Method for inhibiting growth of mould hyphae in silage forage grass

Abstract

The invention relates to the technical field of forage grass ensiling fresh-keeping, in particular to a method for inhibiting growth of mould hyphae in ensiling forage grass, which comprises the following steps: 1) preparing targeted straw vinegar: adopting a dry distillation method, cooling and condensing, standing and layering, filtering and adsorbing and purifying by activated carbon, and accurately controlling the content of acetic acid to be 30-40% and the content of phenolic compounds to be 15-20%; 2) adding a composite probiotic protection system: spraying targeting straw vinegar liquid accounting for 0.5-5% of the weight of the raw materials into the silage raw materials, and synchronously adding 0.1-0.3% of a composite protective agent; 3) regulating and controlling the ensiling environment in stages: the temperature is 25-30 DEG C and the humidity is 60-70% in the first 10 days, the temperature is 20-25 DEG C and the humidity is 60-70% in the later 20-35 days, and the total ensiling time is 30-45 days; and 4) dual detection verification: detecting mycotoxin by HPLC-MS / MS, and detecting the number of beneficial bacteria by a plate counting method.Experiments show that the method has an aspergillus flavus and aspergillus fumigatus inhibition rate of more than 65%, a lactobacillus survival rate of more than 90%, no residue of straw vinegar, and low cost, and is suitable for silage of various forage grass such as corn straw, alfalfa and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of forage silage preservation, in particular to a method for inhibiting the growth of mold mycelium in silage forage. BACKGROUND

[0002] At present, the scale development of grain-saving livestock farming, the demand for forage animals (cattle, sheep, etc.) is growing explosively. Silage technology, as the core means of long-term preservation of forage, its safety and quality directly determine the breeding benefit. The core principle of silage is to promote the proliferation of lactic acid bacteria in an anaerobic environment. Lactic acid bacteria decompose forage carbohydrates to produce lactic acid, thereby reducing the environmental pH value and inhibiting the growth of spoilage microorganisms. However, mold contamination during silage is always a problem in the industry. Mold spores (such as Aspergillus flavus and Aspergillus fumigatus spores) are easily carried during the harvesting of silage raw materials. If the temperature and humidity are not properly controlled (such as temperature > 30℃, humidity > 75%) or the sealing is not strict, mold spores will quickly germinate into mycelium, not only competing for nutrients and causing forage spoilage, but also producing highly toxic metabolites such as aflatoxin B1 and fumonisin F1. These toxins, when ingested by herbivorous animals, can cause liver damage, immune suppression, and even acute poisoning and death. According to statistics, the mortality rate of cattle and sheep caused by moldy silage is 5-8% per year, causing tens of billions of economic losses.

[0003] The existing silage mold inhibition technology has significant defects, the most core problem of which is "lack of selective protection of beneficial bacteria". The current technology mainly relies on two types of additives: one is chemical preservatives (such as potassium sorbate, sodium benzoate), which can exert broad-spectrum antibacterial effect by damaging microbial cell membranes, but this type of substance cannot distinguish between mold and beneficial bacteria. Experimental data shows that the number of lactic acid bacteria in silage corn stalks with 0.5% sodium benzoate is reduced by 40-50% compared with the control group, and the lactic acid production is reduced by 30%, which in turn leads to an increase in the pH value of the silage environment, increasing the risk of spoilage. Moreover, chemical preservatives are prone to residual in forage (such as sodium benzoate residual amount can reach 0.3-0.5 mg / kg), which can accumulate in animals' bodies when fed for a long time, and even be transmitted to humans through dairy products and meat products, posing a food safety hazard. The other is a single biological additive (such as calcium propionate, plant essential oil), which has lower toxicity than chemical preservatives, but still has the problem of "too wide inhibition spectrum": for example, the number of yeast in 1% calcium propionate treated silage alfalfa is reduced by 35%, while yeast can assist in the decomposition of cellulose, and its reduction will lead to a 10-15% reduction in the degradation rate of forage crude fiber, affecting animal digestion and absorption.

[0004] In addition, the existing straw vinegar application technology also does not solve the "beneficial bacteria protection" problem. As a natural extract, straw vinegar has a certain mold inhibitory effect, but the traditional extraction process is rough (only simple condensation, without removing heavy phenols), resulting in complex composition - the tar and heavy phenols (such as cresol) contained in the crude straw vinegar not only stimulate the cell wall of mold, but also damage the peptidoglycan structure of lactic acid bacteria, making the survival rate of lactic acid bacteria less than 60%; and the existing technology has not optimized the proportion of effective ingredients, when the acetic acid content is less than 25%, the inhibition rate of mold is less than 30%, and when the acetic acid content is higher than 40%, the lactic acid bacteria will be inactivated. At the same time, the existing technology lacks systematic verification of the "mold inhibition-beneficial bacteria protection" effect, most of which only detect the number of mold hyphae, without establishing beneficial bacteria activity detection standards (such as lactic acid bacteria number, lactic acid production), and cannot judge the fermentation quality of silage forage grass.

[0005] More importantly, the existing technology does not clarify the mechanism of "inhibiting mold without affecting beneficial bacteria", resulting in a lack of theoretical support for the technical solution. There are essential differences in the cell structure of mold and beneficial bacteria: the main component of mold cell wall is chitin (containing β-1,4-glucoside bond), while the cell wall of lactic acid bacteria is peptidoglycan (containing β-1,3-glucoside bond), and the cell wall of yeast is glucan and mannan. If an inhibitor that can specifically act on chitin can be designed, and a substance that can protect peptidoglycan and glucan is added, "targeted inhibition of mold and directional protection of beneficial bacteria" can be achieved. However, the existing technology does not take advantage of this structural difference, but still uses the "non-discriminatory attack" inhibition idea, resulting in a reversal of the technical effect.

[0006] In summary, the existing technology cannot meet the four major needs of "high-efficiency mold inhibition, protection of beneficial bacteria, no residue, and low cost" at the same time, and there is an urgent need to develop a technical solution based on "structural difference targeting", which can solve the contradiction between mold inhibition and beneficial bacteria protection in silage forage grass by precisely regulating the composition of inhibitors and the protection agent system, and promote the green and sustainable development of animal husbandry. SUMMARY

[0007] The present application aims to overcome the defects of the prior art and specifically solve the following core problems: the existing additives (chemical preservatives, single biological agents) cannot distinguish between mold and beneficial bacteria, resulting in inhibition of mold while damaging lactic acid bacteria and yeast, reducing silage fermentation efficiency and forage nutritional value; traditional straw vinegar liquid has complex components (contains heavy phenols and tar), which not only inhibits beneficial bacteria but also affects the palatability of forage, and the proportion of effective components is out of control, the mold inhibition effect is unstable; the existing technology lacks a mechanism design for inhibiting mold and protecting beneficial bacteria, and a dual-effect detection standard has not been established (only mold is detected, not beneficial bacteria), which cannot ensure silage quality; chemical preservatives are high in cost (adding cost per ton of silage raw materials increases by 50-80 yuan) and have residual risks, which do not meet the requirements of green animal husbandry. The ultimate goal is to provide a silage forage mold mycelium growth inhibition method that is "targeted mold inhibition, directional bacteria protection, residue-free, and low-cost", which realizes mold inhibition rate ≥65%, beneficial bacteria survival rate ≥90% by clarifying the mechanism, optimizing process parameters, and establishing detection standards, while reducing silage cost and protecting the health of herbivores.

[0008] The technical solution adopted by the present application to solve its technical problems is: a method for inhibiting the growth of mold mycelium in silage forage, comprising the following steps: a) preparing a targeted finished straw vinegar liquid: crushing crop straw (at least one of corn straw, wheat straw or rice straw) to 5-10 mm, placing it in a dry distillation furnace, and heating it to 400-600°C at a rate of 5°C / min under air isolation (vacuum degree -0.08~-0.09MPa) for 2-3h to produce volatile gases by pyrolysis of the straw; pass the gas into a -5~0°C ethylene glycol condensation tower, collect the crude straw vinegar liquid after condensation; let the crude liquid stand at 25-30°C for 48-72h, take the middle layer aqueous phase after layering, and filter it through a 0.22μm organic filter membrane; add 5-8% of granular activated carbon (particle size 0.5-1mm) by weight to the filtrate, stir and adsorb at 25°C, 150-200r / min for 12-16h, remove the activated carbon by filtration again, and obtain the targeted finished straw vinegar liquid, which contains 30-40% acetic acid, 15-20% phenolic compounds (phenol, guaiacol), 8-12% ketones (acetone, butanone), and pH 3.0-3.5; b) raw material pretreatment and mixed with composite additives: remove moldy parts of the silage forage (corn stalks, alfalfa or grass), cut to 2-3 cm, adjust the initial moisture content to 65-70%; evenly spray the target finished straw vinegar liquid obtained in step a) accounting for 0.5-5% of its weight into the raw material, while adding 0.1-0.3% of the composite probiotic protective agent (fucoidan and chitosan are compounded at a mass ratio of 1:1) accounting for the weight of the raw material. 0.1-0.3%, use an atomizing nozzle (atomizing particle size 50-100 μm) to spray and continuously turn the raw material to ensure that the additive evenly covers the surface of the raw material. After spraying, stand for 10-15 min to allow the additive to penetrate; c) stage by stage silage environment regulation: layer by layer load the raw material treated in step b) into the silo, compact every 30-40 cm thick with a compactor to remove air, and after filling, use "double sealing" (0.12 mm polyethylene film for the inner layer, 0.5 mm canvas for the outer layer, and edge soil compaction) ; After sealing, regulate the environment in stages: control the temperature to 25-30℃ by electric heating sheet for the first 10 days (rapid fermentation period), and control the temperature to 20-25℃ by natural cooling combined with a ventilation valve for the next 20-35 days (stable period), and maintain the humidity to 60-70% by saturated sodium chloride solution throughout the process, the total silage time is 30-45 days (30 days for corn stalks, 35 days for alfalfa, and 45 days for grass) ; d) dual effect detection: after silage, samples are collected using the five-point sampling method; ① Mycotoxin detection: after freeze-drying and crushing, HPLC-MS / MS (chromatographic column C18 column 2.1 mm x 150 mm 1.8 μm, mobile phase methanol-0.1% formic acid water solution 70:30, ESI+ ion source, MRM mode) is used to detect aflatoxin B1, fumonisin F1 and ochratoxin A, to ensure that the content is ≤GB13078-2017 limit; ② Beneficial bacteria detection: plate counting method, MRS medium 37℃ anaerobic culture for 48h to count lactic acid bacteria, PDA medium 28℃ aerobic culture for 72h to count yeast, to ensure that the number of lactic acid bacteria is ≥10 6 CFU / g, the number of yeast is ≥10 4 CFU / g.

[0009] Specifically, the initial water content of the crop straw in step a) is controlled to be 10-15%.

[0010] Specifically, the working pressure of the atomizing nozzle in step b) is 0.2-0.3 MPa, to ensure that the coverage uniformity of the additive on the surface of the raw material is ≥95%.

[0011] Specifically, the sample pretreatment process for mycotoxin detection in step d) is as follows: 5 g of the crushed sample is weighed, 20 mL of a methanol-water (80:20) mixture is added, ultrasonic extraction is carried out at 300 W for 30 min, and centrifugation is carried out at 8000 r / min for 10 min to obtain the supernatant; 5 mL of n-hexane is added to the supernatant, oscillation is carried out for 10 min, centrifugation is carried out at 8000 r / min for 5 min, the upper layer is discarded, defatting is repeated twice, and the lower layer is filtered through a 0.22 mu m filter membrane to obtain the test solution.

[0012] Specifically, after the activated carbon adsorption in step a), the content of heavy phenols in the filtrate is detected by a UV spectrophotometer to be less than or equal to 0.5 mg / L, so as to ensure that the harmful substances are removed.

[0013] Specifically, the addition amount of the composite probiotic protective agent in step b) is 0.2% of the weight of the raw material, and the purity of trehalose is greater than or equal to 99%, and the deacetylation degree of chitosan is greater than or equal to 85%.

[0014] Specifically, in step c), if the temperature in the cellar exceeds 30 DEG C in the first 10 days, a ventilation valve is opened (each time for 5 min, and the interval is 2 h) to reduce the temperature; and if the temperature is lower than 20 DEG C in the last 20-35 days, an electric heating sheet (power 500 W) is turned on to supplement the temperature.

[0015] Specifically, in step d), when counting the lactic acid bacteria, the sample is diluted by 1:10 to 10 -8 , 10 -6 , 10 -7 , 10 -8 dilution, 0.1 mL of each dilution is coated, and 3 parallel samples are set in each group to take the average value.

[0016] The beneficial effects of the present application are as follows: Specifically, the present application realizes precise regulation through "targeted straw vinegar liquid + composite protective agent" - acetic acid (30-40%) in the straw vinegar liquid can destroy the chitin cell wall of the mold (cause the cell wall to break and collapse), and phenols (15-20%) can inhibit the chitin synthase activity of the mold, thereby blocking the growth of the mycelium; and the composite protective agent (trehalose + chitosan) can form a protective film on the surface of the lactic acid bacteria and the yeast, thereby avoiding the damage of acetic acid and phenols to the cell wall of the beneficial bacteria. Experimental data show that the inhibition rates of the present application on aspergillus flavus, aspergillus fumigatus and aspergillus terreus are 68%, 72% and 65% respectively, the survival rate of lactic acid bacteria is 92%, and the survival rate of yeast is 88%, which are much better than the prior art (the survival rate of lactic acid bacteria in the chemical preservative group is only 55%); Residue-free and high safety: The targeted straw vinegar is derived from crop straw (agricultural waste), and no chemical synthetic substances are added during the extraction process. Harmful components are removed by activated carbon adsorption. After ensiling, acetic acid and phenols can be partially degraded by forage microorganisms (residual amount <0.1 mg / kg), and the complex protective agent (trehalose, chitosan) can be digested and absorbed by animals without any toxic side effects. HPLC-MS / MS detection shows that the content of aflatoxin B1 in the treatment group is only 10-15 μg / kg, which is much lower than the national standard limit (50 μg / kg), completely solving the problem of toxin pollution. Low cost and high resource utilization: The raw material of straw vinegar is crop straw (300-400 L of crude liquid can be extracted from each ton of straw, and 150-200 L of finished product can be obtained after purification), and the cost of raw material is only 1 / 5 of that of propionic acid. The cost of adding per ton of ensiling raw material is reduced by 60-80 yuan. At the same time, the resource utilization of straw reduces the pollution caused by burning (0.8 tons of CO2 can be produced by burning each ton of straw), which meets the "double carbon" target. Stable process and wide applicability: The phased environmental regulation can adapt to the needs of different seasons and regions (the temperature control time in the first stage can be shortened to 8 days in summer, and it can be extended to 12 days in winter), and it is suitable for corn straw, alfalfa, and other forages. Improving the nutritional value of forage: The activity of beneficial bacteria can promote the transformation of forage nutrients - lactic acid bacteria can convert carbohydrates into lactic acid to improve palatability; yeast can decompose crude fiber, increasing the crude fiber degradation rate by 12-15%; experiments show that the crude protein content of ensiled corn straw in the treatment group is 8.5-9.0% (increased by 12% compared with before ensiling), the lactic acid content is 1.8-2.0% (increased by 45% compared with the control group), and the animal feed intake increases by 15-20%. BRIEF DESCRIPTION OF DRAWINGS

[0017] The application will be further described below in conjunction with the drawings and examples.

[0018] Figure 1 : Comparison of Aspergillus flavus mycelium ultrastructure between the control group (without adding targeted straw vinegar) and the treatment group (adding 2% targeted straw vinegar) (1000x); Left graph (control group): Mycelium cell wall is complete and smooth without damage; Right graph (treatment group): Mycelium cell wall is broken and collapsed, and the contents are leaked; Figure 2 : Comparison of Aspergillus fumigatus mycelium ultrastructure between the control group and the treatment group (5000x); Left graph (control group): Mycelium internal organelles (such as mitochondria) are complete; Right graph (treatment group): Holes appear in the mycelium, and organelles are disintegrated; Figure 3 : Beta diversity of fungal ITS sequencing results of the control group CG and the treatment group TG, indicating the reliability of the repetition; Figure 4: The species level fungal species distribution venn diagram of the fungal ITS sequencing results of the control group CG and the treatment group TG, indicating the detected fungal species between several samples; Figure 5 : The species level fungal enrichment of the fungal ITS sequencing results of the control group CG and the treatment group TG, indicating the fungal species enriched in each sample; Figure 6 : The species level difference fungal scatter diagram of the fungal ITS sequencing results of the control group CG and the treatment group TG, wherein the aspergillus flavus, aspergillus fumigatus and aspergillus terreus marked in red are harmful fungi of the aspergillus genus, and can produce toxins in different degrees; after the treatment group TG, the above three aspergillus fungi are significantly reduced, indicating that the addition of 5% straw vinegar can significantly reduce the abundance and expression level of harmful aspergillus fungi. DETAILED DESCRIPTION

[0019] In order to make the technical means, creative features, purposes and effects realized by the present application easy to understand, the present application will be further described below in combination with specific embodiments.

[0020] As shown in Figures 1-6 , the method for inhibiting the growth of mold mycelium in silage forage of the present application is based on the design of core technology based on the difference in cell wall structure of mold and beneficial bacteria, and realizes "mold inhibition without damaging bacteria" through the combination scheme of "targeted straw vinegar preparation + composite probiotic protective agent addition + staged environmental regulation + double effect detection", which is as follows: 1. Preparation of targeted finished straw vinegar (the core is precise regulation of ingredients to achieve specific inhibition of mold) 1) Raw material pretreatment: select corn straw, wheat straw or rice straw (initial moisture content 10-15%, avoid incomplete dry distillation due to too high water content), crush to 5-10mm, ensure uniform particles and increase pyrolysis contact area; 2) Dry distillation: load the crushed straw into a dry distillation furnace (model GL-100), close the door and vacuum to -0.08~-0.09MPa, completely isolate air (prevent straw oxidation and combustion, ensure pure pyrolysis products); start the heating device, heat at a rate of 5℃ / min to 400-600℃ (temperature too low, insufficient production of acetic acid, phenolic and ketone, too high, more tar produced), keep for 2-3h, pyrolyze cellulose and hemicellulose in the straw into volatile gases containing acetic acid, phenolic and ketone; 3) Condensation collection: introduce the volatile gas into a condensation tower through a high-temperature resistant pipe, pass -5~0℃ ethylene glycol solution into the condensation tower (low temperature can ensure that the gas is fully condensed into liquid), the gas is cooled and condensed to form crude straw vinegar, which is collected in a sealed polyethylene container (to avoid oxidation of the crude liquid with air); 4) Fractionation purification (key step, remove harmful components, retain targeted antifungal components): ① Static stratification: place the crude straw vinegar liquid in a constant temperature environment of 25-30°C for 48-72h, and use the density difference of different components to realize stratification. The upper layer is light oil phase (contains a small amount of terpenes, no antifungal effect), the middle layer is water phase (contains effective components such as acetic acid, phenols, ketones, etc.), and the lower layer is tar phase (contains heavy phenols, asphalt, which can damage beneficial bacteria). The middle layer of water phase is extracted by siphon method, and the upper and lower layers are discarded. ② Filtration and impurity removal: filter the middle layer of water phase through a 0.22μm organic filter membrane (pore size can trap suspended impurities and part of the macromolecular tar) to remove solid particles and colloidal substances, and obtain a clear filtrate. ③ Activated carbon adsorption: add 5-8% of granular activated carbon (particle size 0.5-1mm, specific surface area ≥1000m 2 / g, strong adsorption capacity) to the clear filtrate, and stir and adsorb at 25°C, 150-200r / min for 12-16h. Activated carbon can specifically adsorb residual heavy phenols (such as cresol, dimethyl phenol) and tar. The filtrate after adsorption is detected by ultraviolet spectrophotometer to ensure that the content of heavy phenols is ≤0.5mg / L. ④ Secondary filtration: filter again through a 0.22μm organic filter membrane to remove activated carbon particles, and obtain the targeted finished straw vinegar liquid. 5) Component verification: use high performance liquid chromatography (HPLC) to detect the components of the finished product to ensure that the acetic acid content is 30-40% (acetic acid can destroy the chitin cell wall of mold), the phenolic compound (phenol, guaiacol) content is 15-20% (phenols can inhibit the enzyme activity of mold), the ketone compound (acetone, butanone) content is 8-12% (ketones can help reduce the environmental pH value), and the pH value is 3.0-3.5 (this pH value is safe for lactic acid bacteria and has strong inhibitory effect on mold).

[0021] 2. Design and addition of composite probiotic protective agent (the core is to protect beneficial bacteria and offset the potential impact of straw vinegar liquid) 1) Protective agent selection basis: design a protection system based on the cell wall structure of beneficial bacteria. The cell wall of lactic acid bacteria is peptidoglycan, the cell wall of yeast is glucan, and trehalose can combine with peptidoglycan and glucan through hydrogen bonds to form a "moisture-resistant stress-resistant film", reducing the stimulation of straw vinegar liquid to the cell wall. Chitosan has a positive charge, which can combine with the negative charge group on the surface of the cell membrane of beneficial bacteria to form a physical barrier, preventing acetic acid and phenols from entering the cell interior. In addition, trehalose and chitosan have a synergistic effect when compounded (mass ratio 1:1), and the protection effect is better than that of a single protective agent. 2) Raw material pretreatment: Selecting silage forage (corn stalks, alfalfa or grass), removing dry leaves, weeds and moldy parts (to avoid excessive initial mold contamination); using a chopper to cut the raw material to 2-3 cm (too short is easy to compact and clump, too long is not easy to exclude air); measuring the initial moisture content of the raw material, if the moisture is <65%, spray distilled water to adjust to 65-70% (this moisture content is beneficial to lactic acid bacteria fermentation, and can promote the penetration of additives); 3) Compound additive mixing: ① According to the proportion of 0.5-5% of the weight of the raw material, the target finished straw vinegar liquid is taken (adjust according to the degree of mold contamination: take 0.5-2% for light contamination, take 3-5% for heavy contamination), add an equal volume of distilled water to dilute (reduce the concentration to avoid local high concentration damage to beneficial bacteria); ② According to the proportion of 0.1-0.3% of the weight of the raw material, the compound probiotic protective agent (trehalose purity ≥99%, chitosan deacetylation degree ≥85%) is taken, added to the diluted straw vinegar liquid, stirred at 25°C, 300r / min for 5-10min, until the protective agent is completely dissolved (to ensure uniform dispersion); ③ Use atomizing nozzle (working pressure 0.2-0.3MPa, atomizing particle size 50-100μm) to spray the compound additive on the raw material, and continuously turn the raw material with a turner (spray 1 circle for every 1 turn), to ensure that the coverage uniformity of the additive on the surface of the raw material is ≥95%; after spraying, stand for 10-15min, to allow the additive to penetrate into the interior of the raw material (to avoid staying only on the surface).

[0022] 3. Stage silage environment regulation (the core is to match the growth characteristics of mold and beneficial bacteria, and to optimize the conditions for mold inhibition and bacteria protection) 1) Filling and sealing: The treated raw material is filled into a concrete silo (5m long x 3m wide x 2m high) layer by layer, and is compacted by a compactor (pressure 5-8MPa) every 30-40cm thick to exclude air in the gap between the raw materials (anaerobic environment is the premise of lactic acid bacteria proliferation), until the raw material is filled in the silo (10-15cm higher than the silo opening, to prevent sealing from being not tight due to settlement in the later period); the sealing adopts "double-layer structure": the inner layer is 0.12mm polyethylene film (good sealing property, puncture-resistant), which ensures complete coverage of the raw material, and the edge extends to 50cm outside the silo; the outer layer is 0.5mm canvas (wear-resistant, to prevent the film from being damaged by external force), and the edge of the canvas is compacted with wet soil (soil moisture ≥20%, to enhance the sealing property); one temperature sensor (accuracy ±0.5°C) and one humidity sensor (accuracy ±2%) are installed in the middle and four corners of the silo, respectively, and the sensors are connected to the controller to monitor and automatically adjust the environmental parameters in real time; 2) Temperature and humidity control in stages: ① The first stage (the first 10 days, the rapid fermentation period): This stage is the key period of lactic acid bacteria proliferation, and higher temperature is needed to promote lactic acid bacteria metabolism. The temperature in the pit is regulated to 25-30°C through the use of external electric heating sheets (power 1000W, one set every 2m), and the lactic acid bacteria reproduction rate at this temperature is 2-3 times that at 20°C. At the same time, the humidity is maintained at 60-70% by placing saturated sodium chloride solution (relative humidity 75% at 25°C) in the pit. If the humidity is >70%, the controller automatically opens the pit top ventilation valve (5 minutes of ventilation each time, with an interval of 2 hours) to reduce the humidity (excessive humidity is easy to breed mold). In this stage, the acetic acid and phenols in the straw vinegar liquid can quickly inhibit the germination of mold spores, avoiding the competition for nutrients between mold and lactic acid bacteria; ② The second stage (the last 20-35 days, the stable period): In this stage, lactic acid bacteria have proliferated a lot, and the lactic acid content has reached 1.5-2.0%. The temperature needs to be reduced to inhibit the extension of mold hyphae. The electric heating sheets are turned off, and the temperature is regulated to 20-25°C through natural cooling combined with the ventilation valve. At this temperature, the growth rate of mold hyphae is reduced by more than 50%, while lactic acid bacteria still maintain high activity. The humidity is still maintained at 60-70%, and the sealing condition is checked manually every 3 days. If the film is found to be damaged, it is repaired in time with adhesive tape and the soil is compacted again; 3) Control of ensiling time: The total ensiling time is adjusted according to the type of raw materials. Corn straw has low fiber content, and the quality can be stabilized after 30 days of ensiling. Alfalfa has high protein content, and 35 days are needed to ensure that the protein is not degraded. Sheep grass has high crude fiber content, and 45 days are needed to promote fiber decomposition and ensure palatability.

[0023] 4. Double effect detection (the core is to verify the effect of "mold inhibition without damaging bacteria", and to establish quality standards) 1) Sample collection: After ensiling, five-point sampling method (east, west, south, north, and center of the pit body) is used to collect samples, 500g from each position, mixed and then divided into 100g, and divided into two parts (one part for toxin detection, and the other part for microbial detection); 2) Mycotoxin detection (HPLC-MS / MS method): ① Sample pretreatment: The toxin detection sample was placed in a freeze dryer (temperature -50℃, vacuum degree 10Pa) and dried for 24h, then crushed and passed through an 80 mesh sieve (to ensure uniformity and sufficient extraction); 5g of the crushed sample was weighed, 20mL of a methanol-water mixture (volume ratio 80:20, methanol can effectively extract fat-soluble toxins) was added, and the mixture was placed in an ultrasonic cleaner (power 300W, temperature 25℃) for ultrasonic extraction for 30min (ultrasonic extraction can destroy cell structure and improve extraction rate); the extract was centrifuged in a centrifuge (8000r / min) for 10min, and the supernatant was taken; 5mL of n-hexane was added to the supernatant (to remove fat and avoid interference with detection), and after oscillation for 10min, the mixture was centrifuged (8000r / min, 5min), and the upper n-hexane phase was discarded; the defatting was repeated twice, and the lower layer solution was filtered through a 0.22μm organic filter membrane to obtain the sample solution; ② Detection conditions: the chromatographic column was Agilent ZORBAX SB-C18 (2.1mm x 150mm, 1.8μm, good separation effect); the mobile phase A was 0.1% formic acid aqueous solution, the mobile phase B was methanol, and the gradient elution program was 0-5min 30%B, 5-10min 30-70%B, 10-15min 70%B; the flow rate was 0.3mL / min, the column temperature was 35℃, the injection volume was 5μL; the ion source was an electrospray ion source (ESI+), the spray voltage was 3.5kV, and the ion source temperature was 300℃; the target toxins were detected by multiple reaction monitoring mode (MRM): aflatoxin B1 (parent ion 313.1, daughter ions 285.1 / 249.1), fumonisin F1 (parent ion 722.3, daughter ions 334.1 / 316.1), and patulin A (parent ion 404.1, daughter ions 358.1 / 239.1); ③ Judgment criteria: if the contents of the three toxins are all ≤ the limit value of GB13078-2017 "Feed Hygiene Standard" (aflatoxin B1 ≤ 50μg / kg, fumonisin F1 ≤ 1000μg / kg, patulin A ≤ 10μg / kg), the mold inhibition effect is determined to be qualified; 3) Beneficial bacteria detection (plate counting method): ① Lactic acid bacteria counting: the microbial detection sample was diluted by gradient dilution (dilution liquid was sterile normal saline containing 0.85% NaCl to maintain osmotic pressure) at a ratio of 1:10, and diluted to 10 -8 ; 0.1mL of bacterial solution at three dilution degrees of 10 -6 , 10 -7 , 10 -8 was taken and spread on MRS medium (designed for lactic acid bacteria, containing lactose, yeast extract and other nutrients); the medium was placed in an anaerobic incubator (37℃, anaerobic environment: 95% N2+5% CO2) and cultured for 48h; the number of colonies was counted by plate counting instrument, and the number of lactic acid bacteria per gram of sample was calculated; Yeast count: Take 10 -5 , 10 -6 , 10 -7 mL of the diluted bacterial solution, and spread it on PDA medium (containing glucose, potato extract, suitable for yeast growth); put it into a constant temperature incubator (28°C, aerobic environment) for 72h; count the number of colonies, and calculate the number of yeast per gram of sample; Determination criteria: if the number of lactic acid bacteria is ≥10 6 CFU / g, and the number of yeast is ≥10 4 CFU / g, it is determined that the beneficial bacteria protection effect is qualified; 4) Auxiliary detection: measure the lactic acid content (using high performance liquid chromatography) and crude fiber content (using acid washing method) of the silage grass, if the lactic acid content is ≥1.5%, and the crude fiber content is reduced by 8-12% compared with before silage, further verify that the metabolic activity of the beneficial bacteria is normal.

[0024] Example 1: Silage treatment based on corn straw (verify the mold inhibition and bacteria protection effect) Preparation of experimental materials 1) Silage raw material: select fresh corn straw (variety "Zhengdan 958", within 12h after harvesting, plant height 2.5m, leaf blade emerald green, no mold), remove the roots and withered leaves, cut to 2-3cm with a grass cutter; use the drying method (105°C drying to constant weight) to measure the initial moisture content of 68% (meet the requirement of 65-70%, no need to adjust); use the gradient dilution plate method to detect the initial microorganism quantity of the raw material: lactic acid bacteria 1.2×10 5 CFU / g, yeast 8.5×10 3 CFU / g, mold 3.2×10 3 CFU / g (lighter degree of pollution); 2) Preparation of target finished straw vinegar: select corn straw (moisture content 12%), crush to 8mm, load into a dry distillation furnace; vacuum to -0.085MPa, heat to 500°C at 5°C / min, keep for 2.5h; lead out the gas and condense the crude liquid with -2°C ethylene glycol; stand for 60h at 28°C, siphon the middle layer of water phase; filter with 0.22μm filter membrane, then add 7% granular activated carbon (particle size 0.8mm, specific surface area 1200m 2 / g), stir and adsorb for 14h at 25°C and 180r / min; filter twice to get the finished product, HPLC detection of components: acetic acid 35%, phenols 18%, ketones 10%, pH value 3.2, heavy phenols content 0.3mg / L; 3) Compound probiotic protective agent: purchase food-grade trehalose (purity 99.5%, Shandong Futian Biological Technology) and chitosan (degree of deacetylation 88%, Zhejiang Jinshell Biological), mix them according to the mass ratio of 1:1, and reserve; 4) Instruments and equipment: dry distillation furnace (GL-100), HPLC-MS / MS instrument (Agilent 6460), anaerobic incubator (YQX-II), plate counter (Scan 1200), atomizing nozzle (working pressure 0.25MPa, atomizing particle size 80μm).

[0025] Experimental Design and Procedure 1) Group design: Set up 3 treatment groups, each with 1000kg of corn stalk raw material: ① Experimental group: Add 2% targeted straw vinegar solution + 0.2% compound protective agent; ② Chemical preservative group: 0.5% sodium benzoate added (existing technology comparison); ③ Blank control group: No additives were added; 2) Additive mixing: ① Experimental group: Measure 20kg of finished straw vinegar solution and dilute it with 20kg of distilled water; measure 2kg of compound protective agent and add it to the diluted solution, stir at 25℃ and 300r / min for 8min until dissolved; spray the raw material with an atomizing nozzle while turning it over, and let it stand for 12min after spraying. ② Chemical preservative group: Take 5 kg of sodium benzoate, dissolve it in 50 kg of distilled water, and spray it onto the raw material in the same way; ③ Blank control group: Sprayed with 50kg of distilled water; 3) Filling and sealing: All three groups were filled into concrete silage pits of the same specifications (5m long × 3m wide × 2m high), and compacted every 35cm thick layer (pressure 6MPa). After filling, the pits were sealed with double layers (inner layer 0.12mm polyethylene film, outer layer 0.5mm canvas, and compacted soil at the edges). Four temperature and humidity sensors were installed in each pit and connected to a controller. 4) Staged temperature control: ① Experimental group: The temperature was controlled at 28±2℃ for the first 10 days (electric heating element power 1000W), and the temperature was controlled at 23±2℃ for the next 30 days (natural cooling). The humidity was 65±2% throughout the process (adjusted by saturated sodium chloride solution). ② Chemical preservative group and blank control group: The same staged temperature control scheme was used (to exclude the influence of environmental differences); 5) Silage time: Corn stalks should be ensiled for 30 days; 6) Sample Testing: After ensiling, samples were taken at five points to test the following indicators: ① Mycotoxin detection: The samples were processed according to the above HPLC-MS / MS method, and the results showed that the content of aflatoxin B1 in the experimental group was 11.8 μg / kg, the content of fumonisin F1 was 235 μg / kg, and the content of ochratoxin A was 3.2 μg / kg; the corresponding values in the chemical preservative group were 28.5 μg / kg, 350 μg / kg, and 6.8 μg / kg; and the corresponding values in the blank control group were 92.5 μg / kg, 850 μg / kg, and 15.6 μg / kg; the toxin content in the experimental group was much lower than that in the other two groups, and met the national standard. ②Beneficial bacteria count: Lactic acid bacteria: 1.5 x 10 7 CFU / g in the experimental group, 5.8 x 10 6 CFU / g in the chemical preservative group, and 8.2 x 10 5 CFU / g in the blank control group; the number of lactic acid bacteria in the experimental group was 2.6 times that of the chemical preservative group and 18.3 times that of the blank control group; Yeast: 2.3 x 10 4 CFU / g in the experimental group, 1.2 x 10 4 CFU / g in the chemical preservative group, and 6.5 x 10 3 CFU / g in the blank control group; the number of yeast in the experimental group was significantly higher than that in the other two groups; ③Mold count: 85 CFU / g in the experimental group, 210 CFU / g in the chemical preservative group, and 1.2 x 10 4 CFU / g in the blank control group; the mold inhibition rate in the experimental group was 99.3%, and that in the chemical preservative group was 98.2%, but the experimental group also ensured the activity of beneficial bacteria; ④Nutrient content detection: Crude protein content: 8.8% in the experimental group, 7.2% in the chemical preservative group, and 6.5% in the blank control group; the experimental group increased by 35.4% compared with the blank control group; Lactic acid content: 1.9% in the experimental group, 1.1% in the chemical preservative group, and 0.8% in the blank control group; the lactic acid production in the experimental group was 2.4 times that in the blank control group; Crude fiber content: 28.3% in the experimental group, 31.5% in the chemical preservative group, and 33.8% in the blank control group; the experimental group decreased by 16.3% compared with the blank control group.

[0026] Example 2: Silage processing based on alfalfa (optimization of composite preservative concentration) Experimental material preparation 1) Silage raw material: Fresh alfalfa (variety "Zhongmo No. 3", harvested at the initial flowering stage, leafy and free of pests and diseases), cut to 2.5 cm; the initial moisture content was 72%, and it was adjusted to 68% by airing for 1 h (25°C, ventilation); initial microbial detection: lactic acid bacteria 8.5 x 104 CFU / g, yeast 6.2 x 10 3 CFU / g, mold 2.8 x 10 3 CFU / g; 2) Targeted finished straw vinegar: same as Example 1 (35% acetic acid, 18% phenols, 10% ketones); 3) Compound protective agent: four concentration gradients are set: 0.1%, 0.2%, 0.3%, 0.4% (all trehalose: chitosan = 1:1); 4) Experimental design: the alfalfa raw material is divided into 5 groups (500 kg each), respectively: ① Blank protection group: 2% straw vinegar is added, no protective agent is added; ② Protective agent 0.1% group: 2% straw vinegar + 0.1% protective agent is added; ③ Protective agent 0.2% group: 2% straw vinegar + 0.2% protective agent is added; ④ Protective agent 0.3% group: 2% straw vinegar + 0.3% protective agent is added; ⑤ Protective agent 0.4% group: 2% straw vinegar + 0.4% protective agent is added; Each group is ensiled in a small silo bag (50 kg / bag, polyethylene material) to avoid differences in silo environment; Experimental steps 1) Additive mixing: the straw vinegar and protective agent are weighed according to the proportion in each group, diluted and sprayed (parameters are the same as in Example 1), and left for 15 min; 2) Bagging and sealing: 10 silo bags are filled in each group, 50 kg per bag, manually compacted (3 times per bag), and the bag opening is sealed (sealed by a heat sealer); 3) Environmental control: all silo bags are placed in a constant temperature and humidity incubator, and the temperature is controlled according to the phased scheme: 28°C for the first 10 days, 23°C for the last 25 days, and the humidity is 65%; 4) Silage time: the alfalfa is ensiled for 35 days; 5) Detection index: after the ensiling is completed, 3 bags of samples are taken from each group, and the following indexes are detected: ① Survival rate of beneficial bacteria: Survival rate of lactic acid bacteria (relative to the initial number of lactic acid bacteria): blank protection group 65%, 0.1% group 78%, 0.2% group 92%, 0.3% group 93%, 0.4% group 92%; it can be seen that when the concentration of protective agent is ≥0.2%, the survival rate of lactic acid bacteria is stable at more than 92%; Survival rate of yeast: blank protection group 62%, 0.1% group 75%, 0.2% group 88%, 0.3% group 89%, 0.4% group 87%; similarly, it is stable when the concentration is ≥0.2%; Mold inhibition rate: 60% for the blank control group, 68% for the 0.1% group, 72% for the 0.2% group, 73% for the 0.3% group, and 72% for the 0.4% group; the inhibition rate reached more than 72% when the concentration was greater than or equal to 0.2%; ③Cost of protective agent: 2.5 yuan per ton of raw materials for the 0.2% group, 3.8 yuan for the 0.3% group, and 5.0 yuan for the 0.4% group; the 0.2% group had the lowest cost, and there was no significant difference in effect compared to high concentrations; ④Palatability of forage grass: using sensory evaluation (color, odor, texture): the 0.2% group of alfalfa was green, had a clear fragrance of lactic acid, and had soft texture; the 0.4% group had a slight chitosan odor due to excessive protective agent, and the palatability decreased; Conclusion: The optimal addition concentration of the compound probiotic protective agent is 0.2% of the raw material weight, which can achieve a lactic acid bacteria survival rate of 92%, a mold inhibition rate of 72%, and the lowest cost and best palatability.

[0027] Example 3: Silage processing based on Chinese wildrye (verification of the necessity of phased temperature control) Experimental material preparation 1) Silage raw material: fresh Chinese wildrye (within 20 hours after harvesting, thick stems and green leaves) was selected and cut into 3 cm pieces; the initial moisture content was 62%, and distilled water was sprayed to adjust it to 67%; the initial microbial detection: lactic acid bacteria 6.8 x 10 4 CFU / g, yeast 5.5 x 10 3 CFU / g, and mold 3.5 x 10 3 CFU / g; 2) Targeted finished straw vinegar and compound protective agent: same as in Example 1, with a protective agent addition of 0.2%; 3) Experimental design: two environmental control schemes were set up for each group of 1000 kg of Chinese wildrye: ①Experimental group (phased temperature control): 28°C for the first 10 days and 23°C for the next 35 days, with a humidity of 65%; ②Control group (static temperature control): 25°C throughout, with a humidity of 65%; both groups were packed into the same silo with consistent sealing methods; Experimental steps 1) Additive mixing and silo packing: same as in Example 1, both groups were added with 2% straw vinegar + 0.2% protective agent; 2) Environmental control: the experimental group was controlled by phased temperature (electric heating film + ventilation valve), and the control group was controlled at 25°C throughout (only ventilation valve); 3) Silage time: Chinese wildrye was silaged for 45 days; 4) Detection indicators: ①Microbial dynamic monitoring: samples were taken at 10 days, 25 days, and 45 days of silage to detect the number of lactic acid bacteria and mold: 10 days of silage: lactic acid bacteria 1.2 x 107 CFU / g, control group 1.0x10 7 CFU / g; the experimental group mold 5.2x10 2 CFU / g, control group 8.5x10 2 CFU / g; the experimental group early lactic acid bacteria proliferate faster, and mold inhibition is more significant; 25 days of ensiling: the experimental group lactic acid bacteria 1.4x10 7 CFU / g, control group 1.1x10 7 CFU / g; the experimental group mold 2.1x10 2 CFU / g, control group 4.3x10 2 CFU / g; 45 days of ensiling: the experimental group lactic acid bacteria 1.3x10 7 CFU / g, control group 9.8x10 6 CFU / g; the experimental group mold 72 CFU / g, the control group 135 CFU / g; the final number of mold in the experimental group is only 53% of the control group; ②Mold toxin content: the experimental group aflatoxin B1 10.5 μg / kg, the control group 22.8 μg / kg; the experimental group has lower toxin content; ③Nutritional ingredients: the experimental group crude protein 9.2%, lactic acid 1.8%, crude fiber 26.5%; the control group crude protein 8.0%, lactic acid 1.3%, crude fiber 29.3%; the experimental group has better nutrient retention; ④Ensiling stability: after opening, the two groups of samples are placed in a 25℃, 70% humidity environment, and the mold time is observed: the experimental group appears a small amount of mold in 15 days, and the control group appears mold in 8 days; the experimental group has better stability, and the spore residue of mold is reduced due to phased temperature control; Conclusion: phased environmental regulation (25-30℃ for the first 10 days, 20-25℃ for the last 20-35 days) can significantly improve the mold inhibition effect and beneficial bacteria activity of Chinese wild ryegrass silage, which is better than the static temperature control scheme, and is an important guarantee for realizing "mold inhibition and bacteria protection".

[0028] In summary, the present application solves the core problem of the prior art "mold inhibition at the expense of bacteria" by "targeted mold inhibition component design + directional bacteria protection system construction + phased environment matching + dual effect verification", and has the advantages of no residue, low cost and wide applicability, and can be widely applied to silage forage treatment for herbivorous animal breeding, and promote the green and sustainable development of grain-saving livestock farming.

[0029] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above embodiments, and the above embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for inhibiting the growth of mold mycelia in silage, characterized in that, Includes the following steps: a) Preparation of targeted finished straw vinegar: Crush crop straw to 5-10mm, place it in a dry distillation furnace, and heat it to 400-600℃ at a rate of 5℃ / min under air-isolated conditions and a vacuum degree of -0.08~-0.09MPa, holding it at this temperature for 2-3h to allow the straw to pyrolyze and produce volatile gases; pass the gas into an ethylene glycol condenser at -5~0℃, collect the crude straw vinegar after condensation; let the crude liquid stand at 25-30℃ for 48-72h, and separate it into layers. Take the middle layer aqueous phase and filter it through a 0.22μm organic filter membrane; add 5-8% by weight of 0.5-1mm granular activated carbon to the filtrate, stir and adsorb for 12-16h at 25℃ and 150-200r / min, filter again to remove the activated carbon, and obtain the targeted finished straw vinegar solution, which contains 30-40% acetic acid, 15-20% phenolic compounds, 8-12% ketone compounds, and pH value of 3.0-3.5; b) Raw material pretreatment and compound additive mixing: Remove moldy parts from the silage raw material, chop it to 2-3cm, and adjust the initial moisture content to 65-70%; spray 0.5-5% of the targeted finished straw vinegar obtained in step a) evenly onto the raw material, and add 0.1-0.3% of the compound probiotic protectant by weight of the raw material. The compound probiotic protectant is a mixture of trehalose and chitosan in a mass ratio of 1:

1. Spray with an atomizing nozzle with a particle size of 50-100μm and continuously turn the raw material to ensure that the additive evenly covers the surface of the raw material. After spraying, let it stand for 10-15 minutes to allow the additive to penetrate. c) Phased silage environment control: The raw materials treated in step b) are loaded into the silage pit layer by layer. Every 30-40cm thick layer is compacted with a compactor to remove air. After filling, double-layer sealing is used, with an inner layer of 0.12mm polyethylene film and an outer layer of 0.5mm canvas. The soil at the edges is compacted. After sealing, the environment is controlled in stages: During the rapid fermentation period of the first 10 days, the temperature is controlled at 25-30℃ using electric heating elements. During the stabilization period of the next 20-35 days, the temperature is controlled at 20-25℃ by natural cooling combined with ventilation valves. The humidity is maintained at 60-70% throughout the process using a saturated sodium chloride solution. The total silage time is 30-45 days. d) Dual-effect testing: After silage, samples were collected using a five-point sampling method; ① Mycotoxin testing: After freeze-drying and pulverizing, the samples were tested for aflatoxin B1, fumonisin F1, and ochratoxin A using HPLC-MS / MS to ensure that the content was ≤ GB13078-2017 limit; ② Beneficial bacteria testing: The plate count method was used, with lactic acid bacteria counted by anaerobic culture at 37℃ for 48h on MRS medium and yeast counted by aerobic culture at 28℃ for 72h on PDA medium, ensuring that the number of lactic acid bacteria was ≥ 10. 6 CFU / g, yeast count ≥10 4 CFU / g.

2. The method for inhibiting the growth of mold mycelia in silage according to claim 1, characterized in that: In step a), the initial moisture content of crop straw is controlled to be 10-15%.

3. The method for inhibiting the growth of mold mycelia in silage according to claim 1, characterized in that: In step b), the working pressure of the atomizing nozzle is 0.2-0.3 MPa to ensure that the additive has a uniform coverage of ≥95% on the surface of the raw material.

4. The method for inhibiting the growth of mold mycelia in silage according to claim 1, characterized in that: The sample pretreatment process for mycotoxin detection in step d) is as follows: Weigh 5g of pulverized sample, add 20mL of methanol-water (80:20) mixture, extract by ultrasonication at 300W for 30min, centrifuge at 8000r / min for 10min and take the supernatant; add 5mL of n-hexane to the supernatant and shake for 10min, centrifuge at 8000r / min for 5min and discard the upper layer, repeat the defatting process twice, and filter the lower layer through a 0.22μm filter membrane to obtain the test solution.

5. The method for inhibiting the growth of mold mycelia in silage according to claim 1, characterized in that: After activated carbon adsorption in step a), the content of heavy phenols in the filtrate is detected by ultraviolet spectrophotometer to be ≤0.5mg / L, ensuring the removal of harmful substances.

6. The method for inhibiting the growth of mold mycelia in silage according to claim 1, characterized in that: In step b), the amount of compound probiotic protectant added is 0.2% of the weight of the raw materials, wherein the purity of trehalose is ≥99% and the degree of deacetylation of chitosan is ≥85%.

7. The method for inhibiting the growth of mold mycelia in silage according to claim 1, characterized in that: In step c), if the temperature inside the cellar exceeds 30°C during the first 10 days, open the ventilation valve and ventilate for 5 minutes each time, with an interval of 2 hours, to lower the temperature; if the temperature is below 20°C during the next 20-35 days, turn on the electric heating element to supplement the temperature.

8. The method for inhibiting the growth of mold mycelia in silage according to claim 1, characterized in that: In step d), when counting lactic acid bacteria, the sample is serially diluted 1:10 to 10. -8 Take 10 -6 10 -7 10 -8 Spread 0.1 mL of each diluted bacterial solution onto a plate, and set up 3 parallel samples for each group and take the average value.

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