A high-temperature resistant antibacterial active fermentation broth and its application in the storage of cash crops

Abstract

The present invention discloses a high-temperature resistant antibacterial active fermentation broth, which is obtained by liquid fermentation of Bacillus velezensis ( Bacillus velezensis ), FS001. After centrifuging to remove the bacterial cells, it can be used directly or after sterilization; the fermentation broth has good high-temperature and acid-base resistance characteristics. When this high-temperature resistant antibacterial active fermentation broth is applied to cash crops and feed storage, the fermentation product has strong inhibitory activity against Aspergillus flavus, and has the effect of extending the storage period. The high-temperature resistant antibacterial active fermentation broth of the present invention has broad application prospects in the storage of cash crops and feeds.

Description

Technical Field

[0001] The present invention belongs to the technical field of applied microbiology, and particularly relates to a high-temperature resistant antibacterial active fermentation broth prepared by liquid fermentation of Bacillus velezensis FS001 ( Bacillus velezensis FS001), and its application in the storage of cash crops. Background Art

[0002] Food, feed, cash crops and their processed products are susceptible to infection by microorganisms (such as bacteria, fungi and actinomycetes, etc.) during storage. Among them, the most harmful are fungi and their mycotoxins. After mycotoxin contamination of animal products (such as animal tissues, eggs, milk, etc.) and grains, it is not easy to remove. It will not only reduce the nutritional value and economic value of the products, but also threaten human health through the food chain. In the links of animal and plant breeding, production, storage, processing, sales, etc., the control of bacterial and fungal diseases still relies on traditional chemical drugs and antibiotics. However, the improper use for a long time, in large doses and over a large area will lead to problems such as drug resistance of pathogenic bacteria, drug residues, and environmental pollution, posing potential safety hazards to human health. Therefore, it is imperative to study alternative solutions to protect food crops. Among these alternatives, biological control by beneficial microorganisms is a possible method to reduce the use of synthetic chemicals and their negative impact on the environment. Scientific evidence related to biological control shows that microorganisms such as yeasts, bacteria and fungi have the ability to mitigate mycotoxins by preventing the presence of mycotoxins in animal feed and animal-derived foods. Summary of the Invention

[0003] Aiming at the problems existing in the prior art, the present invention provides a high-temperature resistant antibacterial active fermentation broth, which is prepared by liquid fermentation of Bacillus velezensis ( Bacillus velezensis ), and after centrifugation to remove the bacterial cells, it can be used directly or after sterilization.

[0004] In order to achieve the above invention purpose, the present invention is realized through the following technical solutions:

[0005] Bacillus velezensis FS001 ( Bacillus velezensis FS001) in the present application has been publicly disclosed before the filing date of the present invention, and its preservation number in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 17946.

[0006] In the present invention, the liquid fermentation medium (GLB) of Bacillus velezensis FS001 is composed of 10 - 15 g of tryptone, 10 - 15 g of sodium chloride, 2 - 10 g of yeast extract, 10 - 20 g of corn starch, and 0.5 - 1.5 g of sodium dihydrogen phosphate. Add water, heat and boil until dissolved, make up the volume to 1 L, adjust the pH to 5.0 - 6.0 after cooling to room temperature, and sterilize at 121 °C under high pressure steam for 20 - 30 min.

[0007] The method for fermenting with Bacillus velezensis FS001 is as follows: (1) Prepare LB medium, sterilize it at 121 °C under high pressure steam for 20 min. Take out Bacillus velezensis FS001 stored in an ultra-low temperature refrigerator at -80 °C, streak and separate it on an LB solid plate for activation for 12 h. Pick a single colony on the plate and inoculate it into a test tube containing 5 mL of liquid LB medium, and culture it at 37 °C and 250 rpm with shaking for 12 h to obtain the seed liquid for subsequent fermentation; (2) Prepare GLB medium, inoculate the seed liquid into a 250 mL conical flask containing 50 mL of GLB medium, and culture it at 31 °C and 250 rpm with shaking for 36 h - 60 h. Centrifuge the fermentation broth at 10000 rpm for 10 min, discard the bacterial precipitate, collect the supernatant, filter and sterilize it through a 0.22 μm water-based filter membrane, and measure its inhibitory activity against Aspergillus flavus.

[0008] The method for measuring the inhibitory activity against Aspergillus flavus: (1) Elute the spores of Aspergillus flavus growing on a PDA plate with 0.01 mol / L phosphate buffer containing Tween 80 to make a uniform spore suspension and count it with a hemocytometer under a microscope; (2) Prepare PDA medium and sterilize it at 121 °C under high pressure steam for 20 min, then cool it to 45 °C - 60 °C and add the spore suspension of Aspergillus flavus to make the spore concentration in the system 1×10 4 -1×10 6 CFU / mL, and then pour the plate evenly; (3) After completely cooling and solidifying, germinate it in a constant temperature incubator at 28 °C for 0 h - 8 h; use an 8 mm puncher to punch holes evenly on the germinated plate, add 100 μL of sterile supernatant, and culture it statically in a constant temperature incubator at 28 °C for 60 h - 72 h, take it out, and measure the diameter of the inhibition zone by the cross method.

[0009] The fermentation product of Bacillus velezensis FS001 of the present invention is treated at 20 °C, 40 °C, 60 °C, 80 °C, and 100 °C, and its activity is not affected. After treatment at 121 °C, its activity is slightly lost, decreasing by 5.8%.

[0010] After the fermentation product of Bacillus velezensis FS001 of the present invention is treated with acids and bases at different levels (pH 3 - 12), the retention rate of its antibacterial activity is above 90%, and the activity is the lowest at pH 12, only decreasing by 8.04%.

[0011] The present invention applies the high-temperature resistant antibacterial active fermentation broth to the storage of corn germ meal: Sterile storage solution a: The fermentation broth is sterilized by high-temperature steam at 121 °C for 20 min, cooled to room temperature, centrifuged at 10000 rpm for 10 min, the thalli are removed, and the supernatant solution a is retained. The solution a is diluted 10 times and 100 times with distilled water to obtain 10-fold and 100-fold solution a. Bacteria-containing storage solution b: The fermentation broth is centrifuged at 10000 rpm for 10 min, the thalli are removed, and the supernatant solution is retained to obtain solution b. The solution b is diluted 10 times and 100 times with distilled water to obtain 10-fold and 100-fold solution b. The storage solution is added to the corn germ meal inoculated with Aspergillus flavus spores after sterilization, and the corn germ meal added with sterile water is used as a blank control. The moisture content is adjusted to 28%, and stored for 15 d. Three replicates are set for each group; the total number of molds and bacteria and the nutritional quality (crude protein, crude fat, crude ash) before, during and after storage are measured, and the inhibition rate of Aspergillus flavus, the disease index and the relative prevention and control effect are compared to evaluate its storage and preservation performance.

[0012] The present invention applies the high-temperature resistant antibacterial active fermentation broth to the storage of peanuts and wheat: Select intact peanut and wheat grains, disinfect the peanuts and wheat with 1% sodium hypochlorite, and then rinse them 3 times with sterile water. Load them into screw-cap bottles, with 100 g of peanuts or wheat in each bottle. Preparation of storage solution: The fermentation broth is sterilized by high-temperature steam at 121 °C for 20 min, cooled to room temperature, centrifuged at 10000 rpm for 10 min, the thalli are removed, and the supernatant solution is retained to obtain storage solution ①. The storage solution ① is diluted 10 times and 100 times with sterile distilled water to obtain 10-fold storage solution ② and 100-fold storage solution ③. Storage setting: Add an equal amount of 10 mL of the storage solution to the treatment group, and add an equal amount of 10 mL of sterile distilled water to the blank control group, and set three replicates. Add 1 mL of 10 6 CFU / mL of Aspergillus flavus spore suspension, and stir evenly. Adjust the moisture content in the system to about 28% with sterile distilled water, then shake the screw-cap bottle to evenly cover the spores on the peanuts and wheat, cover with a breathable sealing film, and store statically in an incubator at 28 °C for 15 d. Count the number of Aspergillus flavus in the peanuts and wheat after storage for 15 d, and calculate the inhibition rate.

[0013] Advantages and technical effects of the present invention: The preparation method of the Bacillus velezensis FS001 fermentation broth of the present invention is simple, and the raw materials are easily obtained. It is flexible to use and not restricted by the object. Whether to use the fermentation broth after high-temperature sterilization can be selected according to the application scenario. When the fermentation broth is used for grains that need to be stored after high-temperature treatment, such as after high-temperature exposure or steaming, it can still play a preservation role; for example, when the fermentation broth is applied to corn, it can ensure that its protein and fat are not damaged. Because the active substances in the fermentation broth can withstand high temperatures of 121°C, have a wide pH tolerance, can tolerate a variety of metal ions (the fermentation broth cannot be stored in iron containers), and have the characteristics of ultraviolet resistance and protease resistance, they can adapt to different harsh conditions in various application scenarios, maintain their antibacterial activity, and play the best effect, so as to extend the shelf life of cash crops, feeds, etc., retain their quality to the greatest extent, and increase the economic value. Description of the Drawings

[0014] Figure 1 It is a schematic diagram of the inhibitory effect of the Bacillus velezensis FS001 fermentation broth on Aspergillus flavus. In the figure, a is the LB medium, b is the blank control, and c is the GLB medium;

[0015] Figure 2 It is the result of the temperature tolerance of the Bacillus velezensis FS001 fermentation broth;

[0016] Figure 3 It is the result of the acid-base tolerance of the Bacillus velezensis FS001 fermentation broth;

[0017] Figure 4 It is the result of the metal ion tolerance of the Bacillus velezensis FS001 fermentation broth;

[0018] Figure 5 It is the result of the ultraviolet tolerance of the Bacillus velezensis FS001 fermentation broth;

[0019] Figure 6 It is the result of the protease tolerance of the Bacillus velezensis FS001 fermentation broth. Detailed Embodiments

[0020] The technical solution of the present invention will be further described in detail below through examples. However, the content of the present invention is not limited thereto. In the examples, the methods are conventional methods unless otherwise specified, and the materials, reagents, etc. are obtained from commercial sources unless otherwise specified;

[0021] Example 1: Preparation of the Fermentation Broth of Bacillus velezensis FS001

[0022] 1. Streak inoculate Bacillus velezensis FS001 stored in DMSO at -80°C onto a solid LB medium and culture it in an incubator at 37°C for 12 h. Then pick a single colony and inoculate it into a liquid LB medium containing 5 mL, and culture it with shaking at 37°C and 250 rpm for 8 h to obtain a fermentation seed solution.

[0023] Prepare the fermentation medium GLB: 10 g of tryptone, 10 g of sodium chloride, 5 g of yeast extract, 15 g of corn starch, 1.0 g of sodium dihydrogen phosphate. Add water and heat to boiling until dissolved, make up the volume to 1 L with deionized water, adjust the pH to 5.0 after cooling to room temperature, and sterilize at 121°C under high pressure steam for 25 min.

[0024] Preparation of FS001 fermentation broth: Inoculate the seed solution into a 250 mL conical flask containing 50 mL of GLB medium, culture it with shaking at 31°C and 250 rpm for 48 h. Centrifuge the fermentation broth at 10000 rpm for 10 min, discard the bacterial precipitate, collect the supernatant, filter and sterilize it through a 0.22 μm water-based filter membrane, and measure its inhibitory activity against Aspergillus flavus.

[0025] 2. Use the agar diffusion method to measure the inhibitory activity against Aspergillus flavus

[0026] Elute the Aspergillus flavus spores growing on the PDA plate with a phosphate buffer solution containing Tween 80 to make a uniform spore suspension and count it. After preparing the PDA medium and sterilizing it at 121°C under high pressure steam for 20 min, cool it to 50°C and add the Aspergillus flavus spore suspension to make the spore concentration in the system 1×10 4 -1×10 6 CFU / mL. Mix well and pour into plates. After completely cooling and solidifying, germinate it in an incubator at 28°C for 6 h. Use an 8 mm punch to punch holes evenly on the germinated plate, add 100 μL of sterile supernatant, and statically culture it in an incubator at 28°C for 48 h. Take it out and measure the diameter of the inhibition zone by the cross method ( Figure 1 )

[0027] Example 2: Characteristics of the fermentation product of Bacillus velezensis FS001

[0028] (1) Thermal stability

[0029] Take an equal amount of the FS001 fermentation broth in Example 1 in a sterile test tube, keep it in a constant temperature water bath at 20, 40, 60, 80, and 100 °C for 30 min, perform high-temperature sterilization treatment at 121 °C for 30 min, take it out immediately after the treatment and cool it to room temperature in an ice bath. Use the untreated fermentation broth as a control, measure its antibacterial activity retention rate. Treating for 30 min at 20, 40, 60, and 80 °C has no significant effect on the activity of inhibiting Aspergillus flavus, and the activity increases by 5.28% after treatment at 100 °C; after treatment at 121 °C and above, the activity is well maintained, being 94.20%, only decreasing by 5.8%, and the change is not significant ( Figure 2 )

[0030] (2) Acid and alkali tolerance

[0031] Use 6 mol / L HCl and 3 moL / L NaOH solutions to adjust the pH of the fermentation broth in Example 1 to 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. After maintaining at room temperature for 1 h, adjust it to pH 7.0. Use the original fermentation broth (pH 7.0 - 7.2) added with an equal amount of sterile water as a control, measure its antibacterial activity retention rate. The results show that the antibacterial activity retention rate of the fermentation broth is above 90%. The activity is the lowest at pH 12, only decreasing by 8.04% ( Figure 3 )

[0032] (3) Metal ion tolerance

[0033] Prepare solutions with concentrations of 100 mmol / L of Ca 2+ , Zn 2+ , Li + , Na + , Mn 2+ , Mg 2+ , Fe 2+ , Fe 3+ , K + , Cu 2+ . After filtering and sterilizing through a 0.22 μm filter membrane, set aside. Take an equal amount of the fermentation broth in Example 1 (900 μL) in centrifuge tubes respectively, and add the above metal ion solutions (100 μL) respectively to make the final concentrations of Ca 2+ , Zn 2+ , Li + , Na + , Mn 2+ , Mg 2+ , Fe 2+ , Fe 3+ , K + , Cu 2+ in the system be 10 mmol / L. Use the fermentation broth added with the same volume of sterile water as a control. After standing at room temperature for 2 h, measure its antibacterial activity; its activity is affected to varying degrees. Among them, the fermentation broth has an impact on Fe3+ Intolerance and loss of activity occur, so iron containers need to be avoided during storage or use; it shows good tolerance to other metal ions in the experiment ( Figure 4 ).

[0034] (4)Ultraviolet tolerance

[0035] The fermentation broth of Example 1 was irradiated at a distance of 30 cm from a UV lamp (30 W, wavelength 253.7 nm) for 10, 20, 30, 40, 50, 60, and 120 min respectively. The untreated fermentation broth was used as a control to measure its antibacterial activity; with the extension of the UV irradiation time, even after 2 h of irradiation, the antibacterial activity of the fermentation broth was still well maintained and basically unchanged ( Figure 5 ).

[0036] (5)Protease tolerance

[0037] Trypsin, pepsin, papain, alkaline protease, acidic protease, neutral protease, and proteinase K were prepared into enzyme solutions with an activity of 100 U / mL using 25 mmol / L phosphate buffer (pH 7.0), and added to an equal volume of the fermentation broth of Example 1. The pH was adjusted to the optimal pH for each enzyme's action, so that the final mass concentration of each enzyme in the system was 10 U / mL. The sterile supernatant with an equal volume of deionized water added was used as a control. After water-bathing in a 37°C water bath for 2 h, the enzymes were inactivated by boiling and then cooled, and the pH was adjusted back to 7.0. It was made up to the same volume with sterile deionized water, and the antibacterial activity retention rate was measured; each protease had no significant effect on the activity of the fermentation broth, and its activity remained above 95% after treatment and was not affected by its degradation ( Figure 6 ).

[0038] Example 3: Application of the fermentation broth of Bacillus velezensis FS001 in the storage of corn germ meal

[0039] (1)Preparation of storage solution

[0040] Sterile storage solution a: The fermentation broth of Example 1 was sterilized by high-temperature steam at 121°C for 20 min, cooled to room temperature, and then centrifuged at 10000 rpm for 10 min. The supernatant was retained after removing the bacteria to obtain it. It was diluted 10-fold and 100-fold with distilled water to obtain 10-fold and 100-fold solution a;

[0041] Bacteria-containing storage solution b: The fermentation broth of Example 1 was centrifuged at 10000 rpm for 10 min, and the supernatant was retained after removing the bacteria to obtain it. It was diluted 10-fold and 100-fold with distilled water to obtain 10-fold and 100-fold solution b;

[0042] (2) Sample treatment: Referring to GB / T 10358-2008 / ISO 771:1977 Determination of moisture and volatile matter content in oilseed cakes and meals, the moisture content in corn germ meal was determined, and the measured moisture content was 7.17%±0.24%. According to the ratio of 8:1, corn germ meal and water were mixed evenly. 90 g of corn germ meal was dispensed into 250 mL screw-cap bottles and sealed with a breathable membrane. It was sterilized with high-temperature steam at 121 °C for 20 min, and after sterilization, it was cooled to room temperature;

[0043] (3) Add 10 mL of different storage solutions (see Table 1) to the corn germ meal treated in step (2). The blank control group was added 10 mL of sterile distilled water, and three replicates were set for each group; then 1 mL of 10 7 CFU / mL Aspergillus flavus spore suspension was added, and it was stirred evenly to make the concentration of Aspergillus flavus spores in the system reach 10 5 CFU / g. According to the original moisture content of corn germ meal measured previously, the moisture content in the system was adjusted to about 28% with sterile distilled water, and then it was stirred evenly. The sealing film was covered, and it was left standing in a constant-temperature incubator at 28 °C for 15 d; The following were added to the experimental group and the control group:

[0044] Table 1 Detailed table of storage content

[0045]

[0046] (4) 90-day storage setting: In experimental group C, 90 g of corn germ meal (not sterilized) was dispensed into 250 mL screw-cap bottles. The experimental group was added 10 mL of sterile storage solution a, and the control group was added 10 mL of sterile distilled water. Three replicates were set for each group. It was stirred evenly and sealed with a breathable membrane, and it was left standing in a constant-temperature incubator at 28 °C for 90 d. The total number of colonies and nutritional quality indicators were measured every 30 d. (Note: The sample corn germ meal in experimental groups A and B had been sterilized, while experimental group C had not been sterilized);

[0047] (5) Mold and bacteria counting: Referring to GB 13092 Determination of total mold count in feeds and GB 13093 Determination of total bacteria count in feeds, the mold and bacteria in corn germ meal at different storage dates were counted; In the 15-day storage group, samples were taken and measured every 3 d, and in the 90-day storage group, samples were taken and measured every 30 d. Each time, 10 g of samples were evenly taken under sterile operation and put into a 250 mL conical flask containing 90 mL of sterile normal saline, and it was shaken and mixed evenly for 30 min to obtain a 1:10 dilution. The remaining operations were the same, and three replicates were done for each group;

[0048] (6) Aspergillus flavus inhibition rate, disease index, and relative control effect: Based on the mold count, calculate the number of Aspergillus flavus in the sample. The Aspergillus flavus inhibition rate is calculated according to formula (1):

[0049] Inhibition rate (%) = (1 - total number of Aspergillus flavus in the treatment group / total number of Aspergillus flavus in the control group) × 100% (1)

[0050] According to the growth and contamination area of Aspergillus flavus in corn germ meal, classify its contamination level:

[0051] Table 2 Classification table of Aspergillus flavus contamination level

[0052]

[0053] Calculate the disease index and control effect (%) according to formulas (2) and (3):

[0054] Disease index = ∑(number of diseased at each level × corresponding disease level number) × 100 / (total number of investigations × highest level number) (2)

[0055] Control effect (%) = (control disease index - treatment disease index) × 100 / control disease index (3)

[0056] (7) Changes in nutritional quality of corn germ meal before and after storage

[0057] Crude protein: Refer to the Kjeldahl method for the determination of protein in foods in GB5009.5 - 2016, and determine the crude protein content of corn germ meal before and after storage in this study.

[0058] Crude fat: Refer to the Soxhlet extraction method for the determination of fat in foods in GB5009.6 - 2016, and determine the crude fat content of corn germ meal before and after storage in this study.

[0059] Ash: Refer to the determination of ash in foods in GB5009.4 - 2016, and determine the crude ash content of corn germ meal before and after storage in this study.

[0060] (8) Result analysis

[0061] a. Total number of bacteria: The results are shown in Table 3. In experimental Group A with aseptic storage solution a added, throughout the storage period, except for occasional contamination by 1 or 2 bacteria (negligible) during sample dilution plate counting in the later stage (6d), there was no significant bacterial growth. For experimental Group B with bacteria-containing storage solution b added, FS001 did not show significant growth but also did not die significantly, and there was no contamination by miscellaneous bacteria during colony counting on the plate. FS001 is an aerobic bacterium that forms highly stress-resistant spores in harsh environments. During storage, all groups were kept static in a ventilated constant-temperature incubator, with no air circulation inside the samples and low moisture being unfavorable for its reproduction;

[0062] Table 3 Total number of bacteria in different treatment groups

[0063] ;

[0064] b. Total number of molds: The results are shown in Table 4. Throughout the storage experiment, no growth of Aspergillus flavus was observed in all experimental groups (Group A and Group B), and no growth of molds or miscellaneous bacteria was seen during mold counting on the plate. Only in the blank control group (CK) was there significant proliferation of Aspergillus flavus, which rapidly increased from 1.26×10 5 CFU / g on the 3rd day to 1.34×10 9 CFU / g. There was no sign of growth of Aspergillus flavus in the experimental groups. It is speculated that during the treatment of samples with storage solution, Aspergillus flavus was inhibited by the antibacterial substances in the FS001 fermentation broth or its spores were damaged, and spore germination was blocked, losing the ability to reproduce, indicating that this fermentation broth has good application potential in the storage of food crops and feeds;

[0065] Table 4 Total number of molds in different treatment groups

[0066] ;

[0067] c. Total number of colonies in experimental Group C: After 90 days of storage, significant mildew occurred in the CK group, i.e., the blank control. The total number of molds increased from 8.63×10 2 CFU / g on the 0th day to 5.41×10 6 CFU / g on the 90th day, an increase of more than 6000 times; the total number of bacteria increased from 2.96×10 3 CFU / g on the 0th day to 6.72×10 4 CFU / g on the 90th day, an increase of 22 times. In Group C, the total number of molds increased from 8.63×10 2 CFU / g on the 0th day to 7.83×10 2 CFU / g on the 90th day, and the total number of bacteria increased from 2.96×10 3 CFU / g on the 0th day to 1.79×10 3CFU / g, with no obvious increase, indicating that the antibacterial active substances in the fermentation broth can better control the microbial growth of corn germ meal during storage, thus reducing the quality deterioration caused by microorganisms;

[0068] Table 5 Total number of colonies at different storage times

[0069]

[0070] d. Aspergillus flavus inhibition rate and relative control effect: As shown in Table 6, since Aspergillus flavus did not grow in the experimental groups (Group A and Group B), therefore, its relative control effect was 100% and the disease index was 0%, which further confirmed the good ability of FS001 fermentation broth to inhibit Aspergillus flavus.

[0071] Table 6 Disease indices in different treatment groups

[0072] 。

[0073] e. Changes in nutritional quality of corn germ meal before and after storage

[0074] The conventional nutritional components (crude protein, crude fat, crude ash) of corn germ meal were measured before and after storage, aiming to evaluate the freshness preservation ability of FS001 fermentation broth, analyze its storage characteristics, and the impact of Aspergillus flavus on the nutritional quality of corn germ meal. The relevant measurement results are shown in Table 7:

[0075] Table 7 Nutritional quality of corn germ meal in different treatment groups

[0076]

[0077] During the entire storage process, no growth of Aspergillus flavus was observed in the experimental groups, and only the blank control group was infected with Aspergillus flavus, with its Aspergillus flavus inhibition rate and relative control effect reaching 100%. Measuring the nutritional quality of corn germ meal before and after storage found that Group A could better ensure that the crude protein, crude fat, and ash of corn germ meal were not damaged and lost; Group B could significantly increase the content of crude protein and crude ash; in the CK group, except for no significant change in crude ash, its crude protein and crude fat were significantly reduced.

[0078] Nutritional quality changes in experimental Group C: As shown in Table 8, comparing the quality of corn germ meal (unsterilized) before and after 90 days of storage and after using the storage liquid, it was found that except for no significant change in crude ash in all three groups, the crude protein and crude fat contents in Group C had no significant change compared with the corn germ meal before storage, while the crude protein in the CK group was significantly reduced by 7.6760% and the crude fat was significantly reduced by 0.6058%. Thus, it can be seen that the FS001 fermentation product can better ensure the quality of corn germ meal during storage and extend the shelf life.

[0079] Table 8 Nutritional Quality of Corn Germ Meal with Different Storage Times

[0080] 。

[0081] Example 4: Application of the Fermentation Broth of Bacillus velezensis FS001 in the Storage of Peanuts and Wheat

[0082] (1) Sample treatment: Select intact peanut and wheat grains, disinfect peanuts and wheat with 1% sodium hypochlorite, then rinse with sterile water 3 times, and put them into screw-cap bottles, with 100 g of peanuts or wheat in each bottle;

[0083] (2) Preparation of storage solution: Sterilize the fermentation broth with high-temperature steam at 121 °C for 20 min, cool to room temperature and then centrifuge at 10000 rpm for 10 min to remove the cells and retain the supernatant to obtain storage solution ①. Dilute it 10-fold and 100-fold with sterile distilled water to obtain storage solutions ② 10-fold and ③ 100-fold.

[0084] (3) Storage setting: Add 10 mL of storage solutions ①, ②, and ③ to the treatment groups in step (1) respectively. The blank control group is added with an equal amount of 10 mL of sterile distilled water, and three replicates are set. Add 1 mL of 10 6 CFU / mL of Aspergillus flavus spore suspension, stir evenly to make the concentration of Aspergillus flavus spores in the system reach 10 4 CFU / g. Adjust the moisture content in the system to about 28% with sterile distilled water, then shake the screw-cap bottle to evenly cover the spores on peanuts and wheat, cover with a breathable sealing film, and store statically in an incubator at 28 °C for 15 d.

[0085] (3) Counting of Aspergillus flavus: Refer to GB 4789.15 to count the molds in peanuts and wheat after 15 d of storage and calculate the inhibition rate. Take 25 g of samples evenly by aseptic operation, put them into a 500 mL conical flask containing 225 mL of normal saline with 0.05% Tween 80, shake and mix evenly for 30 min to evenly elute the Aspergillus flavus spores on the surface to obtain a 1:10 dilution. The remaining operations are the same.

[0086] (4) Experimental results: In the Aspergillus flavus control experiment, after peanuts and wheat were stored at 28 °C for 15 d, the total number of Aspergillus flavus counted is shown in Table 7:

[0087] Table 7 Total Number of Aspergillus flavus and Its Inhibition Rate after Storage of Peanuts and Wheat

[0088]

[0089] During the entire storage process, no growth of Aspergillus flavus was observed in the peanut and wheat experimental groups ①②③, and only the blank control group was infected by Aspergillus flavus. The inhibition rate of Aspergillus flavus reached 100%, indicating that the fermentation broth had good storage effects in storage applications.

Claims

1. Application of a high-temperature resistant antibacterial active fermentation broth in the storage of cash crops or feeds, wherein the high-temperature resistant antibacterial active fermentation broth is prepared by liquid fermentation of Bacillus velezensis ( Bacillus velezensis ), FS001, and removing the bacterial cells by centrifugation. The specific preparation method is as follows: (1) Prepare LB medium, sterilize it at 121 °C under high-pressure steam for 20 min. Take out Bacillus velezensis FS001 stored in an ultra-low temperature refrigerator at -80 °C, streak and separate it on an LB solid plate for activation for 12 h. Pick a single colony on the plate and inoculate it into a test tube containing 5 mL of liquid LB medium, and culture it at 37 °C and 250 rpm for 12 h as the seed liquid for subsequent fermentation; (2) Prepare GLB medium, inoculate the seed liquid into a 250 mL conical flask containing 50 mL of GLB medium, and culture it at 31 °C and 250 rpm for 36 h - 60 h. Centrifuge the fermentation broth at 10000 rpm for 10 min, discard the bacterial precipitate, and collect the supernatant and filter it through a 0.22 μm water-based filter membrane to remove bacteria.

2. According to the application described in claim 1, wherein: The cash crops are corn, wheat or peanuts.

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