Three-dimensional synergistic alfalfa silage additive of sludge oil and preparation method thereof

Abstract

The application discloses a kind of fungus residue oil three-dimensional synergistic alfalfa silage additive and preparation method thereof, belong to the field of forage silage preservation.For the problems such as strong corrosion of chemical preservative in existing alfalfa silage additive, poor compatibility of lactic acid bacteria and essential oil, easy volatile failure of essential oil and waste of oregano distillation residue, the method comprises: part of oregano essential oil distillation residue powder is mixed with lactobacillus plantarum and lactobacillus buchneri at 3:1, and defatted milk powder and sucrose are freeze-dried to obtain fungus residue composite particles;Cinnamon essential oil is embedded with β-cyclodextrin to obtain pH-responsive microcapsules, which release ≤25% in 24h at pH 6.5 and ≥75% at pH 4.0;Then mix the fungus residue particles, the remaining residue powder and the microcapsules, so that the final bacterial powder content is 8-12%, the residue content is 75-80%, and the microcapsule content is 8-10%.The additive is used for alfalfa silage, which can realize rapid acid reduction and long-term synergistic bacteriostasis, and the stable period after opening is more than 30 days.

Description

Technical Field

[0001] This invention relates to the field of forage silage preservation. More specifically, this invention relates to a method for preparing a three-dimensional synergistic alfalfa silage additive using mushroom residue oil. Background Technology

[0002] Alfalfa, as the most widely planted and nutritionally valuable high-quality legume forage in my country and even globally, has always been a research hotspot in the livestock industry for its silage preservation technology. Alfalfa silage refers to the process of preserving fresh alfalfa into silage feed for long-term livestock consumption through microbial fermentation by lactic acid bacteria and other microorganisms under anaerobic conditions. However, alfalfa's inherent characteristics of low soluble carbohydrate content and high buffering capacity make natural fermentation far more difficult than that of conventional silage crops such as corn and sorghum, requiring the use of exogenous additives to achieve high-quality silage.

[0003] Currently, alfalfa silage preservation mainly relies on three technical solutions: chemical preservatives (organic acids such as formic acid and propionic acid), single lactic acid bacteria inoculants, and pure plant essential oils / plant extracts. While chemical preservatives can quickly reduce acidity and inhibit bacteria, they are highly corrosive, easily damage equipment, and endanger the health of operators. Long-term use can also lead to feed residues, which does not align with the policy direction of reducing antibiotics and green farming. Regarding single lactic acid bacteria preparations, homofermentative lactic acid bacteria (such as *Lactobacillus plantarum*) produce acid quickly but have weak anti-mold and anti-secondary fermentation capabilities, while heterofermentative lactic acid bacteria (such as *Lactobacillus brunelli*) have good antibacterial effects but slow fermentation initiation. Neither can simultaneously achieve rapid fermentation and long-term preservation. Commercially available inoculants have limited functions, poor aerobic stability after opening, and a high risk of secondary fermentation and mold growth.

[0004] Numerous studies have been reported on lactic acid bacteria additives. For example, a compound lactic acid bacteria silage additive has been disclosed, which, through the combination of *Lactobacillus plantarum* and *Lactobacillus bruneri*, can increase the lactic acid and acetic acid content of alfalfa silage and inhibit mold growth. A study published in the *Journal of Grassland Science* in 2024 also showed that a compound lactic acid bacteria mixture composed of *Lactobacillus plantarum* and *Lactobacillus bruneri* can significantly affect the quality of alfalfa silage. However, such single-lactic acid bacteria formulations rely solely on microbial fermentation to lower the pH of the silage to inhibit spoilage bacteria. This fails to address the secondary fermentation problem caused by oxygen entry after the silage is opened. Once the packaging is broken, the acid production of the lactic acid bacteria ceases, and the remaining aerobic microorganisms rapidly multiply, leading to silage heating, mold growth, and spoilage, quickly causing the silage's freshness-locking function to fail.

[0005] In the application of plant essential oils, natural extracts such as cinnamon oil and oregano oil possess excellent antibacterial and preservative activities. However, direct addition presents challenges such as high volatility, easy oxidation, and easy decomposition at high temperatures, resulting in significant loss of active ingredients and a short duration of action. More importantly, high concentrations of essential oils can inhibit the activity of lactic acid bacteria, and the two have poor compatibility, making synergistic use in the same additive is impossible. To address the stability issue of essential oils, existing research has employed β-cyclodextrin encapsulation technology to prepare cinnamon oil microcapsules. For example, journals such as *Food Science* have reported methods for preparing sustained-release microcapsules using β-cyclodextrin encapsulation of plant essential oils. However, these studies only focus on the preparation process and release characteristics of the microcapsules themselves, without combining them with lactic acid bacteria and oregano oil distillation residues for application in alfalfa silage.

[0006] Furthermore, the extraction of oregano essential oil produces a large amount of distillation residue, accounting for more than 90% of the fresh weight of the raw material. Traditional disposal methods include landfilling or incineration, which not only wastes resources but also generates greenhouse gases. Although recent studies have revealed that oregano essential oil distillation residue can improve the fermentation quality of alfalfa silage through a triple mechanism of inhibiting harmful bacteria, promoting beneficial bacteria, and activating metabolism, and patents have disclosed methods for directly adding high-carvacrol-type oregano essential oil distillation residue to silage raw materials for fermentation, these technical solutions all involve the single addition of oregano residue without three-dimensional synergistic compounding with lactic acid bacteria and plant essential oil microcapsules.

[0007] In summary, existing alfalfa silage preservation technologies, which involve simple physical mixing of oregano residue, compound lactic acid bacteria, and cinnamon essential oil or conventional slow-release microcapsule treatment, fail to integrate the microbial agents and residue carriers into functional units in terms of spatial structure, nor do they enable the release rhythm of essential oils to actively match the silage fermentation process in terms of time. This results in mutual interference and functional disconnect between components, making it difficult to truly achieve the three-dimensional synergistic effect of "microbial-residue-oil" and limiting further improvement in the quality of alfalfa silage. Summary of the Invention

[0008] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0009] Another objective of this invention is to provide a method for preparing a three-dimensional synergistic alfalfa silage additive based on microbial residue and oil. This method utilizes a microbial residue composite freeze-drying protection technology and pH-responsive cinnamon essential oil microcapsules to achieve three-dimensional synergy among microorganisms, residue, and oil, significantly improving silage quality. Lactic acid bacteria rapidly produce acid in the early stages of silage, the residue continuously inhibits miscellaneous bacteria, and the microcapsules accelerate the release of essential oil in an acidic environment to enhance protection after opening. The three work together to achieve full-cycle freshness preservation.

[0010] To achieve these objectives and other advantages according to the present invention, a method for preparing a three-dimensional synergistic alfalfa silage additive using fungal residue oil is provided, comprising:

[0011] S1. The oregano essential oil distillation residue is prepared into oregano essential oil distillation residue powder; Lactobacillus plantarum powder and Lactobacillus buchneri powder are mixed at a mass ratio of 3:1 to obtain compound lactic acid bacteria powder; a portion of the oregano essential oil distillation residue powder is mixed with the compound lactic acid bacteria powder at a mass ratio of 1:1~2, and based on the total mass of the oregano essential oil distillation residue powder and the compound lactic acid bacteria powder, 5~12% skim milk powder and 1~4% sucrose are added as a protective agent, and the mixture is freeze-dried at low temperature to obtain the fungal residue compound freeze-dried granules;

[0012] S2. Using β-cyclodextrin as the wall material and cinnamon essential oil as the core material, and controlling the core-to-wall ratio at 1:8~10, cinnamon essential oil microcapsules were prepared by a programmed cooling aqueous solution encapsulation method. The cumulative release rate of the prepared cinnamon essential oil microcapsules was ≤25% in a buffer solution at pH 6.5 and ≥75% in a buffer solution at pH 4.0 after 24 hours.

[0013] S3. Mix the mycelium residue composite freeze-dried granules obtained in step S1, the remaining oregano essential oil distillation residue powder, and the cinnamon essential oil microcapsules obtained in step S2. Control the content of each component in the additive after mixing to meet the following requirements: the mass percentage of the composite lactic acid bacteria powder is 8-12%, the total mass percentage of the oregano essential oil distillation residue powder is 75-80%, the mass percentage of the cinnamon essential oil microcapsules is 8-10%, and the remainder is skim milk powder and sucrose. Package the mixture to obtain the mycelium residue oil three-dimensional synergistic alfalfa silage additive.

[0014] Preferably, the preparation process of the oregano essential oil distillation residue powder in step S1 includes: first soaking the oregano essential oil distillation residue in a 2-5% (by mass) citric acid aqueous solution until the moisture content is 40-50%, then stacking it at 40-50°C for 6-12 hours, then drying it with low-temperature hot air until the moisture content is ≤10%, pulverizing it and passing it through a 60-150 mesh sieve, and then sterilizing it by low-temperature irradiation to obtain the oregano essential oil distillation residue powder.

[0015] Preferably, in step S1, the oregano essential oil distillation residue powder is divided into two grades: the oregano essential oil distillation residue powder used to prepare the fungal residue composite freeze-dried particles is obtained by passing through a 100-150 mesh sieve; and the remaining oregano essential oil distillation residue powder in step S3 is obtained by passing through a 60-80 mesh sieve.

[0016] Preferably, the low-temperature freeze-drying process in step S1 includes: first cooling the mixture to -40 to -50°C at a rate of 0.5 to 1.5°C / min and holding it at that temperature for 2 to 4 hours, then rapidly cooling it to -70 to -80°C and holding it at that temperature for 4 to 6 hours; and then sublimation drying for 24 to 48 hours under a vacuum of 10 to 30 Pa and a shelf temperature of -25 to -15°C.

[0017] Preferably, the obtained fungal residue composite freeze-dried particles have a porosity of 40-60%, an average pore size of 5-20 μm, and a bulk density of 0.35-0.50 g / cm³. 3 .

[0018] Preferably, the viable count of the compound lactic acid bacteria powder in step S1 is ≥5×10⁻⁶. 11 CFU / g.

[0019] Preferably, the programmed cooling aqueous solution encapsulation method in step S2 is as follows: β-cyclodextrin is dissolved in hot water at 60~80℃ to form a saturated or near-saturated solution, and cinnamon essential oil is slowly added dropwise under constant temperature stirring for no less than 30 min, while maintaining the temperature and stirring for 2~4 h; then the temperature is lowered to 4~10℃ at a rate of 0.5~1℃ / min, and crystallization is allowed to occur for 12~24 h; the microcapsules are separated, washed with water and 20~30% ethanol (by volume), and then dried under vacuum at ≤40℃.

[0020] Preferably, the mixing in step S3 is carried out in two steps: first, the fungal residue composite freeze-dried granules and the remaining oregano essential oil distillation residue powder are added into the mixing equipment in proportion and mixed for 5-10 minutes, so that the residue powder coats the surface of the fungal residue composite freeze-dried granules to form a coating layer; then, cinnamon essential oil microcapsules are added and the mixing continues for 5-10 minutes.

[0021] This invention further claims a three-dimensional synergistic alfalfa silage additive containing microbial residue and oil, comprising the following components by mass percentage: 8-12% compound lactic acid bacteria powder, 75-80% oregano essential oil distillation residue powder, and 8-10% cinnamon essential oil microcapsules; wherein the compound lactic acid bacteria powder is obtained by mixing Lactobacillus plantarum powder and Lactobacillus brucelli powder in a mass ratio of 3:1, and the cinnamon essential oil microcapsules are prepared by encapsulation with β-cyclodextrin as the wall material and cinnamon essential oil as the core material.

[0022] This invention further claims a method for locking in the freshness of alfalfa silage, comprising: uniformly applying the three-dimensional synergistic alfalfa silage additive prepared by the aforementioned method of preparing alfalfa silage additive with mushroom residue oil at a ratio of 25-45g per ton of alfalfa silage raw material, mixing evenly, and then sealing and ensiling.

[0023] The present invention has at least the following beneficial effects:

[0024] Firstly, this invention premixes a compound lactic acid bacteria powder of Lactobacillus plantarum and Lactobacillus brucelli in a 3:1 ratio with oregano essential oil distillation residue powder in a 1:1~2 ratio, adds skim milk powder and sucrose preservative, and freeze-dries at low temperature to form compound freeze-dried granules of bacterial residue. These granules are then compounded with the remaining residue powder and cinnamon essential oil microcapsules, so that the mass percentage of compound lactic acid bacteria powder in the final additive is precisely controlled at 8~12%. When added to alfalfa silage at 25~45g per ton, the pH can be reduced to below 4.2 within 48 hours, and the aerobic stability period after opening can be extended to more than 30 days, while greatly reducing the end-use cost.

[0025] Secondly, this invention uses a 2-5% citric acid solution to wet and activate the distillation residue of oregano essential oil, and combines this with stacking at 40-50℃ to induce partial degradation of cellulose in the residue and increase the freeness of active ingredients; at the same time, the residue powder is graded into 100-150 mesh fine powder (to participate in freeze-drying granulation to construct the microbial residue composite particle skeleton) and 60-80 mesh coarse powder (to form an external coating layer when finally mixed), realizing the gradient utilization of functional components of the residue, and improving the release efficiency and antibacterial durability of active substances such as carvacrol and thymol in the residue;

[0026] Thirdly, this invention uses a programmed cooling aqueous solution encapsulation method to prepare pH-responsive cinnamon essential oil microcapsules. By controlling the core-to-wall ratio of 1:8~10 and a specific cooling crystallization program, the cumulative release rate of the microcapsules is ≤25% in pH 6.5 buffer solution and ≥75% in pH 4.0 buffer solution over 24 hours. This precisely matches the fermentation process of alfalfa silage. In the initial sealing stage, the microcapsules are stable and do not release in a near-neutral environment, preventing the essential oil from escaping prematurely and inhibiting the activity of lactic acid bacteria. After entering the acidic stable period, the release of cinnamon essential oil is accelerated, strengthening the continuous antibacterial protection after opening. This solves the problem that plant essential oils and lactic acid bacteria cannot be used synergistically in the same system.

[0027] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0029] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0030] Example 1

[0031] Step 1: Pretreatment and grading of oregano essential oil distillation residue:

[0032] 100 kg of fresh oregano essential oil distillation residue with a moisture content of approximately 68% was uniformly sprayed with a 3% (w / w) citric acid aqueous solution and stirred until the residue was fully moistened to a moisture content of 45%. The moistened oregano essential oil distillation residue was then piled at 45°C for 8 hours to induce partial degradation of cellulose and increase the free activity of active ingredients. After piling, the residue was dried using a low-temperature hot air dryer at 55°C until the moisture content was 8.5%. The dried residue was then pulverized. The pulverized powder was passed through a 100-mesh sieve, and the fine powder passing through the 100-mesh sieve was collected as the fine fraction for preparing the mycelial residue composite freeze-dried granules. The coarse powder that did not pass through the 100-mesh sieve was further passed through an 80-mesh sieve, and the powder passing through the 80-mesh sieve was collected as the coarse fraction for forming the outer coating layer during final mixing. Both powders were then subjected to low-temperature irradiation sterilization. After sterilization, the active ingredient retention rate was 87%, yielding fine and coarse oregano essential oil distillation residue powders for later use.

[0033] Step 2: Preparation of compound lactic acid bacteria powder:

[0034] Take commercially available freeze-dried Lactobacillus plantarum powder (live count ≥ 1.0 × 10⁻⁶). 12 CFU / g) and commercially available Lactobacillus brucellosis lyophilized powder (live count ≥8.0×10⁻⁶). 11 The two bacterial powders (CFU / g) were activated separately with sterile physiological saline. The two bacterial powders were then added to a mixer at a mass ratio of *Lactobacillus plantarum*: *Lactobacillus bruschetta* = 3:1 and mixed at low speed for 15 min to obtain a compound lactic acid bacteria powder. The viable count was tested to be ≥5 × 10⁻⁶. 11 CFU / g.

[0035] Step 3: Preparation of the fungal residue composite freeze-dried granules:

[0036] Take the fine powder of oregano essential oil distillation residue obtained in step one and the compound lactic acid bacteria powder obtained in step two, and put them into a mixer at a mass ratio of 1:1, and premix for 10 minutes. Based on the total mass of the oregano essential oil distillation residue powder and the compound lactic acid bacteria powder, 12% skim milk powder and 3% sucrose (as a preservative) were added, and the mixture was continued for 15 minutes until homogeneous. The mixture was spread evenly in a freeze-drying pan, with a thickness not exceeding 15 mm, and freeze-dried at low temperature according to the following procedure: cooling to -45℃ at a rate of 1.0℃ / min and holding for 3 hours; then rapidly cooling to -75℃ at a rate of ≥2℃ / min and holding for 5 hours; then sublimation drying was carried out for 36 hours under a vacuum of 20 Pa and a shelf temperature of -20℃. After freeze-drying, the freeze-dried lumps were gently crushed in an environment with a relative humidity of ≤20%, and granulated through a 10-mesh sieve to obtain the fungal residue compound freeze-dried granules. The porosity of the obtained fungal residue compound freeze-dried granules was tested to be 52%, the average pore size was 12 μm, and the bulk density was 0.42 g / cm³. 3 .

[0037] Step 4: Preparation of pH-responsive cinnamon oil microcapsules:

[0038] A saturated solution of 4.5 kg β-cyclodextrin was prepared by dissolving it in hot water at 70 °C, with a total volume of approximately 25 L. The solution was stirred at a constant temperature and 400 rpm. 0.5 kg cinnamon essential oil (cinnamaldehyde content ≥85%) was slowly added dropwise to the β-cyclodextrin solution using a syringe pump, controlling the adding rate to ensure a adding time of at least 30 min. The mixture was stirred at 70 °C for 3 h for encapsulation. After encapsulation, the temperature was programmed to decrease to 6 °C at a rate of 0.8 °C / min, and the mixture was allowed to stand for crystallization for 18 h to allow complete precipitate separation of the inclusion complex. The microcapsule precipitate was separated by centrifugation, washed twice with deionized water and once with 25% ethanol (v / v), and then vacuum dried at 35 °C to constant weight, yielding 5.03 kg of cinnamon essential oil microcapsules. The cumulative release rate was 22.3% in pH 6.5 phosphate buffer and 78.6% in pH 4.0 citrate-phosphate buffer, meeting the requirements for pH-responsive release.

[0039] Step 5: Compound mixing of alfalfa silage additives

[0040] The freeze-dried granules of fungal residue and the coarse powder of oregano essential oil distillation residue obtained in step one were added to a three-dimensional motion mixer in a certain proportion. The mixing was carried out in two steps: first, the freeze-dried granules of fungal residue and the coarse powder of oregano essential oil distillation residue were added to the mixer and mixed at low speed at room temperature for 8 minutes, so that the coarse powder was evenly coated on the surface of the freeze-dried granules of fungal residue, forming a composite granule with a coarse powder coating layer, a freeze-dried granule skeleton, and an internal bacterial agent gradient structure; then, cinnamon essential oil microcapsules were added, and mixing was continued for 8 minutes, so that the microcapsules were evenly dispersed between the composite granules. After mixing, the three-dimensional synergistic alfalfa silage additive product of fungal residue and oil was obtained. It was vacuum-packed with aluminum foil composite film. The viable bacteria count in the finished additive was tested to be 5.2 × 10⁻⁶. 10 CFU / g.

[0041] Comparative Example 1

[0042] In step three, the compound lactic acid bacteria powder and the fine powder of oregano essential oil distillation residue are mixed and freeze-dried without adding skim milk powder or sucrose. The remaining steps are the same as in Example 1.

[0043] Comparative Example 2

[0044] In step three, the compound lactic acid bacteria powder, oregano essential oil distillation residue fine powder, and protectant are only physically mixed without freeze-drying and granulation, and are directly used for compounding and mixing alfalfa silage additives. The remaining steps are the same as in Example 1.

[0045] Comparative Example 3

[0046] In step three, the compound lactic acid bacteria powder is freeze-dried and then directly mixed with the fine powder of oregano essential oil distillation residue and the protectant without co-freeze-drying. This mixture is used for compounding alfalfa silage additives. The remaining steps are the same as in Example 1.

[0047] The alfalfa silage additives prepared in Example 1 and Comparative Examples 1-3 were placed under accelerated storage conditions at 40°C (simulating high-temperature transportation or storage environment) for 30 days, and the viable bacterial count and survival rate were measured before and after storage. At the same time, the acid production rate (time for pH to drop below 4.2) of each additive group in silage application was measured, and the results are shown in Table 1.

[0048] Table 1 Performance of Alfalfa Silage Additives

[0049] <![CDATA[Viable bacterial count before accelerated storage (×10 10 CFU / g)]]> 5.2 5.0 5.1 5.1 <![CDATA[The number of viable bacteria after accelerated storage for 30 days (×10 10 CFU / g)]]> 4.8 1.3 1.8 0.9 viable bacteria survival rate (%) 92.3 26.3 35.3 17.6 Time required for silage pH to drop to 4.2 46 72 68 78 Final product looseness good Severe caking Adhesion Layering

[0050] Note: Good looseness indicates that the particles are in a free-flowing state with no visible agglomerates, and the passing rate through a 20-mesh sieve is ≥95%; Severe agglomeration indicates that the material is clumped into large, hard lumps (lump size >50 mm), which can only be dispersed by external force, and the passing rate through a 20-mesh sieve is <50%; Adhesion indicates that the material is agglomerated into soft lumps (lump size <50 mm), which can be dispersed by light touch, and the passing rate through a 20-mesh sieve is 50%~95%; Segregation indicates that the components of different densities are clearly separated, with the upper layer being the light component (such as residual powder) and the lower layer being the heavy component (such as bacterial powder sediment), and the RSD (relative standard deviation) value of bacterial powder content in different parts of the sample is >15% during sampling and testing.

[0051] As shown in Table 1, Example 1 achieved a post-storage viable bacteria survival rate of up to 92.3%, significantly better than Comparative Examples 1-3. This indicates that the skim milk powder and sucrose preservative synergistically form a three-dimensional protective network around the bacteria through a co-freeze-drying process, greatly improving the thermal and storage stability of lactic acid bacteria. Simultaneously, the group of this invention achieved a pH reduction to below 4.2 in silage within only 46 hours, exhibiting the fastest acid production rate, indicating that the lactic acid bacteria in the combined freeze-dried granules of bacteria and residue possess higher initial activity and a faster metabolic recovery rate.

[0052] Example 2 (Field Application Trial of Alfalfa Silage)

[0053] Alfalfa grown to the initial flowering stage (approximately 10% budding rate) was harvested on a sunny morning. Immediately after harvesting, it was chopped to 2-3 cm using a hay cutter, and the moisture content was adjusted to 68%, meeting the silage moisture standard. Four treatment groups were established:

[0054] Experimental group (alfalfa silage additive obtained in Example 1): 35 g of the finished additive prepared in Example 1 was added to each ton of alfalfa raw material. The additive was first dissolved in 20 L of clean water and stirred evenly (water temperature ≤30℃). It was then evenly applied to the chopped alfalfa raw material by spraying, stirred evenly, wrapped and ensiled, and stored at room temperature in a sealed container.

[0055] Control group 1 (imported lactic acid bacteria additive): Add commercially available imported lactic acid bacteria additive (main components are Lactobacillus plantarum, Lactobacillus brylerii, and Pediococcus pentosaceus, with a viable count ≥2×10^11 CFU / g) to each ton of alfalfa raw material. Use at the recommended dosage of 10 g / ton according to the label instructions. Apply by spraying and then wrap for silage.

[0056] Control group 2 (chemical preservative): 5 L of 85% formic acid solution (equivalent to 4.25 kg / ton of pure formic acid) was added to each ton of alfalfa raw material, diluted and sprayed, and then wrapped and ensiled.

[0057] Control group 3 (blank control): Sprayed with an equal amount of water, without adding any preservatives, and wrapped for silage.

[0058] Each treatment group had three replicates. The silage bags were 55 cm in diameter and 70 cm in height, each weighing approximately 50 kg, and stored at room temperature (daily average temperature 22–28°C). Samples were taken 48 hours after ensiling to determine pH and lactic acid content. pH was determined by adding 10 g of silage sample to 90 mL of distilled water, homogenizing, and allowing it to stand for 30 min. The supernatant was then measured using a pH meter. Lactic acid content was determined using high-performance liquid chromatography (HPLC). Sensory evaluation was conducted based on industry-standard sensory criteria, comprehensively assessing the odor, appearance, and texture of the silage. The results are shown in Table 2.

[0059] Table 2 Results of field trials of alfalfa silage

[0060] experimental group 4.15±0.05 4.26±0.21 Fragrant aroma, free from mold. Control group 1 4.28±0.07 3.85±0.18 Slight sour taste, normal Control group 2 4.02±0.04 3.42±0.15 pungent sour smell Control group 3 5.82±0.11 1.07±0.09 Foul odor, mold visible on the surface

[0061] Note: Aromatic aroma and absence of mold meet the standards for high-quality silage; a slightly sour smell is normal and indicates average / medium quality silage; a foul odor and visible mold on the surface indicate poor quality silage.

[0062] As shown in Table 2, the pH value of the experimental group dropped below the safe threshold of 4.15 within 48 hours, which was close to that of the chemical preservative group and significantly better than the single lactic acid bacteria group and the blank group. The pungent sour smell of control group 2 in the table may be due to the addition of formic acid. The experimental group had the highest lactic acid content, indicating that the alfalfa silage additive of the present invention can effectively promote the rapid reproduction and acid production of lactic acid bacteria and quickly stabilize the silage environment.

[0063] After 60 days of silage storage, samples were unpacked and taken to determine the crude protein content, dry matter loss rate, and microbial colony count. Crude protein was determined using the Kjeldahl method; the dry matter loss rate was calculated from the weight difference before and after ensiling; and microbial counts were performed using the plate dilution method. The results are shown in Table 3.

[0064] Table 3 Nutrients and microorganisms after silage storage

[0065] experimental group 88.1±1.2 7.6±0.8 8.62±0.15 Not detected 0.09±0.02 Control group 1 81.3±1.8 10.4±1.1 7.88±0.21 2.35±0.32 1.28±0.15 Control group 2 76.3±2.1 12.8±1.3 6.24±0.18 1.92±0.28 0.23±0.04 Control group 3 63.5±2.5 22.4±1.8 5.12±0.22 4.56±0.41 9.35±0.82

[0066] Note: Not detected indicates a mold count <10 CFU / g.

[0067] As shown in Table 3, the experimental group had the highest crude protein retention rate of 88.1%, which was significantly better than the chemical group and the single microbial agent group; the dry matter loss rate was only 7.6%, which was the lowest among all groups; the number of lactic acid bacteria reached 8.62 log10 CFU / g, which was significantly higher than the single microbial agent group and the chemical group; no mold was detected and the butyric acid content was extremely low, which indicates that the protein in the experimental group did not undergo putrefactive degradation and the silage quality was excellent.

[0068] After 60 days of silage storage, the bags were unpacked, and the silage from each treatment group was placed in an open chamber at 25°C (simulating the environment after opening and use). Temperature changes were monitored daily (aeration was considered to begin when the temperature inside the bag increased by ≥2°C compared to the ambient temperature), and sensory changes were recorded. The test results are shown in Table 4.

[0069] Table 4. Results of Aerobic Stability Tests After Opening

[0070] experimental group ≥30 (no putrefaction after 30 days) The fragrance was still present on day 30, and there was no fever. Control group 1 9 A slight musty smell appeared on the 9th day, and significant heat and mold growth were observed on the 12th day. Control group 2 7 The sour taste lessened on day 7, and mold spots appeared on day 10. Control group 3 5 On the 5th day, the patient experienced fever, foul odor, and a large amount of mold on the surface.

[0071] As shown in Table 4, the aerobic stability period of the experimental group after opening was more than 30 days, far exceeding the 9 days of control group 1, the 7 days of control group 2, and the 5 days of the blank group. The results indicate that the pH-responsive cinnamon oil microcapsules in the additive of this invention can continue to release antibacterial active ingredients after the silage is opened, effectively inhibiting the secondary fermentation of aerobic microorganisms and ensuring the quality stability of the silage during its use period.

[0072] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A method for preparing a three-dimensional synergistic alfalfa silage additive using mushroom residue oil, characterized in that, include: S1. The oregano essential oil distillation residue is prepared into oregano essential oil distillation residue powder; Lactobacillus plantarum powder and Lactobacillus buchneri powder are mixed at a mass ratio of 3:1 to obtain compound lactic acid bacteria powder; a portion of the oregano essential oil distillation residue powder is mixed with the compound lactic acid bacteria powder at a mass ratio of 1:1~2, and based on the total mass of the oregano essential oil distillation residue powder and the compound lactic acid bacteria powder, 5~12% skim milk powder and 1~4% sucrose are added as a protective agent, and the mixture is freeze-dried at low temperature to obtain the fungal residue compound freeze-dried granules; S2. Using β-cyclodextrin as the wall material and cinnamon essential oil as the core material, and controlling the core-to-wall ratio at 1:8~10, cinnamon essential oil microcapsules were prepared by a programmed cooling aqueous solution encapsulation method. The cumulative release rate of the prepared cinnamon essential oil microcapsules was ≤25% in a buffer solution at pH 6.5 and ≥75% in a buffer solution at pH 4.0 after 24 hours. S3. Mix the mycelium residue composite freeze-dried granules obtained in step S1, the remaining oregano essential oil distillation residue powder, and the cinnamon essential oil microcapsules obtained in step S2. Control the content of each component in the additive after mixing to meet the following requirements: the mass percentage of the composite lactic acid bacteria powder is 8-12%, the total mass percentage of the oregano essential oil distillation residue powder is 75-80%, the mass percentage of the cinnamon essential oil microcapsules is 8-10%, and the remainder is skim milk powder and sucrose. Package the mixture to obtain the mycelium residue oil three-dimensional synergistic alfalfa silage additive.

2. The preparation method of the three-dimensional synergistic alfalfa silage additive based on fungal residue oil as described in claim 1, characterized in that, The preparation process of the oregano essential oil distillation residue powder in step S1 includes: first soaking the oregano essential oil distillation residue in a 2-5% citric acid aqueous solution until the moisture content is 40-50%, then stacking it at 40-50℃ for 6-12 hours, then drying it with low-temperature hot air until the moisture content is ≤10%, pulverizing it and passing it through a 60-150 mesh sieve, and then sterilizing it by low-temperature irradiation to obtain the oregano essential oil distillation residue powder.

3. The preparation method of the three-dimensional synergistic alfalfa silage additive based on fungal residue oil as described in claim 2, characterized in that, In step S1, the oregano essential oil distillation residue powder is divided into two grades. The oregano essential oil distillation residue powder used to prepare the fungal residue composite freeze-dried particles is obtained by passing through a 100-150 mesh sieve. The remaining oregano essential oil distillation residue powder in step S3 is obtained by passing through a 60-80 mesh sieve.

4. The preparation method of the three-dimensional synergistic alfalfa silage additive based on fungal residue oil as described in claim 3, characterized in that, The low-temperature freeze-drying process in step S1 includes: first cooling the mixture to -40 to -50°C at a rate of 0.5 to 1.5°C / min and holding it at that temperature for 2 to 4 hours; then rapidly cooling it to -70 to -80°C and holding it at that temperature for 4 to 6 hours; and then sublimation drying for 24 to 48 hours under a vacuum of 10 to 30 Pa and a shelf temperature of -25 to -15°C.

5. The preparation method of the three-dimensional synergistic alfalfa silage additive based on fungal residue oil as described in claim 4, characterized in that, The obtained fungal residue composite freeze-dried granules have a porosity of 40-60%, an average pore size of 5-20 μm, and a bulk density of 0.35-0.50 g / cm³. 3 .

6. The preparation method of the three-dimensional synergistic alfalfa silage additive based on fungal residue oil as described in claim 5, characterized in that, The viable count of the compound lactic acid bacteria powder in step S1 is ≥5×10⁻⁶. 11 CFU / g.

7. The preparation method of the three-dimensional synergistic alfalfa silage additive based on fungal residue oil as described in claim 6, characterized in that, The programmed cooling aqueous solution encapsulation method described in step S2 is as follows: β-cyclodextrin is dissolved in hot water at 60~80℃ to form a saturated or near-saturated solution. Cinnamon essential oil is slowly added dropwise under constant temperature stirring for no less than 30 min, and the temperature is maintained while stirring for 2~4 h. Then, the temperature is lowered to 4~10℃ at a rate of 0.5~1℃ / min, and the solution is allowed to stand for crystallization for 12~24 h. The microcapsules are separated, washed with water and 20~30% ethanol (by volume), and then dried under vacuum at ≤40℃.

8. The preparation method of the three-dimensional synergistic alfalfa silage additive based on fungal residue oil as described in claim 7, characterized in that, The mixing described in step S3 is carried out in two steps: first, the fungal residue composite freeze-dried granules and the remaining oregano essential oil distillation residue powder are added into the mixing equipment in proportion and mixed for 5-10 minutes, so that the residue powder coats the surface of the fungal residue composite freeze-dried granules to form a coating layer; then, cinnamon essential oil microcapsules are added and the mixing continues for 5-10 minutes.

9. A three-dimensional synergistic alfalfa silage additive based on fungal residue oil, characterized in that, The product comprises the following components by weight percentage: 8-12% compound lactic acid bacteria powder, 75-80% oregano essential oil distillation residue powder, and 8-10% cinnamon essential oil microcapsules; the compound lactic acid bacteria powder is obtained by mixing Lactobacillus plantarum powder and Lactobacillus buchneri powder in a weight ratio of 3:1, and the cinnamon essential oil microcapsules are prepared by encapsulation with β-cyclodextrin as the wall material and cinnamon essential oil as the core material.

10. A method for preserving the freshness of alfalfa silage, characterized in that, include: The three-dimensional synergistic alfalfa silage additive prepared by the method of preparing the three-dimensional synergistic alfalfa silage additive of mushroom residue oil according to any one of claims 1 to 8 is uniformly applied to the alfalfa silage raw material at a ratio of 25 to 45g per ton of alfalfa silage raw material, and after being mixed evenly, it is sealed and silaged.