Method for biologically reducing mycotoxin in corn byproducts

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for biologically reducing mycotoxin in a corn by-product, which comprises the following steps: fermenting the corn by-product twice, fermenting the corn by-product for the first time by adopting aspergillus oryzae and bacillus licheniformis, and then fermenting for the second time by adopting lactobacillus casei; the fermented product contains neutral protease, alkaline protease, small peptide, L-lactic acid and other beneficial metabolites, so that the microbial environment of animal intestines can be effectively improved, the natural immune function of animal organisms can be regulated and controlled, and the disease resistance and stress resistance can be improved; moreover, the degradation rate of aflatoxin B1 is 70%-80%, the degradation rate of zearalenone is 75%-85%, the degradation rate of vomitoxin is 60%-70%, the three mycotoxins are all effectively degraded, the quality safety is ensured, and the resource utilization rate of corn byproducts is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for biologically reducing fungal toxins in corn by-products. Background Art

[0002] Corn husk and corn germ meal are the main by-products of corn deep processing. They have complex structures and are difficult to be directly digested and absorbed by animals. In addition, zearalenone (ZEN) and deoxynivalenol (DON) are the main mycotoxin contaminants. In addition, they also contain aflatoxin B1 (AFB1), which further limits the application of corn by-products in animal feed.

[0003] For mycotoxins, the detoxification treatment methods used mainly include: physical detoxification, chemical detoxification, and biological enzymatic hydrolysis. Among them, the biological enzymatic hydrolysis mainly uses certain enzymes. For example, the enzymes that can degrade aflatoxin B1 include AFO oxidase, laccase CotA, and manganese peroxidase; the enzymes that degrade zearalenone include lactonase ZHD101 and ZEND, laccase BsCotA, and peroxidoreductase Prx; the enzymes for degrading vomitoxin include DepA, DepB, QDDH, AKR13B2, AKR6D1, DDH, and acetylated 3-AC-DON oxidase. The degradation of enzymes is used to destroy mycotoxins or reduce their toxicity.

[0004] However, currently these enzymes can only degrade one toxin, while corn by-products are commonly contaminated by the coexistence of multiple fungal toxins. Therefore, a safe and effective method is needed to reduce fungal toxins in corn by-products while ensuring utilization rate. Summary of the invention

[0005] To solve the problems raised by the background technology, the present invention provides a method for reducing the content of mycotoxins in corn by-products by microbial fermentation.

[0006] The technical solution of the present invention is as follows:

[0007] The invention provides a method for reducing the content of mycotoxins in corn by-products by microbial fermentation. Aspergillus oryzae SLY-M-15 and Bacillus licheniformis are inoculated into a fermentation medium containing corn husks, sprayed corn husks and corn germ meal, and a first fermentation is performed to obtain a mixed fermentation product. Lactobacillus casei is inoculated into the mixed fermentation product, and a fermentation product is obtained by a second fermentation.

[0008] The deposit number of the Aspergillus oryzae SLY-M-15 is CGMCC NO.40471.

[0009] In the fermented product, the neutral protease activity is above 800 U / g, and the alkaline protease activity is above 500 U / g.

[0010] In the fermented product, the content of small peptides is more than 12%, the content of L-lactic acid is more than 4%, and the content of total acid is more than 5%.

[0011] In the fermented product, the degradation rate of aflatoxin B1 is 70%-80%, the degradation rate of zearalenone is 75%-85%, and the degradation rate of vomitoxin is 60%-70%.

[0012] Based on the weight of the fermentation medium, 1% of the inoculated neutral protease activity of Aspergillus oryzae SLY-M-15 was 5000U / g or more, and 2% of the inoculated viable bacteria count was 3.0×10 10 For Bacillus licheniformis with CFU / mL or above, the number of viable bacteria inoculated with 1.5% is 1.0×10 10 CFU / mL or above Lactobacillus casei.

[0013] The weight percentages of corn husk, sprayed corn husk and corn germ meal in the fermentation medium are 30%-35%, 30%-40% and 30%-35% respectively. The water content of the fermentation medium is 47%-48%. The pH value of the fermentation medium is adjusted to 5.5-6.0 by calcium hydroxide.

[0014] The first fermentation has a temperature of 30°C-37°C and a time of 40-48 hours.

[0015] The second fermentation has a temperature of 37° C. and a time of 60 h to 84 h.

[0016] Beneficial Effects

[0017] The invention ferments corn by-products twice, uses Aspergillus oryzae and Bacillus licheniformis to ferment the corn by-products for the first time, and then uses Lactobacillus casei to ferment the corn by-products for the second time to obtain a fermented product, which not only reduces fungal toxins and improves safety, but is also rich in various nutrients and improves application value.

[0018] The fermentation product obtained by the present invention not only has neutral protease and alkaline protease with high enzyme activity, but also has other beneficial metabolites, such as small peptides and L-lactic acid, which can effectively improve the microbial environment of the animal intestine, regulate the body's natural immune function, and improve the disease resistance and stress resistance; in addition, aflatoxin B1, zearalenone, and vomitoxin are all degraded, achieving the reduction of fungal toxins, ensuring product quality and safety, and improving the resource utilization rate of corn by-products.

[0019] Strain deposit information

[0020] Strain name: Aspergillus oryzae strain SLY-M-15

[0021] Classification: Aspergillus oryzae

[0022] Deposit number: CGMCC NO.40471

[0023] Storage time: January 6, 2023

[0024] Deposit address: General Microbiology Center, China Microbiological Culture Collection Administration (No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) DETAILED DESCRIPTION

[0025] The following examples are intended to illustrate the present invention rather than to further limit the present invention.

[0026] The invention provides a method for reducing the content of mycotoxins in corn by-products by microbial fermentation. Aspergillus oryzae SLY-M-15 and Bacillus licheniformis are inoculated into a fermentation medium containing corn husks, sprayed corn husks and corn germ meal, and a first fermentation is performed to obtain a mixed fermentation product. Lactobacillus casei is inoculated into the mixed fermentation product, and a fermentation product is obtained by a second fermentation.

[0027] The deposit number of Aspergillus oryzae SLY-M-15 is CGMCC NO.40471;

[0028] The invention ferments corn byproducts twice, uses Aspergillus oryzae and Bacillus licheniformis to ferment the corn byproducts for the first time, and then uses Lactobacillus casei to ferment the corn byproducts for the second time. The fermented product not only reduces fungal toxins and improves safety, but also is rich in various nutrients and improves application value.

[0029] In terms of protease activity, in the fermentation product, the neutral protease activity is above 800 U / g, and the alkaline protease activity is above 500 U / g.

[0030] In terms of nutrients, the fermented product has a small peptide content of more than 12%, an L-lactic acid content of more than 4%, and a total acid content of more than 5%.

[0031] In terms of degradation of mycotoxins, in the fermented product, the degradation rate of aflatoxin B1 is 70%-80%, the reduction rate of zearalenone is 75%-85%, and the degradation rate of vomitoxin is 60%-70%.

[0032] The fermentation product obtained by the present invention not only has neutral protease and alkaline protease with high enzyme activity, but also has beneficial metabolites such as small peptides and L-lactic acid, which can effectively improve the microbial environment of the animal intestine, regulate the body's natural immune function, and enhance the disease resistance and stress resistance ability; moreover, aflatoxin B1, zearalenone, and vomitoxin are all degraded, achieving the reduction of fungal toxins, ensuring quality safety, and significantly improving the resource utilization rate of corn by-products.

[0033] In terms of the preparation method, regarding the strain and the amount of strain inoculation, based on the weight of the fermentation medium, 1% of the inoculation was Aspergillus oryzae SLY-M-15 with an activity of more than 5000U / g of neutral protease, and 2% of the inoculation was 3.0×10 10 For Bacillus licheniformis with CFU / mL or above, the number of viable bacteria inoculated with 1.5% is 1.0×10 10 CFU / mL or above Lactobacillus casei.

[0034] Among them, the neutral protease activity is above 5000U / g, which means that after fermenting Aspergillus oryzae SLY-M-15 strain on culture medium, the neutral protease activity in the fermentation product is measured, and Aspergillus oryzae SLY-M-15 with a neutral protease activity of above 5000U / g is selected for inoculation.

[0035] The strains used are mixed for fermentation, which, on the one hand, fully ferments corn by-products and improves the nutritional value of the fermented product; on the other hand, degrades fungal toxins during the fermentation process and improves its resource utilization rate.

[0036] In order to improve the utilization rate of corn by-products, with regard to the amount of raw materials added, preferably, the weight percentages of corn husks, sprayed corn husks, and corn germ meal in the fermentation medium are 30%-35%, 30%-40%, and 30%-35%, respectively, the water content of the fermentation medium is 47%-48%, and the pH value of the fermentation medium is adjusted to 5.5-6.0 with calcium hydroxide.

[0037] Furthermore, in order to fully ferment the corn by-products and improve the nutritional value, in terms of the control of process conditions, the first fermentation has a temperature of 30°C-37°C and a time of 40-48 hours to ensure the fermentation effect of the aerobic stage.

[0038] In addition, the second fermentation has a temperature of 37° C. and a time of 60 h to 84 h to ensure that the anaerobic fermentation is fully completed.

[0039] Applying the fermented product of the present invention to animal feed not only ensures the safety of the feed, but also can provide a variety of nutrients and reduce the cost of feed production.

[0040] Example 1

[0041] Inoculating Aspergillus oryzae SLY-M-15 and Bacillus licheniformis into a fermentation medium containing corn husks, sprayed corn husks, and corn germ meal, performing a first fermentation at 30° C. for 48 hours to obtain a mixed fermentation product, inoculating Lactobacillus casei into the mixed fermentation product, performing a second fermentation at 37° C. for 60 hours to obtain a fermentation product;

[0042] The weight percentages of corn husk, sprayed corn husk and corn germ meal in the fermentation medium are 30%, 40% and 30% respectively, the water content of the fermentation medium is 47%-48%, and the pH value of the fermentation medium is adjusted to 5.5-6.0 with calcium hydroxide.

[0043] Regarding the strains and the amount of strain inoculation, based on the weight of the fermentation medium, 1% of the inoculation was Aspergillus oryzae SLY-M-15 with a neutral protease activity of more than 5000U / g, and 2% of the inoculation was 3.0×10 10 For Bacillus licheniformis with CFU / mL or above, the number of viable bacteria inoculated with 1.5% is 1.0×10 10 CFU / mL or above Lactobacillus casei.

[0044] Example 2

[0045] Inoculating Aspergillus oryzae SLY-M-15 and Bacillus licheniformis into a fermentation medium containing corn husks, sprayed corn husks, and corn germ meal, performing a first fermentation at 33° C. for 45 hours to obtain a mixed fermentation product, inoculating Lactobacillus casei into the mixed fermentation product, performing a second fermentation at 37° C. for 72 hours to obtain a fermentation product;

[0046] The weight percentages of corn husk, sprayed corn husk and corn germ meal in the fermentation medium are 32%, 35% and 33% respectively, the water content of the fermentation medium is 47%-48%, and the pH value of the fermentation medium is adjusted to 5.5-6.0 with calcium hydroxide.

[0047] Regarding the strains and the amount of strain inoculation, based on the weight of the fermentation medium, 1% of the inoculation was Aspergillus oryzae SLY-M-15 with a neutral protease activity of more than 5000U / g, and 2% of the inoculation was 3.0×10 10 For Bacillus licheniformis with CFU / mL or above, the number of viable bacteria inoculated with 1.5% is 1.0×10 10 CFU / mL or above Lactobacillus casei.

[0048] Example 3

[0049] Inoculating Aspergillus oryzae SLY-M-15 and Bacillus licheniformis into a fermentation medium containing corn husks, sprayed corn husks, and corn germ meal, performing a first fermentation at 37° C. for 40 hours to obtain a mixed fermentation product, inoculating Lactobacillus casei into the mixed fermentation product, performing a second fermentation at 37° C. for 84 hours to obtain a fermentation product;

[0050] The weight percentages of corn husk, sprayed corn husk and corn germ meal in the fermentation medium are 35%, 30% and 35% respectively, the water content of the fermentation medium is 47%-48%, and the pH value of the fermentation medium is adjusted to 5.5-6.0 with calcium hydroxide.

[0051] Regarding the strains and the amount of strain inoculation, based on the weight of the fermentation medium, 1% of the inoculation was Aspergillus oryzae SLY-M-15 with a neutral protease activity of more than 5000U / g, and 2% of the inoculation was 3.0×10 10 For Bacillus licheniformis with CFU / mL or above, the number of viable bacteria inoculated with 1.5% is 1.0×10 10 CFU / mL or above Lactobacillus casei.

[0052] Comparative Example 1

[0053] Compared with Example 1, during the first fermentation, only Aspergillus oryzae SLY-M-15 was inoculated, and the second fermentation was not performed. The remaining operations were the same as in Example 1, with the same bacterial inoculum amount as in Example 1 being inoculated, and the same technology and fermentation conditions as in Example 1 being adopted.

[0054] Comparative Example 2

[0055] Compared with Example 1, only Bacillus licheniformis was inoculated during the first fermentation, and no second fermentation was performed. The remaining operations were the same as in Example 1, the same strain inoculation amount was inoculated, and the same process and fermentation conditions were used as in Example 1.

[0056] Comparative Example 3

[0057] Compared with Example 1, Lactobacillus casei was inoculated into a fermentation medium containing corn husks, sprayed corn husks, and corn germ meal, and 1‰ of acidic protease, 0.5‰ of neutral protease, and 0.5‰ of alkaline protease were added. The remaining operations were the same as in Example 1, with the same bacterial inoculum size as in Example 1 being inoculated, and the same process and fermentation conditions as in Example 1 being adopted.

[0058] Comparative Example 4

[0059] Compared with Example 1, only the first fermentation was performed, and the second fermentation was not performed. The remaining operations were the same as those in Example 1, the same strains and strain inoculation amounts were inoculated, and the same processes and fermentation conditions were used.

[0060] Comparative Example 5

[0061] Compared with Example 1, only Aspergillus oryzae SLY-M-15 was inoculated during the first fermentation. The remaining operations were the same as in Example 1, inoculating the same strains and strain inoculum amount as in Example 1, and adopting the same technology and fermentation conditions as in Example 1.

[0062] Comparative Example 6

[0063] Compared with Example 1, only Bacillus licheniformis was inoculated during the first fermentation. The remaining operations were the same as those in Example 1, with the same strains and inoculation amount as in Example 1, and the same process and fermentation conditions as in Example 1.

[0064] Comparative Example 7

[0065] Compared with Example 1, Aspergillus oryzae CICC2035 was inoculated during the first fermentation. The remaining operations were the same as in Example 1, the same strains and strain inoculation amount were inoculated, and the same process and fermentation conditions were adopted as in Example 1.

[0066] Experimental results and analysis

[0067] The fermented products obtained in Examples 1-3 and Comparative Examples 1-7 were crushed respectively, and the protease activity, crude protein, small peptides, L-lactic acid, total acid, mycotoxins (zearalenone (ZEN), deoxynivalenol (DON), aflatoxin B1 (AFB1)) content and degradation rate were measured.

[0068] 1. Protease activity

[0069] The determination method refers to the determination of protease activity in Appendix A.4 of GB1886.174-2016.

[0070] The test results of protease activity in the fermented products obtained in Examples 1-3 and Comparative Examples 1-7 are shown in Table 1.

[0071] Table 1 Detection results of protease activity in different fermentations

[0072]

[0073] As shown in Table 1, the neutral protease and alkaline protease of the fermentation products of Examples 1 to 3 are higher than those of Comparative Examples 3 and Comparative Examples 5 to 7; the neutral protease and alkaline protease of the fermentation products of Examples 1 to 3 are slightly lower than those of Comparative Examples 1 to 2 and Comparative Example 4 due to the enzymatic hydrolysis of the second fermentation. The neutral protease and alkaline protease of the fermentation products of Comparative Examples 5 to 6 are higher than those of Comparative Example 7, but lower than those of Examples 1 to 3, indicating that Aspergillus oryzae SLY-M-15 and Bacillus licheniformis are better than Aspergillus oryzae CICC2035; the neutral protease and alkaline protease of the fermentation product of Comparative Example 3 are the lowest.

[0074] This indicates that the fermentation product obtained by the method of the present application has high enzyme activity of neutral protease and alkaline protease.

[0075] 2. Crude protein, small peptides, L-lactic acid, total acid

[0076] The crude protein determination method refers to the standard GB / T 6432-2018;

[0077] The determination method of small peptides refers to the determination method of peptide content in Appendix B of GB22492-2008;

[0078] The total acid determination method refers to the second method of pH potentiometric titration in GB12456-2021;

[0079] L-lactic acid is measured using a SBA-40E or equivalent biosensor analyzer in accordance with the instrument's operating procedures.

[0080] The test results of crude protein, small peptides, L-lactic acid and total acid in the fermented products obtained in Examples 1-3 and Comparative Examples 1-7 are shown in Table 2.

[0081] Table 2 Detection results of crude protein, small peptides, L-lactic acid and total acid in different fermentations

[0082]

[0083] As shown in Table 2, the content of small peptides, L-lactic acid and total acid in the fermentation products of Examples 1 to 3 is significantly higher than that of Comparative Examples 1 to 7, and the crude protein content does not change significantly. Specifically, Comparative Examples 1 to 2 and Comparative Example 4 only carry out the first fermentation, and the small peptides, L-lactic acid and total acid in the fermentation products are significantly lower than those in Examples 1 to 3; Comparative Examples 5 to 6 use rice koji SLY-M-15 or enzyme-producing lichens for separate fermentation, and the small peptides, L-lactic acid and total acid in the fermentation products are lower than those in Examples 1 to 3; Comparative Example 7 uses Aspergillus oryzae CICC2035 for separate fermentation, and the small peptides, L-lactic acid and total acid in the fermentation products are significantly lower than those in Examples 1 to 3; Comparative Example 3 adds a certain proportion of protease preparations to the fermentation medium, and the small peptides, L-lactic acid and total acid in the fermentation products are significantly lower than those in Examples 1 to 3.

[0084] It can be seen from this that the method of the present application can increase the content of small peptides, L-lactic acid, and total acid in the fermentation product, thereby effectively improving the microbial environment of the animal intestine, regulating the body's immune function, and improving disease resistance and anti-stress capabilities.

[0085] 3. Content and degradation rate of mycotoxins (zearalenone (ZEN), vomitoxin (DON), aflatoxin B1 (AFB1))

[0086] The determination was performed using a multi-flux fluorescence detection platform. Weigh 2.500 g of the fermentation product, add 50% ethanol / water extract, shake it thoroughly with a vortexer for 1 minute, and then let it stand for more than 1 minute. At the same time, place the microporous strip in the microporous of the incubator carrier, draw 100 μL of sample diluent and add it to the microporous, insert the test strip of the corresponding toxin item and place it on the carrier for standby, add 400 μL of sample diluent to the centrifuge tube, and then draw 100 μL of supernatant from the sample extract after standing and add it to the centrifuge tube to mix, and draw 100 μL of the mixed solution into the microporous and blow for 10 seconds to mix thoroughly, close the carrier on the incubator, and drop the test card. After 7 minutes, take out the test card and insert it into the reader to read the result within 1 minute. At the same time, the unfermented corn by-product fermentation medium is used as the initial toxin sample, and the determination method is the same as the fermentation method. To ensure the accuracy of the detection of mycotoxin content, high performance liquid chromatography can be used for verification.

[0087]

[0088] The degradation rates of mycotoxins (aflatoxin B1 (AFB1), zearalenone (ZEN), and deoxynivalenol (DON)) in the fermented products obtained in Examples 1-3 and Comparative Examples 1-7 are shown in Table 3.

[0089] Table 3 Degradation rate of mycotoxins in different fermentations

[0090]

[0091] As shown in Table 3, the degradation rates of aflatoxin B1 (AFB1), zearalenone (ZEN), and vomitoxin (DON) in the fermented products of Examples 1 to 3 are significantly higher than those in Comparative Examples 1 to 7. Specifically, in Comparative Example 1 using Aspergillus oryzae SLY-M-15 and Comparative Example 2 using Bacillus licheniformis for only the first fermentation, the degradation rates of aflatoxin B1 (AFB1), zearalenone (ZEN), and vomitoxin (DON) in the fermented products are lower than those in Examples 1 to 3; in Comparative Examples 5 and Comparative Example 6 using Aspergillus oryzae SLY-M-15 or Bacillus licheniformis for two fermentations, the degradation rates of aflatoxin B1 (AFB1), zearalenone (ZEN), and vomitoxin (DON) in the fermented products are higher than those in Examples 1 to 3. The degradation rates of Examples 1 to 3 are lower than those of Comparative Examples 1 to 2, and are slightly higher than those of Comparative Examples 1 to 2. Comparative Example 7 uses Aspergillus oryzae CICC2035, and the degradation rates of zearalenone (ZEN), deoxynivalenol (DON), and aflatoxin B1 (AFB1) in the fermentation product are all lower than those of Examples 1 to 3, and lower than those of Comparative Examples 5 to 6. Comparative Example 3 adds a certain proportion of protease preparation to the fermentation medium, and the degradation rates of zearalenone (ZEN), deoxynivalenol (DON), and aflatoxin B1 (AFB1) in the fermentation product are much lower than those of Examples 1 to 3.

[0092] Therefore, the method of the present application can significantly improve the degradation rate of aflatoxin B1, zearalenone, and vomitoxin in the fermentation product, reduce the content of mycotoxins in corn by-products, achieve the elimination of mycotoxins, ensure product quality and safety, and significantly improve the resource utilization rate of corn by-products.

Claims

1. A method for biological reduction of mycotoxins in corn by-products, characterized in that: Inoculating Aspergillus oryzae SLY-M-15 and Bacillus licheniformis into a fermentation medium containing corn husks, sprayed corn husks, and corn germ meal, performing a first fermentation to obtain a mixed fermentation product, inoculating Lactobacillus casei into the mixed fermentation product, and performing a second fermentation to obtain a fermentation product; The deposit number of the Aspergillus oryzae SLY-M-15 is CGMCC NO.40471.

2. The method for biological reduction of mycotoxins in corn by-products according to claim 1, characterized in that: In the fermented product, the neutral protease activity is above 800 U / g, and the alkaline protease activity is above 500 U / g.

3. The method for biological reduction of mycotoxins in corn by-products according to claim 1, characterized in that: In the fermented product, the content of small peptides is more than 12%, the content of L-lactic acid is more than 4%, and the content of total acid is more than 5%.

4. The method for biological reduction of mycotoxins in corn by-products according to claim 1, characterized in that: In the fermented product, the degradation rate of aflatoxin B1 is 70%-80%, the degradation rate of zearalenone is 75%-85%, and the degradation rate of vomitoxin is 60%-70%.

5. The method for biological reduction of mycotoxins in corn by-products according to claim 1, characterized in that: Based on the weight of the fermentation medium, 1% of the inoculated neutral protease activity of Aspergillus oryzae SLY-M-15 was 5000U / g or more, and 2% of the inoculated viable bacteria count was 3.0×10 10 For Bacillus licheniformis with CFU / mL or above, the number of viable bacteria inoculated with 1.5% is 1.0×10 10 CFU / mL or above Lactobacillus casei.

6. The method for biological reduction of mycotoxins in corn by-products according to claim 1, characterized in that: The weight percentages of corn husk, sprayed corn husk and corn germ meal in the fermentation medium are 30%-35%, 30%-40% and 30%-35% respectively. The water content of the fermentation medium is 47%-48%. The pH value of the fermentation medium is adjusted to 5.5-6.0 by calcium hydroxide.

7. The method for biological reduction of mycotoxins in corn by-products according to claim 1, characterized in that: The first fermentation has a temperature of 30°C-37°C and a time of 40-48 hours.

8. The method for biological reduction of mycotoxins in corn by-products according to claim 1, characterized in that: The second fermentation has a temperature of 37° C. and a time of 60 h to 84 h.

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