Method for identifying influence of bacillus velezensis on storage quality of mangoes

Abstract

The invention discloses a method for identifying the influence of bacillus velezensis on the storage quality of mangoes, the bacillus velezensis is preserved in China Center for Type Culture Collection on June 17, 2024, and the preservation number of the bacillus velezensis is CCTCC (China Center for Type Culture Collection) NO: M20241234. According to the invention, biocontrol bacteria of main disease pathogenic bacteria after mango harvesting are separated and screened from mango pulp, peel and kernel by adopting a plate confrontation method, identification is carried out by adopting morphology and physiological and biochemical determination in combination with 16S DNA sequence analysis, and the influence of the biocontrol bacteria on the storage quality of the harvested mangoes is researched; the basis is provided for improving the quality and commodity value of the picked mangoes and prolonging the shelf life; results show that the bacterial strain numbered as FP-KT-3 has the best inhibition effect on phomopsis pathogenic bacteria, the bacteriostasis rate of the bacterial strain reaches up to 74.89%, the bacterial strain is identified as bacillus velezensis, and it is indicated that the softening speed of fruits can be obviously slowed down and the flavor and nutritional quality of the fruits can be kept when mangoes are treated by the bacillus velezensis.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a method for identifying the influence of Bacillus Velez on the storage quality of mangoes. Background Art

[0002] Mango belongs to the genus Mangifera in the Anacardiaceae family. It is popular among people for its beautiful appearance and bright color. It is rich in nutrients, including carbohydrates, proteins and fats, as well as a variety of beneficial substances such as vitamins A, C, B1, polyphenols, crude fiber, flavonoids and carotenoids, and is known as the "king of tropical fruits". Since the growth and harvesting period is mostly in a hot and rainy environment, and the fruit is juicy, high in sugar content, and has strong respiration, it is easily attacked by microorganisms after harvesting, causing rotting, sourness, softening and juicing, which makes the taste and appearance poor, reducing its storage quality and commodity value. Therefore, how to effectively maintain the storage quality of mangoes after harvest has become an important issue that needs to be solved urgently.

[0003] At present, there have been extensive studies on the preservation of mangoes after harvest at home and abroad. The main measures include chemical control, biological control, physical control and other means or their combined application. Among them, biological control has shown significant advantages in the control of mangoes after harvest. Including reducing toxic residues, improving fruit safety, no drug resistance, low processing costs and stable control effects, it has attracted widespread attention from researchers at home and abroad. Bacteria are the most commonly studied and widely used type of antagonistic microorganisms.

[0004] As a new species of Bacillus, Bacillus velezii has the characteristics of rapid growth, strong stability, wide ecological niche, and environmental friendliness. It can produce a variety of antibacterial substances, including antibiotics, siderophores, hydrolases, antibacterial volatile organic compounds, and peptides. Therefore, field trials on the disease prevention and growth promotion of this strain have been widely used in a variety of economic crops such as kiwifruit, citrus, strawberry, tomato, and cucumber. Although a large number of studies have reported its potential in biological control, its application in fruit storage and preservation is still rare. In this experiment, an antagonistic biocontrol bacterium was screened from mango, and the changes in soluble solids content, hardness, titratable acid content, and soluble sugar content of mango fruit during storage were studied. The purpose is to explore effective post-harvest preservation treatment methods for mangoes, in order to provide a theoretical reference for the research on mango storage and preservation technology. Summary of the invention

[0005] The invention aims to provide an identification method for the effect of Bacillus Velez on the storage quality of mango, so as to provide a theoretical reference for the research on the storage and preservation technology of mango.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme: a method for identifying the effect of Bacillus velezensis on the storage quality of mango, wherein the Bacillus velezensis was deposited in the China Center for Type Culture Collection on June 17, 2024, and the classification name is: Bacillus velezensis FP-KT-3, Latin name: Bacillus velezensis FP-KT-3, the deposit address is: Wuhan University, Wuhan, China, and its deposit number is CCTCC NO: M20241234. The identification method comprises the following steps:

[0007] Preferably, the method for separating the biocontrol bacteria from the mango peel, pulp and core in step S1 is: Peel: Wash the peel tissue three times with sterile water and grind it into slurry in a sterile mortar. Take 1 mL of slurry and dilute it with 10 mL and 20 mL of sterile water respectively. Finally, take 50 µL of the original slurry and the diluted solution and spread them on LB and NA medium respectively. Incubate for 48 hours and then perform streak purification. Pulp: scrape the mango pulp juice onto LB medium and culture for 48 h before streak purification, or take 1 mL of mango pulp juice and dilute it with 10 mL and 20 mL of sterile water respectively, and finally take 50 µL of the dilution and spread it on NA and LB medium respectively; Core: Use a scalpel to scrape the juice, pulp, and fiber from the core and place them on LB medium for 48 hours before streak purification.

[0008] Preferably, the identification of the biocontrol bacteria in step S3 includes morphological identification, physiological and biochemical identification tests and molecular biological identification of the biocontrol bacteria.

[0009] Preferably, the morphological identification method is to inoculate the biocontrol bacteria onto a NA culture medium plate by streak method, culture at 28°C for 48 hours, observe and record the colony morphology, color, size, edge, surface, transparency, ridge shape, etc. of the biocontrol bacteria, and perform Gram staining and microscopic morphology observation.

[0010] Preferably, the physiological and biochemical identification test includes the following methods: (1) VP test: Inoculate the bacteria into a glucose-peptone water culture medium and culture at 28°C for 2-4 days. Take a certain amount of the culture medium with a pipette, add an equal amount of 40% NaOH and a small amount of creatine, mix well, shake vigorously or heat in a water bath, and observe the color of the culture medium after 20 minutes. If the culture medium turns red, it is positive (+), otherwise it is negative (-); (2) Catalase test: The bacteria to be tested were cultured on NA medium for 24 h, and a few drops of H 2 O 2, observe the bubble generation, if a large number of bubbles are generated within 30 seconds it is positive (+), if no bubbles are generated it is negative (-); (3) Starch hydrolysis test: The fresh strain to be tested is inoculated on a broth peptone medium plate containing 0.2% soluble starch and cultured at 28°C for 3-5 days. After obvious colonies are formed, Lugol's iodine solution is added. If a transparent or white band appears around the strain and is not stained by the iodine solution, it means that the strain has the ability to decompose starch and the reaction is positive (+). If there is no white or transparent band around the colony, the reaction is negative (-). (4) Gelatin hydrolysis test: Take the strain cultured for 24 hours and inoculate it into gelatin medium, and set up a blank control without inoculation. Incubate at 20℃ for 5, 10 and 15 days to observe whether the gelatin liquefies. If part or all of the gelatin clots turn into a flowable liquid, it is a positive gelatin hydrolysis test (+). If there is no depression on the gelatin surface and it is a stable clot, it means that the strain has no ability to liquefy gelatin, which is a negative gelatin hydrolysis test (-). (5) Glucose fermentation test: Take the strain to be tested that has been cultured for 24 hours and inoculate it into a glucose medium. Culture it at 28°C for 3-7 days. If the inoculated bacteria can decompose a certain sugar or alcohol in the medium, it can produce acid, causing the medium to change from purple to yellow, which is positive (+). If it remains purple, it is negative (-). (6) Methyl red test: Inoculate the bacteria to be tested after culturing for 2 days into a glucose-peptone water culture medium and incubate at 28°C for 48-72 hours. After taking out, add 3-5 drops of methyl red reagent. If the culture medium is red, it is positive (+), and if it is yellow, it is negative (-); (7) Aerobic assay: The bacteria to be tested, which have been cultured for 2 days, are inoculated into the bottom of the culture medium using an inoculation needle of 208 mm in length. The results are observed after 3 days. If the colonies grow along the surface of the culture medium, it indicates that the bacteria are aerobic and is positive (+). If the colonies grow along the puncture line, it indicates that the bacteria are anaerobic and is negative (-). (8) Casein hydrolysis test: The bacteria to be tested are spotted on a milk plate after being cultured for 2 days and cultured at 28°C for 3-5 days. Record whether the casein around and below the colonies has been decomposed and become transparent. If transparent, it is positive (+); if opaque, it is negative (-). (9) Phosphate solubilization ability test: The antagonistic bacteria screened were inoculated onto NBRIP medium and cultured at 28°C for 4-5 days. The appearance of a transparent circle was observed. If the bacteria decomposed and became transparent, it was positive (+), and if there was no transparent circle, it was negative (-). (10) Potassium-solubilizing ability test: The antagonistic bacteria screened were inoculated onto Alexander silicate medium and cultured at 28°C for 4-5 days. The presence of a transparent zone was observed. If the zone was decomposed and transparent, it was positive (+), and if there was no transparent zone, it was negative (-). (11) Nitrogen fixation ability test: The antagonistic bacteria screened were inoculated onto Asbby medium and cultured at 28°C for 4-5 days. The presence of a transparent zone was observed. If the zone decomposed and became transparent, it was positive (+); if there was no transparent zone, it was negative (-).

[0011] Preferably, the method for molecular biological identification of the biocontrol bacteria is: using the Trelief® Bacteria Genomic DNA Kit bacterial genomic DNA extraction kit to extract the DNA of the biocontrol bacteria, using the genomic DNA as a template, using the bacterial 16S rDNA gene sequence universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') to perform PCR amplification on the target strain, and performing bidirectional sequencing on the PCR product. The obtained sequencing results are subjected to BLAST comparison analysis of the sequences in the GenBank database, and the sequences of the related model strains are downloaded, and a phylogenetic tree is constructed using MEGA-X software.

[0012] Preferably, the method for measuring the quality-related indicators of the sample in step S4 is as follows: S41. Determination of titratable acid content: accurately weigh 10g pulp homogenate, add 20mL distilled water, shake well, heat in 80℃ water bath for 30min, then transfer to 10mL centrifuge tube, centrifuge at 8000r / min, 4℃ for 10min, take supernatant and make up to 50mL with distilled water, shake well, take 10ml sample solution, transfer to a conical flask, add 2-3 drops of 1% phenolphthalein indicator, titrate with calibrated 0.01mol / LNaOH, titrate until the solution turns slightly red and does not fade within 30s as the endpoint (PH=8.1~8.3), record the amount of NaOH, measure three fruits in each treatment group, repeat the measurement three times for each mango fruit, titrate with distilled water instead of filtrate as blank control, and calculate as follows: titratable acid content = ×100%; where V is the total volume of sample extract (mL); Vs is the volume of filtrate taken during titration (mL); c is the concentration of NaOH solution (mol / L); V1 is the volume of NaOH solution consumed in titrating the filtrate (mL); V0 is the volume of NaOH solution consumed in titrating distilled water (mL); m is the sample mass (g); f is the conversion factor (g / mmol), calculated based on malic acid 0.067; S42, determination of soluble solids: take a certain amount of mango homogenate, centrifuge at 8000rpm for 10min, take an appropriate amount of supernatant and use a refractometer to determine the soluble solids content (%) in each sample, measure three fruits for each treatment, measure each mango fruit three times, and take the average value; S43. Determination of hardness: Use a fruit hardness meter to measure the hardness of mango fruit. Peel off a thin layer of skin (about 2 mm thick and more than 1 mm in diameter) at the equator of the mango. Then take two points at the fruit pedicle and the fruit end equidistant from the equator and measure them again. Measure 3 points for each fruit and calculate the average value, which is the hardness of the fruit (Kg / cm2). S44. Determination of soluble sugar: The total soluble sugar content of the sample was determined by anthrone colorimetry.

[0013] The invention has the advantages that: the biocontrol bacteria of the pathogenic bacteria of the main post-harvest mango diseases are separated and screened from the mango flesh, peel and core by the plate confrontation method, and the identification is carried out by combining morphological, physiological and biochemical determination with 16S DNA sequence analysis, and the influence of the biocontrol bacteria on the storage quality of the post-harvest mango is studied, so as to provide a basis for improving the post-harvest quality and commodity value of the mango and extending the shelf life; the results show that 116 bacterial strains are separated from the mango flesh, peel and core tissues, among which 23 biocontrol strains have antagonistic effects on the pathogenic bacteria Phomopsis sp. of mango stem rot, the strain numbered FP-KT-3 has the best inhibitory effect on the pathogenic bacteria of the genus Phomopsis, and the inhibitory rate thereof is as high as 74.89%, and it is identified as Bacillus velez; 10 3 times (~2×10 8 CFU / mL)、10 4 times (~2×10 7 CFU / mL)、10 5 times (~2×10 6 Mangoes were treated with a suspension of Bacillus Velez-Nematode at 100 CFU / mL. The suspensions of different concentrations significantly maintained the contents of titratable acid, soluble solids and soluble sugar in mangoes in the late storage period, indicating that treatment of mangoes with Bacillus Velez-Nematode could significantly slow down the softening rate of the fruit and maintain the flavor and nutritional quality of the fruit. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The colony morphology (A) and Gram staining (B) of the biocontrol bacterium FP-KT-3; Figure 2 Results of FP-KT-3 physiological and biochemical tests, including: a: protein hydrolysis test, b: starch hydrolysis test, c: catalase test, d: gelatin liquefaction test, e: VP test, f: aerobic determination test, g: methyl red test, h: glucose oxidation and fermentation test; Figure 3 Neighbour-joining phylogenetic tree constructed based on 16S rDNA gene sequence; Figure 4 Effects of biocontrol bacteria on titratable acidity in mango fruit: F1:10 3 (~2×108 CFU / mL), F2: 10 4 times (~2×10 7 CFU / mL), F3: 10 5 times (~2×10 6 CFU / mL), CK: (control); Figure 5 Effect of biocontrol bacteria on mango fruit firmness: F1:10 3 (~2×10 8 CFU / mL), F2: 10 4 times (~2×10 7 CFU / mL), F3: 10 5 times (~2×10 6 CFU / mL), CK: (control); Figure 6 Effects of biocontrol bacteria on soluble solids in mango fruit: F1:10 3 (~2×10 8 CFU / mL), F2: 10 4 times (~2×10 7 CFU / mL), F3: 10 5 times (~2×10 6 CFU / mL), CK: (control); Figure 7 Effects of biocontrol bacteria on soluble sugars in mango fruit: F1:10 3 (~2×10 8 CFU / mL), F2: 10 4 times (~2×10 7 CFU / mL), F3: 10 5 times (~2×10 6 CFU / mL), CK: (control). DETAILED DESCRIPTION

[0015] In order to further explain the technical solution of the present invention, it is described in detail below through specific embodiments.

[0016] The present invention provides a method for identifying the effect of Bacillus Velez on the storage quality of mangoes. The identification method is as follows: 1. Selection and preparation of materials and reagents Kate mango: purchased from Dalduo Supermarket in Baoshan City, Yunnan Province. The product is ripe, the fruit is fresh and intact, and the size is uniform. 1% phenolphthalein indicator: Dissolve 1.0 g of phenolphthalein in 100 mL of ethanol (95%) to obtain 1% phenolphthalein indicator, and store it at room temperature for later use; 0.01mol / L NaOH solution: Take 0.2g of sodium hydroxide, put it in a clean beaker, add distilled water, stir with a glass rod until it is completely dissolved, transfer all the solution to a 500mL volumetric flask, make up to volume, and store at room temperature for later use; Anthrone reagent: 200 mg of anthrone is dissolved in 100 mL of concentrated sulfuric acid. Prepare and use immediately; Glucose standard solution (100 μg / mL): 100 mg of dry glucose, distilled water to make up to 1000 mL for later use.

[0017] 2. Culture medium Table 1 Test medium Table 1 Culture medium 3. Instruments and Equipment BSD-100 constant temperature incubator, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; V-500 visible spectrophotometer, Shanghai Yuanxi Instrument Co., Ltd.; PL203 electronic balance, Mettler-Toledo Instrument (Shanghai) Co., Ltd.; HHS-21-6 electric constant temperature water bath, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; GY-4 fruit hardness tester, Zhejiang Top Instrument Co., Ltd.; Microfuge® 20R desktop microcentrifuge, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory.

[0018] IV. Methods 1. Separation of biocontrol bacteria: Take healthy mangoes without disease spots and wash them with clean water outside the clean bench, then disinfect them with 75% alcohol spray for 2-5 minutes, and then put them on the clean bench for ultraviolet sterilization for 15 minutes, and then separate the biocontrol bacteria from the mango peel, pulp and core in turn.

[0019] Peel: Wash the peel tissue three times with sterile water and grind it into slurry in a sterile mortar. Take 1 mL of the slurry and dilute it with 10 mL and 20 mL of sterile water respectively. Finally, take 50 µL of the original slurry and the diluted solution and spread them on LB and NA culture media respectively, culture for 48 hours, and then perform streak purification.

[0020] Pulp: Scrape the mango pulp juice onto LB medium and culture for 48 hours before streak purification, or take 1mL of mango pulp juice and dilute it with 10mL and 20mL of sterile water respectively, and finally take 50µL of the dilution and spread it on NA and LB medium respectively.

[0021] Core: Use a scalpel to scrape the juice, pulp, and fiber from the core and place them on LB medium for 48 hours before streak purification.

[0022] 2. Screening of biocontrol bacteria: Activate the pathogen of mango stem rot, Phomopsis spp., on PDA plates and culture at 28°C for 5-7 days. Use a 6mm diameter puncher to take a bacterial cake and inoculate it in the center of a new PDA medium. Use a sterilized toothpick to inoculate different biocontrol bacteria around the indicator bacteria. The distance between the inoculation point and the bacterial cake is about 2.5mm. Use a PDA plate inoculated with only pathogens as a blank control and culture at 28°C until the hyphae of the control plate reaches the edge of the plate. Observe the growth of pathogens and whether there is an inhibition zone or inhibition zone around them. Use the same method to screen the strains with antagonistic effects. Use the cross method to measure the colony diameter of the pathogens, and calculate the average value and inhibition rate, where the inhibition rate = (1-colony diameter of the treatment group / colony diameter of the control group) × 100%; 3. Identification of biocontrol bacteria: The methods refer to the "Handbook of Systematic Identification of Common Bacteria" (Dong Xiuzhu and Cai Miaoying, 2001) and "Bergery's Manual of Bacterial Identification", etc., and perform Gram staining and a series of physiological and biochemical tests on the bacteria, and analyze the results.

[0023] Morphological identification: The biocontrol bacteria were inoculated onto NA medium plates by streak method and cultured at 28°C for 48 hours. The colony morphology, color, size, edge, surface, transparency, ridge shape, etc. of the biocontrol bacteria were observed and recorded, and Gram staining and microscopic morphology observation were performed.

[0024] Physiological and biochemical identification tests: (31) VP test: Inoculate the bacteria into a glucose-peptone water culture medium and culture at 28°C for 2-4 days. Take a certain amount of the culture medium with a pipette, add an equal amount of 40% NaOH and a small amount of creatine, mix well, shake vigorously or heat in a water bath, and observe the color of the culture medium after 20 minutes. If the culture medium turns red, it is positive (+), otherwise it is negative (-). (32) Catalase test: The bacteria to be tested were cultured on NA medium for 24 h, and a few drops of H 2 O 2 , observe the bubble generation, if a large number of bubbles are generated within 30 seconds it is positive (+), if no bubbles are generated it is negative (-); (33) Starch hydrolysis test: The fresh strain to be tested is inoculated on a broth peptone medium plate containing 0.2% soluble starch and cultured at 28°C for 3-5 days. After obvious colonies are formed, Lugol's iodine solution is added. If a transparent or white band appears around the strain and is not stained by the iodine solution, it means that the strain has the ability to decompose starch and the reaction is positive (+). If there is no white or transparent band around the colony, the reaction is negative (-). (34) Gelatin hydrolysis test: Take the strain cultured for 24 hours and inoculate it into gelatin medium, and set up a blank control without inoculation. Incubate at 20°C for 5, 10 and 15 days to observe whether the gelatin liquefies. If part or all of the gelatin clots turn into a flowable liquid, it is a positive gelatin hydrolysis test (+). If there is no depression on the gelatin surface and it is a stable clot, it means that the strain has no ability to liquefy gelatin, and it is a negative gelatin hydrolysis test (-). (35) Glucose fermentation test: Take the strain to be tested that has been cultured for 24 hours and inoculate it into a glucose medium. Culture it at 28°C for 3-7 days. If the inoculated bacteria can decompose a certain sugar or alcohol in the medium, it will produce acid, causing the medium to change from purple to yellow, which is positive (+). If it remains purple, it is negative (-). (36) Methyl red test: Inoculate the bacteria to be tested after culturing for 2 days into a glucose-peptone water culture medium and incubate at 28°C for 48-72 hours. After taking out the culture medium, add 3-5 drops of methyl red reagent. If the culture medium turns red, it is positive (+), and if it turns yellow, it is negative (-). (37) Aerobic assay: The bacteria to be tested, which have been cultured for 2 days, are inoculated into the bottom of the culture medium using a 208 mm long inoculation needle. The results are observed after 3 days. If the colonies grow along the surface of the culture medium, it indicates that the bacteria are aerobic and is positive (+). If the colonies grow along the puncture line, it indicates that the bacteria are anaerobic and is negative (-). (38) Casein hydrolysis test: The bacteria to be tested are spotted on a milk plate after being cultured for 2 days and cultured at 28°C for 3-5 days. Record whether the casein around and below the colonies has been decomposed and become transparent. If transparent, it is positive (+); if opaque, it is negative (-). (39) Phosphate solubilization ability test: The antagonistic bacteria screened were inoculated onto NBRIP medium and cultured at 28°C for 4-5 days. The appearance of a transparent zone was observed. If the zone decomposed and became transparent, it was positive (+), and if there was no transparent zone, it was negative (-). (310) Potassium-dissolving ability test: The antagonistic bacteria screened were inoculated onto Alexander silicate medium and cultured at 28°C for 4-5 days. The presence of a transparent zone was observed. If the zone was decomposed and transparent, it was positive (+), and if there was no transparent zone, it was negative (-). (311) Nitrogen fixation ability test: The antagonistic bacteria screened were inoculated onto Asbby medium and cultured at 28°C for 4-5 days. The appearance of a transparent zone was observed. If the zone decomposed and became transparent, it was positive (+); if there was no transparent zone, it was negative (-).

[0025] The method for molecular biological identification of biocontrol bacteria is as follows: the DNA of biocontrol bacteria is extracted using the Trelief® Bacteria Genomic DNA Kit, and the genomic DNA is used as a template to perform PCR amplification of the target strain using the bacterial 16S rDNA gene sequence universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'- GGTTACCTTGTTACGACTT-3'). The PCR product is sent to Kunming Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The sequencing results are subjected to BLAST comparison analysis of the sequences in the GenBank database, and the sequences of related model strains are downloaded, and the phylogenetic tree is constructed using MEGA-X software.

[0026] 4. Mango preservation test treatment: Select fresh mangoes of uniform size, no mechanical damage on the appearance, and uniform maturity, and divide them into 4 groups: 3 times (~2×10 8 CFU / mL)、10 4 times (~2×10 7 CFU / mL)、10 5 times (~2×10 6 Mangoes were immersed in a suspension of Bacillus Velez-Nematode at 20 CFU / mL for 25 min, with sterile water as the control. After drying, the mangoes were packed in a fresh-keeping bag with a thickness of 0.01 mm and placed in a constant temperature box at 25±0.5℃. The quality-related indicators of the samples were measured and analyzed every 4 days.

[0027] (41) Determination of titratable acid content: Accurately weigh 10g of pulp homogenate, add 20mL of distilled water, shake well, heat in a water bath at 80℃ for 30min, then transfer to a 10mL centrifuge tube, centrifuge at 8000r / min and 4℃ for 10min, take the supernatant and dilute to 50mL with distilled water, and shake well. Take 10ml of sample solution, transfer to a conical flask, add 2-3 drops of 1% phenolphthalein indicator, and titrate with calibrated 0.01mol / LNaOH. Titrate until the solution turns slightly red and does not fade within 30s (PH=8.1~8.3), record the amount of NaOH used, measure three fruits in each treatment group, and repeat the measurement three times for each mango fruit. Use distilled water instead of filtrate for titration as blank control. The calculation formula is as follows: Titratable acid content = ×100%; where V is the total volume of sample extract (mL); Vs is the volume of filtrate taken during titration (mL); c is the concentration of NaOH solution (mol / L); V1 is the volume of NaOH solution consumed in titrating the filtrate (mL); V0 is the volume of NaOH solution consumed in titrating distilled water (mL); m is the sample mass (g); f is the conversion factor (g / mmol), calculated based on malic acid 0.067; (42) Determination of soluble solids: Take a certain amount of mango homogenate, centrifuge at 8000 rpm for 10 min, take an appropriate amount of supernatant and use a refractometer to determine the soluble solids content (%) in each sample. Measure three fruits for each treatment, measure each mango fruit three times, and take the average value; (43) Determination of hardness: The hardness of mango fruit was measured using a fruit hardness meter. A thin layer of skin (about 2 mm thick and more than 1 mm in diameter) was peeled off at the equator of the mango. Two points were measured at the fruit pedicle and the fruit end equidistant from the equator. Three points were measured for each fruit, and the average value was calculated as the hardness of the fruit (kg / cm2). (44) Determination of soluble sugar: Anthrone colorimetry is a sensitive, rapid and simple method for determining the total soluble sugar content of a sample. Preparation of glucose standard curve: Take 8 dry and clean test tubes, add different volumes of 100 μg / mL glucose standard solution and distilled water as shown in the table below, and prepare a series of standard glucose solutions of different concentrations. Place them in an ice water bath and cool for 5 min. Then, add 4.0 mL of anthrone reagent to each test tube in sequence, shake and mix thoroughly. After adding each test tube, place it in a boiling water bath and heat it for 10 min. Cover the tube mouth to prevent evaporation. After taking it out, cool it to room temperature in running water. Use a 1 cm thick colorimetric dish and adjust the zero value with tube No. 1 as the blank control. Rapidly measure the light absorption value of each tube at a wavelength of 620 nm. Draw a standard curve with the absorbance value as the ordinate and the standard glucose content (μg) as the abscissa, and find the linear regression equation.

[0028] Table 2: Preparation of standard curve for determination of total soluble sugar by anthrone colorimetric method Sample preparation: Weigh 0.5g pulp homogenate into a test tube using an analytical balance, add 10mL distilled water, and seal with plastic wrap. Shake well and place in a 80℃ hot water bath for 30min, remove and cool to room temperature. Centrifuge at 8000r / min for 10min, take the supernatant and repeat centrifugation once.

[0029] Sample determination: Take 1mL of the supernatant and put it into a clean test tube, add 4mL of anthrone reagent, boil it in a boiling water bath for 10 minutes, take it out and cool it. Using distilled water as a reference, measure the light absorption value of each tube on a spectrophotometer at a wavelength of 620nm, repeat three times, and calculate the average value. The calculation formula is as follows: Total Sugar = 00% M' is the sugar content (μg) obtained from the standard curve; V is the total volume of the sample extract (mL); N is the dilution factor of the sample extract; Vs is the volume of the sample extract taken during the determination (mL); and M is the sample mass (g).

[0030] 5. Data processing: SPSS statistics 16.0 software was used for statistical analysis of the data. The Duncan's multiple comparison method was used to analyze the significance of the differences, and Origin2021 software was used to draw the graphs.

[0031] 5. Results Analysis 1. Isolation and screening of biocontrol bacteria A total of 116 bacterial strains were isolated from mango pulp, peel and core tissues, and 23 biocontrol strains were obtained through plate confrontation test, which had antagonistic effects on the pathogen of mango stem rot Phomopsis sp. In order to further optimize the selection of biocontrol bacteria, 12 biocontrol bacteria with more significant inhibitory effects were selected for rescreening. The results showed that the strain numbered FP-KT-3 had the best inhibitory effect on Phomopsis sp. pathogens, with an inhibition rate of up to 74.89%. Based on the comprehensive antagonistic effect, the strain numbered FP-KT-3 was selected as the research object for subsequent experiments and analysis.

[0032] Table 3 Antagonistic effect of 12 bacteria on mango stem rot fungi measured by plate confrontation method Table 3 Antagonistic effect of 12 strains of bacteriaagainst Phomopsis sp. by plate confrontation method 2. Morphological identification The screened FP-KT-3 strain was streaked on NA medium for 24 h and the colony morphology was observed. Figure 1 As shown, the colonies of this strain are off-white, opaque, dry and slightly raised on the surface, and sticky in texture. Under a microscope, Gram staining of the bacteria is purple, indicating positive, and the cells are rod-shaped.

[0033] 3. Physiological and biochemical characteristics of biocontrol bacteria like Figure 2 As shown, the FP-KT-3 strain can hydrolyze starch, gelatin, protein, catalase, and glucose, all showing positive characteristics, and is negative in VP, nitrogen fixation, potassium solubilization, phosphorus solubilization, and aerobic test tests. The above results, combined with the Manual of Systematic Identification of Common Bacteria, can preliminarily identify this strain as Bacillus sp.

[0034] 4. Molecular identification like Figure 3As shown in the figure, the DNA of strain FP-KT-3 was used as a template, and the 16S rDNA sequence was used for amplification and sequencing to obtain a gene fragment of 1444bp in length. The phylogenetic tree was constructed using MEGA-X software according to the Neighbor-Joining method. The phylogenetic tree results showed that the 16S rDNA gene sequence of strain FP-KT-3 was in the same branch as Bacillus velezensis, showing a support rate of 98%. Combined with the results of morphological and physiological and biochemical characteristics, strain FP-KT-3 can be identified as Bacillus velezensis.

[0035] 5. Effect of Bacillus Velez on titratable acid in mango like Figure 4 As shown in the figure, during the storage of mango fruit, the titratable acid content of each treatment group showed a significant downward trend. Compared with the CK group, the titratable acid content of the F1 and F2 treatment groups decreased relatively slowly in the first 4 days. In the later stage of treatment, both treatment groups were able to effectively maintain a high titratable acid content. Especially on the 16th day, the titratable acid content of the F2 treatment group was significantly higher than that of the control group and other treatment groups (P<0.05). Therefore, the use of F2 concentration of Bacillus Velez suspension to treat mango can effectively maintain its high titratable acid content in the later stage.

[0036] 6. Effect of Bacillus Velez on the firmness of mango like Figure 5 As shown in the figure, during the entire storage period, the hardness of mango fruit gradually decreased with the passage of time. On the 4th day of storage, the hardness of F1 and F2 fruits was 7.22 kg / cm 2 and 6.48kg / cm 2 Significantly higher than F3 and CK fruit hardness 5.23kg / cm 2 and 4.39kg / cm 2 (P<0.05), and there was no significant difference in fruit hardness between the treatment groups and the control group in the other storage time periods tested (P>0.05). Therefore, treating mango with F1-fold and F2-fold concentrations of Bacillus Velez suspension may have a slight effect on delaying the softening of fruit hardness.

[0037] 7. Effect of Bacillus Velez on Soluble Solids in Mango like Figure 6As shown in the results, the content of soluble solids increased in the early stage and decreased in the later stage. When comparing different treatment groups, it was found that the TSS content of mango fruit in the F2 treatment group and the F3 treatment group reached the maximum on the 4th day and then decreased rapidly. The TSS content of the fruit of the F1 treatment group and the control group reached the maximum on the 8th day and then began to decrease. On the 16th day of storage, the soluble solids of the F1 treatment group, the F2 treatment group and the F3 treatment group were 11.44%, 11.77% and 11.82%, respectively, which were significantly higher than the soluble solids content of the control group (10.84%) (P<0.05). Therefore, the use of Bacillus Velez suspension at F1, F2 and F3 concentrations to treat mango can effectively slow down the decline of the soluble solids content of mango and maintain a higher soluble solids content in the fruit in the later stage.

[0038] 8. Effect of Bacillus Velez on Soluble Sugar in Mango The flavor and taste of mango fruit are directly affected by changes in the soluble sugar content, which is the main source of the fruit's sweetness. Figure 7 As shown in the figure, the soluble solid content showed a trend of first increasing, then decreasing, and then increasing again during storage. On the 4th day, the soluble sugar content of both the CK group and the control group reached the highest peak, among which the growth rate of the CK group was the most significant, and its soluble sugar content was as high as 115.69%. However, the soluble sugar content then dropped rapidly, while the other treatment groups showed a relatively gentle downward trend and reached the lowest value on the 12th day. On the 16th day, the soluble sugar content of all treatment groups increased, especially the mango treated with F1 concentration of Bacillus Velez suspension, whose soluble sugar content was significantly higher than that of other groups (P<0.05). Therefore, treating mango with F1 concentration of Bacillus Velez suspension can effectively increase the soluble sugar content in the fruit in the late storage period, thereby possibly improving the flavor and taste of the fruit.

[0039] VI. Conclusion In this study, 23 strains with antagonistic effects on pathogens were obtained through plate confrontation test. Through repeated screening, it was found that the strain numbered FP-KT-3 had the best inhibitory effect on the mango stem rot pathogen Phomopsis, with an inhibition rate of up to 74.89%. Finally, the strain with the best antagonistic effect (numbered FP-KT-3) was used as the research object for the next step of research. After morphological and physiological and biochemical characterization, combined with 16S rDNA sequence analysis, FP-KT-3 was identified as Bacillus velezensis.

[0040] The standards for evaluating mango maturity can be divided into two categories: external and internal. Among them, the internal standards mainly include factors such as the firmness of the flesh, the soluble solids content, the titratable acid content, the pH value, and the soluble sugar content. The hardness of mango fruit is a key indicator to measure its ability to resist pressure, and is closely related to the length of the fruit's storage and transportation period and supply period. The greater the hardness, the more storable the fruit is. Soluble solids, soluble sugars, and titratable acid are important indicators to measure the nutritional value and flavor quality of the fruit. Among them, soluble solids usually include soluble sugars and pectin nutrients, which not only reflect the nutritional value of mangoes, but also affect its taste to a certain extent. Therefore, it can be used as an important indicator to reflect changes in nutrition and flavor. Titratable acid, as the main source of mango sourness, is an important flavor indicator of mango.

[0041] Storage potential plays an important role in mango sales decisions and is the primary indicator of concern to fruit merchants. Studies have shown that Bacillus Velez ZSY-1 has a significant control effect on the softening phenomenon of tomatoes after harvest by effectively reducing the release of ethylene, thereby maintaining its hardness. In this experiment, the hardness of mangoes treated with Bacillus Velez FP-KT-3 showed a gradual downward trend during storage, and this treatment significantly slowed down the softening rate of mango fruits. In addition, this study found that the suspension of Bacillus Velez FP-KT-3 can effectively maintain the content of titratable acid, soluble sugar and soluble solids in mangoes, thereby maintaining the flavor and nutritional quality of mangoes and further improving its storage quality. This is similar to the research results on the preservation effect of Bacillus Velez fermentation liquid on cherry tomatoes and grapes. The study also showed that the treatment delayed the decline in fruit hardness and brittleness, and at the same time, it effectively delayed the decline in titratable acid content, TSS and VC content, and maintained the flavor and nutritional quality of the fruit.

[0042] The above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for identifying the effect of Bacillus Velez on the storage quality of mango, wherein the Bacillus Velez was deposited in the China Center for Type Culture Collection on June 17, 2024, and its deposit number is CCTCC NO: M20241234, characterized in that: The steps include: S1. Separation of biocontrol bacteria: Take healthy mangoes without disease spots and rinse them with clean water outside the clean bench, then spray disinfect with 75% alcohol for 2-5 minutes, and then put them on the clean bench for ultraviolet sterilization for 15 minutes, and then separate the biocontrol bacteria from the mango peel, pulp and core in turn; S2. Screening of biocontrol bacteria: Phomopsis spp., the pathogen of mango stem rot, was activated on a PDA plate and cultured at 28°C for 5-7 days. A 6mm diameter puncher was used to take a bacterial cake and inoculated it in the center of a new PDA medium. A sterilized toothpick was used to inoculate different biocontrol bacteria around the indicator bacteria. The distance between the inoculation point and the bacterial cake was about 2.5 mm. A PDA plate inoculated with only pathogens was used as a blank control. The plate was cultured at 28°C until the hyphae of the control plate reached the edge of the plate. The growth of the pathogen and whether an inhibition zone or inhibition zone was formed around the plate were observed. The strains with antagonistic effects were rescreened using the same method. The colony diameter of the pathogen was measured by the cross method. The average value and inhibition rate were calculated, where the inhibition rate = (1-colony diameter of the treatment group / colony diameter of the control group) × 100%; S3. Identification of biocontrol bacteria: Gram staining and a series of physiological and biochemical tests are performed on the bacteria, and the results are analyzed; S4. Mango preservation test treatment: Select fresh mangoes of uniform size, no mechanical damage on the appearance, and uniform maturity and divide them into 4 groups: 3 times (~2×10 8 CFU / mL)、10 4 times (~2×10 7 CFU / mL)、10 5 times (~2×10 6 Mangoes were soaked in a suspension of Bacillus Velezii with a concentration of 100 CFU / mL for 25 min, and sterile water was used as a control. After drying, the mangoes were packed in a fresh-keeping bag with a thickness of 0.01 mm and placed in a constant temperature box at 25±0.5℃. The quality-related indicators of the samples were measured and analyzed every 4 days. S5. Data processing: SPSS statistics 16.0 software was used for statistical analysis of the data. The Duncan's multiple comparison method was used to determine the significance of the differences. Origin2021 software was used for drawing.

2. The method for identifying the effect of Bacillus Velez on the storage quality of mango according to claim 1, characterized in that: The method for separating the biocontrol bacteria from the mango peel, pulp and core in step S1 is as follows: Peel: Wash the peel tissue three times with sterile water and grind it into slurry in a sterile mortar. Take 1 mL of the slurry and dilute it with 10 mL and 20 mL of sterile water respectively. Finally, take 50 µL of the original slurry and the diluted solution and spread them on LB and NA medium in turn, culture for 48 hours, and then perform streak purification. Pulp: scrape the mango pulp juice onto LB medium and culture for 48 hours before streak purification, or take 1mL of mango pulp juice and dilute it with 10mL and 20mL of sterile water respectively, and finally take 50µL of the dilution and spread it on NA and LB medium respectively; Core: Use a scalpel to scrape the juice, pulp, and fiber from the core and place them on LB medium for 48 hours before streak purification.

3. The method for identifying the effect of Bacillus Velez on the storage quality of mango according to claim 1, characterized in that: The identification of the biocontrol bacteria in step S3 includes morphological identification, physiological and biochemical identification tests and molecular biological identification of the biocontrol bacteria.

4. The method for identifying the effect of Bacillus Velez on the storage quality of mango according to claim 3, characterized in that: The morphological identification method is to inoculate the biocontrol bacteria onto a NA culture medium plate by streaking, culture at 28° C. for 48 hours, observe and record the colony morphology, color, size, edge, surface, transparency, ridge shape, etc. of the biocontrol bacteria, and perform Gram staining and microscopic morphology observation.

5. The method for identifying the effect of Bacillus Velez on the storage quality of mango according to claim 3, characterized in that: The physiological and biochemical identification test includes the following methods: (1) VP test: Inoculate the bacteria into a glucose-peptone water culture medium and culture at 28°C for 2-4 days. Take a certain amount of culture medium with a pipette, add an equal amount of 40% NaOH and a small amount of creatine, mix well, shake vigorously or heat in a water bath, and observe the color of the culture medium after 20 minutes. If the culture medium turns red, it is positive, otherwise it is negative. (2) Catalase test: The bacteria to be tested are cultured on NA medium for 24 h, a few drops of H2O2 are added, and the generation of bubbles is observed. If a large number of bubbles are generated within 30 seconds, it is positive; if no bubbles are generated, it is negative. (3) Starch hydrolysis test: The fresh strain to be tested is inoculated on a broth peptone medium plate containing 0.2% soluble starch and cultured at 28°C for 3-5 days. After obvious colonies are formed, Lugol's iodine solution is added. If a transparent or white band appears around the strain and is not stained by the iodine solution, it means that the strain has the ability to decompose starch and the reaction is positive. If there is no white or transparent band around the colony, the reaction is negative. (4) Gelatin hydrolysis test: Take the strain cultured for 24 hours and inoculate it into gelatin medium, and set up a blank control without inoculation. Incubate at 20℃ for 5, 10 and 15 days to observe whether the gelatin liquefies. If part or all of the gelatin clots turn into flowable liquid, it is a positive result for gelatin hydrolysis. If there is no depression on the surface of the gelatin and it is a stable clot, it means that the strain has no ability to liquefy gelatin, which is a negative result for gelatin hydrolysis. (5) Glucose fermentation test: Take the strain to be tested that has been cultured for 24 hours and inoculate it into a glucose medium. Culture it at 28°C for 3-7 days. If the inoculated bacteria can decompose a certain sugar or alcohol in the medium, it can produce acid, causing the medium to change from purple to yellow, which is a positive result. If it remains purple, it is a negative result. (6) Methyl red test: Inoculate the bacteria to be tested after culturing for 2 days into a glucose-peptone water culture medium and incubate at 28°C for 48-72 hours. After taking out, add 3-5 drops of methyl red reagent. If the culture medium turns red, it is positive, and if it turns yellow, it is negative. (7) Aerobic assay: The bacteria to be tested, which have been cultured for 2 days, are inoculated into the bottom of the culture medium using an inoculation needle of 208 mm in length. The results are observed after 3 days. If the colonies grow along the surface of the culture medium, it indicates that the bacteria are aerobic and the result is positive. If the colonies grow along the puncture line, it indicates that the bacteria are anaerobic and the result is negative. (8) Casein hydrolysis test: The bacteria to be tested are spotted on a milk plate after being cultured for 2 days and cultured at 28°C for 3-5 days. Record whether the casein around and below the colonies has been decomposed and become transparent. If transparent, it is positive, and if opaque, it is negative. (9) Phosphate solubilization ability test: The antagonistic bacteria screened were inoculated onto NBRIP medium and cultured at 28°C for 4-5 days. The appearance of a transparent circle was observed. If the bacteria decomposed and became transparent, it was positive; if there was no transparent circle, it was negative. (10) Potassium-dissolving ability test: The antagonistic bacteria screened were inoculated onto Alexander silicate medium and cultured at 28°C for 4-5 days to observe whether a transparent zone appeared. If the bacteria decomposed and became transparent, it was positive; if there was no transparent zone, it was negative. (11) Nitrogen fixation ability test: The antagonistic bacteria screened were inoculated onto Asbby medium and cultured at 28°C for 4-5 days. The presence of a transparent zone was observed. If the zone decomposed and became transparent, it was positive; if there was no transparent zone, it was negative.

6. The method for identifying the effect of Bacillus Velez on the storage quality of mango according to claim 3, characterized in that: The method for molecular biological identification of the biocontrol bacteria is as follows: using a Trelief® Bacteria Genomic DNA Kit bacterial genomic DNA extraction kit to extract the DNA of the biocontrol bacteria, using the genomic DNA as a template, using bacterial 16S rDNA gene sequence universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') to perform PCR amplification on the target strain, performing bidirectional sequencing on the PCR product, performing BLAST comparison analysis on the sequence obtained in the GenBank database, downloading the sequence of the relevant model strain, and using MEGA-X software to construct a phylogenetic tree.

7. Determination of titratable acid content: accurately weigh 10g pulp homogenate, add 20mL distilled water, shake well, heat in 80℃ water bath for 30min, then transfer to 10mL centrifuge tube, centrifuge at 8000r / min, 4℃ for 10min, take supernatant and make up to 50mL with distilled water, shake well, take 10ml sample solution, transfer to a conical flask, add 2-3 drops of 1% phenolphthalein indicator, titrate with calibrated 0.01mol / LNaOH, titrate until the solution turns slightly red and does not fade within 30s as the end point, PH=8.1~8.3, record the amount of NaOH, measure three fruits in each treatment group, repeat the measurement three times for each mango fruit, titrate with distilled water instead of filtrate as blank control, and calculate as follows: titratable acid content = ×100%; among which, V is the total volume of sample extract; Vs is the volume of filtrate taken during titration; c is the concentration of NaOH solution; V1 is the volume of NaOH solution consumed in titrating the filtrate; V0 is the volume of NaOH solution consumed in titrating distilled water; m is the sample mass; f is the conversion factor, calculated as 0.067 for malic acid; S42, determination of soluble solids: take a certain amount of mango homogenate, centrifuge at 8000rpm for 10min, take an appropriate amount of supernatant and use a refractometer to determine the soluble solids content in each sample, measure three fruits for each treatment, measure each mango fruit three times, and take the average value; S43. Determination of hardness: The hardness of mango fruit is measured by using a fruit hardness meter. A thin layer of skin is peeled off at the equator of the mango, and then two points are measured at the fruit pedicle and the fruit end equidistant from the equator. Three points are measured for each fruit, and the average value is the hardness of the fruit. S44. Determination of soluble sugar: The total soluble sugar content of the sample was determined by anthrone colorimetry.

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