Method for degrading vomitoxin in coconut meal through microbial fermentation

Abstract

The invention discloses a method for degrading vomitoxin in coconut meal through microbial fermentation, and belongs to the technical field of coconut meal fermentation. In order to solve the problems of limitation of microbial strain screening culture, insufficient vomitoxin degradation efficiency and inaccurate fermentation process control, by screening and domesticating microbial strains, the specific enzyme activity is remarkably improved, efficient degradation of vomitoxin is realized, an environment suitable for microbial growth and metabolism is provided, and the vomitoxin degradation efficiency is improved. By combining the synergistic effect of microbial fermentation and an exogenous enzyme preparation, the vomitoxin degradation efficiency is improved to the greatest extent, the limitation of single microbial metabolism is broken through, particularly, remarkable advantages are shown in a high-mycin-polluted complex matrix, the vomitoxin content is remarkably reduced, and the vomitoxin degradation efficiency is improved to the greatest extent. The substrate quality of the coconut meal is maintained, and the efficient, safe and controllable vomitoxin degradation method is constructed through optimization treatment of the coconut meal substrate, microbial screening and domestication and the synergistic effect of exogenous enzymes.

Description

Technical Field

[0001] The invention relates to the technical field of coconut meal fermentation, in particular to a method for degrading vomitoxin in coconut meal by utilizing microbial fermentation. Background Art

[0002] DON is a secondary metabolite produced by Fusarium fungi and widely exists in grains and their by-products (such as wheat, corn, coconut meal, etc.). Due to its strong toxicity and high stability, DON not only seriously affects the safety of livestock and poultry feed, but also poses a potential threat to human health through the food chain.

[0003] At present, the degradation methods for vomitoxin mainly include physical, chemical and biological methods. Although physical methods (such as high temperature and irradiation) can partially reduce the content of vomitoxin, they often lead to nutrient loss or quality deterioration of raw materials; chemical methods (such as oxidant treatment) may introduce residues and pose safety risks; in contrast, biological methods have gradually become a research hotspot due to their advantages such as high efficiency, greenness, and no toxic side effects. However, traditional microbial fermentation degradation technology generally has problems such as low degradation efficiency of strains, harsh action conditions, and poor matrix adaptability, which cannot meet the needs of industrialization. In addition, the degradation efficiency of vomitoxin in complex matrices (such as coconut meal) is even lower, especially when the content of vomitoxin is high, it is often difficult to achieve efficient removal by a single microorganism.

[0004] In actual operation, there are still the following problems:

[0005] Under the existing technology, insufficient raw material pretreatment leads to fermentation instability, and the limitations of microbial strain screening and cultivation result in poor strain adaptability and low efficiency in preparing active bacterial liquid. The concentration and activity of the bacterial liquid are unstable, which affects the colonization of the strain in the early stage of fermentation. The degradation efficiency of vomitoxin is insufficient, the fermentation process control is not precise, and secondary pollution is prone to occur during the processing of fermentation products. The uneven particles affect the digestion and absorption of livestock and the efficiency of feed processing. Summary of the invention

[0006] The object of the present invention is to provide a method for degrading vomitoxin in coconut meal by microbial fermentation, so as to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for degrading vomitoxin in coconut meal by microbial fermentation, comprising the following steps:

[0008] Raw material pretreatment, crushing coconut meal and adjusting the moisture content of coconut meal, adding glucose and urea to the coconut meal, mixing evenly and then sterilizing with high-temperature steam to obtain a sterilized pretreated coconut meal matrix;

[0009] Screening and culturing microbial strains, selecting Trichoderma and Geotrichum candidum that can secrete DON to degrade, and performing acclimation and screening, culturing the screened strains in a liquid culture medium, and keeping them ready for use after the strains' bacterial activity reaches an optimal state, thereby obtaining an active microbial bacterial liquid;

[0010] A solid-state fermentation system is established, wherein an active microbial liquid is inoculated into a sterilized coconut meal matrix, the inoculated coconut meal matrix is ​​placed in a fermentation container, and the matrix is ​​turned over to increase oxygen supply for solid-state fermentation;

[0011] Enzyme preparation addition: after solid-state fermentation, an exogenous enzyme preparation that degrades vomitoxin is added;

[0012] The fermentation products are collected and processed, and after the fermentation is completed, the fermentation products are dried, crushed and evenly mixed to obtain fermented coconut meal;

[0013] The degradation effect of vomitoxin was tested by high performance liquid chromatography to detect the residual amount of vomitoxin in coconut meal before and after treatment to confirm whether the fermentation treatment significantly reduced the content of vomitoxin.

[0014] Furthermore, the raw material pretreatment specifically includes the following steps:

[0015] The coconut meal raw material is processed by crushing equipment to a particle size range of 0.5-2mm to optimize the adsorption area of ​​microorganisms and promote the improvement of enzyme decomposition efficiency;

[0016] Determine the initial moisture content of the crushed coconut meal, and adjust the moisture content by adding purified water and drying according to the requirement of a final moisture content of 40%-60%. After uniform mixing, let it stand for 30 minutes to balance the humidity, so as to ensure the uniform water absorption of the coconut meal and provide an ideal environment for subsequent fermentation, avoiding the problem of strain inactivation or over-fermentation caused by excessive local humidity;

[0017] According to the weight ratio of coconut meal matrix, add 0.5%-2% glucose and urea, mix the coconut meal, glucose and urea evenly, and control the mixing time to 5-10 minutes to ensure the uniform distribution of glucose and urea in the matrix, so as to further improve the fermentation efficiency;

[0018] The evenly mixed coconut meal matrix is ​​placed in a high temperature sterilization container and sterilized by high temperature steam. The sterilization temperature is controlled at 121°C and the sterilization time is 20-30 minutes. After the sterilization is completed, the matrix is ​​cooled to room temperature for use.

[0019] Furthermore, the microbial strain screening and cultivation specifically includes the following steps:

[0020] Collect samples from the natural environment, preliminarily screen Trichoderma and Geotrichum candidum as target strains by plate dilution coating method, transfer the preliminarily screened strains to culture medium, control the culture temperature at 25-30°C, culture time for 3-5 days, and detect the detoxification enzyme activity by transparent circle method, expand the strain screening range, increase the probability of obtaining high-quality strains, and screen out strains with strong degrading ability to vomitoxin;

[0021] The primary screening strains were placed in a culture medium containing 1-5 μg / mL of vomitoxin and continuously subcultured for 48 hours per generation, with 5-8 generations. The screening strains not only had improved tolerance, but also significantly improved degradation efficiency.

[0022] Among them, the criteria for continuous passage and domestication screening of strains are good bacterial growth and the ability to significantly secrete detoxification enzymes;

[0023] The selected excellent strains were inoculated into liquid culture medium at an inoculation volume of 5%-10% of the culture medium volume. The culture conditions were controlled at 25-30°C and 150-200rpm shaking culture for 24-48 hours until the bacterial concentration reached 1×10 7 -1×10 8 CFU / mL, ensuring rapid reproduction and high efficiency after inoculation;

[0024] The enzyme activity of the strain is detected by colorimetry and the status of the strain is confirmed to obtain active microbial culture liquid, which further ensures that the strain is in good condition and provides highly active culture liquid for solid-state fermentation.

[0025] Furthermore, the natural environment includes soil, humus and plant residues, and the liquid culture medium contains glucose and urea as main nutrients.

[0026] Furthermore, the solid-state fermentation system is established, and specifically includes the following steps:

[0027] The active microbial liquid obtained in the microbial strain screening culture is evenly inoculated into the sterilized coconut meal matrix, and the inoculation amount is controlled to be 5%-10% of the weight of the coconut meal matrix. Sterile tools are used to fully mix the liquid so that it is evenly distributed in the matrix to avoid competition from contaminating bacteria and improve degradation efficiency. The liquid is evenly distributed in the matrix to avoid differences in fermentation results due to uneven inoculation.

[0028] The inoculated coconut meal substrate is placed in a semi-enclosed fermentation container, and the fermentation conditions are adjusted to a temperature of 25-35° C., a relative humidity of 70%-90%, and an initial fermentation time of 24-48 hours;

[0029] After the initial fermentation, the fermentation substrate is turned over once every 12-24 hours to allow the bottom and surface substrates to be fully exchanged. The total fermentation time is controlled to be 5-7 days. The fermentation temperature and humidity are regularly checked during the fermentation period. The temperature range and humidity control provide a suitable fermentation environment to improve the enzyme secretion efficiency of the strain. Regularly turning the substrate promotes oxygen penetration, avoids anaerobic bacteria contamination, and further enhances the degradation capacity of vomitoxin.

[0030] Furthermore, the enzyme preparation addition specifically includes the following steps:

[0031] The dosage of exogenous enzyme preparation is calculated according to the total weight of coconut meal substrate in the fermentation system, and 0.1%-1% of the substrate weight of enzyme preparation is dissolved in sterile water to prepare a uniform enzyme solution, which ensures the degradation effect, reduces the addition cost, ensures that the enzyme is fully in contact with the substrate, and exerts the maximum degradation effect;

[0032] 48 hours after the start of solid-state fermentation, the prepared enzyme solution was evenly sprayed on the surface of the fermentation substrate, and the substrate was fully turned over using sterile tools to form a synergistic system of bacteria and enzymes, which greatly increased the degradation rate of vomitoxin.

[0033] After adding the exogenous enzyme preparation, the fermentation temperature is maintained at 25-35°C, the relative humidity is 70%-90%, and the frequency of substrate stirring is increased to once every 12 hours to enhance the uniformity of the enzyme solution distribution and the oxygenation effect of the substrate.

[0034] Furthermore, the exogenous enzyme preparation is one of peroxidase and peptidase.

[0035] Furthermore, the fermentation product collection and processing specifically includes the following steps:

[0036] When the fermentation process reaches 5-7 days, monitor the status of the fermentation product;

[0037] Random sampling and testing of the degradation effect of DON were performed. When there was no obvious excessive mold growth on the surface of the fermentation product, the humidity was moderate, and the DON residue was significantly reduced and reached the degradation target, the fermentation was confirmed to be over, ensuring that the DON residue reached the degradation target, confirming that the fermentation quality was qualified, and ensuring the stability of the degradation process;

[0038] The fermented coconut meal substrate is taken out from the fermentation container and spread in a drying device for drying. The drying temperature is controlled at 50-60°C for 12-24 hours until the moisture content of the substrate is reduced to 12%-15%, which effectively prolongs the storage time and prevents secondary contamination or corruption caused by excessive moisture.

[0039] The dried fermentation product is processed by a pulverizing device to a particle size range of 0.5-2 mm, and the pulverized fermentation product is fully mixed. The pulverizing process improves the physical properties of the matrix, facilitates subsequent feed processing and use, and improves the digestion and absorption rate of livestock.

[0040] Furthermore, the fermentation product status includes temperature, humidity, odor and bacterial activity parameters.

[0041] Furthermore, the detection of the degradation effect of vomitoxin specifically includes the following steps:

[0042] Randomly sample the coconut meal matrix before and after fermentation, and control the sample quality to 10-20g;

[0043] The sampled coconut meal was crushed to a particle size of less than 1 mm, 3 g of the sample was accurately weighed, 20 mL of acetonitrile-water solution with a volume ratio of 84:16 was added, and the sample was placed in an oscillator and oscillated at 150-200 rpm for 30 minutes. The extract was kept aside to ensure the reliability of the test data. The oscillation extraction technology was used to quickly and efficiently obtain vomitoxin in the sample, providing accurate samples for subsequent testing;

[0044] The extract was filtered through a 0.22 μm filter membrane, and the filtered sample was injected into a high performance liquid chromatograph for detection. A C18 chromatographic column was used, and the mobile phase was an acetonitrile-water solution with a volume ratio of 50:50. The flow rate was 1.0 mL / min, the detection wavelength was 218 nm, and the column temperature was set to 30°C. The peak area and retention time of vomitoxin were recorded, and the content of vomitoxin was calculated by comparing with the standard curve. The separation and sensitivity of vomitoxin detection were improved by using a C18 chromatographic column and an optimized mobile phase.

[0045] The changes in the content of vomitoxin in the coconut meal matrix before and after treatment were compared, and the degradation rate was calculated. When the degradation rate reached more than 90% and the residual vomitoxin was lower than the national and industry standards, the degradation and fermentation treatment was confirmed to be qualified. The data was compared with the vomitoxin content in the matrix before and after treatment to provide a guarantee for feed safety.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1. In the solid-state fermentation system, the present invention optimizes the metabolic environment of microorganisms by accurately adjusting the fermentation conditions, further enhances the degradation ability of the strains, and adjusts the fermentation temperature and humidity, especially when the humidity is controlled at 90%, to provide a more suitable growth environment for the strains. At the same time, by increasing the frequency of stirring during the fermentation process, the aeration of the substrate is significantly improved, the generation of anaerobic conditions is avoided, and the growth of miscellaneous bacteria is effectively inhibited, shortening the fermentation cycle to 6 days, while the degradation rate of vomitoxin is increased to 95%, which can more fully stimulate the metabolic potential of the microbial strains and further enhance the synergistic effect of the exogenous enzyme preparations.

[0048] 2. The enzyme preparation combination of the present invention can not only improve the molecular cleavage efficiency of vomitoxin, but also further degrade the intermediate metabolites through the dual enzyme action, thereby ensuring the thoroughness of the degradation process. The substrate is fully mixed after each addition of the enzyme preparation, which significantly improves the distribution uniformity and effect of the enzyme, effectively avoids the waste of enzyme activity, and further prolongs the action time of the enzyme. Through the high performance liquid chromatography detection method, the accurate quantification of the vomitoxin residue is achieved. During the detection process, the dynamic monitoring of the fermentation state further ensures the stability and controllability of the fermentation process, and improves the safety and consistency of the fermentation product.

[0049] 3. The present invention significantly improves the specific enzyme activity of microbial strains by screening and domesticating them, and realizes efficient degradation of vomitoxin. By optimizing the raw material processing process and fermentation conditions, an environment suitable for microbial growth and metabolism is provided, and the activity and reproduction capacity of the bacteria are improved. The synergistic effect of microbial fermentation and exogenous enzyme preparations is combined to maximize the degradation efficiency of vomitoxin, breaking through the limitation of single microbial metabolism, and showing significant advantages in complex matrices contaminated with polymycin. It can not only significantly reduce the content of vomitoxin, but also maintain the matrix quality of coconut meal, so that it has a wider application potential in the field of animal feed. Through the optimization of the coconut meal matrix, the screening and domestication of microorganisms, and the synergistic effect of exogenous enzymes, an efficient, safe and controllable vomitoxin degradation method is constructed. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is a schematic diagram of the method flow of the present invention. DETAILED DESCRIPTION

[0051] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0052] Example 1

[0053] like Figure 1 As shown, the method of utilizing microbial fermentation to degrade vomitoxin in coconut meal of this embodiment comprises the following steps:

[0054] Raw material pretreatment, crushing coconut meal and adjusting the moisture content of coconut meal, increasing the surface area of ​​coconut meal, facilitating the adsorption and action of microorganisms, improving the uniformity of solid-state fermentation matrix, providing good conditions for the penetration and diffusion of fermentation liquid, adjusting the moisture content to provide suitable humidity conditions for the growth of microorganisms, avoiding the influence of dryness or over-humidity on the activity of strains, adding glucose and urea to coconut meal, mixing evenly and then sterilizing with high-temperature steam to obtain a sterilized pretreated coconut meal matrix, glucose as a carbon source, providing a sufficient energy source for the strain, promoting bacterial metabolism and growth, urea as a nitrogen source, promoting the secretion of detoxification enzymes, further enhancing the degradation ability of vomitoxin, high-temperature steam killing potential bacteria and molds in coconut meal, reducing the possibility of contamination during the fermentation process, improving the safety of raw materials, and providing a sterile environment for subsequent microbial fermentation;

[0055] Screening and culturing microbial strains, selecting Trichoderma and Geotrichum candidum that can secrete deoxynivalenol to degrade, and specifically selecting strains that can secrete enzymes that degrade deoxynivalenol, significantly improving degradation efficiency, using the transparent circle method to detect enzyme activity, ensuring that the screened strains have detoxification capabilities, and conducting acclimation screening, continuously passage in a culture medium containing deoxynivalenol to enhance the strains' adaptability and enzyme secretion efficiency, the acclimated strains can maintain a good growth state, while improving tolerance and degradation capabilities to deoxynivalenol, culturing the screened strains in a liquid culture medium, and keeping them ready for use after the strains' cell viability reaches an optimal state, obtaining active microbial bacterial liquid, liquid culture ensuring large-scale reproduction of the strains, and controlling the cell viability to reach an optimal state, ensuring that the inoculated strains can quickly adapt to the solid-state fermentation environment, and improving degradation efficiency at the initial stage of fermentation;

[0056] A solid-state fermentation system is established, and active microbial liquid is inoculated into the sterilized coconut meal matrix, and sterile tools are used for uniform inoculation to ensure that the liquid is evenly distributed in the matrix to avoid local insufficient fermentation or inactivation problems. The inoculation amount is controlled at 5%-10% of the weight of the matrix to provide sufficient bacterial resources and improve the fermentation efficiency. The inoculated coconut meal matrix is ​​placed in a fermentation container, and the matrix is ​​turned over to increase oxygen supply for solid-state fermentation. The fermentation temperature and humidity are regulated to provide the best growth conditions for the strain. The semi-enclosed design avoids excessive contamination by miscellaneous bacteria, while maintaining a good ventilation environment to promote aerobic metabolism of the strain. After the initial fermentation, the matrix is ​​turned over every 12-24 hours to promote oxygen exchange in the matrix and avoid the adverse effects of anaerobic conditions on fermentation. The turning makes the bacterial body more evenly distributed and improves the overall degradation effect of the matrix.

[0057] Enzyme preparation addition: after solid-state fermentation, add exogenous enzyme preparations that degrade vomitoxin, add peroxidase or peptidase to make up for the insufficient degradation efficiency of simple microorganisms and accelerate the decomposition of vomitoxin. The use time of enzyme preparations is coordinated with the action of strains to form an efficient degradation system of microorganisms and enzymes. Spray the enzyme solution evenly and stir it fully to ensure that the enzyme is evenly distributed on the surface of the substrate to avoid incomplete degradation caused by low local enzyme concentration.

[0058] The fermentation products are collected and processed. After the fermentation is completed, the fermentation products are dried, crushed and evenly mixed to obtain the fermented coconut meal. By monitoring the temperature, humidity and substrate surface state, it is dynamically determined whether the fermentation has achieved the best degradation effect to ensure the fermentation stability and safety. The fermentation products are dried to a moisture content of 12%-15% to extend the storage period of the product and improve the product quality stability. They are crushed to a specific particle size range to improve the uniformity of the particles and enhance the palatability and nutrient absorption effect as a feed raw material;

[0059] The degradation effect of vomitoxin was tested by using high performance liquid chromatography to detect the residual amount of vomitoxin in coconut meal before and after treatment to confirm whether the fermentation treatment significantly reduced the content of vomitoxin. The residual amount of vomitoxin was accurately quantified through standardized detection methods to ensure the scientificity and reliability of the degradation effect. The standard curve was used to correct the error to provide accurate data support for process optimization. The content of vomitoxin before and after fermentation was compared to confirm that the degradation rate reached more than 90%. At the same time, it was verified that the safety of the fermentation product met national and industry standards.

[0060] In the above embodiments, the crushing and moisture adjustment of the raw materials not only optimize the physical structure of the fermentation matrix, but also provide a good environment for the growth of microorganisms; by adding glucose and urea, sufficient carbon and nitrogen sources are provided for the growth of the strain, and the secretion capacity of the enzyme is improved. High-temperature steam sterilization not only kills the miscellaneous bacteria that may affect the fermentation, but also further reduces the mold burden of the coconut meal itself. The solid-state fermentation process not only adapts to the characteristics of coconut meal as a solid raw material, but also creates an environment suitable for the growth of microorganisms by precisely controlling the temperature and humidity. During the fermentation process, the matrix is ​​regularly turned over to increase the oxygen supply, effectively avoiding the problem of excessive mold growth under anaerobic conditions. At the same time, the supplementation of exogenous enzyme preparations further enhances the degradation efficiency of vomitoxin, and the synergistic effect of the organisms and enzymes before and after.

[0061] Example 2

[0062] In order to further solve the problems of fermentation instability caused by insufficient raw material pretreatment, poor strain adaptability due to limitations in microbial strain screening and cultivation, low efficiency in preparing active bacterial solution, unstable concentration and activity of bacterial solution, and affecting the colonization of strains in the early stage of fermentation, this embodiment optimizes the steps of raw material pretreatment, microbial strain screening and cultivation, and establishment of a solid-state fermentation system. The specific optimization contents are as follows:

[0063] Raw material pretreatment specifically includes the following steps:

[0064] The coconut meal raw material is processed into a particle size range of 0.5-2mm by a crushing equipment. By controlling the particle size range, the adsorption area of ​​microorganisms is further optimized, and the efficiency of enzyme decomposition is promoted. The uniform particle size avoids the problem of uneven moisture distribution caused by different particle sizes and improves the fermentation stability of the substrate;

[0065] Determine the initial moisture content of the crushed coconut meal, and adjust the moisture content by adding pure water and drying according to the requirement that the final moisture content is 40%-60%. After uniform mixing, let it stand for 30 minutes to balance the humidity. After adjusting the moisture content to 40%-60%, let it stand for 30 minutes to balance the humidity, so as to ensure the uniformity of water absorption of the coconut meal, provide an ideal environment for subsequent fermentation, and avoid the problem of strain inactivation or over-fermentation caused by excessive local humidity;

[0066] According to the weight ratio of coconut meal matrix, add 0.5%-2% glucose and urea, mix coconut meal, glucose and urea evenly, and control the mixing time to 5-10 minutes. Control the mixing time to effectively prevent uneven distribution of raw materials due to too short mixing time, or agglomeration of particles due to too long mixing time, which affects the sterilization effect, and ensure the uniform distribution of glucose and urea in the matrix, so as to further improve the fermentation efficiency;

[0067] The evenly mixed coconut meal matrix is ​​placed in a high temperature sterilization container and sterilized by high temperature steam. The sterilization temperature is controlled at 121°C and the sterilization time is 20-30 minutes. After the sterilization is completed, the matrix is ​​cooled to room temperature for use to ensure thorough sterilization while avoiding the destruction of the nutritional components of the coconut meal due to long high temperature time. It is cooled to room temperature for use to avoid affecting the survival rate of the active microbial liquid due to high temperature before strain inoculation.

[0068] Microbial strain screening and cultivation specifically includes the following steps:

[0069] Collect samples from the natural environment, where the natural environment includes soil, humus and plant residues, preliminarily screen Trichoderma and Geotrichum candidum as target strains by plate dilution coating method, transfer the preliminarily screened strains to culture medium, control the culture temperature at 25-30°C, culture time for 3-5 days, and detect the activity of detoxification enzymes by transparent circle method. By sampling from the natural environment, the range of strain screening is expanded, the probability of obtaining high-quality strains is increased, and the activity of detoxification enzymes of strains is visually detected by transparent circle method, and strains with strong ability to degrade vomitoxin are screened out;

[0070] The primary screening strains were placed in a culture medium containing 1-5 μg / mL of vomitoxin for continuous subculture and domestication, with each generation culture time being 48 hours and the number of subcultures being 5-8 generations. The criteria for continuous subculture and domestication of the screening strains were that the bacterial body grew well and could significantly secrete detoxification enzymes. The strains were domesticated using a culture medium containing vomitoxin to enhance the strains' adaptability and detoxification ability in the actual fermentation matrix. After 5-8 generations of subculture, the screening strains not only had improved tolerance, but also significantly improved degradation efficiency.

[0071] The selected excellent strains were inoculated into a liquid culture medium, wherein the liquid culture medium had glucose and urea as the main nutrients, the inoculation amount was 5%-10% of the culture medium volume, the culture conditions were controlled at 25-30°C, 150-200rpm shaking culture for 24-48 hours, until the bacterial concentration reached 1×10 7 -1×10 8 CFU / mL, the enzyme activity of the strain is detected by colorimetry and the status of the strain is confirmed to obtain active microbial culture. The concentration of the culture solution is controlled by liquid oscillation culture to ensure rapid reproduction and high efficiency after inoculation. The enzyme activity of the strain is detected by colorimetry to further ensure that the strain is in good condition and provide highly active culture solution for solid-state fermentation.

[0072] The solid-state fermentation system is established, and specifically includes the following steps:

[0073] The active microbial liquid obtained in the microbial strain screening culture is evenly inoculated into the sterilized coconut meal matrix, and the inoculation amount is controlled to be 5%-10% of the weight of the coconut meal matrix. Sterile tools are used to fully mix the liquid so that it is evenly distributed in the matrix. A reasonable inoculation amount ensures the dominant position of the strain in the early stage of fermentation, avoids competition from contaminating bacteria, and improves the degradation efficiency. The liquid is evenly distributed in the matrix after full mixing to avoid differences in fermentation results due to uneven inoculation;

[0074] The inoculated coconut meal substrate is placed in a semi-enclosed fermentation container, and the fermentation conditions are adjusted to a temperature of 25-35°C and a relative humidity of 70%-90%. The initial fermentation time is controlled at 24-48 hours. The temperature range and humidity control provide a suitable fermentation environment, improve the enzyme secretion efficiency of the strain, and regularly turn the substrate to promote oxygen penetration, avoid anaerobic bacteria contamination, and further enhance the degradation ability of vomitoxin;

[0075] After the initial fermentation, the fermentation substrate is turned over every 12-24 hours to allow the bottom and surface substrates to be fully exchanged. The total fermentation time is controlled to be 5-7 days, and the fermentation temperature and humidity are regularly checked during the fermentation period.

[0076] In the above embodiment, in the solid-state fermentation system, the metabolic environment of the microorganisms is optimized by precisely adjusting the fermentation conditions, and the inoculation amount is increased to 10%, further enhancing the degradation ability of the strain; the adjustment of the fermentation temperature and humidity, especially the humidity control at 90%, provides a more suitable growth environment for the strain. At the same time, by increasing the frequency of stirring during the fermentation process, the aeration of the matrix is ​​significantly improved, the generation of anaerobic conditions is avoided, and the growth of miscellaneous bacteria is effectively inhibited, shortening the fermentation cycle to 6 days, and the degradation rate of vomitoxin is increased to 95%, which can more fully stimulate the metabolic potential of the microbial strain and further enhance the synergistic effect of the exogenous enzyme preparation.

[0077] Example 3

[0078] In order to further solve the problems of insufficient degradation efficiency of DON, inaccurate fermentation process control, secondary pollution in the fermentation product processing, uneven particles affecting livestock digestion and absorption and feed processing efficiency, this embodiment optimizes the steps of enzyme preparation addition, fermentation product collection and processing, and DON degradation effect detection. The specific optimization contents are as follows:

[0079] The enzyme preparation addition specifically also includes the following steps:

[0080] According to the total weight of the coconut meal substrate in the fermentation system, the amount of the exogenous enzyme preparation is calculated, and 0.1%-1% of the enzyme preparation by weight of the substrate is dissolved in sterile water to prepare a uniform enzyme solution, wherein the exogenous enzyme preparation is one of peroxidase and peptidase, and the enzyme preparation is added at 0.1%-1% by weight of the substrate, which not only ensures the degradation effect but also reduces the addition cost, and the enzyme solution is dissolved and then sprayed to ensure that the enzyme is fully in contact with the substrate to exert the maximum degradation effect;

[0081] 48 hours after the start of solid-state fermentation, the prepared enzyme solution was evenly sprayed on the surface of the fermentation substrate, and the substrate was fully turned over using sterile tools. Enzyme preparations were added in the middle and late stages of microbial solid-state fermentation to form a synergistic system of bacteria and enzymes, which greatly increased the degradation rate of vomitoxin.

[0082] After adding the exogenous enzyme preparation, the fermentation temperature is maintained at 25-35°C, the relative humidity is 70%-90%, and the frequency of substrate stirring is increased to once every 12 hours. Increasing the stirring frequency can enhance the distribution uniformity of the enzyme solution and the oxygenation effect of the substrate.

[0083] The fermentation product collection and processing specifically includes the following steps:

[0084] When the fermentation process reaches 5-7 days, monitor the status of the fermentation product, wherein the status of the fermentation product includes temperature, humidity, odor and bacterial activity parameters;

[0085] Random sampling was conducted to test the degradation effect of DON. When there was no obvious excessive mold growth on the surface of the fermentation product, the humidity was moderate, and the DON residue was significantly reduced and reached the degradation target, the fermentation was confirmed to be over. Random sampling was conducted when the fermentation was completed to ensure that the DON residue reached the degradation target. At the same time, the matrix status was monitored to confirm that the fermentation quality was qualified. The fermentation parameters were dynamically adjusted through scientific monitoring methods to ensure the stability of the degradation process.

[0086] The fermented coconut meal substrate is taken out from the fermentation container and spread in a drying device for drying. The drying temperature is controlled at 50-60°C for 12-24 hours until the moisture content of the substrate is reduced to 12%-15%, which effectively prolongs the storage time and prevents secondary contamination or corruption caused by excessive moisture.

[0087] The dried fermentation product is processed by a pulverizing device to a particle size range of 0.5-2 mm, and the pulverized fermentation product is fully mixed. The pulverizing process improves the physical properties of the matrix, facilitates subsequent feed processing and use, and improves the digestion and absorption rate of livestock.

[0088] The degradation effect detection of vomitoxin specifically includes the following steps:

[0089] Randomly sample the coconut meal matrix before and after fermentation, and control the sample quality to 10-20g;

[0090] The sampled coconut meal was crushed to a particle size of less than 1 mm, 3 g of the sample was accurately weighed, 20 mL of acetonitrile-water solution with a volume ratio of 84:16 was added, and the sample was placed in an oscillator and oscillated at 150-200 rpm for 30 minutes. The extract was set aside, the sampling quality and the ratio of the extract were controlled, the operation was standardized to avoid measurement errors, and the reliability of the test data was ensured. The oscillation extraction technology was used to quickly and efficiently obtain vomitoxin in the sample, providing accurate samples for subsequent testing;

[0091] The extract was filtered through a 0.22 μm filter membrane, and the filtered sample was injected into a high performance liquid chromatograph for detection. A C18 chromatographic column was used, and the mobile phase was an acetonitrile-water solution with a volume ratio of 50:50. The flow rate was 1.0 mL / min, the detection wavelength was 218 nm, and the column temperature was set to 30°C. The peak area and retention time of vomitoxin were recorded, and the content of vomitoxin was calculated by comparing with the standard curve. The separation and sensitivity of vomitoxin detection were improved by using a C18 chromatographic column and an optimized mobile phase.

[0092] The changes in the content of vomitoxin in the coconut meal matrix before and after treatment were compared, and the degradation rate was calculated. When the degradation rate reached more than 90% and the residual vomitoxin was lower than the national and industry standards, the degradation and fermentation treatment was confirmed to be qualified. The data compared the content of vomitoxin in the matrix before and after treatment, confirming that the degradation rate was as high as more than 90%, while ensuring that the residual concentration was lower than the national standard, providing protection for feed safety.

[0093] In the above embodiments, the enzyme preparation combination can not only improve the molecular cleavage efficiency of vomitoxin, but also further degrade the intermediate metabolites through the dual enzyme action, thereby ensuring the thoroughness of the degradation process. The substrate is fully mixed after each addition of the enzyme preparation, which significantly improves the distribution uniformity and effect of the enzyme, effectively avoids the waste of enzyme activity, and further prolongs the action time of the enzyme. The high performance liquid chromatography detection method is used to achieve accurate quantification of the vomitoxin residue. During the detection process, the fine processing of the sample and the establishment of the standard curve not only improve the accuracy of the test results, but also provide a scientific basis for evaluating the degradation effect of the fermentation process. Through the dynamic monitoring of the fermentation state, the stability and controllability of the fermentation process are further ensured, which not only improves the safety and consistency of the fermentation product, but also provides important technical guarantees for future industrial applications.

[0094] The technical solution of the present invention is further described through the following control groups and application examples:

[0095] Control group:

[0096] Raw material processing: The coconut meal was simply crushed, the particle size range was uneven, the moisture content was not adjusted, no glucose and urea were added, and it was not sterilized.

[0097] Bacteria treatment: No bacterial solution was inoculated.

[0098] Fermentation conditions: No fermentation was carried out, just left to stand.

[0099] Exogenous enzyme treatment: No exogenous enzymes were added.

[0100] Application Example 1:

[0101] Raw material processing: Coconut meal is crushed to a particle size of 0.5-2mm, the moisture content is adjusted to 40%-60%, 1% glucose and urea are added, and it is sterilized and set aside.

[0102] Bacteria treatment: inoculate active microbial liquid, the inoculation amount is 10% of the weight of coconut meal substrate.

[0103] Fermentation conditions: Place in a fermentation container, 25°C, 80% humidity, fermentation cycle is 5 days, turn over every 24 hours.

[0104] Exogenous enzyme treatment: No exogenous enzymes were added.

[0105] Application Example 2:

[0106] Raw material processing: Coconut meal is crushed to a particle size of 0.5-2mm, the moisture content is adjusted to 40%-60%, 1% glucose and urea are added, and it is sterilized and set aside.

[0107] Strain treatment: Use the active bacterial liquid of Trichoderma and Geotrichum candidum after screening and domestication.

[0108] Fermentation conditions: Place in a fermentation container, 30°C, 85% humidity, fermentation cycle is 7 days, turn over every 12 hours.

[0109] Exogenous enzyme treatment: No exogenous enzymes were added.

[0110] Application Example 3:

[0111] Raw material processing: Coconut meal is crushed to a particle size of 0.5-2mm, the moisture content is adjusted to 40%-60%, 1% glucose and urea are added, and it is sterilized and set aside.

[0112] Strain treatment: Use optimized bacterial solution.

[0113] Fermentation conditions: Place in a fermentation container, 30°C, 85% humidity, fermentation cycle is 7 days, turn over every 12 hours.

[0114] Exogenous enzyme treatment: exogenous enzymes were added after 48 hours of fermentation, with the addition amount being 0.5% of the weight of the substrate, and the fermentation was stirred every 12 hours in the later stage.

[0115] The deoxynivalenol in coconut meal was degraded according to different conditions of the control group, application example 1, application example 2 and application example 3, and after the degradation was completed, the coconut meal in the control group, application example 1, application example 2 and application example 3 was sampled and tested to determine the degradation effect. The specific test and determination results are shown in Table 1:

[0116]

[0117] As shown in Table 1, the coconut meal in the control group was not treated in any way, and the degradation rate of vomitoxin was only 2.15%, and the microbial biomass was low, indicating that the natural microbial activity in the coconut meal could not significantly degrade vomitoxin, and the moisture content and matrix state remained stable without substantial changes; in Application Example 1, the untamed microbial culture was inoculated, and the degradation rate was increased to 48.33%, and the microbial biomass increased significantly, but it was still lower than that of other optimized treatment groups. The data showed that the fermentation process had a preliminary effect on the degradation of vomitoxin, but no significant optimization was achieved; in Application Example 2, the degradation rate was significantly increased to 78.52% by screening the tamed microbial culture, indicating that the metabolic efficiency and specific degradation ability of the strain for vomitoxin were optimized after taming, and the microbial biomass increased significantly to 4.87×10 7 CFU / mL, indicating that the microorganisms in the matrix reproduce actively, indicating that the domesticated strains contribute significantly to the degradation efficiency of vomitoxin, while maintaining the stability of the matrix; Application Example 3, based on Application Example 2, combined with exogenous enzyme preparations for synergistic degradation, the degradation rate was further increased to 95.63%, indicating that the introduction of exogenous enzymes can significantly enhance the fermentation effect, and the microbial biomass was maintained at 1.02×10 8 The high level of CFU / mL, uniform moisture content and optimal matrix quality state verified the advantages of the synergistic effect of microbial metabolism and enzyme preparations, especially the stronger detoxification effect in complex mycin environments.

[0118] By screening and domesticating microbial strains, the specific enzyme activity was significantly improved, and the efficient degradation of vomitoxin was achieved. The degradation rate was increased from 48.33% of ordinary strains to 78.52% of domesticated strains, and finally reached 95.63% by combining with exogenous enzymes. By optimizing the raw material processing process and fermentation conditions, a suitable environment for microbial growth and metabolism was provided, the bacterial activity and reproduction ability were improved, and the microbial concentration was increased from 1.32×10 3 The CFU / mL was increased to 1.02×10 8 CFU / mL.

[0119] The synergistic effect of combining microbial fermentation with exogenous enzyme preparations has maximized the degradation efficiency of vomitoxin, breaking through the limitations of single microbial metabolism, especially showing significant advantages in complex matrices contaminated with highly mycotic mycorrhizal compounds. It can not only significantly reduce the content of vomitoxin, but also maintain the matrix quality of coconut meal (there is no significant deterioration in indicators such as odor, color, and texture), giving it a wider application potential in the field of animal feed. Through the optimization of the coconut meal matrix, microbial screening and domestication, and the synergistic effect of exogenous enzymes, an efficient, safe, and controllable vomitoxin degradation method has been constructed.

[0120] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for degrading vomitoxin in coconut meal by microbial fermentation, characterized in that: The following steps are involved: Raw material pretreatment, crushing coconut meal and adjusting the moisture content of coconut meal, adding glucose and urea to the coconut meal, mixing evenly and then sterilizing with high-temperature steam to obtain a sterilized pretreated coconut meal matrix; Screening and culturing microbial strains, selecting Trichoderma and Geotrichum candidum that can secrete DON to degrade, and performing acclimation and screening, culturing the screened strains in a liquid culture medium, and keeping them ready for use after the strains' bacterial activity reaches an optimal state, thereby obtaining an active microbial bacterial liquid; A solid-state fermentation system is established, wherein an active microbial liquid is inoculated into a sterilized coconut meal matrix, the inoculated coconut meal matrix is ​​placed in a fermentation container, and the matrix is ​​turned over to increase oxygen supply for solid-state fermentation; Enzyme preparation addition: after solid-state fermentation, an exogenous enzyme preparation that degrades vomitoxin is added; The fermentation products are collected and processed, and after the fermentation is completed, the fermentation products are dried, crushed and evenly mixed to obtain fermented coconut meal; The degradation effect of vomitoxin was tested by high performance liquid chromatography to detect the residual amount of vomitoxin in coconut meal before and after treatment to confirm whether the fermentation treatment significantly reduced the content of vomitoxin.

2. The method for degrading vomitoxin in coconut meal by microbial fermentation as claimed in claim 1, characterized in that: The raw material pretreatment specifically further comprises the following steps: The coconut meal raw material is processed by a crushing device to a particle size range of 0.5-2 mm; Determine the initial moisture content of the crushed coconut meal, and adjust the moisture content by adding purified water and drying according to the requirement that the final moisture content is 40%-60%, and let it stand for 30 minutes after evenly mixing to balance the humidity; According to the weight ratio of the coconut meal matrix, 0.5%-2% glucose and urea are added, and the coconut meal, glucose and urea are evenly mixed, and the mixing time is controlled within 5-10 minutes; The evenly mixed coconut meal matrix is ​​placed in a high temperature sterilization container and sterilized by high temperature steam. The sterilization temperature is controlled at 121°C and the sterilization time is 20-30 minutes. After the sterilization is completed, the matrix is ​​cooled to room temperature for use.

3. The method for degrading vomitoxin in coconut meal by microbial fermentation as claimed in claim 1, characterized in that: The microbial strain screening and culturing further comprises the following steps: Collect samples from the natural environment, preliminarily screen Trichoderma and Geotrichum candidum as target strains by plate dilution coating method, transfer the preliminarily screened strains to culture medium, control the culture temperature at 25-30°C, culture time for 3-5 days, and detect the detoxification enzyme activity by transparent circle method; The primary screening strains were placed in a culture medium containing 1-5 μg / mL of DON and continuously subcultured for 48 hours per generation, with 5-8 generations; Among them, the criteria for continuous passage and domestication screening of strains are good bacterial growth and the ability to significantly secrete detoxification enzymes; The selected excellent strains were inoculated into liquid culture medium at an inoculation volume of 5%-10% of the culture medium volume. The culture conditions were controlled at 25-30°C and 150-200rpm shaking culture for 24-48 hours until the bacterial concentration reached 1×10 7 -1×10 8 CFU / mL; The enzyme activity of the strain was detected by colorimetry and the status of the strain was confirmed to obtain active microbial culture liquid.

4. The method for degrading vomitoxin in coconut meal by microbial fermentation as claimed in claim 3, characterized in that: The natural environment includes soil, humus and plant residues, and the liquid culture medium has glucose and urea as main nutrient components.

5. The method for degrading vomitoxin in coconut meal by microbial fermentation as claimed in claim 1, characterized in that: The solid-state fermentation system is established, and specifically further comprises the following steps: The active microbial liquid obtained in the microbial strain screening culture is evenly inoculated into the sterilized coconut meal matrix, the inoculation amount is controlled to be 5%-10% of the weight of the coconut meal matrix, and sterile tools are used to fully mix so that the liquid is evenly distributed in the matrix; The inoculated coconut meal substrate is placed in a semi-enclosed fermentation container, and the fermentation conditions are adjusted to a temperature of 25-35° C., a relative humidity of 70%-90%, and an initial fermentation time of 24-48 hours; After the initial fermentation, the fermentation substrate is turned over every 12-24 hours to allow the bottom and surface substrates to be fully exchanged. The total fermentation time is controlled to be 5-7 days, and the fermentation temperature and humidity are regularly checked during the fermentation period.

6. The method for degrading vomitoxin in coconut meal by microbial fermentation as claimed in claim 1, characterized in that: The enzyme preparation addition specifically also includes the following steps: The amount of exogenous enzyme preparation was calculated based on the total weight of the coconut meal substrate in the fermentation system, and 0.1%-1% of the enzyme preparation by weight of the substrate was dissolved in sterile water to prepare a uniform enzyme solution; 48 hours after the start of solid-state fermentation, spray the prepared enzyme solution evenly on the surface of the fermentation substrate, and use sterile tools to fully turn the substrate; After the exogenous enzyme preparation is added, the fermentation temperature is maintained at 25-35°C, the relative humidity is maintained at 70%-90%, and the frequency of substrate turning is increased to once every 12 hours.

7. The method for degrading vomitoxin in coconut meal by microbial fermentation as claimed in claim 6, characterized in that: The exogenous enzyme preparation is one of peroxidase and peptidase.

8. The method for degrading vomitoxin in coconut meal by microbial fermentation as claimed in claim 1, characterized in that: The fermentation product collection and processing specifically further comprises the following steps: When the fermentation process reaches 5-7 days, monitor the status of the fermentation product; Random sampling was performed to test the degradation effect of DON. The fermentation was confirmed to be complete when there was no obvious excessive mold growth on the surface of the fermentation product, the humidity was moderate, the residual amount of DON was significantly reduced and the degradation target was reached; The fermented coconut meal substrate is taken out from the fermentation container and spread in a drying device for drying at a temperature of 50-60°C for 12-24 hours until the moisture content of the substrate is reduced to 12%-15%; The dried fermentation product is processed by a pulverizing device to a particle size range of 0.5-2 mm, and the pulverized fermentation product is fully mixed.

9. The method for degrading vomitoxin in coconut meal by microbial fermentation as claimed in claim 8, characterized in that: The fermentation product status includes temperature, humidity, odor and bacterial activity parameters.

10. The method for degrading vomitoxin in coconut meal by using microbial fermentation as claimed in claim 1, characterized in that: The detection of the degradation effect of DON specifically further comprises the following steps: Randomly sample the coconut meal matrix before and after fermentation, and control the sample quality to 10-20g; The sampled coconut meal was crushed to a particle size of less than 1 mm, 3 g of the sample was accurately weighed, 20 mL of acetonitrile-water solution with a volume ratio of 84:16 was added, and the mixture was placed in an oscillator and extracted at 150-200 rpm for 30 minutes, and the extract was used for later use; The extract was filtered through a 0.22 μm filter membrane, and the filtered sample was injected into a high performance liquid chromatograph for detection. A C18 chromatographic column was used, and the mobile phase was an acetonitrile-water solution with a volume ratio of 50:

50. The flow rate was 1.0 mL / min, the detection wavelength was 218 nm, and the column temperature was set to 30°C. The peak area and retention time of vomitoxin were recorded, and the content of vomitoxin was calculated by comparing with the standard curve. The changes in the content of vomitoxin in the coconut meal matrix before and after treatment were compared, and the degradation rate was calculated. When the degradation rate reached more than 90% and the residual vomitoxin was lower than the national and industry standards, the degradation and fermentation treatment was confirmed to be qualified.

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