Microbial agent preparation control method and system for aquaculture

Abstract

This invention provides a method and system for controlling the preparation of microbial agents for aquaculture, belonging to the field of fermentation engineering technology for microbial agent preparation. The method includes: preparing hydrolysate culture media of old feed, hydrolysate culture media of new feed, and a mixed hydrolysate culture media of old and new feed; culturing and recording lactic acid bacteria and Bacillus aggregates based on the hydrolysate culture media, and screening subsets of lactic acid bacteria and Bacillus; subjecting the lactic acid bacteria subset and Bacillus subset to dual-substrate acclimatization culture, short-range co-adaptation culture, and fermentation propagation, respectively, to obtain lactic acid bacteria ferment and Bacillus ferment; preparing lactic acid bacteria protective components and Bacillus dormant components, and then compounding and formulating them to obtain microbial agents for aquaculture. This invention enables the strain screening, culture medium selection, hydrolysate acclimatization, fermentation propagation, and formulation structure to match the changes in intestinal substrates during the high-density aquaculture feed change period, improving the suitability of the agent for use during the feed change period.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology for the preparation of microbial agents, and specifically to a method and system for controlling the preparation of microbial agents for aquaculture. Background Technology

[0002] With the increasing scale and density of aquaculture, aquatic animals such as fish, shrimp, and crabs are facing growing pressures in terms of feeding intensity, feed utilization, and maintaining intestinal homeostasis. Microbial agents, as common functional products in aquaculture, are typically prepared by screening beneficial microorganisms such as lactic acid bacteria and Bacillus, followed by cultivation, fermentation, compounding, and formulation. They are then used through mixing with feed, periodic feeding, or short-term intensive feeding.

[0003] Most existing compound microbial agents for aquaculture use fixed strain combinations and fixed compounding ratios. For example, pre-screened lactic acid bacteria and Bacillus are fermented and propagated separately, then mixed with a carrier in a fixed ratio to produce powders, granules, or liquid formulations. This type of preparation is suitable for routine aquaculture health care scenarios, but it suffers from insufficient adaptability during high-density aquaculture feed change periods, especially in scenarios where the proportion of new feed is increased in a short period. In high-density aquaculture, farms may switch from old to new feed in a short time due to changes in feed supply, feed number adjustments, switching of aquaculture stages, replacement of protein sources, or cost changes. Differences in the nutritional structure of the old and new feeds, such as crude protein, crude fat, and available carbohydrates, will cause changes in the available substrate in the aquatic animals' intestines. If ordinary fixed-ratio microbial agents are still used, lactic acid bacteria may be unsuitable for the intestinal carrying capacity at the initial stage of feed change due to excessively rapid acid production, and Bacillus may not be able to participate in the decomposition of the new feed substrate in a timely manner because it has not been adapted to the new substrate. Existing preparation methods typically focus on the number of viable bacteria in the strain, the ability to produce acid in a single substance, the activity of a single enzyme, or the stability during conventional storage. However, these methods are difficult to develop into compound microbial agents that match the changes in the intestinal substrate during the feed change period in high-density aquaculture.

[0004] Therefore, how to develop microbial agents for aquaculture that are suitable for supporting intestinal homeostasis and decomposing new substrate during the feed change period in high-density aquaculture is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] Therefore, it is necessary to provide a method and system for controlling the preparation of microbial agents for aquaculture, addressing the mismatch between traditional fixed-ratio compound microbial agents and changes in substrate between old and new feeds during high-density aquaculture.

[0006] To achieve the above objectives, a first aspect of the present invention provides a method for controlling the preparation of microbial agents for aquaculture, the method comprising: Prepare culture media of hydrolysate from old material, culture media of hydrolysate from new material, and culture media of hydrolysate from a mixture of old and new material; Obtain a collection of lactic acid bacteria and a collection of Bacillus, and inoculate the collection of lactic acid bacteria and the collection of Bacillus into a culture system containing the old material hydrolysate culture medium, the new material hydrolysate culture medium and the new and old material mixed hydrolysate culture medium for culture and record the culture. Based on the culture records, a subset of lactic acid bacteria is obtained by screening from the set of lactic acid bacteria, and a subset of Bacillus is obtained by screening from the set of Bacillus; wherein, the subset of lactic acid bacteria includes lactic acid bacteria whose viable growth is stable and whose acid production changes are within a preset mild range in the culture system of mixed new and old materials hydrolysate, and the subset of Bacillus includes Bacillus whose spores recover stably and whose substrate decomposition performance meets preset requirements in the culture system of new material hydrolysate; The lactic acid bacteria subset and the Bacillus subset were subjected to dual-substrate acclimatization culture to obtain lactic acid bacteria culture adapted to the new substrate and Bacillus culture adapted to the new substrate. The lactic acid bacteria culture adapted to the new substrate and the Bacillus culture adapted to the new substrate were subjected to short-term co-adaptation culture, and the retained lactic acid bacteria culture and Bacillus culture were fermented and propagated separately to obtain lactic acid bacteria ferment and Bacillus ferment. The lactic acid bacteria fermentation product was prepared as a lactic acid bacteria protective component, and the Bacillus fermentation product was prepared as a Bacillus dormant component. The two components were then combined and formulated to obtain a microbial agent for aquaculture.

[0007] A second aspect of the present invention provides a control system for the preparation of microbial agents for aquaculture, the system being used to execute the above-described method for controlling the preparation of microbial agents for aquaculture, the system comprising: The hydrolysate culture medium preparation module is used to prepare hydrolysate culture medium made from recycled materials, hydrolysate culture medium made from virgin materials, and hydrolysate culture medium made from a mixture of recycled and virgin materials. The strain acquisition module is used to acquire sets of lactic acid bacteria and Bacillus strains; The culture recording module is used to inoculate the lactic acid bacteria collection and the Bacillus collection into a culture system containing the old material hydrolysate culture medium, the new material hydrolysate culture medium and the new and old material mixed hydrolysate culture medium for culture recording; The strain screening module is used to screen a subset of lactic acid bacteria from the lactic acid bacteria set and a subset of Bacillus from the Bacillus set based on the culture records. An acclimatization and cultivation module is used to perform dual-substrate acclimatization and cultivation on the lactic acid bacteria subset and the Bacillus subset respectively, to obtain lactic acid bacteria cultures adapted to the new substrate and Bacillus cultures adapted to the new substrate. The fermentation and propagation module is used to ferment and propagate lactic acid bacteria culture and Bacillus culture retained after short-term co-adaptation culture, respectively, to obtain lactic acid bacteria ferment and Bacillus ferment; The formulation module is used to prepare the lactic acid bacteria fermentation product into a lactic acid bacteria protective component, the Bacillus fermentation product into a Bacillus dormant component, and to formulate the lactic acid bacteria protective component and the Bacillus dormant component in combination to obtain a microbial agent suitable for aquaculture during the feed change period of high-density aquaculture.

[0008] The above-mentioned method and system for preparing and controlling microbial agents for aquaculture involves recording and screening the cultures of lactic acid bacteria and Bacillus strains using hydrolyzed old feed culture media, hydrolyzed new feed culture media, and mixed hydrolyzed new and old feed culture media. Through dual-substrate acclimatization culture, short-term co-adaptation culture, fermentation propagation, and biphasic compound formulation treatment, the prepared compound microbial agents can be better adapted to the use scenarios during the feed change period of high-density aquaculture.

[0009] Compared with existing methods of preparing compound bacterial agents using fixed strains, fixed culture media, and fixed compounding ratios, this invention has at least the following beneficial effects: First, it can screen mild acid-producing lactic acid bacteria and new substrate-adapted Bacillus based on old material hydrolysate culture media and new material hydrolysate culture media; Second, through dual-substrate acclimatization culture, the strains adapt to the substrate change in advance during the preparation stage; Third, through short-range co-adaptation culture, it screens out strain combinations that significantly inhibit each other in the same preparation; Fourth, by forming a biphasic compound bacterial agent through lactic acid bacteria protective components and Bacillus dormant components, the lactic acid bacteria are released first after feeding, and the Bacillus is subsequently revived in a later stage. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or related technologies, the accompanying drawings used in the description of the embodiments of the present invention or related technologies will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without any creative effort.

[0011] Figure 1 This is a flowchart illustrating the steps of a method for preparing and controlling microbial agents for aquaculture according to one embodiment of the present invention.

[0012] Figure 2 This is a schematic diagram illustrating the screening and domestication culture of a collection of lactic acid bacteria and a collection of Bacillus in one embodiment of the present invention.

[0013] Figure 3 This is a structural principle block diagram of a microbial agent preparation and control system for aquaculture according to one embodiment of the present invention. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings, tables, and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0015] The method for preparing and controlling microbial agents for aquaculture provided in this invention can be applied to the preparation of microbial agents during the feed change period in high-density aquaculture of fish, shrimp, crabs, or other aquatic animals. The high-density aquaculture feed change period can be understood as a short-term phase in aquaculture where the biomass per unit water volume is high, involving switching from old feed to new feed, or from one feed type to another. This feed change can be a one-time change or a gradual increase in the proportion of new feed over several days, and is not limited to a specific feeding method.

[0016] It should be noted that the "lactic acid bacteria collection" in the embodiments of the present invention may include lactic acid bacteria strains isolated from the intestines of aquatic animals, healthy aquaculture environments, or verified safe sources. For example, it may include lactic acid bacteria with potential for aquatic applications such as *Lactobacillus*, *Pediococcus*, and *Lactococcus*, but is not limited to the above genera. The "Bacillus collection" in the embodiments of the present invention may include Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens with potential for aquatic applications, but is not limited to the specific species mentioned above.

[0017] It should also be noted that, in the embodiments of the present invention, "old feed hydrolysate culture medium" refers to a culture medium or substrate obtained by processing the old feed used before feed change through crushing, mixing with water, enzymatic hydrolysis, enzyme inactivation, filtration, and sterilization; "new feed hydrolysate culture medium" refers to a culture medium or substrate obtained by processing the new feed to be used after feed change through the same or similar treatments; "mixed old and new feed hydrolysate culture medium" refers to a culture medium obtained by mixing the old feed hydrolysate culture medium and the new feed hydrolysate culture medium in a preset ratio. The above-mentioned culture media are used to simulate the changes in substrates that can be utilized by bacterial strains in the intestine during feed change and are not limited to a specific fixed hydrolysis process.

[0018] The preparation route of this invention is as follows: First, the old material and the new material are respectively prepared into hydrolysate culture media. Then, the lactic acid bacteria aggregate and the Bacillus aggregate are cultured and recorded and strains are screened using the old material hydrolysate culture media, the new material hydrolysate culture media, and the mixed old and new material hydrolysate culture media. Subsequently, the lactic acid bacteria subset and the Bacillus subset are subjected to dual-substrate acclimatization culture, short-range co-adaptation culture, and fermentation propagation. Finally, the lactic acid bacteria fermentation product and the Bacillus fermentation product are prepared into a biphasic compound bacterial agent.

[0019] Specifically, such as Figure 1-2As shown in the embodiment of the present invention, a method for preparing and controlling microbial agents for aquaculture includes the following steps: Step S1: Prepare hydrolysate culture medium from old material, hydrolysate culture medium from new material, and hydrolysate culture medium from a mixture of old and new material.

[0020] Specifically, old feed samples and new feed samples were obtained separately. The old feed samples were the feed actually used in the farm before the feed change, and the new feed samples were the feed to be used after the feed change. Both old and new feed samples were pulverized so that they could pass through a 20- to 80-mesh sieve. The pulverized samples were then added to sterile water at a mass-to-volume ratio of 1:5 to 1:15, mixed thoroughly, and then subjected to enzymatic hydrolysis.

[0021] In one feasible implementation, 100g of old feed sample is taken, pulverized, and added to 900mL of sterile water. After thorough mixing, neutral protease and amylase are added, and the mixture is enzymatically hydrolyzed at 50℃ to 55℃ for 1.5 to 2.5 hours. After hydrolysis, the enzymes are inactivated at 90℃ to 100℃ for 8 to 15 minutes. The mixture is then filtered and sterilized to obtain the old feed hydrolysate culture medium. The new feed hydrolysate culture medium can be prepared using the same method. It should be noted that the type of enzyme, hydrolysis time, and hydrolysis temperature can be adjusted according to the feed type, as long as a usable substrate suitable for strain culture is formed.

[0022] In another feasible implementation, the old and new feed samples can be subjected to combined enzymatic hydrolysis using one or more of protease, amylase, and lipase. When the main difference between the old and new feeds is the difference in crude protein content, the proportion of protease can be increased; when the main difference is the difference in starch or available carbohydrate content, the proportion of amylase can be increased; and when the main difference is the difference in crude fat content, an appropriate amount of lipase can be added. These treatments are used to improve the simulation of the actual substrate change in the hydrolysate culture medium.

[0023] Furthermore, the old feed hydrolysate culture medium and the new feed hydrolysate culture medium are mixed according to a preset ratio to obtain a mixed old and new feed hydrolysate culture medium. For example, a low-new-feed-ratio mixed hydrolysate culture medium can be obtained by a ratio of 70% old feed hydrolysate culture medium and 30% new feed hydrolysate culture medium; alternatively, a high-new-feed-ratio mixed hydrolysate culture medium can be obtained by a ratio of 40% old feed hydrolysate culture medium and 60% new feed hydrolysate culture medium. The specific mixing ratio can be determined based on the feed change ratio, feed differences, and actual preparation needs.

[0024] In one specific implementation, data on feed preparation corresponding to the feed change period in high-density aquaculture can be obtained to determine the proportion of new feed hydrolysate culture medium in the mixed new and old feed hydrolysate culture medium. The feed preparation data may include the new feed replacement ratio, information on the differences in feed structure between old and new feed, high-density aquaculture load information, and information on loose feces in the early stages of feed change.

[0025] Specifically, information on the differences in feed composition between old and new feed can be obtained by comparing the crude protein, crude fat, and available carbohydrate content on the labels or in the test reports of the old and new feeds. In one feasible implementation, the feed composition difference value... It can be determined in the following ways: ; in, This indicates the difference in feed composition between old and new feed. Indicates the crude protein content of the new feed; Indicates the crude protein content of the old feed; Indicates the crude fat content of the new feed; Indicates the crude fat content of old feed; This indicates the available carbohydrate content of the new feed; This indicates the available carbohydrate content of the old feed. To prevent extremely small positive numbers with a denominator of zero, the available carbohydrate content can be determined based on the content of starch, gelatinized starch, soluble sugars, or other carbon sources in the feed that can be utilized by the intestinal microorganisms of aquatic animals.

[0026] To facilitate unified data processing, in one executable implementation, the new material replacement ratio is... The value ranges from 0 to 1; feed structure difference value Normalization can be performed according to a preset upper limit, so that its value ranges from 0 to 1; High-density aquaculture load level It can be calculated based on the ratio of actual biomass to the recommended upper limit of biomass, and normalized to 0 to 1; the looseness level of feces in the early stage of feed change. Levels 0, 1, 2, and 3 can be converted to 0, 0.33, 0.67, and 1, respectively. The above normalization method is only an example; any method that allows different data to be compared and used within the same range is acceptable.

[0027] In one executable implementation, the refueling load value can be determined based on the above data. : ; in, Indicates the refueling load value. The closer it is to 1, the greater the degree of change in the gut substrate during the current feeding period; Indicates the percentage of new material used to replace old material; Indicates the difference in feed composition; Indicates the load level of high-density aquaculture; This indicates the degree of looseness of the feces in the early stages of feed replacement; This represents the coefficient indicating the impact of the new material replacement ratio on the material replacement load. This represents the coefficient indicating the impact of differences in feed structure on feed change load; This represents the coefficient indicating the impact of high-density aquaculture load level on feed change load; This represents the coefficient indicating the influence of the fecal looseness level on the feed change load. This represents the operation of the natural exponent. The aforementioned... , , , The value can be determined based on the species being raised, the type of feed being changed, and production experience; for example, it can be selected within the range of 0.1 to 2.0.

[0028] It should be noted that the above-mentioned material replacement load values It is used to assist in determining the mixing ratio of hydrolysate, the acclimatization culture ratio, and the compound formulation parameters, and does not change the preparation route of this invention, which is mainly based on hydrolysate culture medium, lactic acid bacteria subset, Bacillus subset, acclimatization culture, fermentation propagation, and formulation.

[0029] Table 1. Preparation method of mixed hydrolysate culture medium containing new and old materials ; It should be noted that Table 1 is only an example proportion. In practical applications, if the main difference between the new and old feeds is the change in crude protein, the proportion of protein hydrolysate substrate in the mixed hydrolysate culture medium can be further increased; if the main difference is the change in starch or gelatinized starch, the proportion of available carbohydrate substrate can be further increased.

[0030] Step S2: Obtain the collection of lactic acid bacteria and the collection of Bacillus.

[0031] In this embodiment of the invention, the lactic acid bacteria aggregate can be obtained by isolating intestinal samples. Specifically, intestinal contents of healthy aquatic animals can be collected, serially diluted under aseptic conditions, and spread onto MRS medium. Colonies with different morphologies, Gram staining characteristics consistent with lactic acid bacteria, and passing the initial safety screening are selected to form a lactic acid bacteria aggregate. The initial safety screening may include hemolytic activity observation, abnormal odor observation, or other commonly used screening methods in the art; the specific method does not constitute a limitation of the invention.

[0032] In this embodiment of the invention, the Bacillus aggregate can be obtained from intestinal samples, culture sediment samples, or other sources that have been verified to be safe. Specifically, the samples can be subjected to moderate heat treatment to enrich spore-forming bacteria, and then spread on nutrient agar medium or a medium suitable for Bacillus growth. Strains with different morphologies that have been preliminarily identified as having the characteristics of Bacillus are selected to form a Bacillus aggregate.

[0033] It should be noted that the lactic acid bacteria aggregates and Bacillus aggregates can come from healthy individuals in the same aquaculture area, or from preserved strains that have been validated for suitability for aquaculture. For strains used in commercial formulations, further routine safety evaluations and strain identification can be performed, such as morphological identification, physiological and biochemical identification, or molecular biological identification.

[0034] Step S3: Record the culture of the lactic acid bacteria aggregate and the Bacillus aggregate.

[0035] Specifically, the lactic acid bacteria aggregate and the Bacillus aggregate are respectively inoculated into a culture system containing the old material hydrolysate culture medium, the new material hydrolysate culture medium, and the new and old material mixed hydrolysate culture medium for culture and recording.

[0036] When recording the culture of lactic acid bacteria, each strain was inoculated separately into hydrolyzed culture medium containing old substrate, hydrolyzed culture medium containing new substrate, and a mixed hydrolyzed culture medium containing both old and new substrate. During the culture process, the growth of viable bacteria, changes in acid production, changes in the acidity of the culture system, and the onset time of lactic acid bacteria activity were recorded. Specifically, viable bacteria growth could be recorded using plate counting; changes in acid production could be reflected by changes in total acid or acidity before and after culture; changes in the acidity of the culture system could be reflected by pH changes; and the onset time of lactic acid bacteria activity could be the time required to reach a stable acid-producing state after culture.

[0037] In one executable implementation, the lactic acid bacteria response record may include a mild acid production index. Substrate transition adaptation index Low Stimulus Stability Index And the onset time of lactic acid bacteria Among them, subscript Used to represent the first in the set of lactic acid bacteria A strain of lactic acid bacteria. Mild acid production index. The substrate transition fitness index can be determined by normalizing the pH decrease and total acid increase per unit culture time. The low-stimulation stability index can be determined based on the ratio of the viable cell growth rate of the lactic acid bacteria in a mixed hydrolysate culture medium containing both new and old materials to its viable cell growth rate in a hydrolysate culture medium containing only the old material. The onset time of lactic acid bacteria can be determined based on how close the pH decrease at the end of the lactic acid bacteria culture is to the preset mild pH decrease range; This can be the time required for the lactic acid bacteria to reach a stable acid-producing state after entering the substrate.

[0038] In one executable implementation, if... Indicates lactic acid bacteria The pH decrease within the preset incubation time, in order to This indicates the increase in total acid. It can be by and We obtain the result by normalizing and then weighting. If we use... Indicates lactic acid bacteria The growth rate of viable bacteria in a mixed hydrolysate culture medium containing both new and old materials, This indicates the amount of viable bacteria growing in the hydrolysate culture medium. According to Confirmed. If based on This indicates the preset mild pH decrease value, in order to Indicates the allowable deviation range, then According to and The degree of similarity between them is determined. The above indicators can be normalized to 0 to 1, with higher values ​​indicating better performance.

[0039] When culturing and recording Bacillus aggregates, each Bacillus strain was inoculated separately into fresh substrate hydrolysate medium, a mixed fresh and old substrate hydrolysate medium, and a fecal simulated substrate medium. During cultivation, spore recovery, substrate decomposition, adaptation to the new substrate, and the onset time of Bacillus effectiveness were recorded. Spore recovery can be represented by the number of vegetative cells or colony formation within a certain period after inoculation; substrate decomposition can be represented by protein zones, starch hydrolysis zones, fat hydrolysis zones, or changes in soluble nitrogen in the culture medium; adaptation to the new substrate can be represented by the growth rate and endpoint bacterial count in the fresh substrate hydrolysate medium; the onset time of Bacillus effectiveness is the time required for Bacillus to transition from the spore state to the effective substrate decomposition state.

[0040] In one executable implementation, the Bacillus response record may include a substrate hydrolysis index. spore recovery index Adaptability Index of New Materials Onset time of Bacillus Among them, subscript Used to represent the first in the Bacillus set Bacillus strain. Substrate enzymatic hydrolysis index. The spore recovery index can be determined by normalizing one or more of the following: protein hydrolysis zone diameter, starch hydrolysis zone diameter, fat hydrolysis zone diameter, or increase in soluble nitrogen in the culture medium; The adaptability index of the new material can be determined based on the number of colonies or vegetative cells that recover growth within a preset time after inoculation. The onset time of Bacillus can be determined by the ratio of its viable cell growth in fresh hydrolysate culture medium to its viable cell growth in ordinary nutrient culture medium. It can be set to the time required for the substrate to decompose.

[0041] The fecal simulated substrate culture medium can be prepared from hydrolysate of fresh feed residue, sterilized intestinal contents simulation material, and a buffer salt system. It is used to simulate the state of incompletely digested substrate entering the later part of the intestine or being excreted with feces after feed change. This culture medium is used to examine the recovery and decomposition ability of Bacillus in the substrate environment after feed change, and is not limited to a specific formulation.

[0042] Table 2. Culture records of lactic acid bacteria and Bacillus. ; It should be noted that the data in Table 2 are merely exemplary data used to illustrate the embodiments of the present invention. In practical applications, the criteria for retention, reserve, or rejection can be adjusted according to the aquatic animal species, feed type, target live bacteria count of the formulation, and production conditions.

[0043] Step S4: Screen the subsets of lactic acid bacteria and Bacillus.

[0044] Specifically, based on the culture records, a subset of lactic acid bacteria is obtained from the set of lactic acid bacteria, and a subset of Bacillus is obtained from the set of Bacillus. The subset of lactic acid bacteria includes lactic acid bacteria whose viable growth is stable and whose acid production changes are within a preset mild range in the culture system of mixed new and old materials hydrolysate. The subset of Bacillus includes Bacillus whose spores recover stably and whose substrate decomposition performance meets preset requirements in the culture system of new material hydrolysate.

[0045] In one embodiment, stable viable cell growth can be defined as the number of viable cells increasing by no less than 1.0 order of magnitude after 12 hours of culture, or the ratio of the number of viable cells to the number of cells increasing in the hydrolysate culture medium is no less than 0.7; stable spore recovery can be defined as the number of colonies forming reaching a preset proportion of the initial spore inoculation amount within 8 hours after inoculation.

[0046] In this embodiment of the invention, when screening a subset of lactic acid bacteria, priority is given to lactic acid bacteria that exhibit stable viable growth in a mixed hydrolysate culture medium of old and new feed, whose acid production changes are within a preset mild range, and whose onset time meets the requirements for use during the feed change period. The preset mild range can be determined based on the aquatic animal species, feed type, and the buffering capacity of the culture system. For example, in one embodiment, the pH decrease of the lactic acid bacteria after 12 hours of culture can be controlled within the range of 0.4 to 0.9.

[0047] In one specific implementation, the material replacement load value can be used as a reference. Determining the optimal acid production level of lactic acid bacteria : ; in, Indicates the current refueling load value The optimal acid production level for lactic acid bacteria; This indicates the minimum acid production requirement for lactic acid bacteria under low feed change load. This indicates the maximum permissible acid production requirement for lactic acid bacteria under high feed change load; Indicates the refueling load value; This represents the adjustment coefficient that indicates how acid production requirements change with the feed load. This represents an extremely small positive number to prevent the denominator from being zero. The above method is used to screen a subset of lactic acid bacteria, but it does not limit the selection of a subset of lactic acid bacteria to this formula.

[0048] In this embodiment of the invention, when screening a subset of Bacillus species, priority is given to retaining Bacillus species that exhibit stable spore recovery in the hydrolysate culture medium of fresh feed, demonstrate good substrate decomposition performance in the fecal simulated substrate culture medium, and whose onset time meets the requirements for use during the feed change period. For example, in feed change scenarios where the crude protein content of the fresh feed increases significantly, Bacillus species with good protein hydrolysis performance can be prioritized; in feed change scenarios where the available carbohydrate content of the fresh feed increases, Bacillus species with good starch hydrolysis performance can be prioritized.

[0049] In one specific implementation, the material replacement load value can be used as a reference. Determining the optimal substrate enzymatic hydrolysis level for Bacillus : ; in, Indicates the current refueling load value The optimal enzymatic hydrolysis capacity of Bacillus subtilis; This indicates the minimum enzymatic hydrolysis requirement for Bacillus under low feed load conditions; This indicates the maximum enzymatic hydrolysis requirement for Bacillus under high feed rate conditions; This represents the adjustment coefficient that indicates how the enzymatic hydrolysis requirement changes with the feed load. This indicates the material change load value.

[0050] In one feasible implementation, the fitness values ​​of individual lactic acid bacteria strains, the fitness values ​​of individual Bacillus strains, and co-adaptation culture performance can be used as screening criteria. Among these, the fitness values ​​of individual lactic acid bacteria strains... It can be determined in the following ways: ; in, Indicates lactic acid bacteria Adaptation values ​​for the current material change scenario; Indicates lactic acid bacteria The mild acid production index; This indicates the suitable acid production level of lactic acid bacteria under the current feed change load; Indicates the allowable deviation in acid production; Represents a very small positive number; Indicates lactic acid bacteria The substrate transition adaptation index; This indicates the basic requirements for substrate transition adaptation; This indicates the strength of the influence of the substrate transition adaptation index on the aptability value of lactic acid bacteria; Indicates lactic acid bacteria The low-stimulation stability index.

[0051] In one executable implementation, when the adaptability value of a single lactic acid bacteria strain is not lower than a first lactic acid bacteria screening threshold, the corresponding lactic acid bacteria is identified as a candidate strain in the lactic acid bacteria subset; when the adaptability value of a single lactic acid bacteria strain is lower than the first lactic acid bacteria screening threshold but not lower than a second lactic acid bacteria screening threshold, the corresponding lactic acid bacteria is used as a backup lactic acid bacteria strain; when the adaptability value of a single lactic acid bacteria strain is lower than the second lactic acid bacteria screening threshold, the corresponding lactic acid bacteria is removed. Specifically, the first lactic acid bacteria screening threshold can be 0.70, and the second lactic acid bacteria screening threshold can be 0.55. The above thresholds are only examples and can be adjusted according to the aquaculture species, feed change intensity, culture medium buffering capacity, and production batch validation results.

[0052] Bacillus single strain aptance value It can be determined in the following ways: ; in, Indicates Bacillus Adaptation values ​​for the current material change scenario; Indicates Bacillus The substrate enzymatic hydrolysis index; This indicates the suitable enzymatic hydrolysis capacity of Bacillus under the current material replacement load; Indicates the allowable deviation width of enzymatic hydrolysis; Represents a very small positive number; The spore recovery index represents the spore resuscitation index of Bacillus. This indicates the basic requirements for spore resuscitation; This indicates the strength of the effect of the spore recovery index on the Bacillus adaptability value; Indicates Bacillus The new material adaptability index.

[0053] In one executable implementation, when the adaptability value of a single Bacillus strain is not lower than the first Bacillus screening threshold, the corresponding Bacillus strain is identified as a candidate strain in the Bacillus subset; when the adaptability value of a single Bacillus strain is lower than the first Bacillus screening threshold but not lower than the second Bacillus screening threshold, the corresponding Bacillus strain is used as a backup Bacillus strain; when the adaptability value of a single Bacillus strain is lower than the second Bacillus screening threshold, the corresponding Bacillus strain is removed. Specifically, the first Bacillus screening threshold can be 0.70, and the second Bacillus screening threshold can be 0.55.

[0054] It should be noted that the above fitness values ​​are used to determine whether a strain is suitable for subsequent acclimatization and formulation steps. In actual production, strain screening can also be performed using grading tables, threshold tables, or a combination of manual verification and test data.

[0055] Step S5: Perform dual-substrate acclimatization culture and short-term co-adaptation culture on the lactic acid bacteria subset and the Bacillus subset.

[0056] Specifically, the lactic acid bacteria subset and the Bacillus subset are subjected to dual-substrate acclimatization culture to obtain lactic acid bacteria culture adapted to the new substrate and Bacillus culture adapted to the new substrate; the lactic acid bacteria culture adapted to the new substrate and the Bacillus culture adapted to the new substrate are subjected to short-term co-acclimatization culture, and the retained lactic acid bacteria culture and Bacillus culture are fermented and propagated to obtain lactic acid bacteria ferment and Bacillus ferment.

[0057] In this embodiment of the invention, dual-substrate acclimatization culture refers to using a culture medium composed of old substrate hydrolysate and new substrate hydrolysate to separately acclimatize a subset of lactic acid bacteria and a subset of Bacillus. The proportion of new substrate hydrolysate in the lactic acid bacteria acclimatization culture medium is lower than that in the Bacillus acclimatization culture medium. This is because lactic acid bacteria are mainly used for mild and stable support in the early stages of substrate change, while Bacillus is mainly used for the decomposition of new substrate in the later stages.

[0058] In one executable implementation, the proportion of virgin material hydrolysate culture medium in the lactic acid bacteria acclimatization culture medium is... It can be determined in the following ways: ; The proportion of fresh material hydrolysate in the Bacillus acclimatization culture medium It can be determined in the following ways: ; in, This indicates the proportion of virgin material hydrolysate in the lactic acid bacteria acclimatization culture medium; This indicates the proportion of fresh material hydrolysate in the Bacillus acclimatization culture medium; This indicates the material change load value. The above method of determining the ratio is only one feasible implementation method. In practical applications, the corresponding ratio range can also be set according to the material change adaptation level.

[0059] Table 3. Data on the preparation of dual-substrate acclimatization culture medium ; In one feasible implementation, a subset of lactic acid bacteria is inoculated into a lactic acid bacteria acclimatization medium at an inoculum of 1% to 5%, and cultured at 30°C to 38°C for 10 to 18 hours. After culture, the viable cell count and pH changes are recorded. A subset of Bacillus is inoculated into a Bacillus acclimatization medium at an inoculum of 1% to 5%, and cultured at 30°C to 37°C for 12 to 24 hours. Spore formation and substrate degradation are observed in the later stages of culture. The above culture temperature, inoculum size, and time can be adjusted according to the characteristics of the strain.

[0060] In this embodiment of the invention, short-range co-adaptation culture is used to confirm whether the domesticated lactic acid bacteria and Bacillus are suitable for inclusion in the same compound formulation. Specifically, lactic acid bacteria cultures adapted to the new substrate and Bacillus cultures adapted to the new substrate are added to a mixed hydrolysate culture system of old and new substrates at a preset inoculation amount and cultured for 6 to 12 hours, recording the viability of the two types of bacteria. If both remain stable, fermentation and propagation proceed; if one significantly decreases, a backup strain is used or the priority of the combination is reduced.

[0061] In one specific implementation, the appropriate timing for the sequential action of lactic acid bacteria and Bacillus can be determined based on their onset times. The appropriate time interval between the onset of action of lactic acid bacteria and the onset of action of Bacillus is defined as follows: It can be determined in the following ways: ; in, This indicates the appropriate time interval between the onset of lactic acid bacteria and the onset of Bacillus thuringiensis under the current feed load; This indicates the longer allowable continuity time under low refueling load; This indicates the shorter connection time required under high refueling load; This indicates the feed change load value. This suitable time interval can be used to determine the degree of release delay of dormant components of Bacillus during subsequent biphasic formulation.

[0062] In one feasible implementation, the lactic acid bacteria can be determined based on the aptability values ​​of individual lactic acid bacteria strains, the aptability values ​​of individual Bacillus strains, short-term co-adaptation culture performance, and the relationship between onset time. With Bacillus Combined adaptation values : ; Among them, the safe acid production constraint item for: ; in, Indicates lactic acid bacteria With Bacillus The combined adaptation value; Indicates lactic acid bacteria The individual plant fit value; Indicates Bacillus The individual plant fit value; Indicates lactic acid bacteria With Bacillus Co-adaptation index in short-range co-adaptation culture; This represents the influence coefficient of the co-adaptation index on the group fit value; Indicates Bacillus Onset time; Indicates lactic acid bacteria Onset time; This indicates the appropriate inoculation time for the two strains under the current refueling load; Indicates the allowable width of the continuation time deviation; Represents a very small positive number; This indicates a safety constraint on acid production. This represents the suppression coefficient when lactic acid bacteria produce acid exceeding the safe range; Indicates lactic acid bacteria The mild acid production index; This indicates the maximum safe acid production limit under the current refueling load; This means that when the lactic acid bacteria acid production index exceeds the safe acid production limit, the excess portion is taken; otherwise, 0 is taken.

[0063] Furthermore, when the fit value of the combination of lactic acid bacteria and Bacillus is not lower than the combination screening threshold, the corresponding combination of lactic acid bacteria and Bacillus is identified as a strain combination that can enter short-range co-adaptation culture; when the fit value of the combination is lower than the combination screening threshold but not lower than the reserve combination threshold, the corresponding strain combination is used as a reserve combination; when the fit value of the combination is lower than the reserve combination threshold, the corresponding strain combination is eliminated. Specifically, the combination screening threshold can be 0.65, and the reserve combination threshold can be 0.50.

[0064] In one executable implementation The viability of lactic acid bacteria and Bacillus can be determined based on their viability retention rates after short-term co-adaptation culture. For example, if the lactic acid bacteria retention rate is 92% and the Bacillus retention rate is 95%, the smaller of the two values ​​can be taken as the viability retention rate. The baseline value; if the retention rate of any strain is lower than the preset value, the corresponding combination will not be included in subsequent fermentation and propagation. The above method enables the results of short-range co-adaptation culture to be used for subsequent compound formulation.

[0065] Step S6: Fermentation propagation and compound formulation.

[0066] Specifically, the lactic acid bacteria fermentation product is prepared as a lactic acid bacteria protective component, and the Bacillus fermentation product is prepared as a Bacillus dormant component, which are then compounded and formulated to obtain a microbial agent for aquaculture.

[0067] In this embodiment of the invention, fermentation propagation includes lactic acid bacteria fermentation propagation and Bacillus fermentation propagation. Lactic acid bacteria fermentation propagation can be carried out using a lactic acid bacteria fermentation medium containing hydrolysates of both fresh and old substrates, controlling the fermentation endpoint to a mild acid-producing state. Bacillus fermentation propagation can be carried out using a Bacillus fermentation medium containing a higher proportion of hydrolysates of the fresh substrate to maintain spore formation ability and adaptation to the new substrate.

[0068] In one feasible implementation, the endpoint for lactic acid bacteria fermentation propagation can be set to a viable count of not less than 1.0 × 10^9 CFU / mL, and the pH drop at the end of the culture should be within a preset mild range. The endpoint for Bacillus fermentation propagation can be set to a spore formation rate of not less than 80%, and the ability to recover growth within a preset time in a retest using fresh hydrolysate culture medium. It should be noted that the above values ​​are merely examples, and those skilled in the art can make reasonable adjustments based on the product formulation and fermentation equipment conditions.

[0069] Table 4 Fermentation batch data ; In this embodiment of the invention, the compound formulation process includes determining the compounding ratio, preparing a lactic acid bacteria protective component, preparing a Bacillus dormant component, and forming a biphasic compound bacterial agent. Specifically, the total proportion of Bacillus can be determined based on the feed change load value, the looseness level of the feces, and the bacterial culture records. and the total percentage of lactic acid bacteria In one executable implementation: ; ; in, This indicates the total percentage of Bacillus in the live bacteria blend; This indicates the total percentage of lactic acid bacteria in the live bacteria blend; Indicates the refueling load value; This indicates the degree of looseness of the feces in the early stages of feed replacement; This indicates taking the smaller value; This indicates taking the larger value. Using the above method, when the feed change load is high and the feces are noticeably loose, the proportion of Bacillus can be increased to enhance the decomposition capacity of the new feed substrate; at the same time, the proportion of Bacillus should be limited to no more than 0.75% to preserve the supporting role of lactic acid bacteria in intestinal homeostasis during the early stages of feed change.

[0070] In specific formulation, Bacillus fermentation material can be mixed with a storage-resistant carrier to prepare a dormant Bacillus component. The storage-resistant carrier may include one or more of zeolite powder, bentonite, microcrystalline cellulose, starch, and alginate carrier. In one embodiment, after centrifugation or concentration of the Bacillus fermentation material, it is mixed with microcrystalline cellulose, starch, and a small amount of binder, and then core particles are formed by wet granulation or extrusion spheronization. The particle size can be from 0.5 mm to 2.0 mm.

[0071] Subsequently, a release delay layer is formed outside the core particles. The release delay layer may be formed of hydroxypropyl methylcellulose, chitosan, sodium alginate, gelatin, or a combination thereof. The thickness of the release delay layer may be from 0.2 mm to 1.0 mm. To match the Bacillus release time with the aforementioned suitable inoculation time, the Bacillus release delay thickness is... It can be determined in the following ways: ; in, Indicates the release delay thickness of the Bacillus preparation layer; Indicates the minimum release delay thickness; Indicates the maximum release delay thickness; This indicates the appropriate time interval between the onset of lactic acid bacteria and the onset of Bacillus thuringiensis under the current feed load; This indicates a shorter connection time under high refueling load; This indicates a longer continuity time under low refueling load. This represents a very small positive number. The above method for determining the thickness is only one feasible implementation method; the thickness of the release delay layer can also be determined based on the granulation equipment, carrier material, and actual release tests.

[0072] Lactic acid bacteria fermentation material is mixed with a protective carrier to prepare a lactic acid bacteria protective component. The protective carrier may include one or more of skim milk powder, trehalose, maltodextrin, soluble starch, and chitosan derivatives. The lactic acid bacteria protective component can be disposed on the outer side of the release delay layer, or it can form an outer layer component together with an easily disintegrating carrier, allowing the lactic acid bacteria protective component to be released earlier after feeding. Since lactic acid bacteria are sensitive to high temperatures, the material temperature is preferably controlled to not exceed 40°C during granulation or coating, and the drying temperature can be controlled within the range of 30°C to 40°C.

[0073] In another embodiment, when preparing the powder, the lactic acid bacteria protective component can be prepared as a rapidly dispersible powder, and the Bacillus dormant component can be prepared as micro-encapsulated particles, and then mixed in a preset ratio to form a biphasic powder. In this biphasic powder, the lactic acid bacteria protective component can be released earlier after mixing, and the Bacillus dormant component can be reactivated in a subsequent stage.

[0074] Table 5. Compound formulation parameters ; It should be noted that the data in Table 5 are exemplary data used to illustrate how the present invention converts the material change load value into compound formulation parameters. In practical applications, the data can be adjusted according to the carrier material, granulation equipment, and usage method.

[0075] Table 6 Small-scale validation data ; As shown in Table 6, in the context of feed change, the biphasic composite microbial agent prepared in this embodiment of the invention can improve feed intake retention and reduce loose feces in the short term after feed change. This is because the lactic acid bacteria protective component is released early after feeding, providing gentler intestinal homeostasis support; the dormant Bacillus component subsequently reactivates and participates in the decomposition of the new substrate, making the use of the microbial agent more aligned with the changes in the intestinal substrate during the feed change period.

[0076] Based on the same inventive concept, embodiments of the present invention also provide a control system for the preparation of microbial agents for aquaculture. For example... Figure 3 As shown, the system includes a hydrolysate culture medium preparation module, a strain acquisition module, a culture recording module, a strain screening module, an acclimatization culture module, a fermentation and propagation module, and a formulation module.

[0077] Specifically, the hydrolysate culture medium preparation module is used to prepare hydrolysate culture media made from recycled materials, hydrolysate culture media made from virgin materials, and mixed hydrolysate culture media made from recycled and virgin materials. Specifically, this module may include a pulverizing device, an enzymatic hydrolysis container, a filtration unit, a sterilization unit, and a mixing unit, or it can be completed manually in combination with conventional pre-fermentation treatment equipment. The strain acquisition module is used to acquire a collection of lactic acid bacteria and a collection of Bacillus. The culture recording module is used to inoculate the lactic acid bacteria and the Bacillus collection into culture systems containing the recycled hydrolysate culture medium, the virgin hydrolysate culture medium, and the mixed hydrolysate culture medium for culture recording. The strain screening module is used to screen for a subset of lactic acid bacteria from the lactic acid bacteria collection and a subset of Bacillus from the Bacillus collection based on the culture records. The acclimatization culture module is used to perform dual-substrate acclimatization culture on the subsets of lactic acid bacteria and the subsets of Bacillus, respectively. The fermentation propagation module is used to ferment and propagate the lactic acid bacteria culture and the Bacillus culture retained after short-term co-adaptation culture, respectively. The formulation module is used to prepare lactic acid bacteria fermentation products into lactic acid bacteria protective components, Bacillus fermentation products into Bacillus dormant components, and to formulate the lactic acid bacteria protective components and the Bacillus dormant components in combination.

[0078] The various modules in the aforementioned aquaculture microbial agent preparation control system can be implemented, in whole or in part, through a combination of production management software, fermentation equipment control units, data recording terminals, manual operating consoles, and hardware such as fermentation tanks, mixing tanks, and granulation equipment. The above module division is for illustrative purposes only and does not imply that the actual equipment must be divided according to the exact same physical structure. Without affecting the technical effect of this invention, the modules can be integrated or set up separately.

[0079] It should be understood that although the processes involved in the embodiments described above are presented in a step-by-step order, these steps are not necessarily performed in that order. Unless otherwise expressly stated herein, there is no strict order restriction on the execution of these steps. For example, the preparation of hydrolysate culture medium and the propagation of bacterial strains can be performed in parallel, as can the domestication culture of lactic acid bacteria and the domestication culture of Bacillus. As long as the data transfer relationship between the steps meets the implementation requirements of the embodiments of the present invention, it should be considered to fall within the protection scope of the present invention.

[0080] It should also be noted that the terms "first," "second," etc., used in this document are used only to distinguish different objects and are not used to limit the order or importance. The terms "comprising," "including," or any other variations thereof, as used in this document, are intended to cover non-exclusive inclusion, such that a process, method, system, or product that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to the process, method, system, or product.

[0081] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of this invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, such as changing the specific lactic acid bacteria strain, adjusting the Bacillus strain, changing the preparation method of the hydrolysate, changing the carrier material, or adjusting the formulation form. These all fall within the scope of protection of this invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A method for controlling the preparation of microbial agents for aquaculture, characterized in that, The method includes the following steps: Prepare culture media of hydrolysate from old material, culture media of hydrolysate from new material, and culture media of hydrolysate from a mixture of old and new material; Obtain a collection of lactic acid bacteria and a collection of Bacillus, and inoculate the collection of lactic acid bacteria and the collection of Bacillus into a culture system containing the old material hydrolysate culture medium, the new material hydrolysate culture medium and the new and old material mixed hydrolysate culture medium for culture and record the culture. Based on the culture records, a subset of lactic acid bacteria is obtained by screening from the set of lactic acid bacteria, and a subset of Bacillus is obtained by screening from the set of Bacillus; wherein, the subset of lactic acid bacteria includes lactic acid bacteria whose viable growth is stable and whose acid production changes are within a preset mild range in the culture system of mixed new and old materials hydrolysate, and the subset of Bacillus includes Bacillus whose spores recover stably and whose substrate decomposition performance meets preset requirements in the culture system of new material hydrolysate; The lactic acid bacteria subset and the Bacillus subset were subjected to dual-substrate acclimatization culture to obtain lactic acid bacteria culture adapted to the new substrate and Bacillus culture adapted to the new substrate. The lactic acid bacteria culture adapted to the new substrate and the Bacillus culture adapted to the new substrate were subjected to short-term co-adaptation culture, and the retained lactic acid bacteria culture and Bacillus culture were fermented and propagated separately to obtain lactic acid bacteria ferment and Bacillus ferment. The lactic acid bacteria fermentation product is prepared as a lactic acid bacteria protective component, and the Bacillus fermentation product is prepared as a Bacillus dormant component. The two components are then combined and formulated to obtain a microbial agent for aquaculture.

2. The method for preparing and controlling microbial agents for aquaculture according to claim 1, characterized in that, Preparation of hydrolysate culture media made from recycled materials, hydrolysate culture media made from virgin materials, and mixed hydrolysate culture media made from recycled and virgin materials includes: Obtain old material samples and new material samples respectively, and crush and mix the old material samples and the new material samples with water respectively; The old material sample and the new material sample after adding water were subjected to enzymatic hydrolysis to obtain the old material hydrolysate and the new material hydrolysate, respectively. The old material enzymatic hydrolysate and the new material enzymatic hydrolysate are subjected to enzyme inactivation, filtration, and sterilization treatments, respectively, to obtain the old material hydrolysate culture medium and the new material hydrolysate culture medium; The old material hydrolysate culture medium and the new material hydrolysate culture medium are mixed in a preset ratio to obtain the new and old material mixed hydrolysate culture medium.

3. The method for preparing and controlling microbial agents for aquaculture according to claim 1, characterized in that, The mixing ratio of the old material hydrolysate culture medium and the new material hydrolysate culture medium in the new and old material mixed hydrolysate culture medium is determined by the following method: Acquire data for feed preparation during the feed change period in high-density aquaculture; wherein, the data for feed preparation includes the replacement ratio of new feed, information on the differences in feed structure between old and new feed, information on high-density aquaculture load, and information on loose feces in the early stage of feed change; Based on the data used for preparing the substrate change, a substrate change adaptation level was determined to characterize the degree of change in intestinal substrate during the substrate change period. The proportion of new material hydrolysate culture medium in the new and old material mixed hydrolysate culture medium is determined according to the material replacement adaptation level; wherein, the higher the material replacement adaptation level, the higher the proportion of new material hydrolysate culture medium in the new and old material mixed hydrolysate culture medium.

4. The method for preparing and controlling microbial agents for aquaculture according to claim 1, characterized in that, Based on the culture records, a subset of lactic acid bacteria is obtained by screening from the lactic acid bacteria set, including: Extract the viable growth, acid production changes, culture system acidity changes, and lactic acid bacteria onset time of each lactic acid bacteria from the culture records in the old material hydrolysate culture medium, the new material hydrolysate culture medium, and the new and old material mixed hydrolysate culture medium; The substrate transition adaptation performance is determined based on the growth of live bacteria; the mild acid production performance is determined based on the changes in acid production and the changes in acidity of the culture system; and the effectiveness of lactic acid bacteria in substrate changes is determined based on the onset time of lactic acid bacteria. Lactic acid bacteria that meet the preset requirements for substrate transition adaptation, mild acid production, and efficacy are identified as candidate strains for the lactic acid bacteria subset. The lactic acid bacteria subset is obtained by screening from the candidate strains of the lactic acid bacteria subset.

5. The method for preparing and controlling microbial agents for aquaculture according to claim 1, characterized in that, Based on the culture records, a subset of Bacillus bacteria is obtained by screening from the Bacillus collection, including: Extract the spore recovery status, substrate decomposition performance, new material adaptation performance, and spore onset time of each Bacillus from the culture records in the new material hydrolysate medium, the new and old material mixed hydrolysate medium, and the fecal simulated substrate medium; The spore recovery performance is determined based on the spore recovery status, the substrate enzymatic hydrolysis performance is determined based on the substrate decomposition performance, the new substrate adaptation performance is determined based on the new substrate adaptation performance, and the effective performance of Bacillus in the new substrate is determined based on the effective time of Bacillus. Bacillus strains that meet the preset requirements in terms of spore resuscitation performance, substrate enzymatic digestion performance, new substrate adaptation performance, and efficacy performance are identified as a subset of candidate strains of Bacillus. The Bacillus subset was obtained by screening from the candidate strains of the Bacillus subset.

6. The method for preparing and controlling microbial agents for aquaculture according to claim 1, characterized in that, The lactic acid bacteria subset and the Bacillus subset were subjected to dual-substrate acclimatization culture, including: Based on the data on feed preparation during the feed change period in high-density aquaculture, the first mixing ratio of new feed hydrolysate culture medium and old feed hydrolysate culture medium in lactic acid bacteria acclimatization culture medium was determined, and the second mixing ratio of new feed hydrolysate culture medium and old feed hydrolysate culture medium in Bacillus acclimatization culture medium was determined. The subset of lactic acid bacteria is inoculated into a lactic acid bacteria acclimatization medium prepared according to the first mixing ratio, and cultured to obtain the lactic acid bacteria culture adapted to the substrate change. The subset of Bacillus bacteria was inoculated into a Bacillus acclimatization culture medium prepared according to the second mixing ratio, and cultured to obtain the Bacillus culture adapted to the new substrate; In the second mixing ratio, the proportion of virgin hydrolysate culture medium is higher than that in the first mixing ratio.

7. The method for preparing and controlling microbial agents for aquaculture according to claim 1, characterized in that, The short-range co-adaptation culture includes: The lactic acid bacteria culture adapted to the new substrate and the Bacillus culture adapted to the new substrate were inoculated into a mixed hydrolysate culture system of old and new substrates for co-culture. Record the viability of lactic acid bacteria and Bacillus during co-culture to obtain co-adaptation culture records; Based on the co-adaptation culture records, strain combinations with a significant decrease in the number of live lactic acid bacteria or Bacillus were removed, while strain combinations in which the number of live lactic acid bacteria and Bacillus remained stable and with low mutual inhibition were retained.

8. The method for preparing and controlling microbial agents for aquaculture according to claim 1, characterized in that, The preserved lactic acid bacteria culture and Bacillus culture were fermented and propagated separately to obtain lactic acid bacteria ferment and Bacillus ferment, including: The preserved lactic acid bacteria culture was inoculated into a lactic acid bacteria fermentation medium for fermentation and propagation. At the end of fermentation, the number of viable lactic acid bacteria and the change in acidity were recorded to obtain the lactic acid bacteria fermentation product. The preserved Bacillus culture was inoculated into Bacillus fermentation medium for fermentation and propagation. At the end of fermentation, the spore formation rate and the performance of the new substrate were recorded to obtain the Bacillus ferment. Lactic acid bacteria fermentation products whose viable count and acidity change meet the preset requirements are identified as lactic acid bacteria fermentation products for compound formulation. Bacillus fermentation products that meet the preset requirements for spore formation rate and performance in retesting of new substrate are identified as Bacillus fermentation products for compound formulation.

9. The method for preparing and controlling microbial agents for aquaculture according to claim 1, characterized in that, The formulation of the compound preparation specifically includes: The Bacillus ferment is mixed with a storage-resistant carrier to prepare core particles containing Bacillus dormancy components; A release delay layer is formed outside the core particles; The lactic acid bacteria fermentation product is mixed with a protective carrier to prepare a lactic acid bacteria protective component, and the lactic acid bacteria protective component is disposed on the outside of the release delay layer or together with the release delay layer to form an outer layer component. Particles containing the lactic acid bacteria protective component and the Bacillus dormant component are subjected to low-temperature drying to obtain a biphasic composite bacterial agent; wherein the lactic acid bacteria protective component is released first after feeding, and the Bacillus dormant component is subsequently revived after the release of the lactic acid bacteria protective component.

10. A control system for preparing microbial agents for aquaculture, characterized in that, The system is used to execute the method for preparing and controlling microbial agents for aquaculture as described in any one of claims 1-9, the system comprising: The hydrolysate culture medium preparation module is used to prepare hydrolysate culture medium made from recycled materials, hydrolysate culture medium made from virgin materials, and hydrolysate culture medium made from a mixture of recycled and virgin materials. The strain acquisition module is used to acquire sets of lactic acid bacteria and Bacillus strains; The culture recording module is used to inoculate the lactic acid bacteria collection and the Bacillus collection into a culture system containing the old material hydrolysate culture medium, the new material hydrolysate culture medium and the new and old material mixed hydrolysate culture medium for culture recording; The strain screening module is used to screen a subset of lactic acid bacteria from the lactic acid bacteria set and a subset of Bacillus from the Bacillus set based on the culture records. An acclimatization and cultivation module is used to perform dual-substrate acclimatization and cultivation on the lactic acid bacteria subset and the Bacillus subset respectively, to obtain lactic acid bacteria cultures adapted to the new substrate and Bacillus cultures adapted to the new substrate. The fermentation and propagation module is used to ferment and propagate lactic acid bacteria culture and Bacillus culture retained after short-term co-adaptation culture, respectively, to obtain lactic acid bacteria ferment and Bacillus ferment; The formulation module is used to prepare the lactic acid bacteria fermentation product into a lactic acid bacteria protective component, the Bacillus fermentation product into a Bacillus dormant component, and to formulate the lactic acid bacteria protective component and the Bacillus dormant component in combination to obtain a microbial agent suitable for aquaculture during the feed change period of high-density aquaculture.

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