A kind of Bacillus Velez IFST-Xd-4 and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a Bacillus Velez subtilis IFST‑Xd‑4 and an application thereof. The deposit number of the Bacillus Velez subtilis IFST‑Xd‑4 is: CGMCC No.29967. The application includes: degrading anti-nutritional factors and zearalenone, and the anti-nutritional factors include one or more of crude fiber, neutral detergent fiber, acid detergent fiber or phytic acid. The present invention screened and obtained a strain of Bacillus Velez subtilis, and after research, it was found that it has the ability to degrade anti-nutritional factors and zearalenone in corn husks. The Bacillus Velez subtilis IFST‑Xd‑4 provided by the present invention can be used to inhibit harmful substances and microorganisms in corn husks, improve antioxidant capacity, and thereby improve the feeding value of corn husks, which is of great significance in the field of improving the utilization rate of by-products in the corn processing industry.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to Bacillus Velez IFST-Xd-4 and application thereof. Background Art

[0002] Corn is one of the main food crops with a high annual output. A part of it is used for industrial production, and about 30% of processing by-products are produced. Among the by-products of corn processing, corn husk accounts for the highest proportion, about 14%. The crude protein content of corn husk is 11.8%, the crude fat content is 6%, and it is rich in dietary fiber (80%), corn fiber oil (3.5%), feruloyl oligosaccharides and other active substances. It has high nutritional value and can be used as a rich source of feed raw materials for feed production. However, corn husk is very susceptible to fungal toxin contamination, which seriously threatens livestock safety. Pollution surveys show that the detection rate of zearalenone (ZEN) in corn husk is as high as 89%. ZEN can bind to estrogen receptors and cause symptoms similar to estrogen excess. It mainly harms the reproductive system of animals. It also has immunotoxicity and can cause acute and chronic poisoning in animals, causing abnormal reproductive function and even death of animals. In addition, anti-nutritional factors such as non-starch polysaccharides and phytic acid phosphorus in corn husks can also affect the digestion and absorption of nutrients by animals.

[0003] Solid-state fermentation mainly uses agricultural by-products as raw materials. Under the premise of meeting the nutritional level of microorganisms, by creating a suitable humidity, temperature and acid-base environment, the relevant components in the feed raw materials are degraded and transformed by microbial fermentation, and high-quality feed products with good palatability, high digestibility, and can improve animal immunity are produced. Corn husks are rich in carbon sources and are a potential nutrient. Through microbial fermentation, macromolecular substances such as crude fiber in corn husks can be decomposed into small molecular substances that are easily used and absorbed by animals. At the same time, the mycotoxin content in corn husks can be effectively reduced, and the feeding value of corn husks can be improved. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a Bacillus Velez subtilis IFST-Xd-4 and application thereof.

[0005] The present invention screened a microorganism in the soil that can degrade harmful substances in corn husks and improve antioxidant capacity, and the microorganism was identified as Bacillus Velez through physical and chemical properties. The present invention further carried out biological preservation of the microorganism, and the preservation information is as follows:

[0006] Deposit number: CGMCC No.29967; Classification name: Bacillus velezensis; Deposit unit: General Microbiology Center of China Culture Collection Administration; Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: March 8, 2024.

[0007] In a first aspect, the present invention provides a bacterial agent, including the Bacillus Velez IFST-Xd-4 or its fermentation product. The fermentation product of the present invention can be the supernatant obtained by fermentation of the microorganism (excluding the microorganism itself) or the whole product including the microorganism itself.

[0008] Furthermore, the bacterial agent is a solid bacterial agent, a liquid bacterial agent or a gel bacterial agent, and the total viable count of the Velez subtilis IFST-Xd-4 in the bacterial agent is 1×10 7 - 10 cfu / g.

[0009] In a second aspect, the present invention provides the use of the Bacillus Velezii IFST-Xd-4 or the bacterial agent in degrading zearalenone.

[0010] The present invention further provides the use of the Bacillus Velezii IFST-Xd-4 or the bacterial agent in any of the following:

[0011] i) degrading anti-nutritional factors; the anti-nutritional factors include one or more of crude fiber, neutral detergent fiber, acid detergent fiber or phytic acid;

[0012] ii) Improve antioxidant capacity;

[0013] iii) inhibiting microorganisms; said microorganisms include Escherichia coli, Staphylococcus aureus and Salmonella.

[0014] Furthermore, the improvement of antioxidant capacity is to increase the scavenging rate of ABTS and DPPH free radicals.

[0015] The present invention further provides the use of the Bacillus Velezii IFST-Xd-4 or the bacterial agent in improving the feeding value of corn husk.

[0016] The present invention verifies through experiments that after inoculating Bacillus Velez subtilis IFST-Xd-4 into corn husks, the levels of zearalenone and anti-nutritional factors in the corn husks can be effectively reduced, thereby significantly improving the feeding value of the corn husks.

[0017] In a third aspect, the present invention provides a fermentation method of Bacillus Velezii, comprising:

[0018] The peanut meal and corn husk are mixed, and then inoculated with the seed liquid of the Bacillus Velez IFST-Xd-4 and fermented.

[0019] Furthermore, the mass ratio of the peanut meal to the corn husk is 1:(1-5).

[0020] Furthermore, the inoculation amount of the Bacillus Velez IFST-Xd-4 is 5-20 v / w%.

[0021] Furthermore, the fermentation conditions include: fermentation time is more than 36 hours, and fermentation temperature is 30-42°C.

[0022] The present invention has the following beneficial effects:

[0023] The present invention screens and isolates a strain of Bacillus Velez, which is named Bacillus Velez IFST-Xd-4. The Bacillus Velez IFST-Xd-4 provided by the present invention has significantly strong ability to degrade zearalenone, remove ABTS and DPPH free radicals, inhibit microorganisms and degrade anti-nutritional factors (crude fiber, neutral detergent fiber, acid detergent fiber and phytic acid, etc.). The Bacillus Velez IFST-Xd-4 provided by the present invention can be used to degrade harmful substances in corn husks, improve the feeding value of corn husks, and is conducive to improving the utilization rate of corn industry processing by-products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is a graph showing the degradation rate of ZEN degradation by Bacillus Velez subtilis IFST-Xd-4 provided in Example 1 of the present invention changing over time.

[0026] Figure 2 This is a diagram for optimizing the ratio of corn husk and peanut meal provided in Example 2 of the present invention.

[0027] Figure 3 This is an optimization diagram of the solid-state fermentation time of Bacillus Velez subtilis IFST-Xd-4 provided in Example 3 of the present invention.

[0028] Figure 4 This is an optimization diagram of the solid-state fermentation temperature of Bacillus Velez IFST-Xd-4 provided in Example 4 of the present invention.

[0029] Figure 5 This is an optimization diagram of the inoculation amount of Bacillus Velez IFST-Xd-4 provided in Example 5 of the present invention.

[0030] Figure 6 This is a graph showing the results of the scavenging rates of ABTS and DPPH free radicals by Bacillus Velez IFST-Xd-4 provided in Example 7 of the present invention.

[0031] Figure 7 This is a graph showing the inhibition results of Bacillus Velez subtilis IFST-Xd-4 on microorganisms provided in Example 8 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0034] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources, for example:

[0035] Example 1 Degradation of ZEN Standard by Bacillus Velez

[0036] This embodiment provides a kind of Bacillus Velezii ( Bacillus velezensis )IFST-Xd-4, and its application in the degradation of zearalenone (ZEN), including:

[0037] 1. Preparation of Bacillus Velez subtilis liquid: inoculate Bacillus Velez subtilis IFST-Xd-4 (inoculation amount 1%) into seed culture medium, culture at a shaking speed of 180 rpm at 37°C for 24 h until the OD600 value is greater than 3.0 to obtain seed liquid, wherein the formula of the seed culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, prepared in distilled water.

[0038] 2. Degradation of ZEN standard: Take 20 mL of cultured bacterial solution, add ZEN with a final concentration of 2 µg / mL, and degrade at 37°C and 180 rpm. Take samples every 12 hours and add an equal amount of methanol to terminate the reaction.

[0039] 3. ZEN extraction and detection: The sample obtained in step 2 was centrifuged at 8000rpm for 10min, and 2mL of supernatant was taken and passed through the ZEN immunoaffinity column. The flow rate was adjusted to 1-2 drops / s until the liquid completely passed through the immunoaffinity column; 10mL of pure water was passed through the affinity column at a flow rate of 1-2 drops / s to clean the affinity column; finally, 1mL of anhydrous methanol was used to elute the affinity column at a flow rate of 1-2 drops / s, and the eluate was collected in a 1.5mL centrifuge tube, filtered with a 0.22µm organic phase nylon membrane, and then loaded into a 2mL chromatographic injection vial to obtain a sample solution.

[0040] 4. ZEN detection: The sample solution obtained in step 3 was detected by HPLC. The HPLC detection conditions were as follows: mobile phase acetonitrile: water = 7:3; flow rate 1 mL / min; chromatographic column C18 (250 mm × 4.6 mm, 5 µm); excitation wavelength 2740 nm, detection wavelength 450 nm; column temperature 30 °C; injection volume 20 µL.

[0041] The results are as follows Figure 1 As shown: Bacillus Velez IFST-Xd-4 can achieve the best degradation effect on 2µg / mL ZEN after 48h, and the degradation rate can reach more than 90%.

[0042] Example 2 Optimization of the ratio of corn husk and peanut meal

[0043] In this example, Bacillus Velez IFST-Xd-4 is applied to ZEN degradation in corn husks and corn meal, including:

[0044] 1. Preparation of seed solution: Inoculate Bacillus Velez IFST-Xd-4 (inoculation amount 1%) into the seed culture medium, culture at a shaking speed of 180 rpm at 37°C for 24 h until the OD600 value is greater than 3.0 to obtain the seed solution, wherein the formula of the seed culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and distilled water.

[0045] 2. Fermentation culture: peanut meal and corn husk are mixed in the ratio of 1:1, 1:2 and 1:4 (mass ratio), 500g of the mixed raw materials are weighed, and the same mass of water is added. After fully mixing, sterilize at 121℃, cool to room temperature, inoculate the seed solution in step 1 according to the inoculation amount of 5% (v / w) of the raw materials, mix well, place in a 37℃ incubator, and ferment for 72 hours. Stir and mix the fermentation raw materials in the clean bench every 12 hours. Take samples at 0h and 72h respectively.

[0046] 3. ZEN extraction and detection: The sample obtained in step 2 was added with 80% methanol solution at a ratio of 1:5 (w / v), ultrasonically extracted for 30 min, centrifuged at 8000 rpm for 10 min, 5 mL of supernatant was taken and passed through the ZEN immunoaffinity column, and the flow rate was adjusted to 1-2 drops / s until the liquid completely passed through the immunoaffinity column; 10 mL of pure water was passed through the affinity column at a flow rate of 1-2 drops / s to clean the affinity column; finally, 1 mL of anhydrous methanol was used to elute the affinity column at a flow rate of 1-2 drops / s, and the eluate was collected in a 1.5 mL centrifuge tube, filtered with a 0.22 µm organic phase nylon membrane, and then loaded into a 2 mL chromatographic injection vial to obtain a sample solution.

[0047] 4. ZEN detection: The sample solution obtained in step 3 was detected by HPLC. The HPLC detection conditions were as follows: mobile phase acetonitrile: water = 7:3; flow rate 1 mL / min; chromatographic column C18 (250 mm × 4.6 mm, 5 µm); excitation wavelength 2740 nm, detection wavelength 450 nm; column temperature 30 °C; injection volume 20 µL.

[0048] Test results such as Figure 2 As shown: when the ratio of peanut meal and corn husk is 1:1, the degradation rate of zearalenone in corn husk is the highest, reaching 95%.

[0049] Example 3 Fermentation time optimization

[0050] The present invention further optimizes the fermentation time for Bacillus Velezii IFST-Xd-4, including:

[0051] 1. Preparation of seed solution: Inoculate Bacillus Velez IFST-Xd-4 (inoculation amount 1%) into the seed culture medium, culture at a shaking speed of 180 rpm at 37°C for 24 h until the OD600 value is greater than 3.0 to obtain the seed solution, wherein the formula of the seed culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and distilled water.

[0052] 2. Fermentation culture: Mix peanut meal and corn husk in a ratio of 1:1, weigh 500g of the mixed raw materials, add equal weight of water, mix thoroughly, sterilize at 121℃, cool to room temperature, inoculate the seed solution in step 1 at 5% (v / w) of the raw materials, mix thoroughly, place in a 37℃ incubator, and ferment for 96 hours. Stir and mix the fermentation raw materials thoroughly in a clean bench every 12 hours. Take samples at 0h, 24h, 48h, 72h and 96h, dry and crush.

[0053] 3. ZEN extraction and detection: The sample obtained in step 2 was added with 80% methanol solution at a ratio of 1:5 (w / v), ultrasonically extracted for 30 min, centrifuged at 8000 rpm for 10 min, 5 mL of supernatant was taken and passed through the ZEN immunoaffinity column, and the flow rate was adjusted to 1-2 drops / s until the liquid completely passed through the immunoaffinity column; 10 mL of pure water was passed through the affinity column at a flow rate of 1-2 drops / s to clean the affinity column; finally, 1 mL of anhydrous methanol was used to elute the affinity column at a flow rate of 1-2 drops / s, and the eluate was collected in a 1.5 mL centrifuge tube, filtered with a 0.22 µm organic phase nylon membrane, and then loaded into a 2 mL chromatographic injection vial to obtain a sample solution.

[0054] 4. ZEN detection: The sample solution obtained in step 3 was detected by HPLC. The HPLC detection conditions were as follows: mobile phase acetonitrile: water = 7:3; flow rate 1 mL / min; chromatographic column C18 (250 mm × 4.6 mm, 5 µm); excitation wavelength 2740 nm, detection wavelength 450 nm; column temperature 30 °C; injection volume 20 µL.

[0055] Test results such as Figure 3 As shown: After 48 hours of solid-state fermentation, the degradation effect of ZEN in corn husk by Bacillus Velez IFST-Xd-4 was the best, and the degradation rate reached 98%.

[0056] Example 4 Fermentation temperature optimization

[0057] The present invention further optimizes the fermentation temperature for Bacillus Velez IFST-Xd-4, including:

[0058] 1. Preparation of seed solution: Inoculate Bacillus Velez IFST-Xd-4 (inoculation amount 1%) into the seed culture medium, culture at a shaking speed of 180 rpm at 37°C for 24 h until the OD600 value is greater than 3.0 to obtain the seed solution, wherein the formula of the seed culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and distilled water.

[0059] 2. Fermentation culture: peanut meal and corn husk are mixed in a ratio of 1:1, 500g of the mixed raw materials are weighed, and the same mass of water is added. After fully mixing, sterilize at 121℃, cool to room temperature, inoculate the seed solution in step 1 according to the inoculation amount of 5% (v / w) of the raw materials, mix well, and place them in 20℃, 25℃, 28℃, 30℃, 37℃, 42℃, 45℃, and 50℃ incubators for solid-state fermentation for 48h. Every 12h, the fermentation raw materials are fully stirred and mixed in the clean bench. Samples are taken at 0h and 48h respectively.

[0060] 3. ZEN extraction and detection: The sample obtained in step 2 was added with 80% methanol solution at a ratio of 1:5 (w / v), ultrasonically extracted for 30 min, centrifuged at 8000 rpm for 10 min, 5 mL of supernatant was taken and passed through the ZEN immunoaffinity column, and the flow rate was adjusted to 1-2 drops / s until the liquid completely passed through the immunoaffinity column; 10 mL of pure water was passed through the affinity column at a flow rate of 1-2 drops / s to clean the affinity column; finally, 1 mL of anhydrous methanol was used to elute the affinity column at a flow rate of 1-2 drops / s, and the eluate was collected in a 1.5 mL centrifuge tube, filtered with a 0.22 µm organic phase nylon membrane, and then loaded into a 2 mL chromatographic injection vial to obtain a sample solution.

[0061] 4. ZEN detection: The sample solution obtained in step 3 was detected by HPLC. The HPLC detection conditions were as follows: mobile phase acetonitrile: water = 7:3; flow rate 1 mL / min; chromatographic column C18 (250 mm × 4.6 mm, 5 µm); excitation wavelength 2740 nm, detection wavelength 450 nm; column temperature 30 °C; injection volume 20 µL.

[0062] Test results such as Figure 4 As shown: There was no significant difference in the degradation of ZEN in corn husk by solid-state fermentation of Bacillus Velez IFST-Xd-4 at 30-42℃, and the degradation effect could reach more than 90%.

[0063] Example 5 Optimization of inoculum size

[0064] The present invention further optimizes the inoculation amount for Bacillus Velezii IFST-Xd-4, including:

[0065] 1. Preparation of seed solution: Inoculate Bacillus Velez IFST-Xd-4 (inoculation amount 1%) into the seed culture medium, culture at a shaking speed of 180 rpm at 37°C for 24 h until the OD600 value is greater than 3.0 to obtain the seed solution, wherein the formula of the seed culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and distilled water.

[0066] 2. Fermentation culture: Mix peanut meal and corn husk in a ratio of 1:1, weigh 500g of the mixed raw materials, add equal weight of water, mix thoroughly, sterilize at 121℃, cool to room temperature, inoculate the seed solution in step 1 at an inoculation rate of 1%, 3%, 5%, 10%, 15% and 20% (v / w) of the raw materials, mix thoroughly, place in a 37℃ incubator, and ferment for 48h. Stir and mix the fermentation raw materials thoroughly in a clean bench every 12h. Take samples at 0h and 48h respectively.

[0067] 3. ZEN extraction and detection: The sample obtained in step 2 was added with 80% methanol solution at a ratio of 1:5 (w / v), ultrasonically extracted for 30 min, centrifuged at 8000 rpm for 10 min, 5 mL of supernatant was taken and passed through the ZEN immunoaffinity column, and the flow rate was adjusted to 1-2 drops / s until the liquid completely passed through the immunoaffinity column; 10 mL of pure water was passed through the affinity column at a flow rate of 1-2 drops / s to clean the affinity column; finally, 1 mL of anhydrous methanol was used to elute the affinity column at a flow rate of 1-2 drops / s, and the eluate was collected in a 1.5 mL centrifuge tube, filtered with a 0.22 µm organic phase nylon membrane, and then loaded into a 2 mL chromatographic injection vial to obtain a sample solution.

[0068] 4. ZEN detection: The sample solution obtained in step 3 was detected by HPLC. The HPLC detection conditions were as follows: mobile phase acetonitrile: water = 7:3; flow rate 1 mL / min; chromatographic column C18 (250 mm × 4.6 mm, 5 µm); excitation wavelength 2740 nm, detection wavelength 450 nm; column temperature 30 °C; injection volume 20 µL.

[0069] Test results such as Figure 5 As shown in the figure: the inoculation amount has a significant effect on the degradation of ZEN in corn husk by solid-state fermentation of Bacillus Velez subtilis IFST-Xd-4, but when the inoculation amount is between 5% and 20%, the degradation rate of ZEN can reach more than 97%.

[0070] Example 6 Effect of Bacillus Velez on Anti-nutritional Factors in Corn Husk

[0071] The present invention further detects the effect of Bacillus Velez IFST-Xd-4 on anti-nutritional factors in corn husk, including:

[0072] 1. Preparation of seed solution: Inoculate Bacillus Velez IFST-Xd-4 (inoculation amount 1%) into the seed culture medium, culture at a shaking speed of 180 rpm at 37°C for 24 h until the OD600 value is greater than 3.0 to obtain the seed solution, wherein the formula of the seed culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and distilled water.

[0073] 2. Fermentation culture: Mix peanut meal and corn husk in a ratio of 1:1, weigh 500g of the mixed raw materials, add equal weight of water, mix thoroughly, sterilize at 121℃, cool to room temperature, inoculate the seed solution in step 1 at 5% (v / w) of the raw materials, mix thoroughly, place in a 37℃ incubator, and ferment for 72 hours. Stir and mix the fermentation raw materials thoroughly in a clean bench every 12 hours. Take samples at 0h, 24h, 48h and 72h, dry and crush.

[0074] 3. Detection of anti-nutritional factors in corn husk:

[0075] (1) The extraction and determination methods of crude fiber refer to GB / T6434-2022 Determination of crude fiber content in feed, GB / T20806-2006 Determination of neutral detergent fiber (NDF) in feed and NY / T1459-2022 Determination of acid detergent fiber in feed.

[0076] (2) The extraction method of phytic acid is as follows:

[0077] Weigh 10g of the sample obtained in step 2 and place it in a conical flask, add 40mL of sodium sulfate-hydrochloric acid extraction solution (sodium sulfate concentration is 10%, hydrochloric acid concentration is 1.2%), shake and extract for 2h, centrifuge the extract at 5000rpm for 5min, collect the supernatant, and dilute to 50mL with sodium sulfate-hydrochloric acid extraction solution, filter through rapid filter paper for use. Take 0.5g of anion exchange resin (AG1-X4106µm-250µm, ion exchange capacity 3.5mmol / g) and wet-load it into an empty column tube, wash the ion exchange column with 15mL of 0.7mol / L sodium chloride solution and 20mL of water respectively. Take 5mL of filtrate, add 1mL of 30g / L sodium hydroxide solution, dilute to 30mL with water, mix well and transfer to the activated ion exchange column, then rinse the exchange column with 15mL of water and 15mL of 0.05mol / L sodium chloride solution at a flow rate of 1mL / min, and discard the effluent. Finally, elute with 25 mL of 0.7 mol / L sodium chloride solution, collect the eluate, and make up to 25 mL.

[0078] The detection method of phytic acid is as follows: accurately pipette 5 mL of the constant volume eluate obtained in the previous section into a 10 mL colorimetric tube, add 4 mL of ferric chloride-sulfosalicylic acid reaction solution, mix well, let stand for 20 minutes, pipette part of the mixed solution into a 1 mL colorimetric dish, measure the absorbance at 500 nm, and determine the phytic acid content according to the standard curve. Repeat the experiment three times and take the average value of the results.

[0079] The results are shown in Table 1:

[0080] Solid-state fermentation of Bacillus Velez IFST-Xd-4 can significantly reduce the fiber content in corn husk. The crude fiber, neutral detergent fiber and acid detergent fiber contents in corn husk were reduced from the initial 39.71±1.23%, 58.31±2.34% and 27.24±2.13% to 26.19±1.98%, 47.88±2.54% and 21.07±2.09%, respectively, and the degradation rates were 34.04%, 17.89% and 22.65%, respectively.

[0081] Solid-state fermentation of Bacillus Velez IFST-Xd-4 can significantly reduce the phytic acid content in corn husk from the initial 10.21±0.54 mg / g to 7.51±0.99 mg / g, with a degradation rate of 26.44%.

[0082] Table 1 Effects of solid-state fermentation of Bacillus Velez IFST-Xd-4 on anti-nutritional factors in corn husk

[0083]

[0084] Example 7 Determination of the free radical scavenging ability of corn husks by solid-state fermentation of Bacillus velez

[0085] The present invention further detects the changes in the antioxidant capacity of corn husk after solid-state fermentation of Bacillus Velez IFST-Xd-4, including:

[0086] 1. Preparation of seed solution: Inoculate Bacillus Velez IFST-Xd-4 (inoculation amount 1%) into the seed culture medium, culture at a shaking speed of 180 rpm at 37°C for 24 h until the OD600 value is greater than 3.0 to obtain the seed solution, wherein the formula of the seed culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and distilled water.

[0087] 2. Fermentation culture: Mix peanut meal and corn husk in a ratio of 1:1, weigh 500g of the mixed raw materials, add equal weight of water, mix thoroughly, sterilize at 121℃, cool to room temperature, inoculate the seed solution in step 1 at 5% (v / w) of the raw materials, mix thoroughly, place in a 37℃ incubator, and ferment for 72 hours. Stir and mix the fermentation raw materials thoroughly in a clean bench every 12 hours. Take samples at 0h, 24h, 48h and 72h, dry and crush.

[0088] 3. The free radical scavenging ability determination method of fermented corn husk can refer to the Solebow ABTS free radical scavenging ability detection kit and DPPH free radical scavenging ability detection kit.

[0089] ABTS free radical scavenging ability test: crush the dried sample, pass it through a 30-50 mesh sieve, weigh 0.05g of the sample, add 1mL of the extract, place it in a 40℃ water bath for 30min, centrifuge it at 1000rpm for 10min, take the supernatant and place it on ice for testing. Preheat the spectrophotometer or microplate reader for 30min, adjust the wavelength to 405nm, the blank tube is 10µL of distilled water plus 20µL of reagent 4 plus 170µL of ABTS working solution, the assay tube is 10µL of supernatant plus 20µL of reagent 4 plus 170µL of working solution, the control tube is 10µL of supernatant plus 190µL of reagent 1, and the positive control is 10µL of VC solution of different concentrations plus 20µL of reagent 4 plus 170µL of ABTS working solution. Free radical scavenging rate determination of positive control D%=[(A 空白 -A 阳性对照 ) / A 空白 ]×100%, the free radical scavenging rate of the sample is determined by D%=[[A 空白 - (A 测定 -A 阳性对照 )] / A 空白 ]×100%.

[0090] DPPH free radical scavenging ability test: crush the dried sample, pass it through a 30-50 mesh sieve, weigh 0.05g of the sample, add 1mL of the extract, place it in a 40℃ water bath for 30min, centrifuge it at 1000rpm for 10min, take the supernatant and place it on ice for testing. Preheat the spectrophotometer or microplate reader for 30min, adjust the wavelength to 515nm, the blank tube is 10µL of the extract plus 190µL of the working solution, the assay tube is 10µL of the supernatant plus 190µL of the working solution, the control tube is 10µL of the supernatant plus 190µL of the reagent 1, and the positive control is 10µL of the reagent 3 plus 190µL of the working solution. The free radical scavenging rate of the positive control is determined as D% = [(A 空白 -A 阳性对照 ) / A 空白 ]×100%, the free radical scavenging rate of the sample is determined by D%=[[A 空白 - (A 测定 -A 阳性对照 )] / A 空白 ]×100%.

[0091] The results are as follows Figure 6 As shown in the data, after solid-state fermentation of corn bran with Bacillus Velez IFST-Xd-4, its ability to scavenge ABTS and DPPH free radicals was significantly improved, indicating that its antioxidant capacity was improved. The ability of corn bran to scavenge ABTS free radicals before and after fermentation was significantly stronger than that to scavenge DPPH free radicals, but the ability of corn bran to scavenge DPPH free radicals enhanced by solid-state fermentation was significantly higher than that to scavenge ABTS free radicals.

[0092] Example 8 Determination of the ability of Bacillus Velezii to inhibit pathogenic bacteria

[0093] The present invention further detects the inhibitory ability of Bacillus Velezii IFST-Xd-4 on common pathogenic bacteria in feed, including:

[0094] 1. Preparation of seed solution of Bacillus Velez IFST-Xd-4: inoculate Bacillus Velez IFST-Xd-4 (inoculum size 1%) into seed culture medium, culture at 37°C for 24 h at a shaking speed of 180 rpm until the OD600 value is greater than 3.0 to obtain seed solution, wherein the formula of the seed culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, prepared in distilled water.

[0095] 2. Pathogenic bacteria culture: Three common pathogenic bacteria (Salmonella, Escherichia coli and Staphylococcus aureus) were selected as indicator bacteria, and the pathogenic bacteria were inoculated into NB medium at a 1% inoculation rate. The shaking speed was 180 rpm and the culture was carried out at 37°C for 24 hours to obtain the pathogenic bacteria seed solution. The formula of NB medium was: peptone 10 g / L, beef extract powder 3 g / L, sodium chloride 5 g / L, distilled water, pH 7.2±0.2.

[0096] 3. Determination of antibacterial properties of Bacillus Velez IFST-Xd-4: Select 2mL of fresh bacterial solution and add it to 100mL of NB solid culture medium, mix well and pour into a culture dish as an indicator bacteria plate. Use a sterile hole puncher to punch holes for later use. Take the activated Bacillus Velez IFST-Xd-4 seeds, take 50µL of bacterial solution, add it to the middle of the indicator bacteria plate, culture at 37℃, and observe the diameter of the inhibition zone.

[0097] The results are as follows Figure 7 As shown, Bacillus Velez IFST-Xd-4 can significantly inhibit the growth of three pathogenic bacteria, and the antibacterial effect is significantly enhanced with the extension of culture time; among them, Bacillus Velez IFST-Xd-4 has the strongest inhibitory effect on Escherichia coli, followed by Staphylococcus aureus, and the inhibitory effect on Salmonella is relatively poor.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Bacillus velezensis IFST-Xd-4, characterized in that: The deposit number of the Bacillus Velez IFST-Xd-4 is: CGMCC No.29967.

2. A bacterial agent, characterized in that The invention comprises the Bacillus Velez subtilis IFST-Xd-4 or a fermentation product thereof according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent is a solid bacterial agent, a liquid bacterial agent or a gel bacterial agent, and the total viable count of the Velez subtilis IFST-Xd-4 in the bacterial agent is 1×10 7-10 cfu / g.

4. Use of the Bacillus Velezii IFST-Xd-4 according to claim 1, or the bacterial agent according to claim 2 or 3 in degrading zearalenone.

5. Use of the Bacillus Velezii IFST-Xd-4 according to claim 1, or the bacterial agent according to claim 2 or 3 in any of the following: i) degrading anti-nutritional factors; the anti-nutritional factors include one or more of crude fiber, neutral detergent fiber, acid detergent fiber or phytic acid; ii) Improve antioxidant capacity; iii) inhibiting microorganisms; said microorganisms include Escherichia coli, Staphylococcus aureus and Salmonella.

6. Use of the Bacillus Velez IFST-Xd-4 according to claim 1 or the bacterial agent according to claim 2 or 3 in improving the feeding value of corn husk.

7. A fermentation method of Bacillus Velez, characterized in that: include: The peanut meal and corn husk are mixed, inoculated with the seed liquid of the Bacillus Velez subtilis IFST-Xd-4 according to claim 1, and then fermented.

8. The fermentation method according to claim 7, characterized in that The mass ratio of the peanut meal to the corn husk is 1:(1-5).

9. The fermentation method according to claim 7 or 8, characterized in that: The inoculation amount of the Bacillus Velez IFST-Xd-4 is 5-20 v / w%.

10. The fermentation method according to claim 7 or 8, characterized in that: The fermentation conditions include: a fermentation time of more than 36 hours and a fermentation temperature of 30-42°C.

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