CN117264795A

CN117264795A - Pacific bacillus for degrading alternariol, biological agent and application thereof

Info

Publication number: CN117264795A
Application number: CN202210711530.1A
Authority: CN
Inventors: 赵丽红; 张建云; 张新悦; 郭永鹏; 刘立萌
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-06-22
Filing date: 2022-06-22
Publication date: 2023-12-22

Abstract

The invention provides Pacific bacillus (Bacillus pacifiicus) ANSB901 and a biological agent prepared from the Pacific bacillus, and also provides application of the Pacific bacillus and the biological agent thereof in the biodegradation of the cross-linked spore phenol toxin. The strain can be used for providing strain resources for biological detoxification of the alternariol in foods and feeds, and the application of the strain can reduce the harm of the alternariol to human and animals and improve the food safety.

Description

Pacific bacillus for degrading alternariol, biological agent and application thereof

Technical Field

The invention belongs to the technical field of microorganism application, and mainly relates to Pacific bacillus for degrading alternaria phenol, a biological preparation and application thereof.

Background

Alternariol (AOH) is a toxic secondary metabolite produced by alternaria. In 1953, the toxins were isolated from Alternaria Tenuis by Raistrick et al, and were one of the highest detectable rates. In recent years, researchers have detected different levels of cross-linked sporophore in crops (corn, wheat, barley, sorghum, etc.), fruits (apples, grapes, citrus tangerines, pomegranates), vegetables (tomatoes, oilseed rape, cabbages, etc.). In vitro and in vivo experiments show that the alternariol has genetic toxicity, cytotoxicity, mutagenicity, estrogen-like toxicity and carcinogenicity, and has potential harm to human and animals.

Currently, most studies on alternaria toxins aim at investigating their biosynthesis, physiological toxicity, detection methods and pollution conditions. The research on the detoxification technology of alternariol in foods and feeds is mainly focused on physical degradation. Wang et al (2022) reduced AOH by 56.0% after 2 minutes of treatment of AOH in jujube juice by cold plasma. The AOH has serious pollution in foods and grain feeds, and causes potential harm to human beings and animals. Therefore, the research and application of the alternaria alternata toxin removal technology can greatly improve the safety of grain feed and food and ensure the physical health of people and animals. Current research on biodegradation of AOH is scarce and only individual microbial strains have been found to be able to degrade AOH. The invention aims to screen beneficial microorganisms capable of efficiently degrading alternariol and apply the beneficial microorganisms to biological detoxification of the alternariol in foods and feeds. Finally, the invention overcomes the defects, and the screened strain has high degradation effect on the alternariol, is suitable for Pacific bacillus (Bacillus pacifiicus) ANSB901 for degrading the alternariol in fruits, vegetables and feeds, and is expected to be used as a new strain resource for biological detoxification of the alternariol in the food and feed industry.

Disclosure of Invention

The invention aims to provide a Pacific bacillus (Bacillus pacifiicus) ANSB901 strain which is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for the year 2022, month 06 and 13, and has a preservation address of CGMCC No.25071, which is a microbiological institute of China national academy of sciences No. 3, north Chen West Lu No. 1, the Korean region of Beijing.

The Pacific bacillus has the following characteristics:

(1) Colony characteristics: the colony morphology surface is convex, the colony is round, opaque, the edge is neat, and gram staining is positive (figure 1); under an optical microscope (FIG. 2), the strain was rod-shaped and covered with flagella, and the size was 20.0 to 22.5 μm (length). Times.10.0 to 11.5 μm (width).

(2) Molecular characteristics: and (3) carrying out molecular biological identification on the strain ANSB901, and amplifying by using a universal primer to obtain a 16Sr DNA sequence. The band was detected using agarose gel (FIG. 3), and the size of the spliced band after sequencing was 1539bp. Sequencing results were aligned by BLAST and a phylogenetic tree was drawn (FIG. 4), with the closest affinity of strain ANSB901 to strain Bacillus pacifiicus MCCC A06182 (NR_ 157733.1), with a similarity of 99.60%, identified and designated as Pacific Bacillus Bacillus pacifiicus.

(3) Active component for degrading AOH: the experimental results show that the active component of Pacific bacillus ANSB901 for degrading AOH is mainly in the cell of the thallus, and the degradation activity of the active component on AOH is 94% (figure 5).

(4) Time profile of active component degradation of AOH: the degradation rate of the Pacific bacillus ANSB901 active component on AOH increases with the increase of time within 24 hours, the degradation starting time is slower within 0-4 hours, the degradation rate of 12 hours on AOH is 67%, and the degradation rate of 24 hours on AOH is 98.4% (figure 6).

(5) Optimum temperature range for active component degradation AOH: the degradation rate of the Pacific bacillus ANSB901 active component to AOH at 20-50 ℃ is about 70 percent (figure 7).

(6) Optimal pH range for active component degradation AOH: the Pacific bacillus ANSB901 active ingredient had approximately 100% AOH degradation at pH 9.0 and 10.0, about 70% AOH degradation at pH 7.0 and 8.0, and decreased AOH degradation at pH 6.0, but maintained 58% degradation (fig. 8).

The invention also provides a biological agent of the Pacific bacillus ANSB901 for detoxication of the alternaria alternata phenol and a preparation method thereof. The preparation method is characterized in that Pacific bacillus ANSB901 is subjected to activation culture and then centrifuged to obtain Pacific bacillus thalli, and the thalli are collected after washing to obtain Pacific bacillus biological preparations; or,resuspending the collected thallus with protecting agent to reach concentration of 10 ⁸ CFU/mL, culturing the suspension for a period of time, and freeze-drying to obtain the Pacific bacillus fungus powder biological agent.

The protective agent can be one or more of skimmed milk powder, sucrose, corn gluten meal, soybean protein concentrate, bran and starch.

The Pacific bacillus ANSB901 has the following uses:

(1) Preparing a biological agent for detoxification of the alternariol;

(2) Is used for removing the cross-linked sporophore in apples, hawthorns, tomatoes, grapes, citrus, pomegranates, peaches, oranges, flour, corn, sorghum, rice and processed products thereof, nuts and processed products thereof, feed and feed raw materials.

The Pacific bacillus ANSB901 can degrade the AOH in a wider temperature (20-50 ℃) and pH (6.0-10.0) range, and the fermentation broth can degrade 90% of the AOH (10 mug/mL) when cultured under the optimal culture condition (pH=7.0, 37 ℃ and fermentation time of 24 hours). The Pacific bacillus ANSB901 is used for removing the alternariol in food or feed, and has very wide application prospect.

The invention further provides application of the Pacific bacillus ANSB901 biological preparation in degrading the alternariol in the fruit juice, in particular to application of the Pacific bacillus biological preparation in mixing with the apple juice, and after incubation for 24 hours, determining the content of the alternariol in the apple juice, and test results show that the Pacific bacillus biological preparation can degrade 40.24% of the alternariol in the apple juice (10 mug/mLAOH).

The invention also provides application of the Pacific bacillus ANSB901 biological preparation in degrading alternaria phenol in mildewed corn, in particular to inoculation of alternaria spore (ATCC 66918) for producing alternaria phenol in corn, then addition of Pacific bacillus ANSB901 fermentation liquor or biological preparation thereof for co-incubation for 24 hours, and the determination result shows that the degradation rate of 50 mug/kg and 25 mug/kg of AOH in mildewed corn by the fermentation liquor is 54.90% and 76.03% respectively.

By implementing the specific invention content, the following beneficial effects can be achieved:

the Pacific bacillus Bacillus pacifiicus ANSB901 provided by the invention is bacillus, has a good technical effect of degrading the alternariol in food and feed, and has a good effect of degrading the alternariol in the food and feed in practical application by fermenting a biological agent prepared from the strain with apple juice and fermenting strain fermentation liquor with mildewed corn.

Description of biological preservation

ANSB901, classification naming: pacific bacillus, bacillus pacifiicus, deposited in China general microbiological culture Collection center (CGMCC) at 13/06/2022, with the deposit number of CGMCC No.25071, which is the institute of microbiological research, national academy of sciences, no. 3, 1, chaoyang, beijing, city.

Drawings

FIG. 1 shows colony (A) and cell (B) morphology (100K) of Pacific Bacillus ansB 901;

FIG. 2 is a scanning electron microscope image (5.0K) and (45K) of Pacific Bacillus ansB 901;

FIG. 3 is an electrophoretogram of the 16S rDNA PCR amplification product of Pacific bacillus ANSB 901;

FIG. 4 is a phylogenetic tree of Pacific Bacillus ANSB 901;

FIG. 5 is a determination of the active components of Pacific Bacillus ANSB901 degradation AOH;

FIG. 6 is a graph showing the time profile for degradation of AOH by the active component of Pacific Bacillus ansB 901;

FIG. 7 is a graph showing the effect of different temperatures on the degradation of AOH by Pacific Bacillus ANSB901 active components;

FIG. 8 is an effect of different pH on the degradation of AOH by Pacific Bacillus ANSB901 active components.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.

EXAMPLE 1 isolation and screening of alternariol degrading bacteria

About 5g of samples such as food, mildewed feed, animal chyme, excrement and the like are weighed respectively, added into 100mL of sterile Phosphate Buffer Solution (PBS), and vortexed and oscillated by a vortexing instrument. The supernatant is diluted in gradient by PBS, and the supernatant is respectively coated on LB culture medium plates by a plate coating method and cultured in a biochemical incubator at 37 ℃; after colony growth, single colony is picked out, fermentation culture is carried out, then the steps are repeated, and the LB plate is streaked and purified for a plurality of times. The single strain number after culture and purification is mixed with 20% glycerol solution according to the ratio of 1:1 (V/V) and then stored in a refrigerator at-20 ℃ for later use.

Inoculating the preserved strain into a bacterial bottle filled with LB liquid medium according to 1% (V/V) for activation to obtain strain fermentation liquor. Taking 990 mu L of secondary activated strain fermentation broth, mixing with 10 mu LAOH standard (1000 mu g/mL) in 2mL of EP by vortex, sealing a blank by using a sealing film and culturing for 24h in a shaking table with the rotating speed of 180r/min at 37 ℃ on a LB culture medium without bacteria and an AOH standard. After the incubation, the supernatant was collected by centrifugation using an equal volume of chromatographic grade methanol solution, vortexed and mixed, and filtered using a 0.22 μm filter membrane. And detecting the AOH content in the sample by using a high performance liquid chromatography-fluorescence detection method, and calculating the AOH degradation rate.

And finally, 91 strains of bacteria are screened out, wherein 68 strains exist for the bacteria with the AOH degradation rate less than 20%, 1 strain of bacteria has very high degradation capacity for AOH, and the degradation rate can reach 90%, and the number is ANSB901.

EXAMPLE 2 morphology and 16S rDNA identification of the Acidocella-l degrading Strain

The degrading bacteria are streaked in LB agar medium, cultured at constant temperature of 37 ℃ and observed in colony morphology and color. Gram staining and identifying bacteria, describing operation by referring to a Soxhinbao gram staining kit, sucking 10 mu L of activated bacterial liquid by using a pipetting gun, uniformly smearing the bacterial liquid on a clean glass slide, dripping crystal violet solution for primary staining after the bacterial liquid is naturally dried, flushing the glass slide by using a washing bottle filled with distilled water after 1min, and absorbing excessive water by using filter paper; dripping iodine solution for dyeing for 1min, and repeating the washing step; dropping a proper amount of decolorizing liquid on the glass slide, gently shaking the glass slide, washing with water after 30s, and sucking excessive water with filter paper without purple shedding; dripping a red dye liquor for counterstaining for 1min, and repeating the washing step; after the slide glass was naturally dried, 1. Mu.L of distilled water was added dropwise to the edge portion of the slide glass, and the slide glass was covered with a light cover glass. Observing the shape and the color of bacteria by an oil lens, and if the strain is gram-positive bacteria, making the strain dark purple to purple; if the strain is a gram-negative bacterial, it will appear red to reddish.

According to morphological observation, the surface of the strain ANSB901 is convex, and the colony is round, opaque, neat in edge and positive in gram staining, as shown in FIG. 1, which is a colony morphology and gram staining picture of the strain ANSB 901; the strain was rod-shaped and covered with flagella, and the size was 20.0-22.5 μm (length). Times.10.0-11.5 μm (width) as observed by a scanning electron microscope (FIG. 2).

Carrying out molecular biological identification on the bacterial strain ANSB901, amplifying by using a universal primer, taking a proper amount of degradation bacterial liquid, centrifuging for 2min at 12000r/min, extracting genome DNA of the bacterial strain according to the specification of a bacterial genome DNA extraction kit, and amplifying by using a bacterial 16S rDNA universal primer. And (3) detecting the amplified product in 1% agarose gel electrophoresis, observing whether the band is clear and bright, and judging whether the PCR is successful. The purified PCR reaction product is sent to the manufacturer for sequencing. The sequencing results were uploaded to the NCBI database and aligned in Gene Bank using its BLAST function, and the alignment was used to construct the phylogenetic tree of the strain using Mega7.0 software.

The size of the spliced strip after sequencing is 1539bp, which is the 16Sr DNA sequence (figure 3) detected by agarose gel electrophoresis. Sequencing results were compared by BLAST, and a phylogenetic tree (FIG. 4) was drawn, with the closest affinity of strain ANSB901 to strain Bacillus pacifiicus MCCC A06182 (NR_ 157733.1), and the similarity was 99.60%. Thus, this strain was identified and named Pacific bacillus Bacillus pacifiicus ANSB901.

EXAMPLE 3 determination of degradation of AOH active Components by Bacillus pacific ANSB901

Strain ANSB901 was inoculated in a 250mL conical flask at an inoculum size of 1% and incubated at constant temperature for 24h. And (3) centrifuging the cultured fermentation liquor at a high speed at a temperature of 4 ℃, wherein the supernatant after centrifugation is the supernatant of the fermentation liquor, and placing the supernatant in a refrigerator at the temperature of 4 ℃ for standby. The precipitate after centrifugation is resuspended by using PBS buffer solution with pH of 7.4, the cell solution after resuspension is centrifuged at 4 ℃, the cell is repeatedly washed for three times, and the cell resuspension after washing is the cell solution.

The test results show that the active component of Pacific bacillus ANSB901 for degrading the AOH is mainly in the cells of the thalli, and the degradation rate of the active component on the AOH is 94 percent (figure 5) which is obviously higher than that of the supernatant of the fermentation broth (the degradation rate of the AOH is 26 percent).

EXAMPLE 4 time curve for degradation of AOH by Pacific Bacillus ANSB901 cell heavy suspension

Taking 990 mu L of bacterial cell heavy suspension of Pacific bacillus ANSB901, adding 10 mu L of AOH standard (1000 g/mL), respectively reacting for 4, 8, 12 and 24 hours at 37 ℃ and stopping methanol, and detecting the content of AOH in the sample by using high performance liquid chromatography.

From the graph (fig. 6), the Pacific bacillus ANSB901 active component can realize better degradation to the AOH, the degradation rate is increased along with the time increase within 24 hours, the degradation starting time is slower within 0-4 hours, the degradation rate of the Pacific bacillus ANSB901 active component to the AOH is about 67%, and the degradation rate of the Pacific bacillus ANSB901 active component to the AOH within 24 hours is 98.4%.

EXAMPLE 5 Effect of culture temperature on degradation of AOH by Pacific Bacillus ANSB901 active ingredient

Taking 990 mu L of prepared Pacific bacillus ANSB901 bacterial cell heavy suspension, adding 10 mu L of AOH standard (1000 mu g/mL), respectively reacting at 20, 30, 37, 40, 45 and 50 ℃ and pH 7.0 for 24 hours, stopping methanol, and detecting the content of AOH in the sample by using high performance liquid chromatography.

The results show that the degradation rate of the Pacific bacillus ANSB901 active component on AOH at 20-50 ℃ is about 70% (figure 7).

EXAMPLE 6 Effect of pH on degradation of AOH by Pacific Bacillus ANSB901 active ingredient

The pH of the bacterial cell heavy suspension is adjusted to 6.0, 7.0, 8.0, 9.0 and 10.0 by using a citric acid-sodium citrate buffer solution, a phosphate buffer solution and a glycine-sodium hydroxide buffer solution, 990 mu L of the bacterial cell heavy suspension with different pH values are respectively taken, 10 mu LAOH standard substances (1000 mu g/mL) are added for reaction at 37 ℃ for 24 hours, methanol is stopped, and the content of AOH in a sample is detected by using high performance liquid chromatography.

The results show that the Pacific bacillus ANSB901 active component has approximately 100% AOH degradation rate at pH 9.0 and 10.0, about 70% AOH degradation rate at pH 7.0 and 8.0, and reduced AOH degradation rate at pH 6.0, but still maintained 58% degradation rate (fig. 8).

EXAMPLE 7 preparation of Pacific Bacillus ANSB901 biological preparation for detoxification of alternariol

Pacific bacillus ANSB901 was inoculated in an inoculum size of 1% to LB medium, and cultured for 24 hours under constant temperature conditions. And (3) centrifuging the cultured fermentation liquor at a high speed at 4 ℃, re-suspending the centrifuged precipitate by using PBS buffer solution with pH of 7.4, centrifuging the re-suspended bacterial cell solution at 4 ℃, and repeatedly washing the bacterial cells for three times to obtain the Pacific bacillus ANSB901 biological preparation.

Alternatively, the cells are resuspended to a concentration of 1X 10 with a protective agent ⁸ CFU/mL. The protective agent is 150g/L skimmed milk powder and 100g/L sucrose. The suspension is pre-cultured for 60min at the temperature of 30 ℃, pre-frozen for 2-5h at the temperature of-20 ℃, and finally freeze-dried to obtain the Pacific bacillus ANSB901 bacterial powder biological agent.

EXAMPLE 8 preparation of Pacific Bacillus ANSB901 biological preparation for detoxification of Acidocella

Alternatively, the cells are resuspended to a concentration of 1X 10 with a protective agent ⁸ CFU/mL. The protective agent is 120g/L corn protein powder and 100g/L soybean protein concentrate powder. Pre-culturing the suspension at 35deg.C for 60min, pre-freezing at-20deg.C for 2-5 hr, and freeze drying to obtain Pacific bacillus ANSB901 fungus powder biological preparation.

EXAMPLE 9 investigation of the effects of Pacific Bacillus Americana ASNB901 on the detoxification of AOH in apple juice

Adding AOH into apple juice to make the final concentration of the apple juice be 10mg/mL, and mixing the apple juice and Pacific bacillus ANSB901 bacterial liquid according to the following ratio of 1:1 were placed in an Erlenmeyer flask and incubated at 37℃and 200rpm for 24h. The samples were tested for AOH content by the HPLC method established above after incubation with no addition of Pacific Bacillus ANSB901 as a control.

The test results showed that the following 1:1 (V/V) and the liquid culture temperature is 37 ℃, and after 24 hours of fermentation, the degradation rate of the Pacific bacillus ANSB901 on the AOH in the apple juice containing 10 mug/mL of AOH is 40.24%.

EXAMPLE 10 investigation of the effects of Pacific Bacillus ANSB901 on AOH detoxification in mildewed corn

Firstly, crushing AOH mildewed corn, diluting the mildewed corn with corn without AOH to prepare mildewed corn with the AOH content of 25 mug/kg, incubating corn flour and Pacific bacillus ANSB901 fermentation liquor according to the feed liquid ratio of 1:9, carrying out shaking culture for 24 hours at the temperature of 37 ℃ under the condition of 220r/min, detecting the AOH content in a sample by an HPLC method, and calculating the degradation rate of the AOH. The mildew corn sample without bacterial liquid is used as a control group. Each sample was set up with 3 replicates.

The test results show that the degradation rate of the Pacific bacillus ANSB901 on the AOH in 25 mug/kg mildewed corn is 76.03%.

EXAMPLE 11 investigation of the effects of Pacific Bacillus Pacific ANSB901 on AOH detoxification in mildewed corn

Firstly, crushing AOH mildewed corn, diluting the mildewed corn with corn without AOH to prepare mildewed corn with AOH content of 50 mug/kg respectively, incubating corn flour and Pacific bacillus ANSB901 fermentation liquor according to a feed liquid ratio of 1:9, carrying out shaking culture for 24h at 37 ℃ under 220r/min, detecting the content of AOH in a sample by an HPLC method, and calculating the degradation rate of the AOH. The mildew corn sample without bacterial liquid is used as a control group. Each sample was set up with 3 replicates.

The test result shows that the degradation rate of the Pacific bacillus ANSB901 on the AOH in 50 mug/kg mildewed corn is 54.90 percent.

The foregoing discussion and examples have been provided merely to illustrate specific embodiments of the invention and are in no way intended to limit the true scope of the invention, which is defined by the claims.

Claims

1. Pacific bacillus (Bacillus pacifiicus) ANSB901 with preservation number of CGMCC No.25071.

2. A method for preparing a alternariol biodegradation agent by using the Pacific bacillus ANSB901 according to claim 1, which is characterized in that the Pacific bacillus ANSB901 is subjected to activation culture and then centrifuged to obtain thalli, and the thalli are collected after washing to obtain a Pacific bacillus viable bacteria preparation.

3. The method according to claim 2, wherein the collected cells are further resuspended to a cell concentration of 10 with a protectant solution ⁸ CFU/mL, culturing the suspension for a period of time, and freeze-drying to obtain the Pacific bacillus fungus powder biological agent.

4. The method of claim 3, wherein the protective agent is one or more of skimmed milk powder, sucrose, corn gluten meal, soy protein concentrate, bran, and starch.

5. A biological agent comprising the bacillus pacific ANSB901 of claim 1.

6. Use of the strain of Pacific bacillus ANSB901 according to claim 1 or the biological preparation of Pacific bacillus ANSB901 according to claim 5 for the biodegradation of alternariol.

7. A method of using the strain of bacillus pacificus ANSB901 according to claim 1 or the biological preparation of bacillus pacificus ANSB901 according to claim 5 for removing alternariol in food and/or feed.

8. The method of claim 7, wherein the food product includes, but is not limited to, apples, haws, tomatoes, grapes, citrus tangerines, pomegranates, peaches, oranges, flours, corns, sorghum, rice and processed products thereof and nuts and processed products thereof.

9. The method of claim 7, wherein the feed includes, but is not limited to, apple pomace fermented feed, silage, corn and various animal feed raw materials and compound feeds of processing byproducts thereof.

10. Use of the strain of Pacific bacillus ANSB901 according to claim 1 or the biological preparation of Pacific bacillus ANSB901 according to claim 5 for the preparation of a food and/or feed additive.