CN114921368A - Bacillus mojavensis YL-RY0310 and culture method and application thereof - Google Patents
Bacillus mojavensis YL-RY0310 and culture method and application thereof Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- 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
-
- 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
-
- 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
Abstract
The invention provides a newly separated Bacillus mojavensis (Bacillus mojavensis) YL-RY0310, and a culture method and application thereof, wherein the Bacillus mojavensis (Bacillus mojavensis) YL-RY0310 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 2022, 4 and 6 days, and the preservation number is CGMCC NO. 24646.
Description
Technical Field
The invention relates to the technical field of fruit and vegetable mycotoxin prevention and control, and particularly relates to bacillus mojavensis YL-RY0310 and a culture method and application thereof.
Background
China is the largest apple producing country in the world, the yield accounts for more than 50% of the apple yield in the world, is the largest fruit variety except melons, and has an important position in the fruit industry in China. However, penicillium expansum (p.expandasu) causes serious penicillium expansum disease in the picking, storage and transportation processes of apples, and a great amount of Patulin (PAT, C) which has potential threats to human health are accumulated in fruits while huge economic losses are caused 7 H 6 O 4 )。
PAT was reported as a genotoxic compound by the World Health Organization (WHO) as early as 2005, and both the WHO and the U.S. food and drug administration (USAFDA) limited the detection content of PAT in food to 50 μ g/kg. Since pregnant women and infants belong to the high risk group exposed to PAT, this standard is raised to 10 μ g/kg in Japan, and the European Commission has limited the maximum daily allowable PAT intake to 0.4 μ g/kg. Although the apple planting area and the apple yield are at the top of the world all the year round in China, the export quantity is strictly limited due to the toxin detection rate. Studies have shown that the presence of patulin is detected in 8% of the 25 apple juice samples; the contamination rate of patulin in apple juice samples in different processing seasons of Shaanxi reaches 96.7-100%, and the level is about 6.30-8.90 mu g/kg; in recent two years, researchers have investigated 137 kinds of fruit products sold and consumed in the market of China, and the contamination rate of patulin is 30.7%, and the concentration range of the patulin is from 10 to 276.9 mug/kg. Therefore, the prevention and control of the contamination of the patulin to the fruits and vegetables become an urgent problem to be solved.
Chemical bactericides are always important methods for preventing and controlling pathogenic bacteria from infecting fruits, but as intensive research shows, most of the chemical bactericides have potential cancerogenic risks, and the long-term use of the chemical bactericides can also aggravate environmental pollution and cause pathogenic bacteria to generate drug resistance. Relatively speaking, the biological bactericide which is safe, environment-friendly and efficient is more scientific, reasonable and urgent.
Disclosure of Invention
In view of the above, the main object of the present invention is to provide a strain of bacillus mojavensis YL-RY0310 capable of inhibiting the growth of penicillium expansum (p.expansum) and efficiently inhibiting the production of patulin, and a culture method and applications thereof, which have the effects of inhibiting the growth of penicillium expansum in post-harvest apples and inhibiting the production of patulin in vivo.
The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme. According to the invention, the Bacillus mojavensis (Bacillus mojavensis) YL-RY0310 has been preserved in China general microbiological culture Collection center (CGMCC) at 4-6.2022 with the preservation number of CGMCC NO. 24646.
The Bacillus mojavensis (Bacillus mojavensis) YL-RY0310 is a biocontrol strain YL-RY0310 which is firstly separated from soil of a jujube garden with 244 groups of Xinjiang Hotan Jade, has good inhibition effect on expanding penicillium and patulin in apples, and is identified as the Bacillus mojavensis through 16S rRNA.
The purpose of the invention and the technical problem to be solved can be realized by adopting the following technical scheme. The culture method of the Bacillus mojavensis YL-RY0310 provided by the invention comprises the following steps:
inoculating the strain YL-RY0310 to NA solid culture medium by an inoculating needle for activation, culturing for 24-48h in an incubator at 25-40 ℃, picking out a single colony of the Bacillus mojavensis by the inoculating needle, transferring the single colony to NB liquid culture medium, culturing for 12-24h in a shaking incubator at 25-40 ℃ at the speed of 125-150r/min, absorbing culture solution accounting for 1-5% of the total volume of the liquid culture medium, transferring to fresh NB liquid culture medium, culturing for 24-72h at 25-40 ℃ at the speed of 125-150r/min to obtain YL-RY0310 fermentation broth, wherein the viable count reaches (1-10) multiplied by 10, and the viable count reaches (1-10) 7 CFU/mL。
The purpose of the invention and the technical problem to be solved can also be realized by adopting the following technical scheme. The application of the Bacillus mojavensis YL-RY0310 in prevention and treatment of fruit and vegetable patulin pollution is provided by the invention.
Preferably, the application of the bacillus mojavensis YL-RY0310 in preventing and treating fruit and vegetable patulin pollution comprises the following steps:
the concentration is (1-10) × 10 7 CFU/mL of fermentation liquid of the Bacillus mojavensis YL-RY0310 is directly sprayed on the surface of the biological sample.
The purpose of the invention and the technical problem to be solved can also be realized by adopting the following technical scheme. The invention provides a biocontrol preparation containing the bacillus mojavensis YL-RY 0310.
Preferably, the biocontrol agent containing the bacillus mojavensis YL-RY0310 is in the form of a liquid, a powder spray, a dry wettable powder or a dry wettable granule.
Preferably, the biocontrol agent contains the above-mentioned Bacillus mojavensis YL-RY0310, wherein the biocontrol agent contains the above-mentioned YL-RY0310 fermentation broth.
Preferably, the biocontrol agent containing the above-mentioned bacillus mojavensis YL-RY0310, wherein the biocontrol agent is a liquid.
Preferably, the biocontrol agent contains the above-mentioned Bacillus mojavensis YL-RY0310, wherein the biocontrol agent contains the above-mentioned YL-RY0310 fermentation broth.
Preferably, the biocontrol preparation containing the bacillus mojavensis YL-RY0310 is characterized in that the final concentration of the bacillus mojavensis YL-RY0310 in the biocontrol preparation is (1-10) x 10 7 CFU/mL。
The purpose of the invention and the technical problem to be solved can also be realized by adopting the following technical scheme. The invention provides an application of a biocontrol agent in preventing and treating fruit and vegetable patulin pollution.
Preferably, the biocontrol agent is used for preventing and controlling fruit and vegetable patulin pollution, wherein the fruit and vegetable patulin pollution prevention and control comprises at least one of inhibition of growth of penicillium expansum or inhibition of patulin synthesis.
Preferably, the biocontrol agent is applied to the prevention and treatment of fruit and vegetable patulin pollution, wherein the application comprises the following steps:
the biocontrol preparation is uniformly sprayed on the surface of a biological sample to inhibit penicillium expansum and patulin.
By means of the technical scheme, the Bacillus mojavensis YL-RY0310 and the culture method and application thereof have at least the following advantages:
according to the invention, a biocontrol bacterium YL-RY0310 with good inhibition effect on expanded penicillium and patulin in apples is separated from soil for the first time, and is identified as the M.haica through 16S rRNA, the thallus of the M.haica YL-RY0310 shows good inhibition effect on the growth of the expanded penicillium in a plate confrontation test, the inhibition rate is 62.57%, and meanwhile, the synthesis inhibition rate of patulin PAT on a confronting plate is 99.81%, so that the biocontrol bacterium YL-RY0310 can be applied to the control of the expanded penicillium and the patulin PAT.
When the bacillus mojavensis YL-RY0310 and the expanded penicillium are cultured in a liquid culture medium, the growth of pathogenic bacteria can be obviously inhibited, the inhibition rate is higher than 95% visually, and the synthesis inhibition rate of patulin reaches 71.66%.
The bacillus mojavensis YL-RY0310 can effectively inhibit the growth of expanded penicillium on apples under the condition of normal-temperature storage, the inhibition rate reaches 64.71%, the prevention effect of the bacillus mojavensis YL-RY0310 on patulin PAT pollution in apples can reach 58.01%, and the prevention and control effect is good, so that the bacillus mojavensis YL-RY0310 can be used for preventing and controlling the pollution of the patulin of apples.
The bacillus mojavensis YL-RY0310 is applied to prevention and control of extended penicillium pollution of apples, and can overcome a series of problems caused by the use of chemical pesticides, thereby being beneficial to pollution-free production of agricultural products and improving the product quality.
The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a detailed description of the preferred embodiments of the present invention.
Drawings
FIG. 1 is a diagram showing the bacteriostatic properties of Bacillus mojavensis YL-RY0310, YL-RY0304, YL-RY0309, YL-RA0201, YL-RA0204, YL-RA0208, YL-RA0210, and CK (extended Penicillium strain inoculated with no antagonistic bacteria) in a control group, which are obtained in example 1 of the present invention;
FIG. 2 is a colony morphology of Bacillus mojavensis YL-RY0310 according to example 1 of the present invention;
FIG. 3 shows the 16S rRNA gene development tree of Bacillus mojavensis YL-RY0310 according to example 1 of the present invention;
FIG. 4 is a graph showing the antagonistic action of Bacillus mojavensis YL-RY0310 on extended penicillium in liquid culture medium in example 2 (CK is control group: only liquid culture extended penicillium; test group is mixed solution of YL-RY0310 bacterial suspension and extended penicillium)
FIG. 5 is a graph showing the antagonistic action of Bacillus mojavensis YL-RY0310 on extended Penicillium Mali in example 3 of the present invention.
Detailed Description
To further illustrate the technical means and effects of the present invention for achieving the predetermined objects, the following detailed description will be given to the specific embodiments, structures, characteristics and effects of the bacillus mojavensis YL-RY0310 and its culturing method and application according to the present invention in combination with the preferred embodiments.
The invention provides a Bacillus mojavensis (Bacillus mojavensis) YL-RY0310 which has been preserved in China general microbiological culture Collection center (CGMCC for short, address: China academy of sciences microorganism research institute No. 3, No.1, West Lu, No.1, North Cheng, south of Beijing City, 4.6.2022, the preservation number is CGMCC NO.24646, and the Bacillus mojavensis (Bacillus mojavensis) is classified and named.
The above Bacillus mojavensis (Bacillus mojavensis) YL-RY0310 is a biocontrol strain YL-RY0310 which is firstly separated from the soil of a jujube garden with 244 groups of Ku Yu, Xinjiang and Tian, has good inhibition effect on Penicillium expansum (P.expansum) and Penicillium expansum elements in apples, is identified as Bacillus mohaiensis by 16S rRNA, shows good inhibition effect on growth of Penicillium expansum in a solid plate confrontation test, and has the inhibition rate of 62.57% and the inhibition rate of PAT of 99.81%. In liquid culture test, the strain also shows better inhibition effect on the growth of expanded penicillium, the inhibition rate is more than 95 percent visually, and the inhibition rate on patulin is 71.66 percent.
The invention also provides a culture method of the bacillus mojavensis YL-RY0310, which comprises the following steps:
inoculating strain YL-RY0310 to NA solid culture medium with inoculating needle, activating, culturing at 25-40 deg.C for 24-48h, picking single colony of Bacillus mojavensis with inoculating needle, transferring to NB liquid culture medium, culturing at 25-40 deg.C in shaking culture chamber at 125-150r/min for 12-24h, sucking culture solution 1-5% of total volume of liquid culture medium, transferring to fresh NB liquid culture medium, and culturing at 25-40 deg.CCulturing at 125-150r/min for 24-72h to obtain YL-RY0310 fermentation broth with viable count of (1-10) × 10 7 CFU/mL。
The invention also provides application of the bacillus mojavensis YL-RY0310 in prevention and treatment of patulin pollution in fruits and vegetables.
In some embodiments of the invention, wherein the applying may comprise the steps of:
the concentration is (1-10). times.10 7 CFU/mL fermentation liquid of the Bacillus mojavensis YL-RY0310 is directly sprayed on the surface of the biological sample, so as to prevent and treat patulin pollution of the biological sample. The spraying manner in this embodiment may be spraying by an automatic sprayer (the spraying rate may be 0.5L/min, for example), or spraying by a plant protection unmanned aerial vehicle (the spraying rate may be 0.43L/min (a single nozzle, taking water as an example), and is not limited specifically herein.
In some embodiments of the invention, wherein the applying may comprise the steps of:
inoculating the surface of the biological sample with the solution with the concentration of (1-10) x 10 7 CFU/mL fermentation liquid of Bacillus mojavensis YL-RY0310, so as to prevent and treat patulin pollution of biological samples. The amount of the inoculation may be, for example, 10. mu.L.
The invention also provides a biocontrol preparation containing the bacillus amyloliquefaciens YL-RY 0310.
In some embodiments of the invention, wherein the biocontrol formulation is in the form of a liquid, a dusting powder, a dry wettable powder or a dry wettable granule. In view of cost saving, the form of the biocontrol agent is preferably made in the form of a liquid.
In some embodiments of the present invention, wherein the biocontrol agent comprises the YL-RY0310 fermentation broth described above.
In some embodiments of the invention, wherein the final concentration of Bacillus mojavensis YL-RY0310 in the biocontrol formulation is (1-10). times.10 7 CFU/mL。
The invention also provides application of the biocontrol agent in preventing and treating fruit and vegetable patulin pollution.
In some embodiments of the present invention, wherein said controlling fruit and vegetable patulin contamination may comprise at least one of inhibiting the growth of penicillium expansum or inhibiting patulin synthesis, such as inhibiting patulin PAT synthesis.
In some embodiments of the invention, wherein the applying comprises the steps of:
the biological control agent is uniformly sprayed on the surface of a biological sample, such as the surface of the whole fruit sample, particularly the positions susceptible to expand penicillium, such as fruit stems, fruit bottoms and the like, so as to prevent and control patulin pollution of the biological sample. The spraying manner in this embodiment may be spraying by an automatic sprayer (the spraying rate may be 0.5L/min, for example), or spraying by a plant protection unmanned aerial vehicle (the spraying rate may be 0.43L/min (a single nozzle, taking water as an example), and is not limited specifically herein.
Unless otherwise specified, the following materials, reagents and the like are commercially available products well known to those skilled in the art; unless otherwise specified, all methods are well known in the art. Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
The present invention will be further described with reference to the following specific examples, which should not be construed as limiting the scope of the invention, but rather as providing those skilled in the art with certain insubstantial modifications and adaptations of the invention based on the teachings of the invention set forth herein.
Example 1 isolation, screening and identification of Bacillus mojavensis YL-RY0310
1. Strain isolation and selection
Separating antagonistic bacteria from soil of the Hotan field Quyu 244 round jujube garden: separating by quartering method, weighing 5g soil, adding 45mL sterile water, shake-culturing at 27 deg.C and 150r/min for 30min, and subjecting the obtained liquid to shaking culture at 10% -1 -10 -6 By gradient dilution of (2), pipetting 100. mu.L, 10. mu.L, respectively -1 -10 -6 The dilution of (6) was spread on NA medium and cultured in an incubator at 27 ℃ for 24 hours. Individual colonies on the plates were picked and streaked on NA medium.
1) Determination of hypha growth bacteriostasis rate
And screening the antagonistic ability by adopting a plate confronting method. Activating the separated and purified strains on an NA culture medium for 24 hours, then lightly scraping the surface of a flat plate by using an inoculating loop, inoculating the single colony to an NB culture medium, and performing liquid shake culture at the temperature of 28 ℃ and the speed of 130r/min for 24 hours to obtain a bacterial suspension. Preparing a spore suspension from 7-day activated Penicillium expansum (a strain is purchased from China agricultural microbial strain preservation management center and is numbered ACCC36188), dripping 10 mu L of stock solution into the center of an NA culture medium, and inoculating 10 mu L of activated antagonistic bacterium suspension for 24 hours at a position 2.5cm away from the center on the same plate; and (3) observing whether the growth of hyphae is inhibited or not by taking a plate which is not inoculated with the antagonistic bacteria and is only inoculated with the extended penicillium as a contrast, measuring the diameter of the extended penicillium on the 7 th day, and calculating the inhibition rate according to the radial growth diameter of the extended penicillium, wherein each strain is in triplicate. And selecting the strains with the highest bacteriostasis rate, and storing for later use. The inhibition was calculated according to the following formula, and the test results are shown in Table 1 and FIG. 1
The inhibition ratio (%) - (C1-C2)/C1X 100%
C1 radial growth diameter of extended Penicillium of control group
C2 radial growth diameter of extended Penicillium of Experimental group
Table 1: inhibition of Penicillium expansum on plates
As can be seen from the experimental results shown in fig. 1 and table 1, the growth inhibition rate of YL-RY0304 on the extended penicillium strain is about 37.45%, the growth inhibition rate of YL-RY0309 on the extended penicillium strain is about 60.60%, the growth inhibition rate of YL-RY0310 on the extended penicillium strain is about 62.57%, the growth inhibition rate of YL-RA0201 on the extended penicillium strain is about 42.75%, the growth inhibition rate of YL-RA0204 on the extended penicillium strain is about 57.82%, the growth inhibition rate of YL-RA0208 on the extended penicillium strain is about 53.94%, and the growth inhibition rate of YL-RA0210 on the extended penicillium strain is about 57.37%. The YL-RY0309, YL-RA0204, YL-RA0208, YL-RA0210 and Bacillus mojavensis YL-RY0310 all have the ability of inhibiting and expanding the growth of penicillium, and the inhibition ability of the Bacillus mojavensis YL-RY0310 is the best.
2) Determination of toxicity inhibiting Properties of plate hypha growth
Placing the plate culture medium with the bacteriostasis rate of more than 50 percent and the hyphae growing for 7 days into a mortar, adding about 75mL of liquid nitrogen, quickly grinding and crushing to 200 meshes, uniformly mixing the parallel groups, randomly sampling, and extracting and detecting the content of the patulin by referring to a method for measuring GB5009.185-2016 (standard food safety food) of patulin.
The conditions are as follows: an XSelect HSS T3 column (5 μm, 4.6X 250mm) was used, the mobile phase was water and acetonitrile (77:23, v/v), the flow rate was 1.0mL/min, and the elution was isocratic. The column temperature was 40 ℃ and the run time for each sample was 10min, with a chromatographic peak at 276 nm. And meanwhile, a standard curve is made according to the patulin standards with different concentrations, the content of the toxin in each experimental group is calculated according to the standard curve, and the inhibition rate is calculated. The toxin content and the inhibition rate are calculated according to the following formulas (1) and (2), and the test results are shown in table 2.
y＝56.828x-637.32 R 2 ＝0.9992 (1)
y: peak area x: concentration (ng/ml)
Toxin inhibition (%) (control group toxin content-experimental group toxin content)/control group toxin content x 100% (2)
Table 2: inhibition of patulin in plates
As can be seen from Table 2, compared with the CK in the control group, YL-RY0309, YL-RY0310, YL-RA0204, YL-RA0208 and YL-RA0210 all have obvious inhibition effects on patulin PAT synthesized by penicillium expansum, wherein the inhibition effect of Bacillus mojavensis YL-RY0310 is the best and reaches 99.81%.
3. Morphological characteristics
Bacterial colony of the bacterial strain YL-RY0310 on the NA culture medium is round and white, the bacterial colony is large, the surface is smooth and dry near the light, the middle is convex, the edge is irregular, the bacterial colony is opaque, and the bacterial colony is flat and easy to pick up. The diameter of the colony is 2-3mm, and the colony morphology is shown in FIG. 2. Gram-positive, rod-shaped, spore-forming.
4. Physiological and biochemical characteristics
The strain YL-RY0310 is suitable for growth on Nutrient Agar (NA) and Nutrient Broth (NB) culture medium, and has a suitable growth temperature range of 4-45 deg.C, an optimum temperature of 37 deg.C, a suitable growth pH value of 3-9, and an optimum pH value of 7.3.
5. Characteristics of molecular biology
Using a bacterial DNA extraction kit, extracting DNA of antagonistic bacteria YL-RY0310 according to the instructions, analyzing adjacent species with BLAST on NCBI, and constructing phylogenetic trees using MEGA 11.0, as shown in fig. 3, homology of antagonistic bacteria YL-RY0310 with Bacillus mojavensis partial 16S rRNA gene strain ifo 15718 reaches 96%, and combining morphological characteristics, physiological and biochemical characteristics of the strain and the results of the BLAST system analysis, the strain YL-RY0310 can be finally identified as Bacillus mojavensis (Bacillus mojavensis). The 16S rRNA sequence of the strain YL-RY0310 is shown in SEQ ID NO. 1.
Example 2 Effect test of Bacillus mojavensis YL-RY0310 on expansion of Penicillium and its toxins in liquid Medium
Sucking 6mL and 6mL of 1X 10 bacterial suspension of 48h activated Bacillus mojavensis YL-RY0310 5 CFU/mL Penicillium expansum spore suspension was mixed and added to different liquid media containing 6mL Potato Dextrose Broth (PDB) (see Table 3 for the composition of control CK and test CK), cultured on a shaker at 28 ℃ for 5 days at 120r/min, the growth of the pathogens was observed, and the patulin PAT in the mixed culture was extracted and detected by HPLC, 3 per group in parallel, according to the following formulaThe inhibition rate was calculated by the following formula, and the test results are shown in fig. 4 and table 4.
Toxin inhibition (%) - (control peak area-experimental peak area)/control peak area × 100%
Table 3: test grouping of Bacillus mojavensis YL-RY0310 on liquid media
Table 4: inhibition rate of antagonistic test of Bacillus mojavensis YL-RY0310 on liquid culture medium on PAT
As shown in figure 4, the Bacillus mojavensis YL-RY0310 has obvious inhibition effect on the growth of liquid-cultured extended penicillium, and the inhibition effect can only be visually observed to exceed 95% through phenomena because the inhibition rate cannot be directly calculated by liquid.
As shown in Table 4, the amount of patulin PAT in the shake culture liquid on day 5 was measured by HPLC, and the inhibition rate of YL-RY0310 bacterial suspension in the experimental group on patulin PAT was 71.66% as compared with the CK in the control group.
Example 3 application of Bacillus mojavensis YL-RY0310 in prevention and treatment of fruit and vegetable patulin pollution
After wiping and sterilizing the surfaces of picked honey-crisp apples (harvested from the co-created forest fruit in Congchun county, Kyoto, Ili) by using 75% alcohol and sterile water in sequence, 10 mu L of YL-RY0310 bacterial suspension activated for 72h (named as experimental group) is inoculated on the equatorial surface of the apples by using a sterile inoculating needle for pricking with a depth of 5 mm. Control CK was inoculated with 10. mu.L of sterile water as a control. After 30min, the control group and the experimental group are respectively divided into 2 × 10 5 CFU/mL spore suspension was re-inoculated with 10. mu.L at apple wounds, and each group was replicated four times. Culturing all apples in a ventilated and lightless room temperature environment for 8 days, measuring the diameters of the scabs, extracting and detecting the PAT content of each group of patulin by using an HPLC method, and calculating the bacteriostasis rateAnd antitoxic effect. The inhibition ratios were calculated according to the following formulas (1) and (2), and the test results are shown in tables 5 and 6 and fig. 5.
Fungal growth inhibition (%) - (C1-C2)/C1X 100% (1)
Toxin inhibition (%) (control group toxin content-experimental group toxin content)/control group toxin content x 100% (2)
C1 radial growth diameter of extended penicillium for control group
C2 radial growth diameter of extended penicillium in experimental group
Table 5: influence of Bacillus mojavensis YL-RY0310 on growth of penicillium expansum with different concentrations on apples
As can be seen from the experimental results in Table 5, the inhibition rate of the Bacillus mojavensis YL-RY0310 on the extended penicillium notatum on the apples is 64.34%, which indicates that the YL-RY0310 has good inhibition effect on the growth of the extended penicillium notatum in the apples.
Table 6: inhibition rate of Bacillus mojavensis YL-RY0310 on synthesis of PAT by penicillium expansum with different concentrations on apples
As shown in Table 6, the content of patulin PAT in apple at day 8 was measured by HPLC when the spore concentration of Penicillium expansum was 2X 10 5 When CFU/mL is adopted, the inhibition rate of YL-RY0310 on PAT is 58.01%, and the inhibition effect is better.
In the description of the present invention, numerous specific details are set forth. It is understood, however, that embodiments of the invention may be practiced without these specific details. In some embodiments, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention can be made, and the same should be considered as the disclosure of the present invention as long as the idea of the present invention is not violated.
While the invention has been described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> Sinkiang academy of agricultural sciences
<120> Bacillus mojavensis YL-RY0310 and culture method and application thereof
<130>
<160>
<170> PatentIn version 3.5
<210> 1
<211> 1424
<212> DNA
<213> Bacillus mojavensis (Bacillus mojavensis)
<400> 1
tgtgtcacct tcggcggctg gctccataaa ggttacctca ccgacttcgg gtgttacaaa 60
ctctcgtggt gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct 120
gatccgcgat tactagcgat tccagcttca cgcagtcgag ttgcagactg cgatccgaac 180
tgagaacaga tttgtgggat tggcttaacc tcgcggtttc gctgcccttt gttctgtcca 240
ttgtagcacg tgtgtagccc aggtcataag gggcatgatg atttgacgtc atccccacct 300
tcctccggtt tgtcaccggc agtcacctta gagtgcccaa ctgaatgctg gcaactaaga 360
tcaagggttg cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa 420
ccatgcacca cctgtcactc tgcccccgaa ggggacgtcc tatctctagg attgtcagag 480
gatgtcaaga cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc 540
ttgtgcgggc ccccgtcaat tcctttgagt ttcagtcttg cgaccgtact ccccaggcgg 600
agtgcttaat gcgttagctg cagcactaag gggcggaaac cccctaacac ttagcactca 660
tcgtttacgg cgtggactac cagggtatct aatcctgttc gctccccacg ctttcgctcc 720
tcagcgtcag ttacagacca gagagtcgcc ttcgccactg gtgttcctcc acatctctac 780
gcatttcacc gctacacgtg gaattccact ctcctcttct gcactcaagt tccccagttt 840
ccaatgaccc tccccggttg agccgggggc tttcacatca gacttaagga accgcctgcg 900
agccctttac gcccaataat tccggacaac gcttgccacc tacgtattac cgcggctgct 960
ggcacgtagt tagccgtggc tttctggtta ggtaccgtca aggtaccgcc ctattcgaac 1020
ggtacttgtt cttccctaac aacagagctt tacgatccga aaaccttcat cactcacgcg 1080
gcgttgctcc gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg 1140
agtctgggcc gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc 1200
gttgccttgg tgagccatta cctcaccaac tagctaatgc gccgcgggtc catctgtaag 1260
tggtagccga agccaccttt tatgtttgaa ccatgcggtt caaacaagca tccggtatta 1320
gccccggttt cccggagtta tcccagtctt acaggcaggt tacccacgtg ttactcaccc 1380
gtccgccgct aacatcaggg agcaagctcc catctgtccg ctcgacttgc atgtatagca 1420
cgcc 1424
Claims (10)
1. A strain of Bacillus mojavensis (Bacillus mojavensis) YL-RY0310 is preserved in China general microbiological culture Collection center (CGMCC) at 4 months and 6 days 2022, with the preservation number of CGMCC NO. 24646.
2. The method of culturing Bacillus mojavensis YL-RY0310, according to claim 1, comprising the steps of:
inoculating the strain YL-RY0310 to NA solid culture medium with an inoculating needle for activation, culturing for 24-48h in an incubator at 25-40 ℃, picking out a single colony of the Bacillus mojavensis with the inoculating needle, transferring to NB liquid culture medium, culturing for 12-24h in a shaking incubator at 25-40 ℃ at the speed of 125-150r/min, sucking culture solution with the total volume of 1-5% of the liquid culture medium, transferring to fresh NB liquid culture medium, culturing for 24-72h at 25-40 ℃ at the speed of 125-150r/min to obtain YL-RY0310 fermentation broth, wherein the viable count reaches (1-10) multiplied by 10, and the number of viable counts reaches (1-10) 7 CFU/mL。
3. The application of the bacillus mojavensis YL-RY0310 in preventing and treating penicillium expansum contamination of fruits and vegetables.
4. The application of claim 3, wherein the application comprises the steps of:
the concentration is (1-10). times.10 7 CFU/mL of fermentation liquid of the Bacillus mojavensis YL-RY0310 is directly sprayed on the surface of the biological sample.
5. A biocontrol agent comprising Bacillus mojavensis YL-RY0310 according to claim 1.
6. The biocontrol formulation of claim 5, wherein said biocontrol formulation is in the form of a liquid, dusting powder, dry wettable powder or dry wettable granules.
7. The biocontrol agent of claim 5, wherein said biocontrol agent comprises said YL-RY0310 fermentation broth.
8. Use of a biocontrol agent as defined in any one of claims 5-7 for the control of patulin contamination in fruits and vegetables.
9. The use of claim 8, wherein said control of fruit and vegetable patulin contamination comprises at least one of inhibition of penicillium expansum growth and inhibition of patulin synthesis.
10. The application of claim 9, wherein the application comprises the steps of: the biocontrol agent is sprayed on the surface of a biological sample to inhibit penicillium expansum and patulin.
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| CN105578884A (en) * | 2013-07-24 | 2016-05-11 | 拜耳作物科学股份公司 | Binary fungicidal composition |
| KR102073487B1 (en) * | 2018-08-31 | 2020-02-04 | 바래봉비료영농조합법인 | Method for manufacturing animal waste compost pellet inoculating bacillus vallismortis bs07m |
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| CN105578884A (en) * | 2013-07-24 | 2016-05-11 | 拜耳作物科学股份公司 | Binary fungicidal composition |
| WO2015036379A1 (en) * | 2013-09-13 | 2015-03-19 | Bayer Cropscience Ag | Fungicidal compositions containing thiazolylisoxazoline fungicide and biological fungicide |
| KR102073487B1 (en) * | 2018-08-31 | 2020-02-04 | 바래봉비료영농조합법인 | Method for manufacturing animal waste compost pellet inoculating bacillus vallismortis bs07m |
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