A method for preparing a feed additive based on ophiopogon root

Abstract

The application discloses a kind of based on preparation feed additive of ophiopogon root, it is related to feed additive technical field, including raw material preparation step, ophiopogon root is pretreated and adds auxiliary carrier, preparation composite enzyme system and preparation composite bacteria, composite enzyme system includes papain and neutral pectinase, composite bacteria includes lactobacillus plantarum, bacillus subtilis and saccharomyces cerevisiae;Enzymolysis step;Fermentation step and drying treatment step.The application is mixed into auxiliary carrier to improve bulkiness in ophiopogon root powder before enzymolysis, then ophiopogon root mixture is soaked and ultrasonic assisted treatment, speed of cell wall rupture is accelerated and ophiopogon root is avoided to appear in the process of enzymolysis fermentation and is gathered, so as to be fully enzymolysed and fermented to ophiopogon root, composite enzyme system and composite bacteria are used to carry out bacteria-enzyme synergistic fermentation, and the extraction rate of effective component in ophiopogon root is effectively improved, and the nutritional effect and palatability of additive are improved.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, specifically to a method for the synergistic fermentation of bacteria and enzymes in the preparation of feed additives based on the rootlets of Ophiopogon japonicus. Background Technology

[0002] As a traditional Chinese medicine, the main medicinal part of Ophiopogon japonicus is its tuberous root, while the fibrous roots are usually discarded as non-medicinal parts, reducing the utilization rate of Ophiopogon japonicus. In order to reduce the waste of Ophiopogon japonicus resources, the fibrous roots are currently used to prepare feed additives. However, because the cell walls of the fibrous roots are relatively tough, traditional preparation methods are difficult to effectively dissolve and extract the effective components in the fibrous roots, thereby reducing the bioavailability of the fibrous roots and the quality and efficacy of the feed additives.

[0003] Patent document CN120616028A discloses the use of Proteobacterium chrysosporum, sucrose, and cellulase for synergistic fermentation of sugarcane pith. This is mainly to improve the storage time and digestibility of sugarcane pith feed. However, it does not take into account the problem that single enzymatic hydrolysis and fermentation methods are difficult to effectively dissolve or extract the effective components in Ophiopogon japonicus rootlets, which in turn affects the extraction rate and nutritional effect of the effective components in Ophiopogon japonicus rootlet feed additives. Summary of the Invention

[0004] The purpose of this invention is to provide a method for the synergistic fermentation of bacteria and enzymes in the preparation of feed additives based on the rootlets of Ophiopogon japonicus, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for the synergistic fermentation of microorganisms and enzymes in the preparation of feed additives based on the rootlets of Ophiopogon japonicus, comprising the following steps: Step 1: Raw material preparation steps, including pretreatment of Ophiopogon japonicus rootlets and addition of auxiliary carriers, preparation of compound enzyme system and preparation of compound microbial culture, wherein the auxiliary carrier is at least one of wheat bran, rice bran and corn cob powder, the compound enzyme system includes papain and neutral pectinase, and the compound microbial culture includes Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae. Step 2: Enzymatic hydrolysis step. The roots of Ophiopogon japonicus are enzymatically hydrolyzed using a compound enzyme system at 45-60℃ for 100-180 min to obtain the enzymatic hydrolysis matrix. Step 3: Substrate adjustment step, adjusting the temperature and pH of the enzymatic hydrolysis substrate to obtain the fermentation substrate; Step 4: Fermentation step, using compound microbial strains to ferment the fermentation substrate at 25-45℃ for 48-72 hours to obtain the additive wet material; Step 5: Drying process. The wet Ophiopogon japonicus root additive is dried, pulverized and sieved at a low temperature of 40-55℃ to obtain the Ophiopogon japonicus root additive.

[0006] Preferably, the pretreatment of the fibrous roots of Ophiopogon japonicus further includes the following steps: Select clean, mold-free, and dry Ophiopogon japonicus fibrous roots as raw materials, and use a pulverizer to pulverize the fibrous root raw materials to 60-80 mesh to obtain fibrous root powder; Add an auxiliary carrier to the fibrous root powder and mix evenly to obtain a mixture of Ophiopogon japonicus fibrous roots; Soak the mixture of Ophiopogon japonicus fibrous roots in warm water for 40-60 minutes to obtain a moistened mixture of fibrous roots. Intermittent ultrasonic-assisted treatment was performed on the mixed moistening material of the fibrous roots to obtain the fibrous root matrix of Ophiopogon japonicus to be enzymatically hydrolyzed.

[0007] Preferably, when the auxiliary carrier is added to the root powder, the particle size of the auxiliary carrier is 5-20 mesh, and the weight ratio of the auxiliary carrier to the root powder in the Ophiopogon japonicus root mixture is (0.5-2):10. The temperature of the warm water is 40-50℃, the soaking time is 40-55 minutes, and the water temperature is maintained during the soaking process. The ultrasonic frequency of the intermittent ultrasonic-assisted treatment is 20-30 kHz, the ultrasonic interval is 10-20 seconds, the treatment temperature is 35-55℃, and the treatment time is 8-15 minutes.

[0008] Preferably, in the complex enzyme system, the mass ratio of papain to neutral pectinase is (2-4):(2-3).

[0009] Preferably, the enzymatic hydrolysis step specifically includes: placing the root matrix of Ophiopogon japonicus to be enzymatically hydrolyzed in an enzymatic hydrolysis device, adding a buffer solution to adjust the pH to 5.5-6.5, then adding a compound enzyme system, and then enzymatically hydrolyzing at 45-55℃ for 100-150 min, and using a stirring rod with a rotation speed of 70-100 rpm to stir during the enzymatic hydrolysis process to obtain the enzymatic hydrolysate.

[0010] Preferably, the buffer solution is a citrate-sodium citrate buffer or a phosphate buffer solution, and the amount of the complex enzyme system added is 1-3% of the weight of the Ophiopogon japonicus root mixture.

[0011] Preferably, the substrate adjustment step specifically includes adjusting the temperature of the enzymatic hydrolysis substrate to 30-40°C by at least one cooling method such as natural cooling, air cooling, or jacket cooling; adjusting the pH of the enzymatic hydrolysis substrate to 6.0-6.5 by adding at least one of lactic acid, citric acid, or phosphate solution; and then adding fermentation regulating additives to the enzymatic hydrolysis substrate and mixing them evenly to obtain the fermentation substrate. The fermentation regulator additive is at least one of glucose, corn flour, soybean meal and honey, and the amount of the fermentation regulator additive added is 0.3 to 0.8% of the weight of the Ophiopogon japonicus root mixture.

[0012] Preferably, the preparation of the composite bacterial strain specifically includes activating and expanding *Lactobacillus plantarum*, *Bacillus subtilis*, and yeast respectively; collecting, washing, and resuspending the cultured cells in physiological saline after centrifugation to obtain *Lactobacillus plantarum* resuspension, *Bacillus subtilis* resuspension, and *Saccharomyces cerevisiae* resuspension; mixing the *Lactobacillus plantarum* resuspension, *Bacillus subtilis* resuspension, and *Saccharomyces cerevisiae* resuspension according to the viable cell count to obtain a composite bacterial cell, wherein the viable cell count ratio of *Lactobacillus plantarum*, *Bacillus subtilis*, and *Saccharomyces cerevisiae* in the composite bacterial cell is (1-3):(1-2):(1-2); and diluting the composite bacterial cell with physiological saline at a NaCl concentration of 0.8-0.9% to obtain a colony formation count of 1*102. 7 ~1*10 9 A complex bacterial strain with a concentration of CFU / mL.

[0013] Preferably, the fermentation step includes transferring the fermentation substrate to a fermentation tank, adding the compound microbial strain, and then fermenting at 30-40°C for 50-72 hours to obtain the additive wet material, wherein the amount of compound microbial strain added is 3-10% of the total weight of the Ophiopogon japonicus root mixture and fermentation conditioning additives.

[0014] Preferably, the drying process specifically includes drying the wet additive material at a drying temperature of 45-60°C to a moisture content of 8-11%, then pulverizing the dried additive and sieving it using a 50-80 mesh sieve, adding silica powder to the pulverized additive and mixing it evenly to obtain the Ophiopogon japonicus root additive, wherein the amount of silica added is 3% of the weight of the Ophiopogon japonicus root additive.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention utilizes a complex enzyme system obtained by mixing papain and neutral pectinase, and prepares a complex microbial body using Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae for the enzymatic hydrolysis and fermentation of Ophiopogon japonicus rootlets. Before enzymatic hydrolysis, the Ophiopogon japonicus rootlets are pulverized and mixed with an auxiliary carrier to improve the fluffiness of the rootlet powder, preventing agglomeration during enzymatic hydrolysis and fermentation. This enhances the contact effect between the enzymatic hydrolysis system, the complex microbial body, and the Ophiopogon japonicus rootlets, thus ensuring thorough enzymatic hydrolysis and fermentation. Furthermore, before enzymatic hydrolysis, soaking the Ophiopogon japonicus rootlet mixture and applying ultrasonic assistance accelerates cell wall rupture, further increasing the extraction rate of effective components in the Ophiopogon japonicus rootlet feed additive and accelerating the enzymatic hydrolysis efficiency.

[0016] 2. This invention obtains a composite enzyme system by mixing papain and neutral pectinase in a mass ratio of 3:2, which is used to enzymatically hydrolyze the fibrous roots of Ophiopogon japonicus. The use of the composite enzyme system is beneficial to improving the destruction effect of the cell wall of the fibrous roots of Ophiopogon japonicus, facilitating the release of effective components in the cell wall, and thus improving the dissolution effect of effective components and small molecules in the fibrous roots of Ophiopogon japonicus.

[0017] 3. This invention prepares a compound microbial body by mixing Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae in a live cell ratio of 3:1.5:1. This compound microbial body is used to dissolve the enzymatically hydrolyzed roots of Ophiopogon japonicus and ferments them through a staged fermentation process. The compound enzyme system promotes synergistic fermentation of bacteria and enzymes, further promoting the release and transformation of effective components in the roots of Ophiopogon japonicus and improving the extraction rate of effective components in the feed additive. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] This invention provides a method for the synergistic fermentation of microorganisms and enzymes in the preparation of feed additives based on the rootlets of Ophiopogon japonicus, comprising the following steps: Step 1: Raw material preparation steps, including pretreatment of Ophiopogon japonicus rootlets and addition of auxiliary carriers, preparation of compound enzyme system and preparation of compound microbial culture, wherein the auxiliary carrier is at least one of wheat bran, rice bran and corn cob powder, the compound enzyme system includes papain and neutral pectinase, and the compound microbial culture includes Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae. The papain activity was 800–1000 U / mg, the neutral pectinase activity was 3000–3500 U / mg, and Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae were preserved at the China Industrial Microbial Culture Collection Center (CICC). The preservation number for Lactobacillus plantarum was CICC 20265, for Bacillus subtilis it was CICC 10067, and for Saccharomyces cerevisiae it was CICC 1769. Step 2: Enzymatic hydrolysis step. The roots of Ophiopogon japonicus are enzymatically hydrolyzed using a compound enzyme system at 45-60℃ for 100-180 min to obtain the enzymatic hydrolysis matrix. Step 3: Substrate adjustment step, adjusting the temperature and pH of the enzymatic hydrolysis substrate to obtain the fermentation substrate; Step 4: Fermentation step, using compound microbial strains to ferment the fermentation substrate at 25-45℃ for 48-72 hours to obtain the additive wet material; Step 5: Drying process. The wet Ophiopogon japonicus root additive is dried, pulverized and sieved at a low temperature of 40-55℃ to obtain the Ophiopogon japonicus root additive.

[0020] Example 1 One embodiment of the present invention provides a method for the synergistic fermentation of microorganisms and enzymes in the preparation of feed additives based on the rootlets of Ophiopogon japonicus, comprising the following steps: Step 1: Pretreatment of the fibrous roots of Ophiopogon japonicus; Select clean, mold-free, and dry Ophiopogon japonicus fibrous roots as raw materials, and use a pulverizer to pulverize the fibrous root raw materials to 60 mesh to obtain fibrous root powder; Add an auxiliary carrier to the root powder and mix evenly. The auxiliary carrier is wheat bran with a particle size of 15 mesh to obtain a mixture of Ophiopogon japonicus root powder. The weight ratio of wheat bran to root powder in the mixture is 1:10. By pulverizing the fibrous roots of Ophiopogon japonicus, the contact area between the fibrous roots and the enzymatic hydrolysis system is increased during the subsequent enzymatic hydrolysis process, which is beneficial to improving the hydrolysis effect. Adding small-particle bran to the pulverized fibrous roots helps to make the enzymatic hydrolysis matrix loose, making it easier for the enzymatic hydrolysis system to contact the fibrous root powder. In addition, the addition of the auxiliary carrier helps to prevent the fibrous root powder from agglomerating after the addition of phosphate buffer solution, thus ensuring that the enzymatic hydrolysis system maintains full contact with the fibrous root powder during the enzymatic hydrolysis process, further improving the enzymatic hydrolysis effect of the enzymatic hydrolysis system on the fibrous roots of Ophiopogon japonicus. Soak the mixture of Ophiopogon japonicus rootlets in warm water at 45℃ for 60 minutes, with a volume ratio of warm water to Ophiopogon japonicus rootlets of 1.2:1. Maintain the water temperature during the soaking process and stir once every 8 minutes. After soaking, the moistened mixture of rootlets is obtained. Intermittent ultrasonic-assisted treatment was performed on the mixed moist material of fibrous roots. The ultrasonic frequency was 30 kHz, the ultrasonic interval was 10 s, the treatment temperature was 45 ℃, and the treatment time was 12 min to obtain the fibrous root matrix of Ophiopogon japonicus to be enzymatically hydrolyzed. Soaking the mixture of Ophiopogon japonicus rootlets in warm water allows the rootlets to absorb water, promoting the formation of pores in the cell walls. This facilitates the entry of the enzymatic hydrolysis system and the extraction of active ingredients from the cell walls. Further, intermittent ultrasonic treatment promotes the formation of microcracks or ruptures in the cell walls, increasing the speed at which the enzymatic hydrolysis system enters the cell walls and reducing the difficulty of cell wall destruction. This shortens the hydrolysis time, improves efficiency, and facilitates more complete extraction of the active ingredients from the Ophiopogon japonicus rootlets. To prepare a composite enzyme system, papain with an enzyme activity of 1000 U / mg and neutral pectinase with an enzyme activity of 3500 U / mg were uniformly mixed at a mass ratio of 3:2 to obtain the composite enzyme system. The preparation of the compound bacterial strains included: activating *Lactobacillus plantarum* in MRS liquid medium at 37°C, then expanding the culture at a 5% inoculum for 15 hours. After expansion, the contents of the culture medium were centrifuged to obtain *Lactobacillus plantarum* cells. The cells were then washed twice with 0.5% physiological saline and resuspended in 0.7% physiological saline to obtain a *Lactobacillus plantarum* resuspension. Additionally, activating *Saccharomyces cerevisiae* in YPD liquid medium at 30°C, then expanding the culture at a 5% inoculum for 20 hours. After expansion, the contents of the culture medium were centrifuged to obtain a *Saccharomyces cerevisiae* resuspension. The materials were centrifuged to obtain Saccharomyces cerevisiae cells. The Saccharomyces cerevisiae cells were then washed twice with 0.5% physiological saline and resuspended in 0.7% physiological saline to obtain a Saccharomyces cerevisiae resuspension. Bacillus subtilis strain was activated in LB liquid medium at 37°C and then expanded at an inoculum of 5% for 15 hours. After the expansion, the materials in the medium were centrifuged to obtain Bacillus subtilis cells. The Bacillus subtilis cells were then washed twice with 0.5% physiological saline and resuspended in 0.7% physiological saline to obtain a Bacillus subtilis resuspension. A composite bacterial cell was prepared by mixing resuspensions of *Lactobacillus plantarum*, *Bacillus subtilis*, and *Saccharomyces cerevisiae* according to the viable cell count, with a viable cell ratio of 3:1.5:1. The composite bacterial cell was then diluted with 0.8% physiological saline (NaCl concentration) to obtain a colony count of 1*102. 9 CFU / mL of compound bacterial strains; Step Two: In the enzymatic hydrolysis step, the root matrix of *Ophiopogon japonicus* to be enzymatically hydrolyzed was placed in an enzymatic hydrolysis device, and a phosphate buffer solution was sprayed onto the root matrix using a nozzle. The pH of the root matrix was adjusted to 6.5, and the root matrix was stirred and mixed with a stirring rod at 80 rpm during the spraying process to ensure uniform mixing of the root matrix and the phosphate buffer solution. Then, a compound enzyme system was added, with the amount of compound enzyme system added being 2% of the weight of the *Ophiopogon japonicus* root mixture. Enzymatic hydrolysis was performed at 50℃ for 120 min, and the stirring rod was used at 100 rpm during the enzymatic hydrolysis process. The enzymatic hydrolysate was obtained after enzymatic hydrolysis. A complex enzyme system consisting of papain and neutral pectinase was used to enzymatically hydrolyze the root matrix of Ophiopogon japonicus, which effectively destroyed the cell walls of the root and promoted the dissolution of polysaccharides, flavonoids and saponins in the root, thus providing sufficient substrate for the fermentation step. Step 3: The substrate adjustment steps specifically include: adjusting the temperature of the enzymatic hydrolysis substrate to 37°C by air cooling; adding phosphate solution to the enzymatic hydrolysis substrate to adjust the pH of the enzymatic hydrolysis substrate to 6.3; adding fermentation regulator additives to the enzymatic hydrolysis substrate and mixing them evenly; wherein the fermentation regulator additives include corn flour and glucose in a mass ratio of 2:1, and the amount of fermentation regulator additives added is 0.6% of the weight of the Ophiopogon japonicus root mixture; and mixing evenly to obtain the fermentation substrate. Adjusting the temperature and pH of the fermentation substrate helps maintain the viable count of the compound microorganisms, thus enabling the fermentation process to continue. Adding fermentation regulators to supplement the carbon source for the compound microorganisms promotes their growth and fermentation during the fermentation process and improves the palatability of the Ophiopogon japonicus root additive. Step Four: The fermentation process involves transferring the fermentation substrate to a fermentation tank, adding a compound microbial culture to the fermentation tank, and adding the compound microbial culture at a rate of 5% of the total weight of the Ophiopogon japonicus root mixture and fermentation conditioning additives. The temperature inside the fermentation tank is adjusted so that the fermentation substrate and compound microbial culture are fermented at 37°C with aeration for 20 hours, and then anaerobic fermented at 35°C in a sealed environment for 45 hours. After the fermentation is completed, the additive wet material is obtained. By mixing Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae and controlling the proportion of live bacteria, a compound microbial strain is obtained. Through a two-stage fermentation method, the first stage of fermentation is carried out in a micro-aerobic environment, allowing Lactobacillus plantarum and Saccharomyces cerevisiae in the compound microbial strain to grow. At the same time, Saccharomyces cerevisiae, as the main bacteria, ferments the fermentation substrate and consumes oxygen. Then, in a closed environment, Lactobacillus plantarum and Bacillus subtilis, as the main bacteria, ferment, which is conducive to obtaining fully fermented wet feed additives. Furthermore, the lactic acid produced during the fermentation process enhances the antibacterial effect and palatability of the wet feed additives, as well as improves the nutritional value of the feed additives. Step 5: In the drying process, after fermentation, the temperature inside the fermentation tank is raised to 75℃ and kept at that temperature for 40 minutes. Then, the wet additive material is taken out and dried at 40℃ using a dryer until the moisture content is 10%. The dried additive is then pulverized using a pulverizing device and passed through a 60-mesh sieve. Silica powder is added to the pulverized additive at a rate of 3% of the weight of the dried and sieved additive. After uniform mixing, the resulting feed additive is obtained. By controlling the moisture content of the Ophiopogon japonicus root additive and adding silica powder, the anti-caking properties of the Ophiopogon japonicus root additive are improved, and its shelf life is extended. Furthermore, using Ophiopogon japonicus roots to prepare the additive increases the utilization rate of agricultural waste.

[0021] Example 2 One embodiment of the present invention provides a method for the synergistic fermentation of microorganisms and enzymes in the preparation of feed additives based on the rootlets of Ophiopogon japonicus, comprising the following steps: Step 1: Pretreatment of the fibrous roots of Ophiopogon japonicus; Select clean, mold-free, and dry Ophiopogon japonicus fibrous roots as raw materials, and use a pulverizer to pulverize the fibrous root raw materials to 60 mesh to obtain fibrous root powder; To prepare a composite enzyme system, papain with an enzyme activity of 1000 U / mg and neutral pectinase with an enzyme activity of 3500 U / mg were uniformly mixed at a mass ratio of 3:2 to obtain the composite enzyme system. The preparation of the compound strains includes activating, expanding, washing and resuspending the Lactobacillus plantarum strain, Bacillus subtilis strain and Saccharomyces cerevisiae strain under the same conditions as in Example 1, to obtain Lactobacillus plantarum resuspension, Bacillus subtilis resuspension and Saccharomyces cerevisiae resuspension; A composite bacterial cell was prepared by mixing resuspensions of *Lactobacillus plantarum*, *Bacillus subtilis*, and *Saccharomyces cerevisiae* according to the viable cell count, with a viable cell ratio of 3:1.5:1. The composite bacterial cell was then diluted with 0.8% physiological saline (NaCl concentration) to obtain a colony count of 1*102. 9 CFU / mL of compound bacterial strains; Step Two: In the enzymatic hydrolysis step, the root matrix of *Ophiopogon japonicus* to be enzymatically hydrolyzed was placed in an enzymatic hydrolysis device, and a phosphate buffer solution was sprayed onto the root matrix using a nozzle. The pH of the root matrix was adjusted to 6.5, and the root matrix was stirred and mixed with a stirring rod at 80 rpm during the spraying process to ensure uniform mixing of the root matrix and the phosphate buffer solution. Then, a compound enzyme system was added, with the amount of compound enzyme system added being 2.2% of the weight of the *Ophiopogon japonicus* root mixture. The enzymatic hydrolysis was carried out at 50℃ for 120 min, and the stirring rod was used at 100 rpm during the enzymatic hydrolysis process. The enzymatic hydrolysate was obtained after enzymatic hydrolysis. Step 3: The substrate adjustment steps specifically include adjusting the temperature of the enzymatic hydrolysis substrate to 37°C by air cooling, adding phosphate solution to the enzymatic hydrolysis substrate, and adjusting the pH of the enzymatic hydrolysis substrate to 6.3 to obtain the fermentation substrate; Step Four: The fermentation process involves transferring the fermentation substrate to a fermentation tank, adding a compound microbial strain to the fermentation tank, and adding the compound microbial strain at a rate of 4.5% of the total weight of the Ophiopogon japonicus root mixture and fermentation conditioning additives. The temperature inside the fermentation tank is adjusted so that the fermentation substrate and compound microbial strain are fermented at 37°C with aeration for 20 hours, and then anaerobic fermented at 35°C in a sealed environment for 45 hours. After the fermentation is completed, the additive wet material is obtained. Step 5: The drying process involves raising the temperature inside the fermentation tank to 75℃ and maintaining this temperature for 40 minutes after fermentation. The wet additive material is then removed and dried at 40℃ using a dryer until the moisture content reaches 10%. The dried additive is then pulverized using a pulverizing device and passed through a 60-mesh sieve. Silica powder is added to the pulverized additive at a rate of 3% of the weight of the dried and sieved additive. After uniform mixing, the resulting product is a *Ophiopogon japonicus* root additive for use in animal feed. Example 3 One embodiment of the present invention provides a method for the synergistic fermentation of microorganisms and enzymes in the preparation of feed additives based on the rootlets of Ophiopogon japonicus, comprising the following steps: Step 1: Raw material preparation steps, including pretreatment of Ophiopogon japonicus rootlets and addition of auxiliary carriers, preparation of compound enzyme system and preparation of compound microbial cells; Step 2: Enzymatic hydrolysis to obtain the enzymatically hydrolyzed matrix; Step 3: Substrate adjustment step, to obtain the fermentation substrate; Step 4: Fermentation step, to obtain wet additive material; Step 5: Drying process to obtain Ophiopogon japonicus root additive; The difference between this embodiment and Embodiment 1 is that in Step 1 of this embodiment, the preparation process of the complex enzyme system involves uniformly mixing papain with an enzyme activity of 1000 U / mg and neutral pectinase with an enzyme activity of 3500 U / mg at a mass ratio of 3:1 to obtain the complex enzyme system. The remaining steps are the same.

[0022] Example 4 One embodiment of the present invention provides a method for the synergistic fermentation of microorganisms and enzymes in the preparation of feed additives based on the rootlets of Ophiopogon japonicus, comprising the following steps: Step 1: Raw material preparation steps, including pretreatment of Ophiopogon japonicus rootlets and addition of auxiliary carriers, preparation of compound enzyme system and preparation of compound microbial cells; Step 2: Enzymatic hydrolysis to obtain the enzymatically hydrolyzed matrix; Step 3: Substrate adjustment step, to obtain the fermentation substrate; Step 4: Fermentation step, to obtain wet additive material; Step 5: Drying process to obtain Ophiopogon japonicus root additive; The difference between this embodiment and embodiment one is that in step one of this embodiment, the preparation process of the composite microbial body includes activating, expanding, washing and resuspending the Lactobacillus plantarum strain, Bacillus subtilis strain and Saccharomyces cerevisiae strain under the same conditions as in embodiment one, to obtain Lactobacillus plantarum resuspension, Bacillus subtilis resuspension and Saccharomyces cerevisiae resuspension. A composite bacterial cell was prepared by mixing resuspensions of *Lactobacillus plantarum*, *Bacillus subtilis*, and *Saccharomyces cerevisiae* according to the viable cell count, with a viable cell ratio of 3:1:1. The composite bacterial cell was diluted with 0.8% physiological saline (NaCl concentration) to obtain a colony count of 1*102. 9 The CFU / mL compound bacterial strain is used, and the remaining steps are the same.

[0023] further, Example 5 One embodiment of the present invention provides a method for the synergistic fermentation of microorganisms and enzymes in the preparation of feed additives based on the rootlets of Ophiopogon japonicus, comprising the following steps: Step 1: Raw material preparation steps, including pretreatment of Ophiopogon japonicus rootlets and addition of auxiliary carriers, preparation of compound enzyme system and preparation of compound microbial cells; Step 2: Enzymatic hydrolysis to obtain the enzymatically hydrolyzed matrix; Step 3: Substrate adjustment step, to obtain the fermentation substrate; Step 4: Fermentation step, to obtain wet additive material; Step 5: Drying process to obtain Ophiopogon japonicus root additive; The difference between this embodiment and Embodiment 1 is that the fermentation steps in this embodiment are as follows: the fermentation substrate is transferred to the fermentation tank, a compound microbial strain is added to the fermentation tank, and the amount of compound microbial strain added is 5% of the total weight of the Ophiopogon japonicus root mixture and the fermentation conditioning additive. The temperature inside the fermentation tank is adjusted so that the fermentation substrate and the compound microbial strain undergo micro-aerobic fermentation at 37°C for 65 hours. After the fermentation is completed, the additive wet material is obtained. The remaining steps are the same.

[0024] Comparative Example 1: One embodiment of the present invention is a method for co-fermentation of bacteria and enzymes to prepare feed additives based on Ophiopogon japonicus rootlets, which specifically includes step one: raw material preparation step, including pretreatment of Ophiopogon japonicus rootlets and addition of auxiliary carrier, preparation of compound enzyme system and preparation of compound bacteria; Step 2: Enzymatic hydrolysis to obtain the enzymatically hydrolyzed matrix; Step 3: Substrate adjustment step, to obtain the fermentation substrate; Step 4: Fermentation step, to obtain wet additive material; Step 5: Drying process to obtain Ophiopogon japonicus root additive; The difference between this embodiment and Embodiment 1 is that the composite enzyme system is obtained by uniformly mixing bromelain with an enzyme activity of 1000 U / mg and neutral pectinase with an enzyme activity of 3500 U / mg in a mass ratio of 3:2. The remaining steps are the same.

[0025] Comparative Example 2: One embodiment of the present invention is a method for co-fermentation of bacteria and enzymes to prepare feed additives based on Ophiopogon japonicus rootlets, which specifically includes step one: raw material preparation step, including pretreatment of Ophiopogon japonicus rootlets and addition of auxiliary carrier, preparation of compound enzyme system and preparation of compound bacteria; Step 2: Enzymatic hydrolysis to obtain the enzymatically hydrolyzed matrix; Step 3: Substrate adjustment step, to obtain the fermentation substrate; Step 4: Fermentation step, to obtain wet additive material; Step 5: Drying process to obtain Ophiopogon japonicus root additive; The difference between this embodiment and Embodiment 1 is that the composite enzyme system is prepared by uniformly mixing papain with an enzyme activity of 1200 U / mg and acidic pectinase with an enzyme activity of 3500 U / mg in a mass ratio of 3:2. The remaining steps are the same.

[0026] Comparative Example 3: One embodiment of the present invention is a method for co-fermentation of bacteria and enzymes to prepare feed additives based on Ophiopogon japonicus rootlets, which specifically includes step one: raw material preparation step, including pretreatment of Ophiopogon japonicus rootlets and addition of auxiliary carrier, preparation of compound enzyme system and preparation of compound bacteria; Step 2: Enzymatic hydrolysis to obtain the enzymatically hydrolyzed matrix; Step 3: Substrate adjustment step, to obtain the fermentation substrate; Step 4: Fermentation step, to obtain wet additive material; Step 5: Drying process to obtain Ophiopogon japonicus root additive; The difference between this embodiment and Embodiment 1 is that, in step one of this embodiment, the preparation of the composite bacterial cell includes activating, expanding, washing, and resuspending Lactobacillus acidophilus (preserved at the China Industrial Microbial Culture Collection Center, preservation number CICC 6055), Bacillus subtilis, and Saccharomyces cerevisiae strains respectively, to obtain Lactobacillus plantarum resuspension, Bacillus subtilis resuspension, and Saccharomyces cerevisiae resuspension. The three resuspensions are then mixed in a live cell ratio of 3:1.5:1 to obtain the composite bacterial cell. The composite bacterial cell is then diluted with 0.8% physiological saline solution to obtain a colony formation count of 1*10⁻⁶. 9 The CFU / mL compound bacterial strain is used, and the remaining steps are the same.

[0027] Comparative Example 4: One embodiment of the present invention is a method for co-fermentation of bacteria and enzymes to prepare feed additives based on Ophiopogon japonicus rootlets, which specifically includes step one: raw material preparation step, including pretreatment of Ophiopogon japonicus rootlets and addition of auxiliary carrier, preparation of compound enzyme system and preparation of compound bacteria; Step 2: Enzymatic hydrolysis to obtain the enzymatically hydrolyzed matrix; Step 3: Substrate adjustment step, to obtain the fermentation substrate; Step 4: Fermentation step, to obtain wet additive material; Step 5: Drying process to obtain Ophiopogon japonicus root additive; The difference between this embodiment and Embodiment 1 is that, in step one of this embodiment, the preparation of the composite bacterial cell includes activating, expanding, washing, and resuspending *Lactobacillus plantarum*, *Bacillus subtilis*, and *Candida utilis* (preserved at the China Industrial Microbial Culture Collection Center, preservation number CICC 1769) strains respectively, to obtain *Lactobacillus plantarum* resuspension, *Bacillus subtilis* resuspension, and *Candida utilis* resuspension. These three resuspensions are then mixed at a viable cell ratio of 3:1.5:1 to obtain the composite bacterial cell. The composite bacterial cell is then diluted with 0.8% physiological saline to obtain a colony formation count of 1*103. 9 The CFU / mL compound bacterial strain is used, and the remaining steps are the same.

[0028] Comparative Example 5: One embodiment of the present invention is a method for co-fermentation of bacteria and enzymes to prepare feed additives based on Ophiopogon japonicus rootlets, which specifically includes step one: raw material preparation step, including pretreatment of Ophiopogon japonicus rootlets and addition of auxiliary carrier, preparation of compound enzyme system and preparation of compound bacteria; Step 2: Enzymatic hydrolysis to obtain the enzymatically hydrolyzed matrix; Step 3: Substrate adjustment step, to obtain the fermentation substrate; Step 4: Fermentation step, to obtain wet additive material; Step 5: Drying process to obtain Ophiopogon japonicus root additive; The difference between this embodiment and Embodiment 1 is that, in step one of this embodiment, the preparation of the composite bacterial cell includes activating, expanding, washing, and resuspending *Lactobacillus plantarum* strains, *Bacillus licheniformis* strains (preserved at the China Industrial Microbial Culture Collection Center, preservation number CICC 10020), and *Saccharomyces cerevisiae* strains, respectively, to obtain *Lactobacillus plantarum* resuspension, *Bacillus licheniformis* resuspension, and *Candida utilis* resuspension. These three resuspensions are then mixed at a viable cell ratio of 3:1.5:1 to obtain the composite bacterial cell. The composite bacterial cell is then diluted with 0.8% physiological saline (NaCl concentration) to obtain a colony formation count of 1*102. 9 The CFU / mL compound bacterial strain is used, and the remaining steps are the same.

[0029] Performance testing The Ophiopogon japonicus root additives prepared in Examples 1 to 4 and Comparative Examples 1 to 5 were tested using the following methods: Take 20g of Ophiopogon japonicus root extract additive and place it in an Erlenmeyer flask. Add 100mL of 70% ethanol solution and reflux at 80℃ for 60 minutes. After cooling, centrifuge the extract at 8000rpm for 15 minutes and collect the supernatant. Measure the polysaccharide content in the supernatant using the phenol-sulfuric acid method according to "Spectrophotometric Method for Determination of Polysaccharides in Natural Plant Feed Raw Materials and Their Extracts" (T / CFIAS 6001-2022). Measure the total flavonoid content using the aluminum nitrate-sodium nitrite colorimetric method according to "Spectrophotometric Method for Determination of Total Flavonoids in Food" (SZDB / Z 349—2019). Measure the total flavonoid content using the vanillin-perchloric acid method according to "GB / T22251-2008". "Determination of Ginsenosides in Health Foods" The total saponin content was determined. The extraction rates of polysaccharides, flavonoids and saponins were calculated based on the weight ratio of the measured total polysaccharide content, total flavonoid content and total saponin content to the Ophiopogon japonicus root additive. The sum of the extraction rates of polysaccharides, flavonoids and saponins was taken as the extraction rate of effective components of Ophiopogon japonicus root. The test was repeated 3 times and the average value was taken. The test results were summarized in the performance test results table. Performance test results table

[0030] As shown in the performance test results table above, a compound enzyme system was obtained by mixing papain and neutral pectinase in a mass ratio of 3:2 and used for enzymatic hydrolysis of Ophiopogon japonicus roots. A mixture of Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae in a viable cell ratio of 3:1.5:1 was used for fermentation of the hydrolyzed Ophiopogon japonicus roots. This improved the dissolution of polysaccharides, flavonoids, and saponins in the roots. Furthermore, pulverizing the roots before enzymatic hydrolysis and adding an auxiliary carrier increased the bulkiness of the root powder, preventing agglomeration during enzymatic hydrolysis and fermentation, and enhancing the contact between the enzymatic hydrolysis system, the compound microorganisms, and the roots. This ensured thorough enzymatic hydrolysis and fermentation of the roots. Additionally, soaking the root mixture and applying ultrasound assistance before enzymatic hydrolysis accelerated cell wall rupture, further increasing the extraction rate of effective components in the Ophiopogon japonicus root feed additive and accelerating the hydrolysis efficiency.

[0031] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for co-fermentation of microorganisms and enzymes in the preparation of feed additives based on Ophiopogon japonicus rootlets, characterized in that, Includes the following steps: Step 1: Raw material preparation steps, including pretreatment of Ophiopogon japonicus rootlets and addition of auxiliary carriers, preparation of compound enzyme system and preparation of compound microbial culture, wherein the auxiliary carrier is at least one of wheat bran, rice bran and corn cob powder, the compound enzyme system includes papain and neutral pectinase, and the compound microbial culture includes Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae. Step 2: Enzymatic hydrolysis step. The roots of Ophiopogon japonicus are enzymatically hydrolyzed using a compound enzyme system at 45-60℃ for 100-180 min to obtain the enzymatic hydrolysis matrix. Step 3: Substrate adjustment step, adjusting the temperature and pH of the enzymatic hydrolysis substrate to obtain the fermentation substrate; Step 4: Fermentation step, using compound microbial strains to ferment the fermentation substrate at 25-45℃ for 48-72 hours to obtain the additive wet material; Step 5: Drying process. The wet Ophiopogon japonicus root additive is dried, pulverized and sieved at a low temperature of 40-55℃ to obtain the Ophiopogon japonicus root additive.

2. The method for co-fermentation of microorganisms and enzymes in preparing feed additives based on Ophiopogon japonicus rootlets according to claim 1, characterized in that: The pretreatment of the fibrous roots of Ophiopogon japonicus also includes the following steps: Select clean, mold-free, and dry Ophiopogon japonicus fibrous roots as raw materials, and use a pulverizer to pulverize the fibrous root raw materials to 60-80 mesh to obtain fibrous root powder; Add an auxiliary carrier to the fibrous root powder and mix evenly to obtain a mixture of Ophiopogon japonicus fibrous roots; Soak the mixture of Ophiopogon japonicus fibrous roots in warm water for 40-60 minutes to obtain a moistened mixture of fibrous roots. Intermittent ultrasonic-assisted treatment was performed on the mixed moistening material of the fibrous roots to obtain the fibrous root matrix of Ophiopogon japonicus to be enzymatically hydrolyzed.

3. The method for co-fermentation of microorganisms and enzymes in preparing feed additives based on Ophiopogon japonicus rootlets according to claim 2, characterized in that: When the auxiliary carrier is added to the root powder, the particle size of the auxiliary carrier is 5-20 mesh, and the weight ratio of the auxiliary carrier to the root powder in the Ophiopogon japonicus root mixture is (0.5-2):

10. The temperature of the warm water is 40-50℃, the soaking time is 40-55 minutes, and the water temperature is maintained during the soaking process. The ultrasonic frequency of the intermittent ultrasonic-assisted treatment is 20-30 kHz, the ultrasonic interval is 10-20 seconds, the treatment temperature is 35-55℃, and the treatment time is 8-15 minutes.

4. The method for co-fermentation of microorganisms and enzymes in preparing feed additives based on Ophiopogon japonicus rootlets according to claim 3, characterized in that: In the complex enzyme system, the mass ratio of papain to neutral pectinase is (2-4):(2-3).

5. The method for co-fermentation of microorganisms and enzymes in preparing feed additives based on Ophiopogon japonicus rootlets according to claim 4, characterized in that: The enzymatic hydrolysis step specifically includes: placing the root matrix of Ophiopogon japonicus to be enzymatically hydrolyzed in an enzymatic hydrolysis device, adding a buffer solution to adjust the pH to 5.5-6.5, then adding a compound enzyme system, and then enzymatically hydrolyzing at 45-55℃ for 100-150 min, and using a stirring rod with a rotation speed of 70-100 rpm to stir during the enzymatic hydrolysis process to obtain the enzymatic hydrolysate.

6. The method for co-fermentation of microorganisms and enzymes in preparing feed additives based on Ophiopogon japonicus rootlets according to claim 5, characterized in that: The buffer solution is a citrate-sodium citrate buffer or a phosphate buffer solution, and the amount of the complex enzyme system added is 1-3% of the weight of the Ophiopogon japonicus root mixture.

7. The method for co-fermentation of microorganisms and enzymes in preparing feed additives based on Ophiopogon japonicus rootlets according to claim 6, characterized in that: The substrate adjustment step specifically includes adjusting the temperature of the enzymatic hydrolysis substrate to 30-40°C by at least one cooling method, such as natural cooling, air cooling, or jacket cooling; adjusting the pH of the enzymatic hydrolysis substrate to 6.0-6.5 by adding at least one of lactic acid, citric acid, or phosphate solution; and then adding fermentation regulating additives to the enzymatic hydrolysis substrate and mixing them evenly to obtain the fermentation substrate. The fermentation regulator additive is at least one of glucose, corn flour, soybean meal and honey, and the amount of the fermentation regulator additive added is 0.3 to 0.8% of the weight of the Ophiopogon japonicus root mixture.

8. The method for co-fermentation of microorganisms and enzymes in preparing feed additives based on Ophiopogon japonicus rootlets according to claim 7, characterized in that: The preparation of the composite bacterial strain specifically includes activating and expanding *Lactobacillus plantarum*, *Bacillus subtilis*, and yeast, respectively; collecting the cultured cells by centrifugation, washing, and resuspending them in physiological saline to obtain *Lactobacillus plantarum* resuspension, *Bacillus subtilis* resuspension, and *Saccharomyces cerevisiae* resuspension; mixing the *Lactobacillus plantarum* resuspension, *Bacillus subtilis* resuspension, and *Saccharomyces cerevisiae* resuspension according to the viable cell count to obtain a composite bacterial cell, wherein the viable cell count ratio of *Lactobacillus plantarum*, *Bacillus subtilis*, and *Saccharomyces cerevisiae* in the composite bacterial cell is (1-3):(1-2):(1-2); and diluting the composite bacterial cell with physiological saline at a concentration of 0.8-0.9% NaCl to obtain a colony formation count of 1*102. 7 ~1*10 9 A complex bacterial strain with a concentration of CFU / mL.

9. The method for co-fermentation of microorganisms and enzymes in preparing feed additives based on Ophiopogon japonicus rootlets according to claim 8, characterized in that: The fermentation step includes transferring the fermentation substrate to a fermentation tank, adding compound microorganisms, and then fermenting at 30-40°C for 50-72 hours to obtain wet additive material, wherein the amount of compound microorganisms added is 3-10% of the total weight of the Ophiopogon japonicus root mixture and fermentation regulator additives.

10. The method for co-fermentation of microorganisms and enzymes in preparing feed additives based on Ophiopogon japonicus rootlets according to claim 9, characterized in that: The drying process specifically includes drying the wet additive material at a drying temperature of 45-60℃ to a moisture content of 8-11%, then pulverizing the dried additive and sieving it using a 50-80 mesh sieve, adding silica powder to the pulverized additive and mixing it evenly to obtain the Ophiopogon japonicus root additive, wherein the amount of silica added is 3% of the weight of the Ophiopogon japonicus root additive.

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