A fungistatic silage additive, feed and use

Abstract

The application provides a kind of silage additive, feed and application of fungus, belongs to the technical field of animal breeding.A kind of silage additive of fungus, including lactobacillus plantarum and the following any one component: quercetin, lactococcus lactis subsp lactis and lactococcus lactis.The application of the silage additive in the preparation of animal feed with whole corn silage as raw material can not change the nutritional quality of whole corn silage feed, improve the aerobic stability of whole corn silage feed, reduce the number of fungi and klebsiella in whole corn silage feed;improve the relative abundance of lactobacillus in whole corn silage feed.It can be seen that the application creates a new type of efficient fungus inhibitor with low price, safety and convenient use, which is of great significance to improve the quality and safety of silage feed and promote the healthy development of animal husbandry.

Description

Technical Field

[0001] This invention belongs to the field of animal husbandry technology, specifically relating to an antifungal silage additive, feed, and its application. Background Technology

[0002] With economic and social development, my country's demand for high-quality dairy products has increased rapidly, leading to a surge in demand for high-quality forage such as whole-plant corn silage. With the construction of large-scale dairy farms, whole-plant corn silage has gradually become dominant. The quality and safety of whole-plant corn silage has become a key factor restricting the improvement of milk yield and quality. Simultaneously, with the increasing national efforts in forage resource development and the promotion of policies related to grain-to-forage conversion, the planting area of ​​silage corn has been continuously expanding, providing ample raw materials for the industrialization of silage. In my country's main whole-plant corn silage production areas, rainfall and heat coincide, making whole-plant corn silage highly susceptible to fungal contamination and aerobic spoilage. However, certain technical bottlenecks remain in areas such as silage raw material control, silage processing technology, and the use of silage additives. Therefore, research and development of whole-plant corn silage additives is of great significance for improving the safety level of silage and the healthy development of animal husbandry in my country.

[0003] Due to technological bottlenecks in silage raw material control, silage processing technology, and the development of supporting facilities and equipment, the problems of poor silage quality and low feed efficiency are particularly prominent. Whole-plant corn silage is prone to fungal contamination. The harvesting period for silage corn is generally in August and September, when temperatures are higher than when grain corn is harvested in October, which easily affects the microorganisms during silage fermentation and promotes fungal proliferation. In 2022, East my country experienced extreme high temperatures for a very long period, severely impacting the harvest of silage corn. Furthermore, harvested whole-plant corn is prone to ear rot. The applicant's related research shows that ear rot has a significant negative impact on the quality and safety of whole-plant corn silage. Pathogens causing ear rot include Penicillium and Fusarium fungi. However, there are currently no reports on feed additives that address the problem of excessive fungal proliferation during the fermentation process of whole-plant corn silage, which seriously affects feed safety. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an antifungal silage additive that keeps the levels of Penicillium and Fusarium in the produced feed at extremely low levels, thereby ensuring the quality and safety of the feed.

[0005] This invention provides an antifungal silage additive, comprising Lactobacillus plantarum and any one of the following components: quercetin, Lactococcus lactis subsp. lactis, and Lactococcus lactis.

[0006] When *Lactobacillus plantarum* and quercetin are combined, the viable count and mass ratio of *Lactobacillus plantarum* and quercetin are (1–10) × 10⁻⁶. 5 CFU: 0.06~0.18g;

[0007] When *Lactobacillus plantarum* and *Lactococcus lactis* subsp. *lactococcus* are combined, the ratio of viable bacteria of *Lactobacillus plantarum* to *Lactococcus lactis* subsp. *lactococcus* is (0.8–1.2):(0.8–1.2).

[0008] When *Lactobacillus plantarum* and *Lactococcus lactis* are combined, the ratio of viable *Lactobacillus plantarum* to viable *Lactococcus lactis* is (0.8–1.2):(0.8–1.2).

[0009] Preferably, the *Lactobacillus plantarum* comprises *Lactobacillus plantarum* strain ZRR;

[0010] The strain ZRR of *Lactobacillus plantarum* has the preservation number CCTCC No: M2016281.

[0011] Preferably, the *Lactococcus lactis* subsp. *lactococcus* is a *Lactococcus lactis* subsp. *lactococcus* that produces nisin.

[0012] The strain preservation number of *Lactococcus lactis* subsp. *lactosporum* that produces nisin is CGMCC No. 1.2030.

[0013] Preferably, the lactococcus lactis is a lactococcus lactis that produces nisin;

[0014] The strain preservation number of the lactococcus lactis that produces nisin is CGMCC No.1.15072.

[0015] This invention provides the application of the silage additive in the preparation of animal feed using whole-plant corn silage as raw material.

[0016] Preferably, when the silage additive is a combination of Lactobacillus plantarum and quercetin, the amount of live Lactobacillus plantarum added to the whole-plant corn silage is (1-10)×10⁻⁶. 5 CFU·g -1 Quercetin accounts for 0.06–0.18% of the total mass of maize silage.

[0017] When the silage additive is a combination of *Lactobacillus plantarum* and *Lactococcus lactis* subsp. *lactococcus* or a combination of *Lactococcus lactis* and *Lactococcus lactis*, the total viable bacteria content of *Lactobacillus plantarum* and *Lactococcus lactis* subsp. *lactococcus* or *Lactococcus lactis* in the whole-plant corn silage is (1–10) × 10⁻⁶. 5 CFU·g -1 .

[0018] Preferably, the dry matter content of the whole-plant corn silage is 35% to 38%.

[0019] Preferably, the application includes one or more of the following:

[0020] 1) It does not change the nutritional quality of whole-plant corn silage;

[0021] 2) Improve the aerobic stability of whole-plant corn silage;

[0022] 3) Reduce the number of fungi in whole-plant corn silage;

[0023] 4) Increase the relative abundance of lactic acid bacteria in whole-plant corn silage;

[0024] 5) Reduce the relative abundance of Klebsiella pneumoniae in whole-plant corn silage.

[0025] Preferably, the fungus includes one of the following: Aspergillus, Penicillium, Fusarium, and yeast.

[0026] This invention provides an animal feed prepared by fermenting whole-plant corn silage using the silage additive as a fermenting agent.

[0027] The antifungal silage additive provided by this invention comprises *Lactobacillus plantarum* and any one of the following components: quercetin, *Lactococcus lactis* subsp. *lactococcus*, and *Lactococcus lactis*; when *Lactobacillus plantarum* and quercetin are combined, the viable count and mass ratio of *Lactobacillus plantarum* and quercetin is (1-10)×10⁻⁶. 5 CFU: 0.06–0.18 g; When *Lactobacillus plantarum* and *Lactococcus lactis* subsp. *lactococcus* are combined, the viable count ratio of *Lactobacillus plantarum* to *Lactococcus lactis* subsp. *lactococcus* is (0.8–1.2):(0.8–1.2); When *Lactobacillus plantarum* and *Lactococcus lactis* are combined, the viable count ratio of *Lactobacillus plantarum* to *Lactococcus lactis* is (0.8–1.2):(0.8–1.2). Regarding *Fusarium* fungi after fermentation of whole-plant corn silage, compared with unfermented silage raw materials, silage additives prepared by combining *Lactobacillus plantarum* with quercetin or with *Lactococcus lactis* significantly reduced the relative abundance of *Fusarium* fungi in the prepared feed; regarding *Penicillium* fungi after fermentation of whole-plant corn silage, silage additives prepared by combining *Lactobacillus plantarum* with quercetin or with *Lactococcus lactis* subsp. *lactococcus* significantly reduced the relative abundance of *Penicillium* in the prepared raw materials.

[0028] Meanwhile, silage additives prepared by combining *Lactobacillus plantarum* with quercetin or *Lactococcus lactis* subsp. *lactocopherol* can effectively reduce the relative abundance of *Aspergillus* spp. in whole-plant corn silage fungi; silage additives prepared by combining *Lactobacillus plantarum* with quercetin or *Lactococcus lactis* can effectively reduce the number of yeasts in whole-plant corn silage fungi; and silage additives prepared by combining *Lactobacillus plantarum* with quercetin or *Lactococcus lactis* subsp. *lactocopherol* or *Lactococcus lactis* can effectively reduce the relative abundance of *Klebsiella* spp. in whole-plant corn silage bacteria.

[0029] Meanwhile, the combination of Lactobacillus plantarum and quercetin to prepare silage additives can effectively increase the relative abundance of Lactobacillus in whole-plant corn silage bacteria and improve the microbial diversity of whole-plant corn silage. Attached Figure Description

[0030] Figure 1 The results show the effects of different additives on the aerobic stability of whole-plant maize silage.

[0031] Figure 2 Curves showing the changes in core temperature and ambient temperature difference between whole-plant corn silage samples after opening for different additive treatment groups;

[0032] Figure 3 The results show the effects of different additives on the relative abundance of Aspergillus species in whole-plant maize silage fungi.

[0033] Figure 4 The results show the effects of different additives on the relative abundance of Fusarium spp. in whole-plant maize silage fungi.

[0034] Figure 5 The results show the effects of different additives on the relative abundance of Penicillium spp. in whole-plant maize silage fungi.

[0035] Figure 6 The results show the effects of different additives on the relative abundance of Lactobacillus spp. in whole-plant maize silage bacteria.

[0036] Figure 7 The results show the effects of different additives on the relative abundance of Klebsiella spp. in whole-plant maize silage bacteria. Detailed Implementation

[0037] This invention provides an antifungal silage additive, comprising Lactobacillus plantarum and any one of the following components: quercetin, Lactococcus lactis subsp. lactis, and Lactococcus lactis.

[0038] When *Lactobacillus plantarum* and quercetin are combined, the viable count and mass ratio of *Lactobacillus plantarum* and quercetin are (1–10) × 10⁻⁶. 5 CFU: 0.06~0.18g;

[0039] When *Lactobacillus plantarum* and *Lactococcus lactis* subsp. *lactococcus* are combined, the ratio of viable bacteria of *Lactobacillus plantarum* to *Lactococcus lactis* subsp. *lactococcus* is (0.8–1.2):(0.8–1.2).

[0040] When *Lactobacillus plantarum* and *Lactococcus lactis* are combined, the ratio of viable *Lactobacillus plantarum* to viable *Lactococcus lactis* is (0.8–1.2):(0.8–1.2).

[0041] In this invention, the *Lactobacillus plantarum* strain preferably includes *Lactobacillus plantarum* strain ZRR; the strain preservation number of *Lactobacillus plantarum* strain ZRR is CCTCC No: M2016281. The *Lactobacillus plantarum* strain ZRR preferably exhibits certain inhibitory effects against *Escherichia coli* (inhibition zone 20-25 mm), *Staphylococcus aureus* (inhibition zone 20-25 mm), and *Aspergillus* (inhibition zone 10-15 mm). The *Lactobacillus plantarum* strain ZRR is *Lactobacillus plantarum* ZRR as published in CN106148249A.

[0042] In this invention, the *Lactococcus lactis* subsp. *lactococcus* is preferably a *Lactococcus lactis* subsp. *lactococcus* that produces nisin. The *Lactococcus lactis* subsp. *lactococcus* with the nisin-producing strain preservation number CGMCC No. 1.2030, originating from Poland, was purchased from the China General Microbiological Culture Collection Center.

[0043] In this invention, the lactococcus is preferably a lactococcus producing nisin. The strain preservation number of the lactococcus producing nisin is preferably CGMCC No. 1.15072, purchased from the China General Microbiological Culture Collection Center.

[0044] In this invention, the quercetin was purchased from Nanjing Rongshengda Experimental Instrument Co., Ltd., brand Macklin, with a purity of 97%.

[0045] In this invention, the preparation method of the silage additive, when Lactobacillus plantarum and quercetin are combined, preferably includes the following steps: activating Lactobacillus plantarum in a skim milk powder solution, and then adding quercetin and mixing; when Lactobacillus plantarum is combined with Lactococcus lactis subsp. lactis or with Lactococcus lactis, preferably, Lactobacillus plantarum is mixed with Lactococcus lactis subsp. lactis or with Lactococcus lactis, and the resulting mixed bacteria are inoculated into the skim milk powder solution for activation. The activation time is preferably 2 hours. The activation temperature is preferably 20–30°C, more preferably 25°C.

[0046] This invention provides an animal feed prepared by fermenting whole-plant corn silage using the silage additive as a fermenting agent.

[0047] This invention provides the application of the silage additive in the preparation of animal feed using whole-plant corn silage as raw material.

[0048] In this invention, the preferred silage additive is a combination of *Lactobacillus plantarum* and quercetin, wherein the amount of live *Lactobacillus plantarum* added to the whole-plant corn silage is (1–10) × 10⁻⁶. 5 CFU·g -1 More preferably 5×10 5 CFU·g -1 The quercetin content in the whole-plant corn silage is 0.06–0.18% by mass, more preferably 0.12%. When the silage additive is a combination of *Lactobacillus plantarum* and *Lactococcus lactis* subsp. *lactocopherol* or a combination of both, the total viable bacteria content of *Lactobacillus plantarum* and *Lactococcus lactis* subsp. *lactocopherol* or *Lactococcus lactis* in the whole-plant corn silage is (1–10) × 10⁻⁶. 5 CFU·g -1 More preferably 5×10 5 CFU·g -1 .

[0049] In this invention, the dry matter content of the whole-plant corn silage is preferably 35%–38%, the crude protein content is 7–9%, and the neutral detergent fiber content is 39–44%. The maturity stage of the whole-plant corn silage is the waxy maturity stage, with the milk line at 1 / 4–1 / 2 of the kernel length. The leaves at the bottom and top of the plant begin to yellow, the ear husks are completely white, and ear rot is quite noticeable at the top of the ear. The preferred harvesting method for the whole-plant corn silage is to cut the whole corn plant and then chop it short, with the cutting height appropriately increased to 8–12 cm and the chopped length 1–2 cm, followed by crushing. The preferred method for determining the dry matter content of the whole-plant corn silage is rapid determination using a microwave oven.

[0050] In this invention, the method for preparing animal feed using whole-plant corn silage as raw material preferably includes diluting a silage additive and spraying it onto the whole-plant corn silage, calculating the remaining water volume to adjust the dry matter content of the raw material to 35%, and sealing and storing the uniformly mixed silage. The storage temperature is preferably 20–30°C, more preferably 25°C. The storage time is preferably 60–90 days, more preferably 75 days.

[0051] In this invention, the application preferably includes one or more of the following:

[0052] 1) It does not change the nutritional quality of whole-plant corn silage;

[0053] 2) Improve the aerobic stability of whole-plant corn silage;

[0054] 3) Reduce the number of fungi in whole-plant corn silage;

[0055] 4) Increase the relative abundance of lactic acid bacteria in whole-plant corn silage;

[0056] 5) Reduce the relative abundance of Klebsiella pneumoniae in whole-plant corn silage.

[0057] In this invention, the fungi preferably include one of the following: Aspergillus, Penicillium, Fusarium, and yeast. The silage additive (Lactobacillus plantarum + quercetin) can increase the content of neutral detergent fiber, hemicellulose, and cellulose, but there is no significant difference compared with the control group; it can also effectively increase the number of lactic acid bacteria and has an inhibitory effect on molds and yeasts; furthermore, the silage additive (Lactobacillus plantarum + quercetin) has the highest aerobic stability, increasing the safety of whole-plant corn silage for more than 2 days and reducing the activity of harmful microorganisms after opening.

[0058] The following detailed description, in conjunction with embodiments, illustrates an antifungal silage additive, feed, and application provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060] Application of compound lactic acid bacteria additives and compound biological agents in whole-plant corn silage

[0061] This experiment was conducted using the same silage raw materials and silage processing level, and was divided into eight groups according to different silage additives:

[0062] Group 1, the control group, did not add any additives. The silage raw materials were sprayed with sterile water at a rate of 20 ml / kg of silage raw materials.

[0063] Group 2, Lactobacillus plantarum group, was supplemented with 100% Lactobacillus plantarum ZRR at a dosage of 5 × 10⁻⁶. 5 CFU·g -1 Weigh out Lactobacillus plantarum ZRR and add it to 2ml of sterilized skim milk powder solution. Activate at room temperature for 2 hours. Add an appropriate amount of water (calculated at 20ml of water per kg of silage raw material) and dissolve evenly. Then use it to spray on the silage.

[0064] Group 3, *Lactococcus lactis* subsp. *lactocopherol*, was supplemented with 100% *Lactococcus lactis* subsp. *lactocopherol* producing nisin, at a dosage of 5 × 10⁻⁶. 5 CFU·g -1 The rest are in the same group, number 2.

[0065] Group 4, Lactococcus lactis group, supplemented with 100% nisin-producing Lactococcus lactis, at a dosage of 5 × 10⁻⁶. 5 CFU·g -1 The rest are in the same group, number 2.

[0066] Group 5, quercetin group, added quercetin to a final concentration of 0.12%, added an appropriate amount of water (calculated at 20ml of water per kg of silage raw material), shook for 10 minutes, and then immediately sprayed into the silage raw material.

[0067] Group 6, Lactobacillus plantarum + quercetin group, contains 5 × 10 5 CFU·g -1 A compound biological additive consisting of Lactobacillus plantarum ZRR and 0.12% quercetin is added to an appropriate amount of water (calculated at 20ml of water per kg of silage raw material), shaken for 10 minutes, and then immediately sprayed into the silage raw material.

[0068] Group 7 consisted of *Lactobacillus plantarum* + *Lactococcus lactis* subsp. *lactocopherol*, in which the ratio of effective viable *Lactobacillus plantarum* ZRR to *Lactococcus lactis* subsp. *lactocopherol* producing nisin was 1:1, and the total addition amount was 5 × 10⁻⁶. 5 CFU·g -1 The rest are in the same group, number 2.

[0069] Group 8 consisted of *Lactobacillus plantarum* and *Lactococcus lactis*, with a live bacteria count ratio of *Lactobacillus plantarum* ZRR to *Lactococcus lactis* producing nisin at 1:1, and an addition amount of 5 × 10⁻⁶. 5 CFU·g -1 The rest are in the same group, number 2.

[0070] Group 9 whole-plant corn raw materials were harvested, crushed, and immediately placed in self-sealing bags for storage at 4℃. Upon returning to the laboratory on the same day, the number of microorganisms was immediately tested. Approximately 300g of the raw materials were weighed on-site, blanched in a local oven at 105℃, and then brought back to the laboratory to be dried at 65℃ until constant weight.

[0071] Whole-plant corn silage preparation: Additives from groups 2-8 are sprayed into the whole-plant corn silage raw material according to their respective proportions. Simultaneously, the remaining water volume after the dry matter content reaches 35% is added to the spray bottle. The mixture is shaken thoroughly and then sprayed onto the whole-plant corn raw material chopped into 1-2 cm pieces. Group 1 is treated with an equal amount of distilled water as a control group. After thorough mixing, the silage raw material is packed into silage bags. Oxygen is removed from the silage bags using a vacuum sealer, and the bags are sealed and stored for 60-90 days at 20-30℃.

[0072] Measurement indicators

[0073] 1. Nutritional quality indicators: crude protein, neutral detergent fiber, acid detergent fiber, acid detergent lignin, hemicellulose, cellulose

[0074] 3. Fermentation quality indicators: pH value

[0075] 4. Microbiological indicators: lactic acid bacteria, yeast, mold

[0076] 5. Microbial diversity indicators: bacterial diversity, fungal diversity

[0077] Sample processing

[0078] 1. After opening the bag and taking a sample, after thoroughly mixing the silage, weigh 20g of silage into an Erlenmeyer flask, add 180ml of deionized water, stir well, and extract at 4℃ for 24h. Then filter with 4 layers of gauze and qualitative filter paper to obtain the extract for pH value determination.

[0079] 2. Take another 20g of fresh sample, add 180ml of 0.85% sterile saline, seal with sealing film, and use it to count lactic acid bacteria, molds, and yeasts.

[0080] 3. After opening the silage, immediately take 50g of fresh sample from each replicate and store it separately at -20℃, and use it as soon as possible for the detection of microbial diversity.

[0081] 4. Weigh approximately 300g of silage and collect it. Blanch it at 105℃ for 0.5h, then dry it in an oven at 65℃ for more than 48h until constant weight. Measure the dry matter content. Crush the dried sample with a high-speed universal pulverizer, sieve it, put it into a polyethylene self-sealing bag, remove the air, seal and store it for nutrient composition determination.

[0082] 5. Weigh approximately 1.7 kg of silage and loosely pack it into a 5L silage container for testing its aerobic stability.

[0083] Determination methods

[0084] 1. Nutritional Quality Analysis: Crude protein (CP) content was determined using the Kjeltec™ 2300 fully automated Kjeltec nitrogen analyzer (FOSS, Denmark) via the Kjeltec method. Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) content were determined using the Van-Soest method with filter bag technology. Hemicellulose content was calculated as the difference between NDF and ADF; cellulose content was also calculated as the difference between ADF and ADL.

[0085] 2. pH value was measured using a glass electrode pH meter (instrument: Mettler Toledo FE20).

[0086] 3. Microbial Counting: Microbial counts were performed using the plate culture method. Serial dilutions were performed using 0.85% sterile physiological saline, and two suitable gradients were selected for plate preparation, with three replicates for each gradient. Lactic acid bacteria, molds, and yeasts were counted using MRS (de Man-Rogosa-Sharpe) agar and Bengal red agar, respectively.

[0087] 4. Aerobic stability: Weigh approximately 1.7 kg of silage and loosely pack it into a 5 L silage bin. Insert a temperature probe into the core of the bin and use a temperature recorder (MDL-1048-A) to measure the temperature of each silage bin and the ambient temperature at regular intervals, once per hour. Calculate the aerobic stability based on the time required for the sample temperature to exceed room temperature by 2°C.

[0088] 4. Microbial diversity analysis: Silage samples were frozen at -20℃ until processing, according to... Total DNA was extracted from the microbial community using the soil DNA kit (Omega Bio-tek, Norcross, GA, US). DNA extraction quality was assessed using 1% agarose gel electrophoresis. DNA concentration and purity were determined using a NanoDrop 2000 and the DNA was sent to Shanghai Meiji Biotechnology Co., Ltd. for microbial community analysis. The V3-V4 region of the bacteria was amplified using primers 338F (5'-ACTCCTACGGGAGGCAGAG-3', SEQ ID NO:1) and 806R (5'-GGACTACHVGGGTWTCTAAT-3', SEQ ID NO:2). For fungi, the ITS region was amplified using primers ITS3F (5'-GCATCGATGAAGAACGCAGC-3', SEQ ID NO:3) and ITS4R (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO:4). Microbial community analysis was performed using the online Majorbio cloud platform (www.majorbio.com).

[0089] Organization and analysis of test records

[0090] A designated person is responsible for collecting and recording experimental data, while Excel spreadsheets are used to organize the relevant data and R language is used to analyze the sample data.

[0091] Table 1. Effects of different additives on the nutritional quality (%DM) of whole-plant maize silage

[0092]

[0093] Table 1 shows that the crude protein, NDF, ADF, and cellulose contents of whole-plant corn silage (Group 1) increased compared to the raw material (Group 9) after ensiling. Groups 3, 5, and 4 had the highest crude protein content, significantly higher than Groups 7 and 9. Groups 4, 6, 7, and 2 had the highest NDF content, significantly higher than Groups 3 and 9. Groups 3 and 5 had the highest ADL content, significantly higher than Groups 1, 6, and 9. Groups 4 and 6 had the highest hemicellulose content, significantly higher than Groups 3, 5, and 9. Groups 2, 4, and 6 had the highest cellulose content, significantly higher than Groups 3, 8, and 9. Although Group 6 had higher contents in several nutritional indicators, there was no significant difference compared to Group 1.

[0094] Table 2. Effects of different additives on dry matter, pH value, and microbial abundance of whole-plant maize silage.

[0095]

[0096] Note: ND indicates not detected.

[0097] Table 2 shows that the whole-plant corn silage processing technology described in this invention successfully controlled the dry matter content of the whole-plant corn silage to around 35%, resulting in a pH value below 3.8 for all treatment groups and excellent overall fermentation. Meanwhile, groups 6 and 2 had the highest lactic acid bacteria counts, significantly higher than groups 9 and 3. After fermentation, the mold count in group 1 of the whole-plant corn silage was lower than that in raw material group 9. The use of all treatment groups resulted in an overall mold count of less than 10 (<1 log). 10 The CFU / g values ​​indicate that all treatments had a certain inhibitory effect on the overall mold population. After fermentation of whole-plant corn silage, yeast counts increased significantly (Group 1 > Group 9). However, similarly, the use of all additives significantly reduced the number of yeast cells in the whole-plant corn silage. Numerically, Group 6 had the lowest yeast count.

[0098] The aerobic stability of whole-plant corn silage is the time required for the core of the silage to reach a temperature 2°C above room temperature after opening. Figure 1 It can be seen that compared with group 1, all treatment groups significantly improved the aerobic stability of whole-plant corn silage, with group 6 exhibiting the highest aerobic stability, exceeding 2 days, greatly increasing the safety of whole-plant corn silage and reducing the activity of harmful microorganisms after opening. Figure 2 It can be seen that the temperature difference between the sample and the ambient temperature reached its maximum within 60-80 hours for all treatment groups. Group 4 had the highest temperature difference, followed by Group 3 and Group 2. The maximum temperature difference of the other treatment groups was lower than that of Group 1.

[0099] from Figure 3It can be seen that compared with group 1, group 4 and group 8 significantly increased the relative abundance of Aspergillus fungi, and were comparable to the relative abundance of Aspergillus in group 9 of whole-plant corn silage. The relative abundance of Aspergillus in groups 1, 2, 5 and 6 were at a low level, with group 5 having the lowest relative abundance of Aspergillus, followed by group 6.

[0100] from Figure 4 It can be seen that, compared with group 9, the relative abundance of Fusarium fungi in group 1 did not decrease after fermentation of whole-plant corn silage. In contrast, groups 5, 6, 8, and 2 significantly reduced the relative abundance of Fusarium fungi, with group 5 showing the best effect and group 6 the second best.

[0101] from Figure 5 It can be seen that the relative abundance of Penicillium fungi generally decreased after silage fermentation of whole-plant corn silage, except for group 8. Compared with group 1, groups 2, 3 and 4 increased the relative abundance of Penicillium, but the relative abundance of Penicillium in groups 5 and 6 remained at an extremely low level.

[0102] from Figure 6 It can be seen that the relative abundance of *Lactobacillus* in group 9 whole-plant corn silage was extremely low. After silage, the relative abundance of *Lactobacillus* in group 1 increased significantly. The relative abundance of *Lactobacillus* in groups 4 and 8 was close to that in the silage raw material. Groups 3 and 7 also significantly reduced the relative abundance of *Lactobacillus* in whole-plant corn silage compared to group 7, indicating that the two types of *Lactococcus lactis* have a strong inhibitory effect on *Lactobacillus* in silage. Groups 2, 5, and 6, however, have a certain promoting effect on *Lactobacillus* in whole-plant corn silage.

[0103] from Figure 7 It can be seen that compared with the whole-plant corn silage raw material in group 9, the Klebsiella spp. bacteria in the silage fermentation of whole-plant corn silage feed increased significantly, especially in the control group in group 1, where the relative abundance of Klebsiella spp. was the highest. The use of additives had a significant inhibitory effect on Klebsiella spp., with the best inhibitory effect in groups 4 and 8, followed by group 6.

[0104] The results showed that groups 3 and 7 contained *Lactococcus lactis* subsp. *lactococcus*, while groups 4 and 8 contained *Lactococcus lactis*, etc. Lactic acid bacteria with antibacterial capabilities exhibited a certain inhibitory effect on harmful bacteria such as *Klebsiella* spp., and while they showed good inhibitory effects on mold numbers, they did not have a specific inhibitory effect on harmful fungi such as *Fusarium* spp., *Aspergillus* spp., and *Penicillium* spp., etc., in groups 5 and 6. The combination of quercetin and *Lactobacillus plantarum* significantly controlled the increase of *Aspergillus* and *Penicillium* spp. in whole-plant corn silage, inhibited *Fusarium* spp., and simultaneously inhibited harmful bacteria such as *Klebsiella* spp., increased the relative abundance of beneficial bacteria such as *Lactobacillus* spp., and improved the microbial diversity of whole-plant corn silage.

[0105] 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 an antifungal silage additive in the preparation of animal feed using whole-plant corn silage as raw material, wherein the silage additive is composed of Lactobacillus plantarum and quercetin; The live bacteria addition amount of *Lactobacillus plantarum* in whole-plant corn silage is (1~10)×10⁻⁶. 5 CFU·g -1 Quercetin accounts for 0.06-0.18% of the total mass of maize silage.

2. The application according to claim 1, characterized in that, The *Lactobacillus plantarum* includes *Lactobacillus plantarum* strain ZRR; The strain ZRR of *Lactobacillus plantarum* has the preservation number CCTCC No: M2016281.

3. The application according to claim 1, characterized in that, The dry matter content of the whole-plant corn silage is 35%~38%.

4. The application according to claim 3, characterized in that, The application also includes one or more of the following: 1) It does not change the nutritional quality of whole-plant corn silage; 2) Improve the aerobic stability of whole-plant corn silage; 3) Reduce the number of fungi in whole-plant corn silage; 4) Increase the relative abundance of lactic acid bacteria in whole-plant corn silage.

5. The application according to claim 4, characterized in that, The fungi include one of the following: Aspergillus, Penicillium, Fusarium, and yeast.

6. The application according to claim 4, characterized in that, The application also includes reducing the relative abundance of Klebsiella spp. in whole-plant corn silage.

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