Lactic acid bacteria and application thereof in degradation of patulin in fruit and vegetable products

Abstract

The invention relates to lactic acid bacteria and application of the lactic acid bacteria to degradation of patulin in fruit and vegetable products, and relates to the technical field of biodegradation, the lactic acid bacteria are lactobacillus brevis, the lactobacillus brevis is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.31161. According to the application of the lactic acid bacteria in degradation of the patulin and the application of the lactic acid bacteria in degradation of the patulin in fruit and vegetable products, a new biodegradation means is provided for degradation of the patulin, the lactic acid bacteria have the advantages of being safe, efficient and environmentally friendly, and meanwhile the quality of the fruit products can be well kept.

Description

Technical Field

[0001] The invention relates to the technical field of biodegradation, in particular to a lactic acid bacterium and application thereof in degrading patulin in fruit and vegetable products. Background Art

[0002] Fruit and vegetable products are an important part of the human diet and are highly praised by consumers for their health benefits such as delaying aging, lowering cholesterol and preventing cardiovascular diseases. However, patulin (PAT), a toxic fungal toxin, will remain in fruits and their products. The intake of PAT will cause immunotoxicity and neurotoxicity and other acute or chronic effects on the human body, endangering the health of consumers and causing serious food safety issues.

[0003] The existing measures for controlling PAT residues in fruits, vegetables and their products mainly include physical and chemical methods, but both have certain drawbacks. Physical methods involve manual picking, high-pressure water washing, refrigeration, juice clarification, filtration, adsorption, pasteurization, radiation and pulsed light treatment, etc. Although they can effectively degrade PAT, they require a lot of manpower and material resources, are expensive, and still affect product quality. Chemical additives include sulfur dioxide, potassium permanganate, ozone, ammonia, ascorbic acid, sodium benzoate and potassium sorbate, etc. Although they can also achieve good degradation effects, these additives will affect the quality of fruit products to varying degrees and excessive addition will be harmful to human health. Therefore, it is necessary to find a safe degradation method that can maintain the quality of fruit products.

[0004] As a new control method, biodegradation is safe, efficient and environmentally friendly, and can better maintain the quality of fruit products. At present, the research on biodegradable PAT at home and abroad is mainly based on two biological sources: microorganisms and enzymes. Studies have shown that yeast and a small amount of mold can degrade PAT, such as Candida guilliermondii , Pichia caribbica and Filamentous fungi FF1-2, but these strains are rarely used in food. Although relevant studies have shown that enzymes can degrade PAT and be applied to food without affecting the quality of food, some studies have found that the degradation capacity of enzymes is limited. For example, porcine pancreatic lipase only degraded 31.26% of PAT (2μg / mL) after 42 hours, which indicates that a certain amount of porcine pancreatic lipase may not be enough to expose the active sites for PAT degradation.

[0005] On this basis, the inventors proposed using a new lactic acid bacteria strain to degrade patulin in fruit and vegetable products, which has far-reaching significance for food safety and human health. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a lactic acid bacterium and an application thereof in degrading patulin in fruit and vegetable products.

[0007] The technical solution is as follows: A lactic acid bacterium, which is Lactobacillus breve ( Levilactobacillus brevis ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.31161 (the deposit date is July 3, 2024), and named LSM01.

[0008] The method for obtaining lactic acid bacteria includes the following steps: Step 1: Get a sample from a specific induction environment, mix the sample with MRS (DeMan, Rogosa and Sharpe) broth, culture it to get an enrichment solution, dilute the enrichment solution and apply it on MRS solid medium in a gradient manner; Step 2, selecting a single colony from the MRS solid culture medium plate for streaking isolation, and after culturing, determining whether it is lactic acid bacteria; Step 3, testing the PAT degradation ability of the obtained lactic acid bacteria strains; Step 4: observe the morphology of the dominant strain and conduct molecular identification; Step 5, testing the PAT degradation ability of the obtained lactic acid bacteria strains; Step six, finally screening out the LSM01 strain with high efficiency in degrading patulin.

[0009] The use of the lactic acid bacteria as claimed in claim 1 in the degradation of patulin. The lactic acid bacteria are inoculated into an MRS broth medium containing patulin, and the temperature is 27-37°C and the concentration of the bacterial solution is 2×10 8 ~ 4×10 8 CFU / mL (colony forming count per milliliter) and cultured for 6 to 24 hours at a pH of 4 to 8 to efficiently degrade patulin, with a degradation efficiency of 95% or more.

[0010] The use of the lactic acid bacteria as claimed in claim 1 in degrading patulin in fruit and vegetable products. The lactic acid bacteria are inoculated into pear juice. Studies have shown that after 48 hours of fermentation, the pear juice can degrade 90.79% of PAT, and other qualities except pH remain almost unchanged, which has practical application value.

[0011] In summary, the present invention includes at least one of the following beneficial technical effects: 1. The present invention provides a new lactic acid bacteria strain, which provides a new biological means for degrading patulin in fruit and vegetable products, has the characteristics of safety, high efficiency and environmental friendliness, and can better maintain the quality of fruit products; 2. The lactic acid bacteria strain (LSM01) provided by the present invention can efficiently degrade patulin under appropriate conditions; 3. The lactic acid bacteria strain (LSM01) provided by the present invention can not only efficiently degrade patulin in specific fruit and vegetable products, but also ensure that other qualities of the fruit and vegetable products remain almost unchanged, making it have practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the ability of different strains to degrade PAT. Different lowercase letters on each column represent significant differences among groups (p<0.05); Figure 2 It is the morphological observation of the dominant strain (LSM01); Figure 3 It is the gel electrophoresis detection of DNA amplification products of the dominant strain (LSM01) and the construction of phylogenetic tree; Figure 4 It is the PAT resistance of LSM01; Figure 5 It is the reaction condition that affects the effect of LSM01 in degrading PAT; Figure 6 is the first-order kinetic analysis of PAT degradation by LSM01; Figure 7 is the PAT degradation chromatogram (A) and the degradation product LC-MS / MS graph (B); Figure 8 It is the change of PAT content of LSM01 in fruit and vegetable products (pear juice) at different fermentation times. DETAILED DESCRIPTION

[0013] The present invention is described in further detail below.

[0014] The following are the materials and reagents used: Fresh Red Fuji apples were purchased from a fruit supermarket next to the Zhejiang Academy of Agricultural Sciences; fermentation raw materials such as naturally fermented kimchi, Shantou fermented sauerkraut, Guizhou sour soup mother water, home-brewed fermented glutinous rice wine, and Chongqing old kimchi mother water were purchased from e-commerce platforms; disposable inoculation loops and coating rods were from Changde Beekman Biotechnology Co., Ltd.

[0015] PAT standard, 100% sterile glycerol solution, Shanghai Yuanye Biotechnology Co., Ltd.; glacial acetic acid, ethyl acetate (chromatographic grade), Shanghai MacLean Biochemical Technology Co., Ltd.; MRS agar, broth culture medium, Gram staining kit, Qingdao Haibo Biotechnology Co., Ltd.; 3% hydrogen peroxide solution, Guangdong Huankai Microbiological Technology Co., Ltd.; DP302 bacterial genomic DNA extraction kit, Tiangen Biochemical Technology Co., Ltd.; DR0101050 rubber tapping recovery kit, Zhejiang Easyside Biotechnology Co., Ltd.

[0016] Regarding the statistical analysis of data, the following explanation is given: The experiment was repeated three times. Excel 2021 was used for data collation and statistics, and GraphPad Prism 9.5 and Origin 2022 were used for analysis of variance, one-way ANOVA analysis and drawing. Different letters indicate significant differences (p<0.05). Embodiment 1

[0017] The acquisition of lactic acid bacteria includes the following steps: First, a standard curve of PAT standard solution should be established in advance. 5 mg of PAT standard was weighed and dissolved in 50 mL of ethyl acetate. After fully dissolved, it was stored in a -20℃ environment. The final concentration of PAT preservation solution was 100 μg / mL. The quantitative PAT preservation solution was blown dry with a nitrogen blower and resuspended in acidified water at pH 4.0. PAT standard solutions with concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1, 5, 10, 50, and 100 μg / mL were prepared. After filtering with a 0.22 μm microporous water filter, the PAT concentration was detected by high performance liquid chromatography (HPLC). Detection conditions: the chromatographic column was SunFire C18, 250 mm×4.6 mm×5 μm, the column temperature was 30℃, the flow rate was 1 mL / min, the mobile phase was ddH2O: acetonitrile = 9:1, the injection volume was 20 μL, the detection wavelength was 276 nm, and the time was 20 min.

[0018] After the PAT standard solution was tested by HPLC according to the above method, the concentration (0, 0.2, 0.4, 0.6, 0.8, 1, 5, 10, 50, 100 μg / mL) of the standard solution was plotted on the x-axis and the PAT retention peak area was plotted on the y-axis to establish a standard curve. After linear fitting, the linear regression equation obtained was y=161786x-60042, R 2 =0.99914, indicating that the two are highly correlated. Therefore, this detection method and this standard curve can be used for PAT content detection in subsequent studies.

[0019] Step 1: Get a sample from a specific inducing environment (kimchi, rotten apples, fermented sauerkraut, rice wine, sour soup or kimchi mother water), take 1 mL of the sample and mix it with MRS broth to make 5 mL of enrichment solution, and culture it at 37℃ for 24 h. The enrichment solution after culture is diluted with sterile saline gradiently (10 -3 ~10 -7 ), take 100 μL of each gradient dilution and evenly spread it on MRS solid culture medium, invert it and culture it in a 37℃ incubator for 36 h, and make three parallels for each gradient.

[0020] Step 2: Select single colonies with different morphologies from the plate for streaking and isolation, and culture at 37°C for 36 h. Perform Gram staining and hydrogen peroxide test on the isolated strains. If the staining is purple and the strain does not produce bubbles in the hydrogen peroxide solution, it is preliminarily determined to be lactic acid bacteria ( Lactobacillus , LAB). The screening strains that passed the preliminary identification (Table 1) were cultured in 5 mL MRS broth (24 h, 37°C) at a 2% inoculum until the third generation, and the bacterial solution was aspirated and mixed with 50% sterile glycerol solution (1:1, v:v) in a cryopreservation tube and stored in a -80°C refrigerator.

[0021] As shown in Table 1, based on the results of Gram staining and hydrogen peroxide tests, 12 potential lactic acid bacteria strains with good growth were isolated from kimchi, corrupt apples, fermented sauerkraut, rice wine, sour soup or kimchi mother water. Subsequent experiments will determine the PAT degradation ability of these 12 isolated strains to screen out the most advantageous PAT-degrading strains.

[0022]

[0023] Step 3: PAT degradation ability test of the obtained lactic acid bacteria strains: activate the frozen strains, dip the bacterial solution in the frozen tube with an inoculation loop, separate and streak on MRS solid medium, culture at 37°C for 36 h, select a single colony with an inoculation loop and culture in 5 mL MRS broth at 37°C for 24 h, and then subculture twice with an inoculation volume of 2% to obtain the test strain bacterial solution. Centrifuge the bacterial solution (10,000 rpm, 4°C, 10 min), discard the supernatant to collect the bacterial cells, and then wash them three times with sterile PBS buffer at pH 7.2 to collect the bacterial cells. The obtained bacteria were resuspended in 1 mL of 10 μg / mL PAT standard solution (pH 4.0), placed in a 37°C constant temperature shaker at a speed of 150 r / min for 24 h, and then sampled and centrifuged (10000 rpm, 4°C, 10 min) to obtain the supernatant. The supernatant was filtered through a 0.22 μm microporous water filter membrane and the PAT concentration was detected by HPLC. The PAT standard solution without resuspending the bacteria was used as a negative control.

[0024] like Figure 1As shown in the figure, when the initial concentration of PAT was 10 μg / mL, the 12 lactic acid bacteria isolated from different food matrices had significant differences in their ability to degrade PAT. Among them, the degradation rate of PAT by 6 strains was higher than 50%, namely LAB 1, LAB 2, LAB 3, LAB 7, and LAB 8. Among them, LAB 2 had the highest degradation rate, reaching 90.21%. The degradation rates of the other 6 strains were lower than 50%, and the lowest was LAB 6, which was only 10.19%. This shows that there are significant differences in the resistance and degradation ability of lactic acid bacteria in different induced environments to PAT. In this study, the dominant strain with the best effect in degrading PAT was LAB 2.

[0025] Step 4: Observe the morphology of the dominant strain LAB 2 and conduct molecular identification: Use an inoculation loop to dip the bacterial solution in the frozen tube of the strain, separate and streak on MRS solid culture medium, and culture at 37°C for 36 hours to observe the morphological characteristics of the bacteria, including colony morphology, cell morphology, and the presence of spores.

[0026] like Figure 2 As shown, LAB 2 grows well on MRS solid medium, with a colony diameter of about 1-2 mm, a smooth and shiny surface; the entire colony is oblate, with a protrusion in the middle, slightly white, slightly transparent, and neat edges. Its individual cells are rod-shaped, belonging to short rods, and the bacteria exist individually or collectively. After staining, it is purple and belongs to Gram-positive bacteria, without flagella and spores.

[0027] Molecular characterization involves the following steps: (1) Extraction of bacterial liquid: When the strain stored in the cryopreservation tube is activated to the third generation, take 5 mL of bacterial liquid and extract DNA according to the method of bacterial DNA extraction kit (TIANGEN, DP302).

[0028] (2) DNA amplification: PCR amplification was performed on DNA samples. The enzyme used for PCR was 2×Taq Plus MasterMixⅡ, and the primers were upstream primer 27-F (5'-AGAGTTTGATCCTGGCTCAG-3') and downstream primer 1492-R (5'-TACGTACCTTGTTACGACTT-3'). The PCR reaction system was 10 μL 2×Taq Plus MasterMixⅡ, 1 μL upstream and downstream primers, 0.2 μg DNA template, and 20 μL ddH2O. The amplification program was pre-deformation at 95℃ for 3 min; deformation at 95℃ for 15 s; annealing at 55℃ for 15 s; extension at 72℃ for 2 min; 35 cycles; and final extension at 72℃ for 1 min.

[0029] (3) Purification: The PCR amplification products were detected by agarose gel electrophoresis. The agarose gel concentration was 1.5%, the electrophoresis voltage was 200 V, and the electrophoresis time was 10 min. After the bands were observed and recorded in the gel imaging system, the gel was cut and recovered using the Easy Gel Extraction & Clean-up Kit (DR0101050). The products were sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing.

[0030] like Figure 3 As shown in the figure, the DNA of LAB 2 strain was amplified by PCR and the amplified product was detected by gel electrophoresis, and a clear band was found at the 1500 bp position. This result was in line with expectations, so the product was recovered by tapping and sequenced to obtain the full 16s rDNA sequence of LAB 2 strain. The sequencing results were compared and analyzed by BLAST on the official website of NCBI, and the results showed that LAB 2 strain was similar to Levilactobacillus brevis ATCC 14869 and Levilactobacillus brevis strain ATCC 14869= DSM 20054 have high homology, 99.86% and 99.51% respectively. Therefore, based on this analysis result, this strain was used as the experimental strain for subsequent experiments in this study, and LAB 2 was renamed Levilactobacillus brevis LSM01 (LSM01).

[0031] Step 5: obtaining the lactic acid bacteria strains L. brevis LSM01 was used to test the degradation capacity of PAT: The strain stored in the cryopreservation tube was activated to the third generation to obtain the test bacterial solution. It was inoculated into MRS broth containing 1, 5, 10, 20, and 100 μg / mL PAT at a 2% inoculation rate and cultured at 37°C for 24 h at a speed of 150 r / min. The OD of the bacterial solution was measured every 4 h using an ELISA reader. 600 Each group was set up with 3 parallels, and the medium without PAT was used as blank control.

[0032] like Figure 4 As shown in the figure, the bacterial growth in the culture medium containing different concentrations of PAT was consistent from 0 to 8 h, and there was no growth inhibition. After 8 h, the growth of the strain in the culture medium with PAT concentrations of 0, 1, 5, and 10 μg / mL remained consistent, and there was no growth inhibition. However, the growth of the strain in the culture medium containing PAT concentrations of 20 and 100 μg / mL was significantly inhibited (p<0.05), indicating that the strain had a good tolerance to PAT when the PAT concentration was less than 20 μg / mL. Therefore, 10 μg / mL will be used as the initial PAT concentration in subsequent studies.

[0033] Step six, finally screening out LSM01 with high efficiency in degrading patulin, which can be used in degrading patulin. Embodiment 2-6

[0034] Tests on temperature conditions in the application of patulin degradation: The strain stored in the cryopreservation tube was activated to the third generation to obtain the test bacterial solution. The plate count results were compared with the bacterial solution OD measured by the microplate reader. 600 Value association; when OD 600 =1.0, the plate count magnitude is 10 8 , specifically about 4.0×10 8 CFU / mL. The bacterial solution was adjusted to OD value of 1.0 and then centrifuged (10000 rpm, 4°C, 10 min), the supernatant was discarded to collect the bacteria, and then washed three times with sterile PBS buffer at pH 7.2 to collect the bacteria.

[0035] The bacteria were placed under different reaction conditions and a single factor experiment was performed, including temperature, initial PAT concentration, bacterial concentration, and pH value. 8 CFU / mL bacterial concentration, pH = 4.0, the temperature of Examples 2-6 is 22, 27, 32, 37, 42 ° C. Then, the mixture was placed in a constant temperature shaker at a speed of 150 r / min for 24 h, and then sampled and centrifuged (10000 rpm, 4 ° C, 10 min) to obtain the supernatant, which was filtered through a 0.22 μm microporous water filter membrane and then tested for PAT concentration by HPLC. The PAT standard solution without resuspending the bacteria was used as a negative control.

[0036] Figure 5 In A, after culturing for 24 h under the condition of an initial PAT concentration of 10 μg / mL, the reaction temperature in the range of 27-37℃ had no significant effect on the degradation of PAT, and all had good degradation effects. Among them, 37℃ could reduce the PAT concentration to 0.41μg / mL and achieve a maximum degradation rate of 95.48%; at 22℃ and 42℃, the degradation effect of the strain on PAT was significantly inhibited (p<0.05), and the degradation rate at 42℃ was the lowest at 81.65%, when the PAT concentration was 1.74 μg / mL. This shows that 37℃ is the most suitable reaction temperature for the test strain, and the temperature of 27℃~37℃ also has a good degradation effect. Although the higher reaction temperature inhibits the activity of the strain in degrading PAT, it also achieves a considerable effect. Embodiment 7-11

[0037] In the application of patulin degradation, the test on the initial PAT concentration is as follows: The strain stored in the cryopreservation tube was activated to the third generation to obtain the test bacterial solution. The plate count results were compared with the bacterial solution OD measured by the microplate reader. 600 Value association; when OD 600 =1.0, the plate count magnitude is 10 8 , specifically about 4.0×10 8 CFU / mL. The bacterial solution was adjusted to OD value of 1.0 and then centrifuged (10000 rpm, 4°C, 10 min) to collect the bacteria, and then washed three times with sterile PBS buffer at pH 7.2 to collect the bacteria.

[0038] The bacteria were placed under different reaction conditions and a single factor test was used, including temperature, initial PAT concentration, bacterial concentration and pH value. 8 CFU / mL bacterial concentration, pH = 4.0, the initial PAT concentrations in Examples 7-11 were 1, 5, 10, 20, and 100 μg / mL, respectively; the culture was then placed in a constant temperature shaker at a speed of 150 r / min for 24 h, and samples were taken. The supernatant was centrifuged (10000 rpm, 4°C, 10 min), filtered with a 0.22 μm microporous water filter membrane, and the PAT concentration was detected by HPLC. The PAT standard solution without resuspending the bacteria was used as a negative control.

[0039] Figure 5 In B, the degradation rate of each group was relatively stable. When the initial concentration of PAT was 1-20 μg / mL, the strain had no significant effect on the degradation rate of PAT. However, when it reached 100 μg / mL, the degradation ability of the strain on PAT decreased significantly ( p <0.05), but still above 95%. According to the results of the strain resistance to PAT in Example 1, the growth of the strain was inhibited at 20 μg / mL, which indicates that L. brevis Although the growth of LSM01 was inhibited in a high-concentration PAT environment, it still showed an excellent PAT degradation effect and could degrade PAT efficiently, which was unexpected for those skilled in the art. Examples 12-16

[0040] Tests on bacterial concentration in the application of patulin degradation: The strain stored in the cryopreservation tube was activated to the third generation to obtain the test bacterial solution. The plate count results were compared with the bacterial solution OD measured by the microplate reader. 600 Value association; when OD 600 =1.0, the plate count magnitude is 10 8 , specifically about 4.0×10 8CFU / mL. The bacterial solution was adjusted to OD value of 1.0 and then centrifuged (10000 rpm, 4°C, 10 min) to collect the bacteria, and then washed three times with sterile PBS buffer at pH 7.2 to collect the bacteria.

[0041] The bacteria were placed under different reaction conditions and a single factor test was performed, including temperature, initial PAT concentration, bacterial concentration and pH value. The temperature was fixed at 37°C, the PAT concentration was 10 μg / mL, and the pH value was 4.0. The bacterial concentrations in Examples 12-16 were 1×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 4×10 8 CFU / mL; then placed in a constant temperature shaker at a speed of 150 r / min for 24 h, sampled, centrifuged (10000 rpm, 4°C, 10 min), took the supernatant, filtered with a 0.22 μm microporous water filter membrane, and then used HPLC to detect the PAT concentration. The PAT standard solution without resuspending the bacteria was used as a negative control.

[0042] Figure 5 C, when the initial PAT concentration was 10 μg / mL, the degradation rate of PAT by the strain was related to the concentration of bacterial solution (0.5-2×10 8 CFU / mL) was significantly positively correlated ( p <0.05), and at 4×10 8 CFU / mL and 1×10 7 CFU / mL, the maximum and minimum degradation rates were 95.03% and 33.50%, respectively. Examples 17-21

[0043] In the application of patulin degradation, the test on pH: The strain stored in the cryopreservation tube was activated to the third generation to obtain the test bacterial solution. The plate count results were compared with the bacterial solution OD measured by the microplate reader. 600 Value association; when OD 600 =1.0, the plate count magnitude is 10 8 , specifically about 4.0×10 8 CFU / mL. The bacterial solution was adjusted to OD value of 1.0 and then centrifuged (10000 rpm, 4°C, 10 min) to collect the bacteria, and then washed three times with sterile PBS buffer at pH 7.2 to collect the bacteria.

[0044] The bacteria were placed under different reaction conditions and a single factor experiment was used, including temperature, initial PAT concentration, bacterial concentration, and pH value. 8 CFU / mL bacterial concentration, pH values ​​(adjusted with HCl and 1% NaOH) in Examples 17-21 were 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively. Then, the culture was placed in a constant temperature shaker at a speed of 150 r / min for 24 h, and the sample was taken after centrifugation (10000 rpm, 4°C, 10 min) to obtain the supernatant, which was filtered through a 0.22 μm microporous water filter membrane and then tested for PAT concentration by HPLC. The PAT standard solution without resuspending the bacteria was used as a negative control.

[0045] Effect of pH on degradation rate Figure 5 As shown in D, when the initial PAT concentration was 10 μg / mL and the pH value was between 4 and 8, the strain had a relatively stable and insignificant effect on the PAT degradation rate, achieving a degradation effect of more than 95%. The most suitable reaction pH condition was 7, which was almost completely degraded. When the pH value was 3, the PAT degradation rate was only 33.30%, and the degradation effect decreased significantly ( p <0.05), which may be due to the excessive acid condition. L. brevis The activity and PAT degradation ability of LSM01 were greatly inhibited. According to previous studies, the degradation rate of PAT by Lactobacillus plantarum also decreased significantly under alkaline conditions (pH 8.0). p <0.05). However, the present invention L. brevis LSM01 was not significantly affected at pH 8, which was also unexpected for those skilled in the art. Examples 22-27

[0046] Kinetic analysis of PAT degradation by LSM01: The strain stored in the cryopreservation tube was activated to the third generation to obtain the test bacterial solution. After the OD value of the bacterial solution was adjusted to 1.0, it was centrifuged (10000 rpm, 4℃, 10 min) and the supernatant was discarded to collect the bacteria. Then the bacteria were washed three times with sterile PBS buffer at pH 7.2 and collected. The bacteria were resuspended in 1 mL 10 μg / mL PAT standard solution (pH 4.0) and then placed in a 37℃ constant temperature shaker with a speed of 150 r / min for 12 h. Samples were taken every 2 h, and the supernatant was centrifuged (10000 rpm, 4℃, 10 min) to obtain the supernatant. The PAT concentration was detected by HPLC after filtering with a 0.22 μm microporous water filter. The PAT standard solution without resuspending the bacteria was used as a negative control.

[0047] Depend on Figure 6A shows that the reaction times of Examples 22-27 are 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h, respectively. L. brevis LSM01 efficiently degraded PAT after 12 h of treatment.

[0048] However, the inventors did not follow the logic that the longer the reaction time, the better the degradation effect, and subsequently shortened the reaction time range to 0-12 h for research (i.e., Examples 28-33, where the corresponding reaction times for Examples 28-33 were 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, respectively). Figure 6 As shown in B, it was found that the efficient degradation of this strain occurred before 6 h, giving a shorter time required for efficient degradation of PAT, with outstanding substantive characteristics and significant progress. C0 / Ct ) is the ordinate, and the reaction time t is the abscissa. The degradation rate of PAT by the strain within 6 h was linearly fitted to obtain the fitting equation ln( C0 / Ct )=0.47205t+0.01274, and R 2 =0.99, e.g. Figure 6 C. This shows that when the initial concentration of PAT is 10 μg / mL, the strain has completed efficient degradation within 6 h, which is unexpected for those skilled in the art. Embodiment 34

[0049] Analysis of PAT degradation products: The degradation products of PAT were identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The chromatographic and mass spectrometric conditions were as follows: the chromatographic column used was InfinityLab Poroshell 120 EC-C18; the column temperature was 30°C; the injection volume was 5.0 μL; the mobile phase was acetonitrile: water = 1:9 isocratic elution, and the flow rate was 0.4 mL / min; the mass spectrometer was analyzed in positive ion mode, and the ion source was Dual-AJS (ESI); the gas flow rate was 30 mL / min; the auxiliary gas rate was 5 mL / min; the spray voltage was 3.5 kV for positive ion; and the ion source temperature was 300°C.

[0050] Depend on Figure 7 A shows that at 0 h, the retention peak of PAT detected by HPLC appeared between 7.7 and 8.2 min, and gradually disappeared with the increase of reaction time. Four retention peaks were found before the PAT peak appeared, and the elution times of peaks 1 to 4 were 4.8, 3.0, 3.5 and 2.2 min, respectively. Among them, the peak area of ​​substance 1 increased synchronously with the decrease of PAT content, which indicates that substance 1 may be L. brevisPotential degradation products of PAT degradation by LSM01.

[0051] PAT and its degradation products were further analyzed and identified using LC-MS / MS technology in positive ion mode. PAT standard [M+H] + =159.14, and found its two main characteristic ion fragments, with mass-to-charge ratios of m / z 97.78 and 125.45. Later, we found that after degradation, PAT [M+H] + =160.18, the ion intensity increased significantly, and two main characteristic ion fragments were found, with mass-to-charge ratios of m / z 116.70 and 131.48, corresponding to a molecular weight of 159. Figure 7 B. Related studies have reported that the degradation products of PAT are mainly Ascladiol, Desoxypatulinic and Hydroascladiol, with relative molecular masses of 156, 156 and 158, respectively. Ascladiol has two isomers, (E)-ascladiol and (Z)-ascladiol. The mass-to-charge ratios of the main characteristic ion fragments of Hydroascladiol (m / z 159.12) are m / z115.06, 131.04 and 141.04, respectively, which are highly similar to our research results. Therefore, we speculate that Hydroascladiol is L. brevis The product of PAT degradation by LSM01. In addition, Hawar's research also showed that Ascladiol is a short-term exposure product after PAT degradation, but after long-term exposure, (E)-ascladiol and (Z)-ascladiol may generate Hydroascladiol mediated by some chemical reaction. The presence of the two isomers of Ascladiol was not found in this identification analysis. Embodiment 35

[0052] Application of LSM01 in specific fruit and vegetable products: The experimental raw materials selected were: Crown pears (purchased from Zhejiang Wenhui Fruit Company).

[0053] Test method: 1. Pear juice preparation: Wash fresh pears → peel and core → cut → squeeze juice → filter (nylon sieve 200 mesh) → add 1% sucrose → sterilize (100℃ water bath, 15min) → refrigerate for later use 2. Pear juice fermentation to degrade patulin (PAT): Use a nitrogen blower to dry the ethyl acetate in the 100 µg / mL PAT standard stock solution, and resuspend it with pear juice to make the PAT concentration in the pear juice 1000 µg / L (because the PAT limit value in the national standard is 50 µg / L, considering the actual exceeding of the limit, the concentration is increased, and the specific concentration after resuspension is about 1278 µg / L). Levilactobacillus brevis LSM01) was washed twice with 0.85% saline and resuspended. The number of bacteria in the bacterial suspension was about 9 log CFU / mL. L. brevis LSM01 was inoculated into pear juice, and its PAT concentration, viable bacterial count, pH, soluble solids, total phenols and Vc content were tested every 12 hours. Pear juice without inoculation of the strain was used as a control.

[0054] Test results: like Figure 8 As shown in the figure, within 12 h of inoculation, the strain efficiently degraded 57.40% of PAT (1278 to 544 µg / L), and the degradation ability weakened subsequently, and finally degraded 90.79% of PAT within 48 h.

[0055] As shown in Table 2, the number of viable bacteria in pear juice increased to 7.85 log CFU / mL within 24 h and then decreased significantly ( p <0.05), the overall viable bacterial count of pear juice was maintained between 7 log CFU / mL and 8 log CFU / mL during the 48 h fermentation period, providing the possibility for the strain to degrade patulin. The pH decreased significantly during the 0-48 h fermentation process ( p <0.05), decreased from the initial 5.02 to 4.47, which increased the acidity of the pear juice. The remaining soluble solids (10.53 to 10.23%), total phenols (1.64 to 1.40 mg / mL) and Vc content (9.91 to 9.45 μg / mL) were slightly weakened within 48 h but did not change significantly. Therefore, after 48 h of fermentation, pear juice could degrade 90.79% of PAT, and other qualities except pH remained almost unchanged, which has practical application value.

[0056]

[0057] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, a person skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as it is within the scope of the present application, it shall be protected by the patent law.

Claims

1. A lactic acid bacterium, characterized in that It is Lactobacillus breve ( Levilactobacillus brevis ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.31161.

2. Use of the lactic acid bacteria as claimed in claim 1 in degrading patulin.

3. The use according to claim 2, characterized in that: The lactic acid bacteria are inoculated into an MRS broth medium containing patulin, and cultured for 6 to 24 hours under the conditions of a temperature of 27 to 37° C., a bacterial liquid concentration of 2×10^8 to 4×10^8, and a pH of 4 to 8, so as to efficiently degrade patulin.

4. Use of the lactic acid bacteria as claimed in claim 1 in degrading patulin in fruit and vegetable products.

5. The use according to claim 4, characterized in that: The lactic acid bacteria are inoculated into pear juice to degrade patulin in the pear juice and maintain the quality of the pear juice.

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