Bacillus licheniformis fermentation liquor and application thereof in mildew prevention and quality improvement of silage

Abstract

The invention belongs to the technical field of biological mildew preventive, and discloses bacillus licheniformis fermentation liquor and application thereof in mildew prevention and quality improvement of silage. The fermentation liquor is prepared by taking bacillus licheniformis CMCC 63516 as a strain under optimized conditions (with an LB culture medium, the inoculum size of 6%, the liquid loading amount of 30% and culture for 18 hours), and the viable count can reach 1.48 * 10 < 9 > CFU / mL. The strain has a remarkable antagonistic effect on toxin-producing aspergillus flavus, can efficiently degrade aflatoxin B1 (the degradation rate gt in 36 hours is 96%), and has good environmental stability. The additive amount of 1.2-4.0% is applied to corn silage, the growth of mould and saccharomycetes can be remarkably inhibited, the content of AFB1 is greatly reduced, and the mould-proof effect is superior to that of a chemical mould-proof agent sodium diacetate. In addition, the fermentation liquor can also effectively improve the quality of silage, increase the content of crude protein and lactic acid, and reduce the pH and the content of ammoniacal nitrogen and detergent fibers. The invention provides a safe, environment-friendly and efficient biological mildew-proof method which has a wide application prospect in the feed industry.

Description

Technical Field

[0001] This invention belongs to the field of biological anti-mold agents, specifically a Bacillus licheniformis fermentation broth and its application in the prevention of mold and improvement of quality in silage. Background Technology

[0002] Silage is the primary roughage for dairy cows, boasting advantages such as high nutritional value, high digestibility, and good palatability. However, it is highly susceptible to mold contamination and spoilage during storage. Aspergillus flavus is one of the most common fungi, producing aflatoxin B1 (AFB1), a highly toxic substance classified as a Group 1 carcinogen by the International Agency for Research on Cancer. When dairy cows consume moldy feed, not only is their own health harmed, but the toxins may also transfer to their milk, posing a serious threat to human health.

[0003] To address the problem of mold growth in feed, chemical mold inhibitors, such as sodium diacetate, formic acid, and propionate, are currently widely used. While these inhibitors are highly effective, they pose problems such as environmental pollution, potential impact on feed palatability, and even potential toxicity to animals. Therefore, the search for safe, environmentally friendly, and effective biological mold inhibitors has become a research hotspot.

[0004] Bacillus licheniformis, as a probiotic, is widely used in medicine, animal feed, and other fields, characterized by rapid growth, strong resistance, and the production of various antibacterial substances. Currently, there are studies on using Bacillus licheniformis as a silage fermentation agent (i.e., promoting lactic acid fermentation), but there are no reports on its use as a silage mold inhibitor (i.e., primarily used to inhibit mold and toxins). Therefore, developing a Bacillus licheniformis-based biological mold inhibitor is of great significance for ensuring the safety of silage and reducing mycotoxin contamination. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing chemical antifungal agents, provide a safe, environmentally friendly, and efficient Bacillus licheniformis fermentation broth, and apply it to the antifungal and quality improvement of silage, providing theoretical and technical support for the research and development of biological antifungal agents.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a Bacillus licheniformis fermentation broth, which is prepared by a method comprising the following steps: (a) Inoculate Bacillus licheniformis seed culture into LB liquid medium; (b) Fermentation culture was carried out under shaking conditions, with the following conditions: inoculum size of 4%-8% (v / v), liquid volume of 20%-40% (v / v), and culture time of 12-24 hours.

[0007] Preferred fermentation conditions are: inoculum size of 6% (v / v), liquid volume of 30% (v / v), and culture time of 18 hours.

[0008] Preferably, the Bacillus licheniformis strain is the strain with accession number CMCC 63516.

[0009] In this invention, the fermentation broth of Bacillus licheniformis was prepared as follows: Using Bacillus licheniformis CMCC 63516 as the starting strain, LB medium was determined to be the optimal fermentation medium through culture medium screening experiments. The fermentation conditions were optimized using single-factor experiments combined with response surface methodology, and the optimal conditions were determined to be: inoculum size 6%, liquid volume 30%, and incubation time 18 hours. Under these conditions, the viable cell count in the fermentation broth reached 1.48 × 10⁻⁶. 9 CFU / mL, which meets the standards for feed additives.

[0010] Secondly, the present invention provides the application of the above-described Bacillus licheniformis fermentation broth in inhibiting the growth of Aspergillus flavus.

[0011] Thirdly, the present invention provides the application of the above-described Bacillus licheniformis fermentation broth in the degradation of aflatoxin B1.

[0012] Antibacterial and detoxifying properties of the fermentation broth: The Bacillus licheniformis fermentation broth of this invention exhibits significant antagonistic effects against toxin-producing Aspergillus flavus. When co-cultured with AFB1, the degradation rate of AFB1 in the fermentation broth can reach over 96.41% within 36 hours. Studies have shown that the detoxifying activity is mainly present in the fermentation supernatant. This fermentation broth exhibits good stability: after 12 hours of UV irradiation, the degradation rate of AFB1 still reaches 89.52%; after heat treatment at 40-121℃, the degradation rate is consistently above 90%; and within the pH range of 6-10, the degradation rate is consistently above 94%.

[0013] Preferably, the Bacillus licheniformis fermentation broth is co-cultured with aflatoxin B1 for 36 hours, and the degradation rate of aflatoxin B1 is not less than 96%.

[0014] Thirdly, the present invention provides the application of the above-described Bacillus licheniformis fermentation broth in the prevention of mold in silage.

[0015] Preferably, the silage is corn silage.

[0016] Preferably, the amount of Bacillus licheniformis fermentation broth added is 1.2%-4.0% (v / w) of the weight of the silage.

[0017] A further preferred embodiment is that the amount of Bacillus licheniformis fermentation broth added is 4.0% (v / w).

[0018] Preferred application: The application includes uniformly spraying or mixing the Bacillus licheniformis fermentation broth into silage, followed by sealing or vacuum sealing for storage.

[0019] Application in mold prevention in silage: The Bacillus licheniformis fermentation broth prepared according to this invention was added to corn silage at a certain proportion. Experiments showed that with increasing addition (1.2%-4.0%), the sensory score of the silage improved, the number of yeasts and molds significantly decreased, and the AFB1 content significantly decreased. The mold prevention effect was optimal when the addition amount was 4.0%. Compared with commonly used chemical mold inhibitors such as sodium diacetate, the Bacillus licheniformis fermentation broth of this invention is more effective in inhibiting the growth of yeasts and molds and reducing AFB1 content.

[0020] Thirdly, the present invention provides a method for improving the quality of silage, comprising the step of adding the above-mentioned Bacillus licheniformis fermentation broth to the silage, wherein the addition can increase the crude protein content and lactic acid content of the silage, and reduce its pH value, ammonia nitrogen content, neutral detergent fiber content and acid detergent fiber content.

[0021] Compared with the prior art, the beneficial effects of the present invention are: 1. Safe and environmentally friendly: The antifungal agent of this invention is derived from the probiotic Bacillus licheniformis, which is non-toxic and leaves no residue, thus avoiding the environmental pollution and animal health risks associated with chemical antifungal agents.

[0022] 2. Dual function of preventing mold and reducing toxicity: It can inhibit the growth of molds such as Aspergillus flavus in silage, and can also efficiently degrade the highly toxic substance aflatoxin B1 that has been produced, ensuring feed safety from both the source and the end.

[0023] 3. Fermentation process optimization: The liquid fermentation conditions of Bacillus licheniformis were optimized using response surface methodology, resulting in a high-concentration fermentation broth, which provides a cost-effective and stable formulation basis for practical applications.

[0024] 4. Significantly improves silage quality: In addition to its anti-mold function, the fermentation liquid of this invention can also improve the fermentation characteristics of silage, increase beneficial substances (such as lactic acid and crude protein), and reduce protein degradation, thereby comprehensively improving the nutritional value and preservation quality of silage.

[0025] 5. Superior application effect compared to some chemical antifungal agents: Under the same experimental conditions, the biological antifungal agent of this invention is superior to sodium diacetate in inhibiting fungi and reducing toxins, demonstrating good substitution potential and application prospects. Attached Figure Description

[0026] Figure 1The effects of different culture media on the viable cell count and spore count of the fermentation broth at 18 h (a) and 24 h (b) are shown in the figure. Different uppercase letters in the figure indicate significant differences in viable cell count in different culture media (p<0.05); different lowercase letters indicate significant differences in spore count in different culture media (p<0.05).

[0027] Figure 2 The effect of different inoculum amounts on the viable cell count and spore count in the fermentation broth.

[0028] Figure 3 The effect of different liquid volumes on the viable cell count and spore count of the fermentation broth.

[0029] Figure 4 The effect of culture time on the number of viable bacteria and spores in the fermentation broth.

[0030] Figure 5 The inhibitory effect of Bacillus licheniformis on toxin-producing Aspergillus flavus is shown; the left side is the blank control group, and the right side is the experimental group.

[0031] Figure 6 The effect of time on the degradation of AFB1.

[0032] Figure 7 The figure shows the degradation effect of different treatment groups on toxins; different uppercase letters in the figure represent significant differences between groups (p<0.05); lowercase letters represent significant differences within groups (p<0.05).

[0033] Figure 8 The UV stability of Bacillus licheniformis fermentation broth.

[0034] Figure 9 The acid-base stability of Bacillus licheniformis fermentation broth.

[0035] Figure 10 The thermal stability of Bacillus licheniformis fermentation broth.

[0036] Figure 11 Silage with different addition amounts during vacuum sealing storage; in the figure, a, b, and c are the experimental results at 3 days, 6 days, and 9 days, respectively; from left to right in a, b, and c are the control, 1.2%, 1.6%, 2%, and 4% addition groups, respectively.

[0037] Figure 12 Silage with different addition amounts during sealed storage; in the figure, a, b, and c are the experimental results at 3 days, 6 days, and 9 days, respectively; from left to right in a, b, and c are the control group, 1.2%, 1.6%, 2%, and 4% addition groups, respectively.

[0038] Figure 13 To compare the dynamics of yeast (a) and mold (b) counts in corn silage with different addition amounts during vacuum sealing storage.

[0039] Figure 14 To compare the dynamics of yeast (a) and mold (b) counts in corn silage with different addition amounts during sealed storage.

[0040] Figure 15 The number of yeasts (a) and molds (b) in corn silage of different treatment groups during vacuum sealing storage.

[0041] Figure 16 The number of yeasts (a) and molds (b) in corn silage of different treatment groups when stored in a sealed container. Detailed Implementation

[0042] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0043] In this invention, Bacillus licheniformis CMCC63516 is purified and cultured from "Zhengchangsheng" Bacillus licheniformis live bacteria capsules (sourced from Northeast Pharmaceutical Group Shenyang First Pharmaceutical Co., Ltd.).

[0044] Aspergillus flavus AS3.870 was obtained from the China Industrial Microbial Culture Collection Center (CICC).

[0045] Example 1: Preparation and Optimization of Fermentation Conditions for Bacillus licheniformis 1. Strain activation and preservation: Take live Bacillus licheniformis capsules (CMCC 63516), aseptically weigh 1g of bacterial powder, dilute with physiological saline, and spread on LB agar plates. After incubation at 37℃ for 24h, pick a loopful of Bacillus licheniformis from the plate and culture it in LB liquid medium. After subculturing, streak it in parallel on LB solid medium. Then examine under a microscope to see if it is a single colony. Once confirmed to be a single colony, mix with 50% glycerol at a ratio of 3:2 and store at -80℃.

[0046] 2. Screening of fermentation media: Three media were designed: (1) LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride; (2) LB medium with added glucose: 10 g / L glucose, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride; (3) A peptone-glucose-calcium chloride medium: 15 g / L glucose, 30 g / L peptone, 1.5 g / L calcium chloride.

[0047] Preparation of Bacillus licheniformis seed culture: One loop of activated Bacillus licheniformis was placed in a 250 mL Erlenmeyer flask containing 50 mL LB medium and cultured with shaking at 37℃ and 180 r / min for 18 h.

[0048] The seed culture (37℃, 180 rpm, shaking culture for 18 h) was inoculated into each culture medium at a 6% inoculum and cultured at 37℃ with shaking at 180 rpm. Samples were taken at 18 h and 24 h, and the viable cell count and spore count were determined using the dilution plating method (spore detection required 80℃ water bath for 15 min before plating). Results are as follows: Figure 1 As shown, LB medium (No. 1) had the highest number of viable bacteria and spores at both time points, so LB medium was selected as the medium for subsequent fermentation.

[0049] 3. Optimization of single-factor experiments: Inoculum size: Under conditions of 40% liquid volume and 18h ​​incubation, inoculum sizes of 2%, 4%, 6%, 8%, and 10% were set. The results showed that the highest viable cell count was achieved at an inoculum size of 6%. Figure 2 ).

[0050] Liquid volume: Under the conditions of 6% inoculum and 18h ​​incubation, liquid volumes of 10%, 20%, 30%, 40%, and 50% were set. The results showed that the highest viable count was achieved at a liquid volume of 30%. Figure 3 ).

[0051] Culture time: Under the conditions of 6% inoculum and 30% liquid volume, the culture time was set to 12h, 18h, 24h, 30h, and 36h. The results showed that the highest viable count was observed after 18h of culture. Figure 4 ).

[0052] 4. Response Surface Methodology Optimization: Based on the single-factor results, a Box-Behnken model was used, with inoculum size (A), liquid volume (B), and incubation time (C) as factors, and the effective viable count as the response value Y, to design a three-factor, three-level response surface methodology experiment. The regression equation was obtained and the optimal conditions were determined using Design-Expert software. The optimized theoretical conditions were: inoculum size 5.71%, liquid volume 26.98%, and incubation time 17.99 h. For ease of operation, these were rounded to 6% inoculum size, 30% liquid volume, and 18 h incubation time. Three validation experiments were conducted under these conditions, and the average viable count was 1.48 × 10⁻⁶. 9 CFU / mL, compared with the predicted value (1.55×10⁻⁶). 9 The CFU / mL value is close to and meets the feed industry standard (>1×10⁻⁶). 9 (CFU / mL). Table 1 shows the validation results.

[0053]

[0054] Example 2: Study on the antibacterial and AFB1 degradation effects of Bacillus licheniformis fermentation broth 1. Preparation of Aspergillus flavus spore suspension: Activated toxin-producing Aspergillus flavus (AS3.870) was inoculated onto PDA slant agar medium and cultured at 28°C for 3 days until abundant spore production. The spores were washed off with sterile physiological saline, filtered, and the concentration adjusted to 10. 5 CFU / mL.

[0055] 2. Antagonistic effect test (plate confrontation method): A suspension of Aspergillus flavus spores was inoculated in the center of an LB agar plate, and then Bacillus licheniformis was inoculated around the perimeter using an inoculation loop. The plate was incubated at 28°C for 7 days. Results are as follows: Figure 5 As shown, after 7 days of inoculation with Bacillus licheniformis around the standard strain of Aspergillus flavus, Bacillus licheniformis inhibited the growth of Aspergillus flavus. Compared with the blank control group, the growth of Aspergillus flavus hyphae on the side closer to Bacillus licheniformis was inhibited, and a clear inhibition edge appeared, proving that Bacillus licheniformis has an antagonistic effect on the growth of Aspergillus flavus.

[0056] 3. Study on the effect of AFB1 degradation: Fermentation broth treatment: The Bacillus licheniformis fermentation broth prepared under optimized conditions (inoculum size 6%, liquid volume 30%, culture time 18h) was centrifuged (8000r / min, 15min) to separate the supernatant (filtered through a 0.22μm filter membrane) and the bacterial cells (washed and resuspended with physiological saline to obtain a bacterial suspension).

[0057] Co-culture degradation: 900 μL of Bacillus licheniformis fermentation broth, supernatant, and bacterial suspension were each added to 100 μL of 20 mg / L AFB1 standard working solution (final AFB1 concentration 2 mg / L). LB medium without the strain was used as a control. The mixtures were co-cultured at 37℃ and 180 rpm. Samples were taken at 12, 24, 36, 48, and 60 h, and the AFB1 content was detected using an aflatoxin B1 enzyme-linked immunosorbent assay (ELISA) kit to calculate the degradation rate.

[0058]

[0059] The sample addition group includes Bacillus licheniformis fermentation broth, supernatant, and bacterial suspension.

[0060] The results are as follows Figure 6 and Figure 7 As shown, the degradation rates of AFB1 in the fermentation broth and supernatant reached 96.41% and 96.64% respectively after 36 h, with no significant difference (p>0.05), and both were higher than those in the bacterial suspension. This indicates that the degradation activity mainly exists in the supernatant. Example 3: Stability Study of Bacillus licheniformis Fermentation Broth 1. UV Stability: The Bacillus licheniformis fermentation broth was irradiated under a UV lamp for different durations (2h, 4h, 6h, 8h, 10h, 12h). 900 μL of each irradiated fermentation broth was added to a 1.5 mL centrifuge tube, followed by 100 μL of 20 mg / L AFB1 standard working solution. LB medium without the strain was used as a control. The mixture was then co-cultured at 37℃ and 180 r / min for 36 h, and the AFB1 degradation rate was measured. The original fermentation broth without UV irradiation was used as a control group.

[0061]

[0062] The results are as follows Figure 8 As shown, the degradation rate gradually decreased with prolonged irradiation time, but the degradation rate still reached 89.52% after 12 hours of irradiation, indicating that the fermentation broth has a certain degree of UV stability.

[0063] 2. Acid-base stability: The pH of the fermentation broth was adjusted to 2, 4, 6, 8, 10, and 12 with 1 mol / L HCl or NaOH, and then returned to the original pH after 24 h of treatment. 900 μL of each acid-base treated fermentation broth was added to a 1.5 mL centrifuge tube, followed by 100 μL of 20 mg / L AFB1 standard working solution. LB medium without the strain was used as a control. The mixture was then co-cultured at 37℃ and 180 r / min for 36 h, and the AFB1 degradation rate was measured.

[0064]

[0065] The results are as follows Figure 9 As shown, the degradation rate of AFB1 in the fermentation broth was above 94% in the pH range of 6-10, with the best effect at pH 8 (95.52%).

[0066] 3. Thermal stability: The fermentation broth was treated at 40, 60, 80, and 100℃ for 1 h, and then sterilized at 121℃ for 20 min, before being allowed to return to room temperature. 900 μL of the heat-treated fermentation broth was added to 1.5 mL centrifuge tubes, followed by 100 μL of 20 mg / L AFB1 standard working solution. LB medium without the strain was used as a control. The tubes were co-cultured at 37℃ and 180 r / min for 36 h, and the AFB1 degradation rate was measured. The original, untreated fermentation broth served as a control group.

[0067]

[0068] The results are as follows Figure 10 As shown, after treatment at various temperatures, the degradation rate of AFB1 in the fermentation broth remained above 90%, indicating that the fermentation broth has good thermal stability. Example 4: Application of Bacillus licheniformis fermentation broth in corn silage for mold prevention 1. Silage Preparation and Treatment: Take corn silage fermented for 60 days and spray it evenly with optimized Bacillus licheniformis fermentation broth at 1.2%, 1.6%, 2.0%, and 4.0% of the silage weight, respectively, and stir well. A control group without added fermentation broth was also set up. The treated silage (100g per bag) was packed into polyethylene bags and sealed using two methods: one group was compacted and then vacuum-sealed, and the other group was compacted and then sealed (without vacuuming). Store at room temperature.

[0069] 2. Sensory evaluation: At 3, 6, and 9 days of storage, sensory evaluation was conducted according to the "Silage Quality Evaluation Standard (1996)" in terms of odor, color, and texture. Sensory evaluation was performed on silage with different amounts of fermentation liquid added. Three people were selected to conduct the evaluation, and the average value was taken. The specific evaluation standards are shown in Table 2.

[0070] Sensory evaluation during vacuum-sealed storage The results of the anti-mold treatment show that ( Figure 11 After vacuum sealing, no obvious mold growth was observed on the surface of the control group and the silage with different addition amounts, and the color was yellowish-brown with no significant difference.

[0071] Preliminary comparison of the sensory quality of each treatment group (Table 3): After 3 days of storage, the sensory evaluation of the control group and all treatment groups was excellent, with a loose and non-sticky texture and a pleasant sweet and sour aroma; after 6 and 9 days of storage, the sensory evaluation of the control group and all treatment groups was good, with a slightly sour aroma and a relatively soft texture, without stickiness. The comparison revealed that when stored in a vacuum-sealed container, the sensory quality of silage with different amounts of fermentation broth added did not differ significantly, but the sensory scores decreased with prolonged storage.

[0072] (2) Sensory evaluation when stored in a sealed container only The antibacterial results show that, when silage is stored in a sealed container, the degree of mold growth varies among the different silage treatment groups. For example... Figure 12 As shown, after 3 days of storage, the control group showed obvious mold growth, the corn silage with 1.2% and 1.6% fermentation liquid added had a small amount of mold growth on the top, the corn silage with 2% fermentation liquid added had slight mold growth on the top, and the corn silage with 4% fermentation liquid added (i.e., enough to spray the entire surface of the silage) showed no obvious mold growth on the surface. After 6 and 9 days of storage, the mold growth in each group was similar: the control group showed obvious mold growth, the corn silage with 1.2%, 1.6%, and 2% fermentation liquid added had a small amount of mold growth on the top, and the corn silage with 4% fermentation liquid added showed no obvious mold growth on the surface.

[0073] As shown in Table 4, a preliminary comparison of the sensory scores of each treatment group under sealed conditions revealed certain differences in sensory quality. After 3 days of storage, the sensory score of the untreated group was good, with a slightly sour odor, a relatively soft texture, and moderate stickiness. All treatment groups with added fermentation liquid received excellent sensory scores, with a loose, non-sticky texture and a pleasant, sweet and sour aroma. With prolonged storage, after 6 and 9 days, both the control group and all treatment groups received good sensory evaluations, but the corn silage with 4% added fermentation liquid had the highest sensory score within the same period.

[0074]

[0075]

[0076]

[0077] 3. Microbial Counting: Samples were taken at corresponding time points (3d, 6d, and 9d of storage), and the total number of molds and yeasts was determined using the dilution plating method according to GB / T 13092-2006. The results showed that, regardless of whether a vacuum was used, the number of yeasts and molds in the group with added fermentation broth was significantly lower than that in the control group (p<0.05), and the group with an addition of 4.0% showed the strongest inhibitory effect. Figure 13 , Figure 14 ).

[0078] 4. AFB1 content determination: Under vacuum sealing conditions, samples were taken at 3 and 9 days of storage, and the AFB1 content was detected by ELISA. The results are shown in Table 5. In the control group without added AFB1, the AFB1 content increased significantly over time, while the AFB1 content in all groups with added fermentation broth was significantly lower than that in the control group, and decreased with increasing addition amount. The AFB1 content in the group with 4.0% added fermentation broth was only 0.016 μg / kg at 9 days, far below the relevant standard (such as DB52 / T 1257.8, which specifies ≤10 μg / kg).

[0079]

[0080] 5. Comparison with chemical antifungal agents: Corn silage fermented for 60 days was mixed with 4% Bacillus licheniformis fermentation broth and 0.1% sodium diacetate antifungal agent, then placed in polyethylene vacuum bags (100 g per bag) and sealed using two different methods. One group was compacted and vacuum-sealed, while the other group was compacted and sealed only, and fermented at room temperature. Uninoculated silage that fermented naturally served as a control. Samples were collected on days 3, 6, and 9 after the start of the experiment to compare their antifungal effects. Sensory evaluation of the silage, determination of total mold count, yeast count, and AFB1 detection methods were the same as above.

[0081] Sensory evaluation (Tables 6 and 7) and microbial count ( Figure 15 , Figure 16 The results showed that the Bacillus licheniformis fermentation broth group had better sensory quality, and the number of yeasts and molds was consistently significantly lower than that of the SDA group (p<0.05). Under vacuum sealing conditions, the AFB1 content in the silage of the Bacillus licheniformis fermentation broth group was also significantly lower than that of the SDA group (Table 8).

[0082]

[0083]

[0084]

[0085] Example 5: Effect of Bacillus licheniformis fermentation broth on the quality of corn silage 1. Silage Treatment: Corn silage fermented for 60 days and 90 days were collected respectively, and Bacillus licheniformis fermentation broth was added at a ratio of 4.0%. After mixing, the silage was vacuum-sealed and stored. Silage fermented naturally without inoculation served as a control group. Samples were taken for analysis at 3, 6, and 9 days of storage. Each sample was divided into two portions. One portion was placed in a 60℃ oven and dried for 48 hours, then pulverized, sieved, and stored in a sealed bag for nutritional index determination. The other portion was stored in a -20℃ refrigerator for fermentation index detection.

[0086] 2. Nutritional Quality Determination: After drying and pulverizing the samples, the dry matter (DM, 105℃ drying method), crude protein (CP, Kjeldahl nitrogen determination method), neutral detergent fiber (NDF), and acid detergent fiber (ADF, Van der Waals method) were determined. The results are shown in Tables 9 and 10. After adding fermentation broth, the dry matter content of the silage decreased slightly (but remained within the excellent range of 27%-29%), while the crude protein content was significantly higher than that of the control group (p<0.05), and the contents of neutral detergent fiber and acid detergent fiber were lower than those of the control group (ADF decreased significantly, p<0.05).

[0087]

[0088]

[0089] 3. Fermentation Quality Determination: Samples were taken from corn silage under different treatments. 10g of corn silage was weighed and added to 90 mL of deionized water. The mixture was extracted at 4℃ for 24 h, and after mixing, it was filtered through gauze to obtain the extract. The extract was used to determine pH, ammonia nitrogen (NH3-N), and lactic acid. The pH value should be measured immediately after the extract is prepared, and the lactic acid and ammonia nitrogen should be detected within 24 h. An appropriate amount of extract was used for pH measurement, and the remaining extract was stored at 4℃. Ammonia nitrogen was determined using the phenol-sodium hypochlorite colorimetric method. Lactic acid was determined using high performance liquid chromatography according to DB15 / T 1458-2018. The results are shown in Tables 11 and 12. The silage group with added fermentation broth had significantly lower pH and ammonia nitrogen content than the control group (p<0.05), while the lactic acid content was significantly higher than the control group (p<0.05), indicating that the fermentation process was improved.

[0090]

[0091]

[0092] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A Bacillus licheniformis fermentation broth, characterized in that, It is prepared by a method including the following steps: (a) Inoculate Bacillus licheniformis seed culture into LB liquid medium; (b) Fermentation culture was carried out under shaking conditions, with the following conditions: inoculum size of 4%-8%, liquid volume of 20%-40%, and culture time of 12-24 hours.

2. The Bacillus licheniformis fermentation broth according to claim 1, characterized in that, The fermentation conditions were: inoculum size of 6%, liquid volume of 30%, and culture time of 18 hours.

3. The Bacillus licheniformis fermentation broth according to claim 1, characterized in that, The Bacillus licheniformis strain is the strain with accession number CMCC 63516.

4. The use of the Bacillus licheniformis fermentation broth according to any one of claims 1-3 in inhibiting the growth of Aspergillus flavus.

5. The use of Bacillus licheniformis fermentation broth according to any one of claims 1-3 in the degradation of aflatoxin B1.

6. The application according to claim 5, characterized in that, The Bacillus licheniformis fermentation broth was co-cultured with aflatoxin B1 for 36 hours, and the degradation rate of aflatoxin B1 was not less than 96%.

7. The application of Bacillus licheniformis fermentation broth according to any one of claims 1-3 in the prevention of mold in silage.

8. The application according to claim 7, characterized in that, The silage is corn silage, and the amount of Bacillus licheniformis fermentation liquid added is 1.2%-4.0% of the weight of the silage.

9. The application according to claim 8, characterized in that, The application includes uniformly spraying or mixing the Bacillus licheniformis fermentation broth into silage, followed by sealing or vacuum sealing for storage.

10. A method for improving the quality of silage, characterized in that, The method includes the step of adding the Bacillus licheniformis fermentation broth according to any one of claims 1-3 to silage, wherein the addition can increase the crude protein content and lactic acid content of the silage, and reduce its pH value, ammonia nitrogen content, neutral detergent fiber content and acid detergent fiber content.

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