CN116590166A - Bacillus belicus and application thereof to storage of dried rice - Google Patents
Bacillus belicus and application thereof to storage of dried rice Download PDFInfo
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- CN116590166A CN116590166A CN202211328685.3A CN202211328685A CN116590166A CN 116590166 A CN116590166 A CN 116590166A CN 202211328685 A CN202211328685 A CN 202211328685A CN 116590166 A CN116590166 A CN 116590166A
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- bacillus
- rice
- velezensis
- belicus
- bacillus velezensis
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B9/00—Preservation of edible seeds, e.g. cereals
- A23B9/16—Preserving with chemicals
- A23B9/24—Preserving with chemicals in the form of liquids or solids
- A23B9/26—Organic compounds; Microorganisms; Enzymes
- A23B9/28—Microorganisms; Enzymes; Antibiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
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- General Health & Medical Sciences (AREA)
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- Medicinal Chemistry (AREA)
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Abstract
The invention provides bacillus belicus and application thereof to storage of dried rice, belonging to the technical field of microorganisms and biological control of stored grain; in the invention, bacillus beleiensis (Bacillus velezensis) E2 is separated and screened from the surface of healthy rice and is preserved in China Center for Type Culture Collection (CCTCC) for 5 months and 10 days in 2022, wherein the preservation number is CCTCC NO: M2022579, and the preservation address is Wuhan in China; the bacillus beijerinckii (Bacillus velezensis) E2 can reduce the content of aflatoxin B1 (AFB 1) in rice, so that the mildewing of stored rice can be effectively controlled, the rise of the fatty acid value of the stored rice can be controlled, the deterioration of the rice can be prevented, and the rice has a good application prospect; the bacillus beijerinckii (Bacillus velezensis) E2 can also be used for preventing other grains mainly mildewed by aspergillus flavus, such as corn, peanut and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms and biological control of stored grain, and particularly relates to bacillus belicus and application thereof to storage of dried rice.
Background
Rice (Oryza sativa L) refers to fruiting body without removing rice hull, and comprises white rice, testa Tritici, embryo bud and rice hull. During the period from planting to storage of rice, the mold in the soil and air (mainly including aspergillus and penicillium, etc.) can enter the ripened or harvested rice by air flow, wind and rain, and is distributed outside and inside the rice. As the rice can be slowly metabolized in the storage process, the temperature and the humidity of the granary are increased, so that the growth and the propagation of pathogenic bacteria such as fungi in the rice are realized, the quality of the rice polluted by the pathogenic bacteria such as fungi is low, and the safety of consumers is threatened. At present, the most common fungi in barns and the most harmful fungi are aspergillus flavus (Aspergillus flavus), which can produce mycotoxins such as aflatoxin.
Conventionally, control of mildewing of stored rice has been largely dependent on physical and chemical means. Common physical methods mainly comprise low-temperature grain storage, gas-conditioned grain storage, irradiation treatment and the like, but the methods are limited by the defects of high cost, complex gas source preparation and the like, and are difficult to widely apply to production practice. The chemical fumigation method is commonly used, and has the advantages of no limitation of regions and seasons, quick response, low cost and the like, and can be used for large-scale control, but chemical agents belong to dangerous goods and dangerous chemicals, and hidden danger is brought to the environment and life safety of people. In addition, in recent years, people call for green grain storage more and more, and the consumption of chemical agents is greatly reduced. Therefore, the research on the grain storage technology which is environment-friendly, safe, effective and economical is particularly urgent.
Biological control methods have been attracting more and more attention in recent years due to their advantages of energy conservation, environmental protection, high efficiency, safety, etc. The broad-spectrum antagonistic bacillus has the advantages of good biological activity, high inhibition activity on various pathogenic microorganisms, convenience for large-scale production and the like, gradually becomes one of the most valuable strains, and various bacillus has high safety to human beings and the environment. However, most bacillus has a narrow antibacterial spectrum, poor stress resistance and poor colonization capability, and is mostly applied to prevention and treatment of plant diseases before harvest, so that the mildew of the stored rice cannot be effectively controlled.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides bacillus beljavensis and application thereof to storage of dried rice. In the invention, antagonistic bacillus is separated and screened from the surface of healthy rice, named as bacillus beleiensis (Bacillus velezensis) E2 and preserved in China Center for Type Culture Collection (CCTCC) for 5 months and 10 days in 2022, wherein the preservation number is CCTCC NO: M2022579, and the preservation address is Wuhan in China; the bacillus beijerinckii (Bacillus velezensis) E2 can reduce the content of aflatoxin B1 (AFB 1) in rice, so that the mildewing of stored rice can be effectively controlled, the rise of the fatty acid value of the stored rice can be controlled, the deterioration of the rice can be prevented, and the rice has a good application prospect; the bacillus beijerinckii (Bacillus velezensis) E2 can also be used for preventing other grains mainly mildewed by aspergillus flavus, such as corn, peanut and the like.
The invention firstly provides bacillus beliae, the antagonistic bacillus is bacillus beliae (Bacillus velezensis) E2, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation number is CCTCCNO: M2022579, the preservation time is 2022, 5 months and 10 days, and the preservation address is Chinese Wuhan.
The invention also provides a microbial preparation, which comprises the bacillus beijerinckii (Bacillus velezensis) E2.
Specifically, the microbial preparation comprises bacillus beijerinckii (Bacillus velezensis) E2 active freeze-dried powder; the freeze-dried powder comprises bacillus bailii (Bacillus velezensis) E2 bacterial precipitation and a protective agent solution.
Specifically, the addition amount of the protective agent solution is equal to the volume of supernatant after the bacillus belicus (Bacillus velezensis) E2 bacterial liquid is centrifuged; the components of the protective agent comprise maltose, skimmed milk powder and sodium glutamate.
Specifically, the protective agent comprises 4-16 g/100mL of maltose, 4-16 g/100mL of skimmed milk powder and 2-14 g/100mL of sodium glutamate as solvents.
The invention also provides application of the bacillus beijerinus (Bacillus velezensis) E2 and/or the microbial preparation in any one of the following:
(1) Application in preventing and controlling aspergillus flavus, gibberella, colletotrichum gloeosporioides, wei Simei Paecilomyces varioti, penicillium italicum, aspergillus ochraceus or penicillium digitatum;
(2) Preventing and controlling mildew of grains, feeds or other raw materials;
(3) Eliminating or inhibiting aflatoxin B1 production in grains, feeds or other raw materials;
(4) Controlling the fatty acid value in grains, feeds or other raw materials;
(5) Use to inhibit low temperature, high temperature, acid and osmotic stress;
(6) Improving the quality of grains, feeds or other raw materials.
Specifically, the application is to prevent and treat stored grains from mildew, wherein the mildew comprises aspergillus flavus mildew and aspergillus niger mildew, and the grains comprise rice, corn and peanut.
Specifically, the application is that bacillus belicus (Bacillus velezensis) E2 reduces the content of aflatoxin B1 in grains, controls the increase of fatty acid value of stored rice, and prevents the grains from deteriorating.
The invention also provides a biological control method for controlling the mildew of the stored rice, which comprises the steps of fully mixing bacillus bailii (Bacillus velezensis) E2 and/or a microbial preparation with grains for storage, and particularly, the amount of bacteria in each gram of rice is 1 x 10 x during the uniform mixing storage -6 ～10 -8 cfu。
Compared with the prior art, the invention has the beneficial effects that:
the bacillus beijerinus (Bacillus velezensis) E2 provided by the invention is obtained by screening from the surface of healthy rice in the laboratory, and has good colonization capability. In addition, the antibacterial agent also has broad-spectrum antibacterial capability and can generate lipopeptides and volatile antibacterial substances.
The bacillus bailii (Bacillus velezensis) E2 provided by the invention has strong low temperature resistance, high temperature resistance, acid resistance and osmotic pressure stress resistance, can resist the granary environment, and is suitable for further development and research. The bacillus beijerinckii (Bacillus velezensis) E2 has positive influence on preventing and controlling stored rice, can effectively reduce the use of chemical bactericides in the rice storage process, provides a certain guiding significance for biological prevention and control of rice mildew, and also provides guidance for storage of other grains mainly formed by aspergillus flavus.
Bacillus bailii (Bacillus velezensis) E2 is prepared into a microbial preparation by a freeze-drying method to prevent and treat the mildew of the stored rice, wherein maltose, skimmed milk powder and sodium glutamate are used as protective agents, and after the protective agents are added, the survival rate of the Bacillus bailii (Bacillus velezensis) E2 is obviously improved and is as high as 91.64 percent. Compared with the preparation method without adding the protective agent, the microbial preparation provided by the invention can more effectively improve the activity of the bacillus after freeze-drying.
The bacillus beijerinckii (Bacillus velezensis) E2 or microbial agent provided by the invention and rice are uniformly mixed and stored to find that: the experimental group has no obvious mildew, while the control group is seriously infected by the mildew, so that the mould number of the stored rice reserves can be obviously reduced, the rise speed of the fatty acid value can be delayed, and the content of AFB1 in the rice can be effectively reduced. The bacillus beijerinckii (Bacillus velezensis) E2 or microbial agent has better biological control effect and has positive effect on guaranteeing the quality of rice.
Drawings
FIG. 1 is a B.bailii (Bacillus velezensis) E2 morphological feature; in the figure, a: colony morphology on LA; b: cell morphology.
FIG. 2 shows the phylogenetic tree (a) of the ITS region of Bacillus beleiensis (Bacillus velezensis) E2 and the phylogenetic tree (b) of Aspergillus flavus (Aspergillus flavus) H3.
FIG. 3 is a graph showing the effect of Bacillus belicus (Bacillus velezensis) E2 fresh bacterial liquid and active lyophilized powder on the amount of Aspergillus flavus (Aspergillus flavus) in rice.
FIG. 4 is a graph showing the effect of Bacillus belicus (Bacillus velezensis) E2 fresh bacterial liquid and active lyophilized powder on fatty acid value in rice.
FIG. 5 is a graph showing the effect of Bacillus belicus (Bacillus velezensis) E2 fresh bacterial liquid and active lyophilized powder on AFB1 content in rice.
FIG. 6 is a graph showing the number of colonization of rice surfaces by Bacillus belicus (Bacillus velezensis) E2.
FIG. 7 is a graph showing the tolerance of Bacillus belicus (Bacillus velezensis) E2 to cold stress.
FIG. 8 is a graph showing the heat stress tolerance of Bacillus belicus (Bacillus velezensis) E2.
FIG. 9 is a graph showing the tolerance of Bacillus belicus (Bacillus velezensis) E2 to osmotic stress.
FIG. 10 is a graph showing the acid stress tolerance of Bacillus belicus (Bacillus velezensis) E2.
FIG. 11 is a contour plot and response surface plot of the interaction of maltose and skimmed milk powder in Bacillus bailii (Bacillus velezensis) E2 active lyophilized powder; in the figure, a: a contour map; b: response surface map.
FIG. 12 is a contour plot and response surface plot of the interaction of maltose and sodium glutamate in Bacillus bailii (Bacillus velezensis) E2 active lyophilized powder; in the figure, a: a contour map; b: response surface map.
FIG. 13 is a contour plot and response surface plot of interaction of skimmed milk powder and sodium glutamate in Bacillus bailii (Bacillus velezensis) E2 active lyophilized powder; in the figure, a: a contour map; b: response surface map.
FIG. 14 is a graph showing the activity change of Bacillus belicus (Bacillus velezensis) E2 active lyophilized powder at different storage temperatures.
Detailed Description
The invention will be further described with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto. The composition of the medium according to the present invention is as follows:
bengalhong medium: 36.6g of Bengalhon, 1000mL of distilled water and sterilizing for 20min at 121 ℃;
PDB medium: 200g of potato is peeled and boiled for 20min, 20g of glucose, 1000mL of distilled water and sterilized at 121 ℃ for 20min;
PDA medium: 200g of potato is peeled and boiled for 20min, 20g of glucose, 20g of agar powder, 1L of distilled water and sterilized at 121 ℃ for 20min;
LA medium: 10g/L NaCl, 10g/L tryptone, 5g/L bacillus powder, 20g/L agar powder, adding distilled water to 1000mL, sterilizing at 121 ℃ for 20min at pH 7.0-7.2
LB medium: 10g/L NaCl, 10g/L tryptone, 5g/L yeast extract, 1000mL distilled water, pH 7.0-7.2, and sterilizing at 121deg.C for 20min.
In the following examples, rice is taken as an example to illustrate the biological control effect of bacillus beleiensis (Bacillus velezensis) E2 on grain mildew, but bacillus beleiensis (Bacillus velezensis) E2 in the present invention can also be used in other grains mainly mildew of aspergillus flavus, including but not limited to corn and peanut.
Example 1: screening and identification of bacillus bailii (Bacillus velezensis) E2
The bacillus beijerinckii (Bacillus velezensis) E2 is obtained by separating the surface of rice harvested from paddy fields of Zhenjiang fruit-wetting agriculture limited company, and the specific obtaining method is as follows:
5g of healthy rice is weighed and suspended in 50mL of sterile water, and is vibrated in a combined shaking table for 30min at 28 ℃ under 180r/min to obtain a suspension. The suspension was diluted in a gradient to a bacterial count of 30-300 per 100. Mu.L, 100. Mu.L of the dilution was pipetted and spread evenly on plates containing LA medium and incubated at 28℃for 2 days. Purifying the strain for multiple times by streak separation after culturing, inoculating the purified bacillus into LB culture medium containing 15% (v/v) glycerol, and storing at-80deg.C for long term.
Identifying morphological characteristics and molecular biology of bacillus obtained by separation, wherein the identification results are shown as follows:
the morphological characteristics of bacillus are shown in fig. 1:
(1) After 36 hours of culture, the colony is regular round, transparent and high in viscosity;
(2) After 48 hours of culture, the bacterial colony is milky, irregularly round, opaque, large in bacterial colony, raised in edge, obvious in surface bulge and smooth in touch;
(3) After gram staining, the colony is purple, the strain is a gram positive strain, the shape is short rod, and spores are generated.
Molecular biological identification of bacillus:
the nucleotide sequence of ITS region is amplified by bacterial 16S rDNA universal primer to isolate bacillus, the sequencing result of the product is input to https:// www.ncbi.nlm.nih.gov/website, homologous sequence is downloaded from NCBI database, and phylogenetic tree is constructed by MEGA6 software as shown in figure 2 a.
Wherein, the nucleotide sequence of the bacterial 16S rDNA universal primer is as follows:
27F：5'-CAGAGTTTGATCCTGGCT-3'(SEQ ID No:1)；
1492R：5'-AGGAGGTGATCCAGCCGCA-3'(SEQ ID No:2)；
the nucleotide sequence of the ITS zone nucleotide is shown as SEQ ID No. 3, and the sequencing result of the product is consistent with the result of SEQ ID No. 3.
SEQ ID No:3：
GCTATAATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGAGCCAGCCGCCGAA
FIG. 2 shows a phylogenetic tree of ITS zone of Bacillus belicus (Bacillus velezensis) E2, and according to the comparative analysis of FIG. 1, the screened bacillus is Bacillus belicus, which is named Bacillus belicus (Bacillus velezensis) E2, and is preserved in China Center for Type Culture Collection (CCTCC), with the preservation number of CCTCC NO: M2022579, the preservation time of 2022, 5 months and 10 days, and the preservation address of Chinese Wuhan.
Example 2: verification of Bacillus bailii (Bacillus velezensis) E2 control of stored rice mildew
In this example, aspergillus flavus was first isolated and purified from the surface of mildewed rice, and then bacillus beijerinckii (Bacillus velezensis) E2 was used as an experimental group, and physiological saline was used as a control group to examine the ability of bacillus beijerinckii (Bacillus velezensis) E2 to antagonize aspergillus flavus, thereby verifying the ability of bacillus beijerinckii (Bacillus velezensis) E2 to control mildewing of stored rice. The specific investigation steps are as follows:
(1) Separating and purifying aspergillus flavus from the surface of mildewed rice:
5g of mildewed rice is weighed and suspended in 50mL of sterile distilled water, and is cultured for 30min at 28 ℃ under 180r/min in a combined shaking table to obtain a suspension. The suspension is diluted in a gradient to 30-300 mould spores per 100. Mu.L, 100. Mu.L of the diluted solution is sucked up and evenly spread on a plate containing the Bengalia red culture medium, and the culture is carried out for 4 days at 28 ℃. Purifying the strain for multiple times by streak separation after culturing, inoculating the purified pathogenic bacteria into PDB culture medium containing 15% glycerol, and storing at-80deg.C for a long time to obtain suspected Aspergillus flavus.
Amplifying ITS region nucleotide sequence of the separated suspected Aspergillus flavus with fungus universal primer, inputting sequencing result of the product into https:// www.ncbi.nlm.nih.gov/website, downloading homologous sequence from NCBI database, constructing phylogenetic tree by MEGA6 software as shown in figure 2 b, and determining that the screened strain is Aspergillus flavus.
Wherein, the general primers of fungi are:
ITS1：TCCGTAGGTGAA(SEQ ID No:4)；
ITS4：TCCTCCGCTTATTGATATGCCCTGCG(SEQ ID No:5)；
the nucleotide sequence of the ITS zone nucleotide is shown as SEQ ID No. 6, and the sequencing result of the product is consistent with SEQ ID No. 6.
SEQ ID No:6
CCGGAGGTATGGGTCTAGCGAGCCACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA
Carrying out morphological characteristic identification on the screened aspergillus flavus:
after culturing on PDA culture medium for 7 days, there are plenty of yellow-green spores on the culture medium, the edge is semi-villiated, and after culturing for 14 days, the spores are ripe and the color becomes dark green.
(2) Preparation of bacillus bailii (Bacillus velezensis) E2 bacterial suspension and aspergillus flavus spore suspension:
preparation of bacillus bailii (Bacillus velezensis) E2 suspension: inoculating Bacillus bailii (Bacillus velezensis) E2 into LB medium, activating twice, culturing at 28deg.C and 180r/min for 18 hr, centrifuging after culturing, re-suspending with sterile water to obtain bacterial suspension, and adjusting the concentration of bacterial suspension to 1×10 by blood cell counting plate 9 cfu/mL. Preparation of aspergillus flavus spore suspension: inoculating a small amount of Aspergillus flavus mycelium in step (1) onto a plate containing PDA culture medium, culturing in a constant temperature incubator at 28deg.C for 7 days, adding appropriate amount of sterile water after culturing, washing spores with sterile cotton swab, removing mycelium with three layers of mirror wiping paper to obtain Aspergillus flavus spore suspension, and adjusting concentration of Aspergillus flavus spore suspension to 1×10 5 Spores/mL for use.
(3) Investigation of the ability of Bacillus belicus (Bacillus velezensis) E2 to antagonize Aspergillus flavus (Aspergillus flavus):
healthy rice is selected, fine impurities such as soil grains and the like are removed by a mesh screen with the aperture of 1mm, rice straw is removed, and then ripe, full and mildew-free rice is taken, sterilized at 121 ℃ for 20min and cooled for standby.
Subpackaging the treated rice in triangular bottles, adding Bacillus bailii (Bacillus velezensis) E2 bacterial suspension and Aspergillus flavus spore suspension into each bottle of an experimental group, and adding normal saline and Aspergillus flavus spore suspension into each bottle of a control group. Wherein, the inoculation amount of bacillus bailii (Bacillus velezensis) E2 bacterial suspension and sterile distilled water is 0.1mL/g, the inoculation amount of aspergillus flavus spore suspension is 0.03mL/g, and the aspergillus flavus spore suspension is stored for 7 days at 37 ℃ in a dark place. Through observation, the control group can observe that the surface of the control group has aspergillus flavus growth, the surface of the bacillus beleiensis (Bacillus velezensis) E2 fresh bacterial liquid treatment group does not have aspergillus flavus growth, and then the total mould number, the fatty acid value and the AFB1 content of the bacillus beleiensis (Bacillus velezensis) E2 fresh bacterial liquid treatment group and the control group are respectively measured.
The method for measuring the total number of moulds, the fatty acid value and the AFB1 content comprises the following steps:
total number of mold: 5g of rice in a control group and a bacillus bailii (Bacillus velezensis) E2 fresh bacterial liquid treatment group are weighed, respectively filled into conical flasks, 45mL of sterile physiological saline is added, shaking is carried out for 20min under the condition of 180r/min to uniformly suspend mould in the sample in the physiological saline, then the mould is diluted in a gradient manner until the number of spores in each 100 mu L is 30-300, the mould is coated in a PDA culture medium, and the mould is counted after culturing for 3 days at 28 ℃.
Fatty acid value: the measurement was carried out according to the method of GB/T20569-2006.
AFB1 content: the content of AFB1 in rice is determined by adopting an enzyme-linked immunosorbent assay, and specific test steps are referred to a PriboFast Aflatoxin B1 (Aflatoxin B1) ELISA rapid detection kit instruction book.
According to the rules of the safety storage of grain and oil, the mould number of the storage is 1 multiplied by 10 5 -9.9×10 5 cfu/g is a critical area for grain storage safety and hazard and is also a critical control area. As can be seen from FIG. 3, the control group had the highest Aspergillus flavus count, reaching 4.4X10 6 cfu/g, which exceeds the area defined by the rules of food and oil safety storage, while the content of Aspergillus flavus in the Bacillus bailii (Bacillus velezensis) E2 fresh bacterial liquid treatment group is 4.92 multiplied by 10 5 cfu/g, within a specified interval.
As can be seen from FIG. 4, the control group had a fatty acid value of 85.67mg/100g, whereas the fresh E2 bacteria treated with Bacillus belicus (Bacillus velezensis) had a fatty acid value of 22.23mg/100g. National standards state that when the fatty acid value of the paddy exceeds 35mg/100g, the paddy is unfavorable, the fatty acid value of the control group exceeds the area, and the Bacillus bailii (Bacillus velezensis) E2 fresh bacterial liquid treatment group still belongs to the paddy favorable to storage. Thus, bacillus beijerinckii (Bacillus velezensis) E2 is effective in extending the shelf life of rice.
As can be seen from FIG. 5, after 7 days of cultivation, the AFB1 content of the control group reached 5.12. Mu.g/kg, while the AFB1 content in the national standard rice was not more than 10. Mu.g/kg, and neither the control group nor the experimental group was out of standard after 7 days of storage at 37℃in the dark, but the AFB1 content of the rice treated with Bacillus belicus (Bacillus velezensis) E2 was the lowest. The bacillus beijerinckii (Bacillus velezensis) E2 fresh bacterial liquid can effectively slow down the growth rate of AFB1 in paddy.
Example 3: investigation of E2 colonization ability of Bacillus bailii (Bacillus velezensis)
In this embodiment, the ability of Bacillus belicus (Bacillus velezensis) E2 to colonize was investigated by measuring the viable count of Bacillus in each tube after different days of culture, and the specific investigation method was as follows:
5g of sterilized rice was added to a 50mL centrifuge tube, followed by a concentration of 1X 10 9 cfu/mL bacillus beijerinckii (Bacillus velezensis) E2 suspension is directly soaked in the surface sterilized rice for 3 hours, and then the rice is taken out to suck the surface liquid. The treated rice grains were placed in an incubator at 37℃for 15 days with rapid culture. In the course of cultivation, 30mL of physiological saline was added to the treated rice every 3 days, followed by cultivation in a shaker (180 r/min) at 37℃for 30min, and after the completion of cultivation, the rice suspension was vigorously vortexed for 1min to obtain a rice suspension, after 10-fold dilution of the rice suspension, 100. Mu.L of the rice suspension was applied to LA medium (three dishes each) and cultivated in the dark at 28℃for 2 days, and then the viable count of Bacillus in each tube after various days of cultivation was measured, and the results are shown in FIG. 5.
FIG. 6 is a graph showing the number of colonization of rice surface by Bacillus belicus (Bacillus velezensis) E2, and it can be seen from the graph that the number of Bacillus belicus (Bacillus velezensis) E2 on rice is rapid within 9 days before inoculationFast growth from initial 1.06×10 6 cfu/g increase to 1.38X10 7 cfu/g; after 9 days of inoculation, the number of bacillus beleidersonii (Bacillus velezensis) E2 on rice tended to stabilize. This demonstrates that the strain can colonize on the surface of rice and has the potential to prevent and control mildew of stored rice.
Example 4: research on bacteriostasis broad-spectrum property of bacillus bailii (Bacillus velezensis) E2
(1) Inhibition of common pathogens by bacillus belicus (Bacillus velezensis) E2:
in the embodiment, the bacteriostasis effect of bacillus berryis (Bacillus velezensis) E2 on common pathogenic bacteria (such as aspergillus flavus, penicillium expansum, gibberella fulvikuroi, colletotrichum gloeosporioides C.gloosporides, penicillium italicum P.itanium, aspergillus ochraceus A.ochraceus, wei Simei Paecilomyces polymorphus P.vispiece and penicillium digitatum) is measured by adopting a plate counter method, and then the bacteriostasis broad spectrum of bacillus berryis (Bacillus velezensis) E2 is examined, wherein the examination method is as follows:
and (3) punching a center hole in the center position of the flat plate, punching 4 holes at equal intervals at a position about 20mm away from the center hole, removing agar in the holes, inoculating pathogenic bacteria spore suspension into the center hole, inoculating bacillus beleiensis (Bacillus velezensis) E2 suspension into the left hole and the right hole, inoculating equal amount of sterile water into the other two holes for comparison, culturing for 4 days at 28 ℃ by using a PDA (personal digital assistant) culture medium, observing the growth condition of pathogenic bacteria, measuring the diameter of hyphae, and calculating the inhibition rate according to the formula of 'antibacterial rate (%) = control group hypha diameter-treatment group hypha diameter/control group hypha diameter multiplied by 100', wherein the inhibition effect of bacillus beleiensis (Bacillus velezensis) E2 is shown in a table 1.
TABLE 1 inhibition of Bacillus bailii (Bacillus velezensis) E2 against different pathogenic bacteria
| Pathogenic bacteria | Inhibition ratio (%) |
| Aspergillus flavus A.flavus | 86.09±0.90 |
| Penicillium expansum P | 28.82±0.98 |
| Gibberella G.fujikuroi | 87.32±1.23 |
| Colletotrichum gloeosporioides C | 76.99±0.68 |
| P. italicum of Italian penicillium | 64.45±1.20 |
| Aspergillus ochraceus A.ochraceus | 53.16±2.80 |
| Wei Simei Pediospora canicola P.vismiae | 73.75±2.23 |
| Penicillium digitatum P | - |
Note that: results are expressed as mean ± standard deviation.
Table 1 shows the inhibition of Bacillus belicus (Bacillus velezensis) E2 against different pathogens, and as can be seen from Table 1, 7 pathogenic bacteria hyphae are inhibited by Bacillus belicus (Bacillus velezensis) E2, P.digitatum does not grow under the treatment of Bacillus belicus (Bacillus velezensis) E2, the inhibition rate cannot be calculated after prolonged culture for 30 days, the inhibition effect of Bacillus belicus (Bacillus velezensis) E2 against P.digitatum is strongest, and the inhibition effect of Bacillus belicus (Bacillus velezensis) E2 against P.expansum is worst, and the inhibition rate is 28.82%. The result shows that the strain E2 has broad-spectrum antibacterial activity.
(2) Inhibition of common pathogens by volatile substances produced by bacillus bailii (Bacillus velezensis) E2:
in the embodiment, the LA plate and the PDA plate are buckled by adopting a plate buckling method, and the sealing film is used for sealing to determine the antibacterial effect of volatile substances generated by bacillus beijerinus (Bacillus velezensis) E2 on common pathogenic bacteria (such as aspergillus flavus A. Flavus, penicillium expansum, gibberella, fujikuroi, colletotrichum gloeosporioides C. Gloeosporides, penicillium italicum P. Itusum, aspergillus ochraceus A. Ochraceus, wei Simei paecilomyces variotis P. Vismii and penicillium digitatum P. Digitatum) by the following investigation method:
experimental group: uniformly coating 100 mu L of bacillus belicus (Bacillus velezensis) E2 bacterial suspension in a plate containing a LA culture medium, and culturing at 28 ℃ for 24 hours to obtain a LA plate for later use; a well (6 mm) was punched in the center of the plate containing PDA medium with a sterile punch, and 40. Mu.L of spore suspension of 8 pathogenic bacteria was inoculated, respectively, to obtain PDA plates. Then, the LA plate and the PDA plate were butted and sealed with a sealing film, and placed in an incubator at 28℃for 4 days, and each experimental group coated with 8 pathogenic spore suspensions was repeated three times.
Control group: the bacillus beijerinus (Bacillus velezensis) E2 bacterial suspension was replaced with 100 μl of sterile water, and the other procedures were unchanged.
The colony diameters of the control group and the experimental group are respectively observed and measured, and the inhibition rate of the volatile substances on common pathogenic bacteria is calculated according to the formula of antibacterial rate (%) =control group hypha diameter-experimental group hypha diameter/control group hypha diameter multiplied by 100, and the calculation results are shown in table 2.
TABLE 2 inhibition of different pathogenic bacteria by volatile substances produced by Bacillus bailii (Bacillus velezensis) E2
| Pathogenic bacteria | Inhibition ratio (%) |
| Aspergillus flavus A.flavus | 60.10±0.34 |
| Penicillium expansum P | 13.57±0.31 |
| Gibberella G.fujikuroi | 9.03±0.76 |
| Colletotrichum gloeosporioides C | 8.38±0.68 |
| P. italicum of Italian penicillium | 17.51±0.29 |
| Aspergillus ochraceus A.ochraceus | 11.52±0.97 |
| Wei Simei Pediospora canicola P.vismiae | 22.94±0.30 |
| Penicillium digitatum P | 29.36±0.68 |
Note that: results are expressed as mean ± standard deviation.
Table 2 shows the inhibitory effects of the volatile substances produced by Bacillus bailii (Bacillus velezensis) E2 on different pathogens, and it can be seen from the table that the Volatile Organic Compounds (VOCs) produced by Bacillus bailii (Bacillus velezensis) E2 have different inhibitory effects on different pathogens. Under the treatment, the diameters of the 8 pathogenic bacteria hyphae are reduced to different degrees, and compared with a control group, the bacterial strain hyphae of an experimental group are sparse and disordered in edges, and the pathogenic bacteria spore generation amount is extremely small. The volatile substances produced by bacillus belicus (Bacillus velezensis) E2 have poor inhibition effect on G.fujikuroi and C.gloeosporioides, and the inhibition rate is lower than 10%. Besides the two pathogenic bacteria, the volatile substances generated by bacillus beijerinus (Bacillus velezensis) E2 have strong inhibition effect on the other 6 moulds, and especially the inhibition rate of A.flavus is as high as 60.10 percent.
Example 5: investigation of stress-resistant Properties of Bacillus bailii (Bacillus velezensis) E2
In this example, stress resistance of bacillus beijerinckii (Bacillus velezensis) E2 was examined by cold stress, heat stress, osmotic stress and acid stress, and specific examination steps are as follows:
(1) Effect of cold stress on bacillus beijerinckii (Bacillus velezensis) E2 survival:
sub-packaging Bacillus bailii suspension into centrifuge tubes, respectively treating at 20deg.C, 4deg.C, 0deg.C and-20deg.C for 1 hr, balancing in water bath at 28deg.C for 10min, and measuring viable count of antagonistic bacteria before and after cold treatment by dilution coating, and calculating bacterial survival rate.
FIG. 7 is a graph showing the tolerance of Bacillus belicus (Bacillus velezensis) E2 to cold stress, and it can be seen from the graph that the survival rate of Bacillus belicus (Bacillus velezensis) E2 decreases with decrease in temperature. The survival rate at 20 ℃ is 99.79%, and the survival rate at 0 ℃ is 53.75%, which shows that bacillus beijerinckii (Bacillus velezensis) E2 has a certain tolerance to cold.
(2) Effect of heat stress on bacillus beijerinckii (Bacillus velezensis) E2 survival:
the bacillus bailii suspension is respectively treated by water bath at 30 ℃,40 ℃, 50 ℃ and 60 ℃ for 30min, and is diluted and coated after being balanced at room temperature for 5min, and the bacterial survival rate is calculated.
FIG. 8 is a graph of the heat stress tolerance of Bacillus belicus (Bacillus velezensis) E2, showing that the higher the temperature, the lower the survival rate of Bacillus belicus (Bacillus velezensis) E2, and the higher the survival rate at 40℃is kept at 76.33%. When the temperature is raised to 50 ℃ or higher, the survival rate of bacillus beleiensis (Bacillus velezensis) E2 is low. This indicates that bacillus beijerinus (Bacillus velezensis) E2 has a certain tolerance to high temperatures.
(3) Influence of osmotic stress on E2 survival of Bacillus bailii (Bacillus velezensis):
LB media with NaCl contents of 4%, 6%, 8% and 10% were prepared, respectively. Inoculating Bacillus bailii (Bacillus velezensis) E2 bacterial suspension with an inoculum size of 2%, culturing at 28 ℃ and 180r/min for 24 hours, diluting and coating, and calculating the survival rate of the Bacillus bailii.
FIG. 9 is a graph showing the tolerance of Bacillus belicus (Bacillus velezensis) E2 to osmotic stress, and it can be seen from the graph that the survival rate of Bacillus belicus (Bacillus velezensis) E2 gradually decreases with increasing NaCl concentration. The NaCl concentration is increased to 8%, the survival rate is still up to 74.14%, and the bacillus beijerinus (Bacillus velezensis) E2 has strong tolerance to osmotic stress.
(4) Effect of acid on bacillus behenii (Bacillus velezensis) E2 survival:
the culture medium was prepared using 0.2mol/L disodium hydrogen phosphate and 0.1mol/L citric acid, the final system of the culture medium was 20mL, 8.82mL, 10.30mL, 12.63mL, 13.85mL of disodium hydrogen phosphate solution was added, 11.18mL, 9.7mL, 7.37mL, 6.15mL of citric acid solution was added to give pH values of 4.5, 5, 6 and 6.5, respectively, bacillus berensis suspension was inoculated into the above culture medium in an inoculum size of 2%, cultured at 28℃for 24 hours at 180r/min, diluted and spread, and bacterial viability was calculated.
FIG. 10 is a graph showing the acid stress tolerance of Bacillus belicus (Bacillus velezensis) E2. It can be seen from the graph that the survival rate of Bacillus belicus (Bacillus velezensis) E2 is slowly reduced with the reduction of pH after the culture of the Bacillus belicus (Bacillus velezensis) E2 in an acidic medium for 24 hours, but the survival rate is as high as 77.21% when the pH is 4.5, which shows that the Bacillus belicus (Bacillus velezensis) E2 has good acid tolerance.
In conclusion, bacillus beijerinckii (Bacillus velezensis) E2 provided by the invention has strong low temperature resistance, high temperature resistance, acid resistance and osmotic pressure stress resistance, can resist the granary environment, and is suitable for further development and research.
Example 6: preparation of Bacillus bailii (Bacillus velezensis) E2 active freeze-dried powder
In this embodiment, bacillus bailii (Bacillus velezensis) E2 active lyophilized powder is taken as an example, and a preparation method of a microbial agent is provided, and the specific preparation process is as follows:
(1) Activation of the strain: and (3) picking bacillus belicus (Bacillus velezensis) E2 preserved in the inclined plane, streaking and inoculating in a LA culture medium, and culturing for 2-3 days at the constant temperature of 28 ℃.
(2) Bacillus bailii (Bacillus velezensis) E2 enrichment culture: inoculating activated bacillus belicus (Bacillus velezensis) E2 into an LB culture medium, shaking and culturing for 18-24 hours at 28 ℃ and 180r/min to obtain seed liquid, then inoculating bacillus belicus (Bacillus velezensis) E2 seed liquid into an LA culture medium according to an inoculum size of 2% (v/v), and culturing for 16 hours at 28 ℃ and 180r/min to obtain enriched bacillus belicus (Bacillus velezensis) E2 culture liquid.
(3) Preparation of bacterial precipitation: taking cultured bacillus bailii (Bacillus velezensis) E2 culture solution, centrifuging for 10min at 4 ℃ and 4000r/min, and pouring out supernatant to obtain bacterial precipitate for later use.
Regarding the selection of centrifugation parameters:
before centrifugation, the number of E2 cells of bacillus beliensis (Bacillus velezensis) in the bacterial liquid is calculated by adopting a dilution coating method, and during centrifugation, the centrifugal parameters are set to 3000, 4000 and 5000 rpm for 10, 15 and 20 minutes respectively. Centrifuging, removing supernatant, resuspending thallus precipitate with normal saline equal to the removed supernatant, gradient diluting to bacterial number of 30-300 per 100 μl, spreading on plate containing LA culture medium, and measuring viable bacteria number. The optimum centrifugation parameters were obtained by calculating the centrifugation yield according to the formula (centrifugation yield=number of E2 cells of Bacillus bailii (Bacillus velezensis) before centrifugation/number of E2 cells of Bacillus bailii (Bacillus velezensis) after centrifugation×100%) from the number of viable bacteria before and after centrifugation.
As a result, it was found that the optimal centrifugation parameters of Bacillus bailii (Bacillus velezensis) E2 bacterial suspension were 4000r/min and 10min, and the centrifugation yield was 93.91%, so that 4000r/min was used for centrifugation for 10min during the preparation of bacterial pellet.
(4) Adding a protective agent and sub-packaging: preparing bacillus beijerinus (Bacillus velezensis) E2 freeze-drying protective agent solution according to a formula, wherein the concentration of each protective agent in the protective agent solution is as follows: maltose 6.53g/100mL, skimmed milk powder 8.40g/100mL, sodium glutamate 8.34g/100mL, and water in balance. Re-suspending the bacterial precipitate with the protective agent solution with the same volume as the supernatant poured in the step (3), mixing uniformly by vortex, balancing for 30min at 28 ℃ under 180r/min, and then sub-packaging in 180mm culture dishes.
(5) Pre-freezing: sealing with fresh-keeping film after packaging, and pre-freezing in refrigerator at-80deg.C for 3 hr.
(6) Vacuum freeze drying: and taking out the sample after 3 hours, quickly punching a hole in the preservative film, and putting the preservative film on a sample table of a freeze dryer, wherein the freeze drying time is 36 hours.
(7) Sealing and preserving: after freeze drying, taking out the sample, grinding the sample into powder, and filling the powder into a penicillin bottle for sealing and preserving to obtain bacillus beijerinus (Bacillus velezensis) E2 active freeze-dried powder.
Example 7: component optimization of bacillus bailii (Bacillus velezensis) E2 active freeze-dried powder
In the embodiment, the bacillus beijerinckii (Bacillus velezensis) E2 active freeze-dried powder components and contents are optimized through a single factor test and a response surface test, and the specific optimization steps are as follows:
(1) Single factor test
In this example, the protectant in 8 was selected to prepare protectant solutions having different mass concentrations, and the types and concentrations of protectant are shown in Table 3. Bacillus bailii (Bacillus velezensis) E2 was resuspended by adding different kinds and concentrations of protective agents according to Table 3, vortexing and balancing, and then the viable count of Bacillus bailii (Bacillus velezensis) E2 was measured. After pre-freezing, freeze-drying is performed, after freeze-drying is finished, an equal volume of physiological saline is added, after activation, dilution and coating are performed, the viable count after freeze-drying is measured, the survival rate (survival rate=viable count before freeze-drying/viable count before freeze-drying×100%) of bacillus bailii (Bacillus velezensis) E2 is calculated, and the influence of different types and different concentrations of protective agents on the freeze-drying survival rate of bacillus bailii (Bacillus velezensis) E2 is shown in table 4.
TABLE 1 type and concentration of lyoprotectant
TABLE 4 influence of different protectants on the lyophilization survival of Bacillus bailii (Bacillus velezensis) E2
As can be seen from Table 4, maltose, sodium glutamate and skimmed milk powder are the 3 protective agents with the best protective effect on Bacillus bailii (Bacillus velezensis) E2.
(2) Response surface test:
according to the single factor test results, 3 kinds of protective agents with the best protective effect on bacillus beijerinckii (Bacillus velezensis) E2 are selected, namely maltose, sodium glutamate and skimmed milk powder. The freeze-drying survival rate of bacillus belicus (Bacillus velezensis) E2 is taken as a response value, the optimization interval of the concentration of the 3 protective agents is selected, design-Expert 13 is used for carrying out response surface test Design of 3 factor 3 level, the Design result is shown in table 5, 19 test points are added in total, analysis processing is carried out on the test result, a binary regression equation is obtained, and the optimal proportion of the protective agents is determined. Response surface test results and analyses are shown in table 6.
TABLE 5 response surface test design factor level Table
TABLE 6 response surface test design and test results
Analysis of table 6 using Design-Expert software, analysis of variance and significance test results are shown in table 7, and multiple regression fitting was performed on the results to obtain multiple regression equations:
Y＝85.62-2.02A+9.41B+0.80C-2.76AB+2.25AC+3.77BC-9.70A 2 -11.00B 2- 9.70C2
in the equation, Y represents the freeze-drying survival rate and A, B, C represents the concentration of maltose, skimmed milk powder and sodium glutamate respectively.
TABLE 7 response surface model analysis of variance
| Source | Sum of squares | Degree of freedom | Mean square | F value | p value | Significance of the invention |
| Model | 4757.13 | 9 | 528.57 | 25.96 | <0.0001 | Is remarkable in |
| A | 55.82 | 1 | 55.82 | 2.74 | 0.1321 | |
| B | 1208.44 | 1 | 1208.44 | 59.36 | <0.0001 | |
| C | 8.81 | 1 | 8.81 | 0.4325 | 0.5272 | |
| AB | 60.85 | 1 | 60.85 | 2.99 | 0.1179 | |
| AC | 40.41 | 1 | 40.41 | 1.99 | 0.1925 | |
| BC | 113.45 | 1 | 113.45 | 5.57 | 0.0426 | |
| A 2 | 1287.36 | 1 | 1287.36 | 63.23 | <0.0001 | |
| B 2 | 1651.37 | 1 | 1651.37 | 81.11 | <0.0001 | |
| C 2 | 1284.89 | 1 | 1284.89 | 63.11 | <0.0001 | |
| Residual error | 183.23 | 9 | 20.36 | |||
| Missing items | 143.33 | 5 | 28.67 | 2.87 | 0.1642 | Is not remarkable |
| Pure error | 39.9 | 4 | 9.98 | |||
| Total dispersion | 4940.36 | 18 | ||||
| Coefficients of | R 2 ＝0.9629 | R Adj 2 ＝0.9258 |
As is clear from Table 7, the regression model has a very high significance (p<0.0001 While the disfigurement term is not significant (p)>0.05 And) indicate that regression analysis is effective. In addition, in the case of the optical fiber,the adjusted determination coefficient (R adj 2 = 0.9258) is close to R 2 The value also confirms the high significance of the model. The response surface analysis results are reliable. A is that 2 、B 2 、C 2 The high F-number of (c) indicates that the variable and the response value Y are not simply linear and are also affected by the interaction between the factors.
In order to better understand the interactive effect of three variables on lyophilization survival, a two-dimensional contour map and a three-dimensional response surface map were generated. As can be seen from fig. 11-13, the two-dimensional contour plot appears to be an ellipse, which indicates that there is an interaction between the interaction terms AB, BC, AC; the curve paraboloids of the three-dimensional response curve graph are all curved surfaces, which indicates that the maximum value exists in the 3 curved surfaces. Analysis is carried out by using Design-Expert software, the optimal formula obtained by prediction is 6.53g/100mL of maltose, 8.40g/100mL of skimmed milk powder and 8.34g/100mL of sodium glutamate, and the highest freeze-drying survival rate is predicted to be 88.02%.
Through verification of the formula, the average freeze-drying survival rate of bacillus beijerinus (Bacillus velezensis) E2 is close to a predicted value and is 91.64%, and the effectiveness of the model is verified.
Example 8: control effect of bacillus bailii (Bacillus velezensis) E2 active freeze-dried powder on pollution of rice aspergillus flavus (Aspergillus flavus)
In this example, the total number of moulds, fatty acid value and AFB1 content were measured by the method described in example 2, and the effect of Bacillus belicus (Bacillus velezensis) E2 active lyophilized powder on contamination of Aspergillus oryzae (Aspergillus flavus) was examined.
Bacillus bailii (Bacillus velezensis) E2 active lyophilized powder is prepared by adjusting to 1×10 with sterile water 9 cfu/mL, other steps are the same as in example 2.
As can be seen from FIG. 3, the Aspergillus flavus content in the rice treated with Bacillus bailii (Bacillus velezensis) E2 active lyophilized powder was 2.75X10 5 cfu/g, and the biocontrol effect of the freeze-dried powder is superior to that of fresh bacterial liquid, because the protective agent in the freeze-dried powder keeps the bailey budsBacillus (Bacillus velezensis) E2 is active and provides a nutrient for it. As can be seen from FIG. 4, the content of the fatty acid value of the freeze-dried powder after treatment is 20.98mg/100g, which is lower than that of the fresh bacterial liquid, and is suitable for storing rice, so that the active freeze-dried powder can effectively prolong the storage life of the rice. As can also be seen in fig. 5, the content of AFB1 in the rice treated with the lyophilized powder was the lowest, which suggests that bacillus belicus (Bacillus velezensis) E2 active lyophilized powder can prevent and control mildewing of stored rice.
Example 9: change in Activity during storage of Bacillus bailii (Bacillus velezensis) E2 active lyophilized powder
In this example, lyophilized E2-active powder of Bacillus belicus (Bacillus velezensis) was stored at 25, 4 and-20deg.C for 14 days, 28 days, 56 days, 64 days and 70 days, respectively, to examine changes in activity during storage of lyophilized E2-active powder of Bacillus belicus (Bacillus velezensis).
0.1g of freeze-dried sample is weighed, 1mL of physiological saline is added for full dissolution, water bath is carried out for 30min at 28 ℃, the bacteria amount per 100 mu L is 30-300, then the solution is coated, the initial survival rate of bacillus bailii (Bacillus velezensis) E2 can be obtained according to the dilution gradient and the times and the colony number on a flat plate, bacillus bailii (Bacillus velezensis) E2 active freeze-dried powder is respectively stored at 25, 4 and 20 ℃, and the survival rate of bacillus bailii (Bacillus velezensis) E2 under different storage conditions is respectively measured at 14 days, 28 days, 56 days, 64 days and 70 days, and the result is shown in figure 14.
As can be seen from FIG. 8, the survival rate of Bacillus belicus (Bacillus velezensis) E2 was decreased with the prolongation of the storage period under the storage conditions of-20, 4 and 25 ℃. The survival rate is reduced slowly within 21 days before storage; after 35 days of storage at-20 ℃, bacillus beijerinus (Bacillus velezensis) E2 survival rates are still as high as 95.15%. The survival rate of the strain E2 stored in the environment of-20 ℃ is highest in any storage period, and after 70 days of storage, the survival rate of the bacillus berryis (Bacillus velezensis) E2 is still up to 56.35%.
Therefore, the optimal storage temperature of the bacillus beijerinus (Bacillus velezensis) E2 active freeze-dried powder is-20 ℃, so that the high activity can be maintained for a long time, and the bacillus beijerinus (Bacillus velezensis) E2 active freeze-dried powder is worthy of further research and popularization.
In conclusion, bacillus bailii (Bacillus velezensis) E2 or bacillus bailii (Bacillus velezensis) E2 active freeze-dried powder provided by the invention has a good biological control effect, can effectively reduce the use of chemical bactericides in the rice storage process, provides a certain guiding significance for biological control of rice mildew, and has a positive effect on guaranteeing the rice quality.
The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.
Claims (9)
1. The bacillus beleiensis is marked as bacillus beleiensis (Bacillus velezensis) E2, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M2022579, the preservation time is 2022, 5 months and 10 days, and the preservation address is Chinese Wuhan.
2. A microbial preparation comprising bacillus bailii (Bacillus velezensis) E2 or a bacterial suspension or a culture or a broth or a fermentation broth thereof according to claim 1.
3. The microbial formulation of claim 2, wherein the microbial formulation comprises bacillus belicus (Bacillus velezensis) E2 active lyophilized powder;
the active freeze-dried powder comprises bacillus bailii (Bacillus velezensis) E2 bacterial precipitation and a protective agent solution.
4. A microbial preparation according to claim 3, wherein the amount of the protectant solution added is equivalent to the volume of supernatant after centrifugation of the bacillus belicus (Bacillus velezensis) E2 bacterial liquid; the components of the protective agent comprise maltose, skimmed milk powder and sodium glutamate.
5. The microbial preparation according to claim 4, wherein the protective agent comprises 4-16 g/100mL of maltose, 4-16 g/100mL of skimmed milk powder and 2-14 g/100mL of sodium glutamate, and the other is solvent water.
6. Use of bacillus belgium according to claim 1 and/or a microbial preparation according to any one of claims 2-5 in any one of the following:
(1) Application in preventing and controlling aspergillus flavus, gibberella, colletotrichum gloeosporioides, wei Simei Paecilomyces varioti, penicillium italicum, aspergillus ochraceus or penicillium digitatum;
(2) Preventing and controlling mildew of grains, feeds or other raw materials;
(3) Eliminating or inhibiting aflatoxin B1 production in grains, feeds or other raw materials;
(4) Controlling the fatty acid value in grains, feeds or other raw materials;
(5) Use to inhibit low temperature, high temperature, acid and osmotic stress;
(6) Improving the quality of grains, feeds or other raw materials.
7. The use according to claim 6, wherein the mould comprises aspergillus flavus mould and the foodstuff comprises rice, maize or peanut.
8. A method for controlling mildew of stored rice, characterized in that the bacillus belicus according to claim 1 and/or the microbial preparation according to any one of claims 2-5 are fully and uniformly mixed with grains for storage.
9. A method for controlling mildew in stored rice according to claim 9, wherein the amount of bacteria per gram of rice during storage is 1 x 10 -6 ～10 -8 cfu。
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| CN117660266B (en) * | 2024-01-29 | 2024-04-09 | 南京农业大学三亚研究院 | Bacillus bailii, antibacterial lipopeptide, biological control microbial agent and application thereof |
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