Application of compound microbial agent combined with organic acid in preparation of silage

Abstract

The application relates to application of a compound bacterial agent combined with organic acid in preparation of silage, and belongs to the technical field of silage fermentation. The application provides application of a compound bacterial agent combined with organic acid in preparation of silage, wherein the compound bacterial agent is composed of lactobacillus plantarum, lactobacillus buchneri and bacillus subtilis in a live bacterial number ratio of (2.8-3.2):(0.8-1.2):(0.8-1.2), and the organic acid is composed of calcium propionate and sodium diacetate in a mass ratio of 1:(0.8-1.2). The compound bacterial agent and the organic acid are added in the preparation process of the silage, so that efficient inhibition and degradation of mycotoxins can be realized, the content of the mycotoxins in the silage is reduced, and the nutritional value and the fermentation effect of the silage can be simultaneously improved.

Description

Technical Field

[0001] This invention belongs to the field of silage fermentation technology, and particularly relates to the application of compound microbial agents combined with organic acids in the preparation of silage. Background Technology

[0002] Silage is a high-quality feed produced by storing fresh green fodder through microbial fermentation. It is widely used in livestock production because it effectively preserves nutrients, extends storage time, and meets the nutritional needs of livestock at different growth stages. However, during the various stages of silage production and fermentation, especially the initial 0-7 day aerobic stage, a large amount of oxygen exists between the raw materials. The plants themselves are still respiring, and aerobic microorganisms such as molds and yeasts are active, easily proliferating and producing mycotoxins such as aflatoxin, zearalenone, and vomitoxin.

[0003] Mycotoxins not only reduce the nutritional value and palatability of silage, but also accumulate in livestock and poultry through the food chain, leading to decreased livestock and poultry production performance, reproductive disorders, immunosuppression, and even affecting the quality and safety of livestock products, endangering human health. Currently, existing silage fermentation technologies mostly reduce or eliminate the harmful effects of toxins by adding adsorbents (such as montmorillonite and yeast cell walls), chemical treatments (such as alkalization and ozonation), or biodegradation (such as specific enzyme preparations). However, adsorbents have low adsorption rates for non-polar toxins such as zearalenone and vomitoxin, and may also adsorb nutrients such as vitamins; chemical detoxification easily damages the nutritional structure of feed and poses a risk of chemical residues; while biodegradation is precise, its effectiveness is unstable and it cannot specifically inhibit the growth and reproduction of molds in the aerobic stage, resulting in poor mycotoxin control.

[0004] Therefore, developing a silage fermentation process that can efficiently reduce the mycotoxin content in silage and improve its nutritional value and fermentation effect has become an urgent technical problem to be solved in the field of silage production. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the application of compound microbial agents combined with organic acids in the preparation of silage, so as to achieve efficient inhibition and degradation of mycotoxins, thereby reducing the mycotoxin content in silage and improving the nutritional value and fermentation effect of silage.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of compound microbial agents combined with organic acids in the preparation of silage. The compound microbial agents are composed of Lactobacillus plantarum, Lactobacillus brunelli and Bacillus subtilis in a viable count ratio of (2.8~3.2):(0.8~1.2):(0.8~1.2). The organic acids are composed of calcium propionate and sodium diacetate in a mass ratio of 1:(0.8~1.2).

[0007] This invention provides a method for preparing silage, comprising the following steps: crushing silage raw materials, filling them into silage facilities, sealing them, and fermenting them; adding compound microbial agents and organic acids during the filling and sealing of the silage raw materials; The compound bacterial agent is composed of Lactobacillus plantarum, Lactobacillus brunelli and Bacillus subtilis in a live count ratio of (2.8~3.2):(0.8~1.2):(0.8~1.2), and the organic acid is composed of calcium propionate and sodium diacetate in a mass ratio of 1:0.8~1.2.

[0008] Preferably, the ratio of the amount of the compound microbial agent added to the mass of the silage raw material is (0.8~1.4) g:1 ton, and the number of viable bacteria in the compound microbial agent is ≥1×10⁻⁶. 9 CFU / g.

[0009] Preferably, the ratio of the amount of calcium propionate added to the mass of the silage raw material is (0.1~1.2) g: 100 g.

[0010] Preferably, the silage material is filled within ≤7 days.

[0011] Preferably, the steps of adding compound microbial agents and organic acids during raw material filling include: filling silage raw materials in layers, mixing the compound microbial agents and organic acids and spraying them onto the surface of each layer of silage raw materials, with each layer having a filling thickness of 20-30 cm; the steps of adding compound microbial agents and organic acids during sealing include: mixing the compound microbial agents and organic acids and spraying them onto the top and end of the silage facility.

[0012] Preferably, mechanical compaction is used during the filling of each layer of silage raw material, with a compaction density of 700 kg / m³. 3 above.

[0013] Preferably, the fermentation time is ≥42 days.

[0014] The present invention also provides a silage, which is prepared using the preparation method described above.

[0015] Preferably, the silage is corn silage.

[0016] The beneficial effects of this invention are: This invention provides the application of compound microbial agents and organic acids in the preparation of silage. By adding compound microbial agents and organic acids during the preparation of silage, the bacteria and their produced enzymes work synergistically to achieve efficient inhibition and degradation of mycotoxins from the source, thereby reducing the mycotoxin content in silage and improving the fermentation quality and nutritional value of silage.

[0017] The compounded organic acid in this invention has a more significant antibacterial effect than a single organic acid, and can avoid the impact of a single organic acid on the palatability of silage. The Bacillus subtilis in the compounded microbial agent can produce antimicrobial peptides, which, together with the compounded organic acid and the original microbial agent, form a "triple antibacterial" effect. At the same time, the enzymes produced can degrade the vomitoxin that has already been produced, breaking through the limitation of traditional technology that "only inhibits but does not degrade", and ensuring the control effect of mold.

[0018] The silage prepared using the organic acids and compound microbial agents of this invention as preservatives has better nutritional value and fermentation quality than silage prepared using other preservatives. Furthermore, the prepared silage promotes animal growth and development, thus improving its overall nutritional value. Attached Figure Description

[0019] Figure 1 A trend graph showing the effect of adding different organic acids on the silage fermentation temperature over time. Detailed Implementation

[0020] This invention provides the application of a compound microbial agent combined with organic acids in the preparation of silage. The compound microbial agent is composed of *Lactobacillus plantarum*, *Lactobacillus brunelli*, and *Bacillus subtilis* in a viable count ratio of (2.8~3.2):(0.8~1.2):(0.8~1.2), preferably (2.8, 3.0, or 3.2):(0.8, 1.0, or 1.2):(0.8, 1.0, or 1.2). The organic acid is composed of calcium propionate and sodium diacetate in a mass ratio of 1:(0.8~1.2), preferably 1:0.8, 1:1.0, or 1:1.2. This invention does not specifically limit the sources of the *Lactobacillus plantarum*, *Lactobacillus brunelli*, *Bacillus subtilis*, calcium propionate, and sodium diacetate; conventional commercially available products in the art can be used.

[0021] The compounded organic acid in this invention has a more significant antibacterial effect than a single organic acid, and can avoid the impact of a single organic acid on the palatability of silage. The Bacillus subtilis in the compounded microbial agent can produce antimicrobial peptides, which, together with the compounded organic acid and the original microbial agent, form a "triple antibacterial" effect. At the same time, the enzymes produced can degrade the vomitoxin that has already been produced, breaking through the limitation of traditional technology that "only inhibits but does not degrade", reducing the mycotoxin content in silage and improving the fermentation quality and nutritional value of silage.

[0022] This invention provides a method for preparing silage, comprising the following steps: crushing silage raw materials, filling them into silage facilities, sealing them, and fermenting them; adding compound microbial agents and organic acids during the filling and sealing of the silage raw materials; the compound microbial agents and organic acids are the same as above, and will not be repeated here.

[0023] In this invention, the silage raw material is preferably fresh, free from mold and rot, and the dry matter content of the silage raw material is preferably 32%-37%, more preferably 32%, 35% or 37%. The silage raw material is crushed, preferably by cutting into segments, the length of which is preferably 1.5-3cm, more preferably 1.5cm, 2cm, 2.5cm or 3cm.

[0024] In this invention, after the silage raw material is crushed, it is filled into a silage facility, preferably a silage pit, silage tower, or silage wrapping equipment. Pre-treatment of the silage facility is preferred during filling. Pre-treatment typically involves thoroughly cleaning the facility to remove residual silage feed, debris, and moldy substances, then disinfecting it with a quicklime solution or disinfectant, and finally drying it before use. This prevents residual mold spores from contaminating the new silage raw material, reducing the risk of mycotoxin generation from the source.

[0025] During filling, it is preferable to fill the silage material into the silage facility in layers, with each layer preferably being 20-30 cm thick, more preferably 20 cm, 25 cm, or 30 cm. Mechanical compaction is preferably used during the filling process, and the compaction density is preferably 700 kg / m³. 3 The above, such as 710kg / m 3 750kg / m 3 Or 800kg / m 3 The key is to compact the pit walls and corners to avoid gaps that could lead to oxygen residue, shorten the aerobic phase, and reduce conditions for mold growth. After each layer is filled, the compound microbial agent and organic acid are mixed and sprayed onto the surface of each layer of silage. In this invention, water is preferably used as a solvent to dilute the mixture of compound microbial agent and organic acid. There is no specific limit to the amount of water used; it can be determined based on the initial dry matter content of the silage, ensuring that the dry matter content of the silage pile after spraying the mixture of compound microbial agent and organic acid is between 30% and 35%. The entire filling process is preferably completed within ≤7 days, more preferably within 3 days, and no longer than 7 days, to prevent the raw materials from becoming moldy due to prolonged exposure to air during the filling period.

[0026] After filling, the silage pit is sealed. Preferably, the sealing process involves first covering the raw material surface with a black and white film, then laying an oxygen-barrier film, and finally a plastic film. The edges of the plastic film are compacted with tires. The top of the silage pit is dome-shaped and extends 50cm above the pit walls. Drainage ditches are dug around the pit to facilitate the drainage of fermentation liquid and prevent rainwater seepage. This invention does not have specific limitations on the type and source of the black and white film, oxygen-barrier film, and plastic film; conventional types and commercially available products in the field can be used. In this invention, during the sealing process, it is preferable to mix the compound microbial agent and organic acid and spray the mixture onto the top and head of the silage facility.

[0027] After sealing, fermentation is carried out. The fermentation time is preferably ≥42 days. During the fermentation period, the sealing condition is checked regularly, and any damaged plastic film is repaired in time to prevent air from entering and causing secondary fermentation and mold growth.

[0028] After the silage is prepared according to the present invention, during the process of opening the silo and taking out the silage, it is preferable to spray organic acid on the section after each extraction to prevent mold growth. The organic acid is prepared by mixing calcium propionate and sodium diacetate in a mass ratio of 1:(0.8~1.2), and the preparation process of the organic acid is the same as the preparation process of the organic acid used for filling and sealing.

[0029] The filling process is a critical period for the proliferation of mycotoxins. Therefore, it is necessary to spray organic acids and compound microbial agents on the surface of the raw materials during this process to precisely inhibit the growth and proliferation of mold during filling. Therefore, the compound microbial agents and organic acids are added during the filling of raw materials. The preferred steps for adding them include: filling the silage raw materials in layers, mixing the compound microbial agents and organic acids and spraying them on the surface of each layer of silage raw materials. The preferred filling thickness of each layer is 20-30 cm.

[0030] In this invention, a pre-treatment spray is performed during sealing, adding compound microbial agents and organic acids. The preferred steps for adding these agents include: spraying the compound microbial agents and organic acids onto the top and end of the silage facility; ensuring that no areas are missed if the top and end of the silage are not properly compacted; inhibiting the growth of mold and the production of mycotoxins on the surface of the raw materials; and laying the foundation for mold control in the subsequent aerobic stage. After spraying, the silage is sealed.

[0031] In this invention, the preferred mass ratio of the compound microbial agent to the silage raw material is (0.8~1.4) g:1 ton, more preferably 0.8 g:1 ton, 1.0 g:1 ton, 1.2 g:1 ton, or 1.4 g:1 ton. The preferred viable count in the compound microbial agent is ≥1×10⁻⁶. 9 CFU / g, more preferably 1×10 9 CFU / g, 2×10 9 CFU / g, 5×10 9 CFU / g, 10×10 9CFU / g or 100×10 9 CFU / g.

[0032] The preferred ratio of the amount of calcium propionate added to the mass of the silage raw material is (0.1~1.2) g:100g, more preferably 0.1g:100g, 0.2g:100g, 0.4g:100g, 0.8g:100g, 1g:100g or 1.2g:100g.

[0033] The amounts of compound microbial agent and calcium propionate given above in this invention refer to the total amount sprayed during the filling and sealing process. That is, after preparing the mixture of compound microbial agent and calcium propionate according to the given amounts, it is placed in the spraying equipment and ready for use. After filling and sealing are completed, the prepared mixture is used up. This invention, by adding compound microbial agent and organic acid during the preparation of silage, utilizes the synergistic technology of bacteria and their produced enzymes to achieve efficient inhibition and degradation of mycotoxins from the source, thereby reducing the mycotoxin content in silage and improving the fermentation quality and nutritional value of silage.

[0034] The present invention also provides a silage, which is prepared using the preparation method described above.

[0035] The silage prepared using the compound microbial agent and organic acids of this invention as preservatives has better nutritional value and fermentation quality than that prepared using other preservatives (such as other organic acids). Furthermore, the prepared silage promotes animal growth and development, thus improving its overall nutritional value.

[0036] In this invention, the silage is preferably corn silage.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Unless otherwise specified, the following embodiments are all conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] In the following examples, Lactobacillus plantarum, Lactobacillus brunelli, and Bacillus subtilis were all provided by China Agricultural University.

[0041] Example 1 Preparation process of corn silage (1) Corn silage raw materials: Select fresh whole-plant corn silage raw materials that are free from mold and rot, chop them into pieces with a length of 1.5~3cm, and ferment them with a dry matter content of 32%-37%.

[0042] (2) Pretreatment of silage facilities When using silage pits as silage facilities, the facilities should be thoroughly cleaned during pretreatment to remove residual silage feed, debris, and moldy substances. Then, they should be disinfected with disinfectant and dried before use. This avoids contamination of new silage raw materials by residual mold spores in the facilities, thus reducing the risk of mycotoxin production from the source.

[0043] (3) Raw material loading and compaction The pretreated raw materials are layered into the silage pit, with each layer being 30cm thick. Mechanical compaction is used during the filling process to ensure that the compaction density reaches 750kg / m³. 3 The key is to compact the pit walls and corners to avoid leaving gaps that could lead to oxygen residue, shorten the aerobic stage, and reduce conditions for mold growth. During the filling process, a mixture of compound microbial agent and organic acid is sprayed on the surface after each layer is filled to precisely inhibit the growth and proliferation of mold during filling. The entire filling process is completed within 7 days to prevent the raw materials from becoming moldy due to prolonged exposure to air during filling.

[0044] (4) Pre-treatment for sealing the cellar Pre-treatment spraying for sealing the silage pit is carried out, focusing on spraying a mixture of compound microbial agent and organic acid on the top area of ​​the silage facility and the head of the silage pit to ensure that the top and head of the pit are not missed, inhibiting the growth of mold and the production of mycotoxins on the surface of the raw materials and in areas with poor compaction, laying the foundation for mold control in the subsequent aerobic stage; after spraying, the sealing treatment in step (5) is carried out.

[0045] (5) Sealed fermentation and post-fermentation management First, cover the raw material surface with a layer of black and white film, then lay an oxygen-barrier film, and finally a plastic film. The edges of the plastic film are secured with tires. The top of the silage pit is made into a dome shape, extending 50cm above the pit walls. Drainage ditches are dug around the pit to facilitate the drainage of fermentation liquid and prevent rainwater seepage. After sealing, fermentation begins. The fermentation cycle is 90 days. During fermentation, the sealing is checked regularly, and any damaged plastic film is repaired promptly to prevent air from entering and causing secondary fermentation and mold growth.

[0046] The total amount of compound microbial agent used in steps (3) and (4) above is as follows: the ratio of the amount of compound microbial agent added to the mass of silage raw material is 1.2g:1 ton, and the number of viable bacteria in the compound microbial agent is ≥1×10⁻⁶. 9 CFU / g, the compound bacterial agent is composed of Lactobacillus plantarum, Lactobacillus brunelli and Bacillus subtilis in a live bacteria ratio of 3:1.1:1.1.

[0047] The organic acids used in steps (3) and (4) above are compounded from calcium propionate and sodium diacetate in a mass ratio of 1:1.1. The total amount of organic acids used in steps (3) and (4) above is calculated based on the amount of calcium propionate used: the mass ratio of the amount of calcium propionate added to the silage raw material is 1.0g:100g.

[0048] When mixing the compound microbial agent and organic acid, dilute with water to ensure that the dry matter content in the silage pile after filling is between 30% and 35% after spraying the mixture of compound microbial agent and organic acid.

[0049] After the above silage is prepared, it should be taken out in a standardized manner after the pit is opened. The daily extraction depth should not be less than 30cm. After extraction, the scattered raw materials at the bottom of the pit should be cleaned up in time to avoid secondary fermentation and the production of mycotoxins. After extraction, organic acid should be sprayed on the surface of the extracted material. The organic acid is a compound of calcium propionate and sodium diacetate in a mass ratio of 1:1.

[0050] Example 2 The difference from Example 1 lies in the composition of the organic acid, the ratio of the compound microbial agent, and the spraying amount; all other aspects are the same as in Example 1. The specific differences are as follows: In this embodiment, the organic acids and their amounts are as follows: 0.2% (0.2g potassium sorbate per 100g corn silage raw material), 0.2% sodium diacetate, 0.2% calcium propionate, 0.2% sodium benzoate, 0.1% sodium diacetate + 0.1% calcium propionate, and 0.1% calcium propionate + 0.1% glycerol.

[0051] The compound microbial agent is composed of Lactobacillus plantarum, Lactobacillus bruneri, and Bacillus subtilis in a ratio of 3:1:1, with an effective viable count of 1×10⁻⁶. 9 The dosage of CFU / g in corn silage is 1g / ton.

[0052] Select the same batch of corn silage raw materials (same dry matter) and set up a control group and 6 treatment groups (potassium sorbate group, calcium propionate group, sodium benzoate group, sodium diacetate group, sodium diacetate group + calcium propionate, calcium propionate + glycerol group). The control group only added compound microbial agent, and the 6 treatment groups added 0.2% potassium sorbate, 0.2% calcium propionate, 0.2% sodium benzoate, 0.2% sodium diacetate, 0.1% sodium diacetate + 0.1% calcium propionate, and 0.1% calcium propionate + 0.1% glycerol to the compound microbial agent of the control group, respectively. According to the group, different organic acids were diluted to obtain organic acids of the same concentration, and mixed with compound microbial agent to prepare additives. The additives were sprayed evenly in (3) and (4) of Example 1 using a spray bottle.

[0053] Detection indicators and results: (1) On day 0 and day 7 of fermentation in step (5), silage samples were collected from the middle of the fermentation tank. Three samples were sent for testing at each time point to detect mycotoxin content. At the same time, temperature changes were monitored (a thermometer was buried during fermentation and the temperature was automatically collected and transmitted back).

[0054] The method for determining mycotoxin content is as follows: a mycotoxin detection kit is used to detect the content of zearalenone and vomitoxin.

[0055] The measurement results are as follows: ① Temperature change: such as Figure 1 As shown, the fermentation temperature change trend was tracked in real time from 0 to 7 days using a temperature sensor. It was found that the temperature of each group first increased and then decreased, and finally remained stable. The potassium sorbate, sodium benzoate, and sodium diacetate + calcium propionate groups were all lower than the control group.

[0056] ② Changes in mycotoxin content at different fermentation times Table 1 shows that, except for the sodium diacetate + calcium propionate group, the levels of zearalenone in 7 days of fermentation were significantly higher than those in 0 days (P < 0.05), indicating that adding 0.1% sodium diacetate + 0.1% calcium propionate during the aerobic fermentation stage can effectively inhibit the increase of mycotoxins in whole-plant corn silage. The levels of vomitoxin in 7 days of fermentation were significantly higher than those in 0 days (P < 0.05), but the vomitoxin content in the 0 group (sodium diacetate + calcium propionate) was lower than in the other groups. Therefore, adding 0.1% sodium diacetate + 0.1% calcium propionate at 0 days of fermentation can effectively inhibit the increase of mycotoxins during the aerobic fermentation stage of silage.

[0057] Table 1. Effects of adding different organic acids on mycotoxins with fermentation days (μg / kg)

[0058] Note: Data in the same column without the same lowercase letter in the superscript indicates a significant difference (P < 0.05), while the presence or absence of the same letter indicates no significant difference (P > 0.05).

[0059] (2) On day 42 of fermentation in step (5), open the container and collect silage samples from the middle of the fermentation tank. Take three samples for testing, mainly to detect fermentation indicators, mycotoxin content and effects on nutrients.

[0060] Nutrients include: dry matter (DM) (drying method, constant temperature drying at 105℃ to constant weight, GB / T 6435), acid detergent fiber (ADF) (Van Soest acid detergent method, AOAC 973.18), neutral detergent fiber (NDF) (Van Soest neutral detergent method, AOAC 2002.04), ash (ASH) (high temperature ignition method, ignition in a muffle furnace at 550~600℃ to constant weight, GB / T6438), and crude fat (EE) (Soxhlet extraction method, ether extraction, GB / T 6433). Fermentation indicators include: pH value (directly measured with a pH meter, measured after mixing the sample with distilled water at a ratio of 1:5 to 1:10), propionic acid (gas chromatography, GC-FID), lactic acid (high performance liquid chromatography, HPLC), acetic acid (gas chromatography, GC-FID), and butyric acid (gas chromatography, GC-FID).

[0061] When testing fermentation indicators, the Fischer score is used (Fischer score method, Fischer score calculation formula = 220 + [2 × DM(%) - 15] - (40 × pH)).

[0062] The mycotoxin content includes vomitoxin and zearalenone, and the detection method is the same as above.

[0063] Test results: ① Nutritional indicators a. Dry matter (DM) The results are shown in Table 2. There were no significant differences in dry matter content among the sodium benzoate group, control group, calcium propionate, sodium diacetate + calcium propionate, and sodium diacetate group, but the sodium benzoate group was significantly higher than the potassium sorbate group and the calcium propionate + glycerol group. The calcium propionate group had the highest dry matter content, around 34.36%, while the calcium propionate + glycerol group had the lowest, around 31.28%. The values ​​for the sodium diacetate group and the sodium diacetate + calcium propionate group were between 33.66 and 33.68. The dry matter content, roughly ranked from lowest to highest, was: calcium propionate group > control group > sodium benzoate group > sodium diacetate group > sodium diacetate + calcium propionate group > potassium sorbate group > calcium propionate + glycerol group.

[0064] b. Acid Detergent Fiber (ADF) The results are shown in Table 2. The sodium diacetate group was significantly higher than the calcium propionate + glycerol group and the potassium sorbate group. The control group, calcium propionate group, and sodium diacetate + calcium propionate group were significantly higher than the calcium propionate + glycerol group. The approximate order of ADF (Advanced Fat Deficiency) was: sodium diacetate group > control group > sodium diacetate + calcium propionate group > calcium propionate group > sodium benzoate group > potassium sorbate group > calcium propionate + glycerol group.

[0065] c. Neutral detergent fiber (NDF) The calcium propionate group, sodium diacetate group, and control group showed significantly higher levels of NDF than the potassium sorbate group and the calcium propionate + glycerol group, but no significant difference compared to the sodium benzoate group and the sodium diacetate + calcium propionate group. There were no significant differences among the sodium diacetate + calcium propionate group, the calcium propionate + glycerol group, the potassium sorbate group, and the sodium benzoate group. The approximate order of NDF from lowest to highest is: sodium diacetate group > control group > calcium propionate group > sodium benzoate group > sodium diacetate + calcium propionate group > potassium sorbate group > calcium propionate + glycerol group.

[0066] d. Ash content (ASH) The calcium propionate + glycerol group and the potassium sorbate group were significantly higher than the other 5 groups. The ASH values, roughly ranked from lowest to highest, were: potassium sorbate group > calcium propionate + glycerol group > sodium diacetate group > calcium propionate group > sodium benzoate group > sodium diacetate + calcium propionate group > control group.

[0067] e. Crude fat (EE) There were no significant differences between the potassium sorbate group and the control group, or between the sodium diacetate + calcium propionate group. There were no significant differences among the control group, sodium benzoate group, sodium diacetate + calcium propionate group, and calcium propionate group, but the differences were significantly higher among the sodium diacetate group and the calcium propionate + glycerol group. There were no significant differences among the sodium benzoate group, calcium propionate group, sodium diacetate group, and calcium propionate + glycerol group.

[0068] Table 2 Effects of different organic acids on nutritional indicators

[0069] Note: Different letters in the superscript of data in the same column indicate significant differences (P < 0.05), while the same or no letters indicate no significant differences (P > 0.05). The same applies to the following table.

[0070] ② Fermentation indicators: There were no significant differences in pH, propionic acid, and lactic acid among the groups. The acetic acid content in the potassium sorbate group was significantly lower than that in the other six groups. Analysis using the Fisher's score method showed that the Fisher's scores of the sodium diacetate + calcium propionate, calcium propionate, sodium diacetate, and sodium benzoate groups were significantly higher than those of the control group (P < 0.05).

[0071] Table 3. Effects on fermentation parameters and Firth score

[0072] The nutritional value of silage was comprehensively evaluated using the fuzzy mathematical membership function method. Positively correlated indicators included DM, EE, propionic acid, lactic acid, acetic acid, and Fischer score; negatively correlated indicators included NDF, ADF, ASH, pH, and butyric acid. The membership function values ​​of all indicators were summed, and the average value was calculated. The silage was then ranked from largest to smallest as follows: Sodium diacetate + calcium propionate > Calcium propionate > Sodium diacetate > Control group > Sodium benzoate > Potassium sorbate > Calcium propionate + glycerol.

[0073] ③ Changes in mycotoxin content The main toxins detected in whole-plant maize silage were vomitoxin and zearalenone. Analysis of Table 4 showed that the vomitoxin content in the calcium propionate group, the sodium diacetate + calcium propionate group, and the sodium diacetate + calcium propionate group were significantly lower than those in the other groups (P < 0.05).

[0074] Table 4. Effects on mycotoxin content

[0075] ④ Summary: 1) Based on the comprehensive evaluation of the nutritional value of silage, the order from highest to lowest is: sodium diacetate + calcium propionate group > calcium propionate group > sodium diacetate group > control group > sodium benzoate group > potassium sorbate group > calcium propionate + glycerol group.

[0076] 2) In terms of toxin production, the calcium propionate group and the sodium diacetate + calcium propionate group showed better results.

[0077] Experimental Example 3 Experiment on the synergistic effect of compound microbial agents and organic acids on the degradation of mycotoxins in whole-plant maize silage A control group and a treatment group were set up. The additive in the control group was 1 g / ton of compound microbial agent (composition same as in Example 1). The treatment group was divided into two groups, and the additives sprayed were 1 g / ton of compound microbial agent + 0.2% sodium diacetate + 0.2% calcium propionate and 1 g / ton of compound microbial agent + 0.2% sodium diacetate, respectively. All other aspects were the same as in Example 1.

[0078] Samples were taken at 42 days of fermentation, using the same sampling method as in Example 2. Fermentation indicators, vomitoxin, and zearalenone were tested using the same testing method as in Example 2.

[0079] Test results Fermentation index results are shown in Table 5. The pH values ​​of both treatment groups were significantly lower than those of the control group (P < 0.05); the lactic acid content of both treatment groups was significantly higher than that of the control group (P < 0.05); the acetic acid content of the group treated with 1 g / ton of compound microbial agent + 0.2% sodium diacetate + 0.2% calcium propionate was significantly higher than that of the control group (P < 0.05), while the propionic acid content was significantly lower than that of the control group (P < 0.05); there was no significant difference in butyric acid content among the groups. This indicates that adding the compound microbial agent of this invention combined with compound organic acids is more effective than adding the compound microbial agent alone.

[0080] Table 5. Effects on fermentation parameters

[0081] The results of vomitoxin and zearalenone detection are shown in Table 6. The vomitoxin and zearalenone levels in the treatment group with 1 g / ton of compound microbial agent + 0.2% sodium diacetate + 0.2% calcium propionate were significantly lower than those in the control group (P < 0.05), indicating that the addition of compound microbial agent and compound organic acid can effectively control the production of mycotoxins during fermentation and can degrade some mycotoxins.

[0082] Table 6. Effects on mycotoxin content

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of compound microbial agents combined with organic acids in the preparation of silage, characterized in that, The compound bacterial agent is composed of Lactobacillus plantarum, Lactobacillus brunelli and Bacillus subtilis in a viable count ratio of (2.8~3.2):(0.8~1.2):(0.8~1.2), and the organic acid is composed of calcium propionate and sodium diacetate in a mass ratio of 1:(0.8~1.2).

2. A method for preparing silage, characterized in that, Includes the following steps: The silage raw materials are crushed, filled into silage facilities, sealed, and fermented; compound microbial agents and organic acids are added during the filling and sealing of the silage raw materials. The compound bacterial agent is composed of Lactobacillus plantarum, Lactobacillus brunelli and Bacillus subtilis in a viable count ratio of (2.8~3.2):(0.8~1.2):(0.8~1.2), and the organic acid is composed of calcium propionate and sodium diacetate in a mass ratio of 1:(0.8~1.2).

3. The preparation method according to claim 2, characterized in that, The ratio of the amount of the compound microbial agent added to the mass of the silage raw material is (0.8~1.4) g:1 ton, and the number of viable bacteria in the compound microbial agent is ≥1×10⁻⁶. 9 CFU / g.

4. The preparation method according to claim 2, characterized in that, The ratio of the amount of calcium propionate added to the mass of the silage raw material is (0.1~1.2) g: 100 g.

5. The preparation method according to claim 2, characterized in that, The silage material is filled within ≤7 days.

6. The preparation method according to claim 2, characterized in that, The steps for adding compound microbial agents and organic acids during raw material filling include: filling the silage raw materials in layers, mixing the compound microbial agents and organic acids and spraying them onto the surface of each layer of silage raw materials, with each layer having a filling thickness of 20-30cm; the steps for adding compound microbial agents and organic acids during sealing include: mixing the compound microbial agents and organic acids and spraying them onto the top and head of the silage facility.

7. The preparation method according to claim 6, characterized in that, Each layer of silage material was mechanically compacted during filling, with a compaction density of 700 kg / m³. 3 above.

8. The preparation method according to claim 2, characterized in that, The fermentation time is ≥42 days.

9. A type of silage, characterized in that, Prepared using the preparation method according to any one of claims 2 to 8.

10. The application according to claim 1, the preparation method according to any one of claims 2 to 8, or the silage according to claim 9, characterized in that, The silage is corn silage.

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