standard deviation. P is less than 0.05 and is used as a judgment standard of difference significance.
4 results and analysis
4.1 weight changes
As can be seen from table 11, the CK group mice continued to gain body weight throughout the duration of the experiment due to the absence of DSS-induced stimulation. During the period of drinking DSS, the body weight of the mice in the model group and the BLCC1-0170 group is increased and then reduced, and the body weight of the mice in the model group and the BLCC1-0170 group is in a descending trend after the day 4 of drinking. At the end of the experiment, the body weight of the mice in the model group is significantly lower than that in the CK group (P < 0.05), and the body weight of the mice in the BLCC1-0170 group is reduced but not significantly different (P > 0.05) compared with that in the CK group, but the body weight is higher than that in the model group. Therefore, the brevibacillus laterosporus BLCC1-0170 has the efficacy of inhibiting the weight loss of mice caused by free drinking of DSS.
TABLE 11 daily body weight (unit: g) of mice during the test
Note: the same or no mark on the shoulder marks of the same row indicates that the difference is not significant (P is more than 0.05), and the different marks on the shoulder marks of the same row indicate that the difference is significant (P is less than 0.05).
4.2 Colon Length changes
After the test was completed, the eyeball was removed and blood was collected, then the cervical spine was taken off and the mouse was sacrificed, the intact colon was dissected 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 for groups of mice
Note: the same or no mark on the shoulder marks of the same row indicates that the difference is not significant (P is more than 0.05), and the different marks on the shoulder marks of the same row indicate that the difference is significant (P is less than 0.05).
Except CK group, colon tissue is intact, feces in intestinal cavity are formed, BLCC1-0170 group, BLCC1-0716 group and model group all have bleeding, and feces in intestinal cavity are reduced. As can be seen from Table 12, the colon length was the longest in the CK group, and the colon length was 3.31cm shorter in the model group than in the CK group. The colon length of the BLCC1-0170 groups is 2.50cm longer than that of the model group, the colon length is improved by 32.8 percent, and the difference is obvious (P is less than 0.05). Colon length in groups BLCC1-0716 was significantly shorter than that in CK group (P < 0.05) and shorter than that in groups BLCC1-0170 (P > 0.05), although longer than that in model group. These results show that Brevibacillus laterosporus BLCC1-0170 can regulate the change of the colon length of mice caused by DSS induction, and the effect is better than that of the BLCC1-0716 group.
4.3 serum cytokine level changes
Measuring the level change of mouse serum cytokines by the above sea enzyme-linked scientific research 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, the serum TNF- α level was highest in the model group, and in the BLCC1-0170 group, 320.05pg/mL was 17.6% lower than that in the model group, and the difference was significant (P < 0.05). Serum IL-6 levels were highest in the model group, and the BLCC1-0170 group was significantly lower than the model group, reduced by 17.2%, and did not differ significantly from the CK group. Serum IL-10 levels were lowest in the CK group and highest in the BLCC1-0170 group, which were 2.8% higher than the model group, but the differences were all insignificant (P > 0.05). In addition, the serum proinflammatory cytokines TNF-alpha and IL-6 of group BLCC1-0170 were at lower levels than those of group BLCC1-0716, and the serum anti-inflammatory cytokine IL-10 was at higher levels than those of group BLCC 1-0716. These results show that Brevibacillus laterosporus BLCC1-0170 can regulate and control the change of the level of the mouse serum inflammatory factor caused by DSS induction, and the effect is better than that of the BLCC1-0716 group.
TABLE 13 mouse serum inflammatory factor level Change (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 set | 388.41±20.12 b | 64.24±1.66 b | 209.28±23.51 |
| Group BLCC1-0170 | 320.05±24.70 a | 53.18±1.88 a | 218.04±23.61 |
| Group BLCC1-0716 | 345.68±26.32 ab | 54.02±3.02 a | 217.44±15.48 |
Note: the same or no mark on the same row of shoulder marks indicates that the difference is not significant (P is more than 0.05), and the difference on the same row of shoulder marks indicates that the difference is significant (P is less than 0.05).
4.4 determination of serum antioxidant index
Malondialdehyde (MDA) is a lipid peroxidation product in vivo, and can indirectly reflect the status of oxygen radical metabolism and the degree of lipid peroxidation in the body; superoxide dismutase (SOD) plays a crucial role in balancing oxidation and antioxidation of the body, and can remove superoxide anions to protect cells from being damaged. The antioxidant indexes of mouse serum are measured by Nanjing-built biological kit, and the measured antioxidant indexes comprise MDA, T-SOD (total superoxide dismutase) and T-AOC (total antioxidant capacity). The results are shown in Table 14.
As can be seen from Table 14, the serum MDA content of the model group is significantly higher than that of the CK group (P is less than 0.05), the increase is 64.7%, and the probable reason is that the stress reaction is generated in the mouse body after the DSS is drunk. 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 T-SOD value is improved by 12.8 percent; the T-AOC value of the BLCC1-0170 groups is 3.2 percent higher than that of the model group. These results show that the Brevibacillus laterosporus BLCC1-0170 has certain intervention and relieving effects on the stress state of the mouse body. However, the indexes of the BLCC1-0716 group in MDA, T-SOD and T-AOC are not obviously different from those of the model group, which shows that the strain BLCC1-0716 has no obvious effect on relieving the stress state of the mouse body.
TABLE 14 determination of mouse serum stress index
Note: the same or different letters in the same row indicate that the difference is not significant (P is more than 0.05), and the different letters in the same row indicate that the difference is significant (P is less than 0.05).
In conclusion, the invention screens 1 endophyte BLCC1-0170 with high chlorogenic acid yield from 45 endophytes separated from eucommia leaves, medlar, honeysuckle and other plants by using a spectrophotometry method and a high performance liquid chromatography, the chlorogenic acid content can reach 11.81 mu g/mL after fermentation optimization, and the chlorogenic acid production capacity is obviously higher than that of the currently known bacillus subtilis. In addition, the bacterial strain is determined to have high chlorogenic acid content, good bacteriostatic ability and good antioxidant ability, has prevention and treatment effects on mouse ulcerative colitis and has certain intervention and relief effects on the stress state of a mouse body through antibacterial performance, antioxidant ability and acid production performance determination of the bacterial strain and mouse in-vivo effect evaluation experiments.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The Brevibacillus laterosporus with high chlorogenic acid yield is named Brevibacillus laterosporus BLCC1-0170, and is preserved in the China center for type culture collection in the university of Wuhan, hubei, china at 20 months and 2022, with the preservation number of CCTCC NO: m2022932.
2. A microbial preparation or feed, comprising Brevibacillus laterosporus and/or a fermented product thereof according to claim 1.
3. Use of Brevibacillus laterosporus and/or a fermentation product thereof according to claim 1 for the preparation of a medicament, a microbial agent or a feed additive having one or more of the following functions;
1) The application in preparing antioxidant medicines, microbial inoculum or feed additives;
2) The application in preparing bacteriostatic drugs, bactericides or feed additives;
3) The application in preparing medicines, microbial inoculum or feed additives for preventing and treating enteritis;
4) The application in preparing medicines, microbial agents or feed additives for improving the antioxidant stress capability of animals.
4. Use according to claim 3, wherein the antioxidant comprises scavenging hydroxyl radicals and/or scavenging DPPH radicals.
5. The use of claim 3, wherein the enteritis is ulcerative colitis.
6. A method for producing chlorogenic acid comprising the fermentative culture of brevibacillus laterosporus as described in claim 1.
7. The method according to claim 6, wherein one or more of glucose, corn starch and corn steep liquor dry powder are used as carbon sources and organic nitrogen sources are used as nitrogen sources during fermentation culture, and the culture temperature is 35-38 ℃.
8. The method according to claim 6, wherein the culture medium is supplemented with copper ions in an amount of 0.001 to 0.005% by mass and the organic nitrogen source in an amount of 0.5 to 2.0% by mass.
9. The method of claim 6, wherein the culture medium is prepared by using glucose as a carbon source and peptone as a nitrogen source, wherein the addition amount of the peptone is 1-2% by mass, and copper sulfate is added, and the addition amount of the copper sulfate is 0.002-0.005% by mass.
10. The method according to claim 6, wherein the composition of the medium during the 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 aerobic culture is carried out, wherein the percentage is mass percentage.
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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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