A composite fermentation product of leaf-eating grass enzyme and a preparation method and application thereof

Abstract

The application discloses a kind of leaf-eating grass enzyme complex fermentation product and its preparation method and application, belong to plant fermentation technical field.The method is inoculated with fermentation strain after being roughly broken to plant raw materials such as leaf-eating grass and wormwood and carries out aerobic pre-fermentation, and obtains bacterial liquid phase and wet solid phase by rough solid-liquid separation;Bacterial liquid phase is anaerobic acidification first, and wet solid phase is temporarily stored in airtight;According to the change of pH and titration acidity after wet grinding round under the condition that different mass ratio of fermentation material and wet solid phase, determine the treatment amount of this round wet solid phase, and after destructing corresponding wet solid phase with part of fermentation material as wet grinding medium, continue anaerobic fermentation in main fermentation container;After acidification state recovers, repeat the above process until wet solid phase is completely processed, then continue anaerobic fermentation and carry out post-processing, obtain liquid or solid-liquid mixed type complex fermentation product.The application makes the destructing of plant tissue and the release process of material match the current acidification conversion state of fermentation system, and can be used for preparing fermented feed or drinking water for breeding animals.

Description

Technical Field

[0001] This invention belongs to the field of plant fermentation technology, specifically referring to a compound fermentation product of leafy grass enzymes, its preparation method, and its application. Background Technology

[0002] Leafy grasses are rich in protein, amino acids, vitamins, minerals, flavonoids, and polyphenols, making them a valuable plant-based nutrient source for food or animal feed. Microbial fermentation promotes the decomposition of plant tissues and the conversion of nutrients, forming organic acids, enzymes, and other fermentation metabolites.

[0003] For example, Chinese patent CN116584580A discloses an energy powder containing raw materials such as edible grass and mugwort. The raw materials such as edible grass and mugwort are ground and then subjected to primary fermentation, followed by multi-stage anaerobic fermentation to achieve the fermentation and transformation of plant raw materials.

[0004] However, for complex fermentation processes that include both aerobic and anaerobic fermentation, the requirements for the state of the materials differ at different fermentation stages. When the plant raw materials are less broken, it is beneficial to maintain the gaps between materials and gas transfer, but the release of substances from within the tissue is insufficient; when the degree of breakage is higher, although it is beneficial to the release of nutrients, it tends to make the materials more compact and allows more soluble components to enter the fermentation system prematurely.

[0005] Moreover, when plant tissues that have undergone preliminary fermentation are concentrated and deeply crushed, soluble sugars, nitrogenous substances and other soluble components will be released in a short period of time. However, the release rate is not necessarily consistent with the subsequent utilization, transformation and acid production rate of microorganisms, which can easily lead to a mismatch between the material release load and the current transformation capacity of the fermentation system.

[0006] Furthermore, the moisture content, tissue maturity, and pre-fermentation state of different batches of plant materials vary, and the actual release load produced by the same crushing intensity and processing time may also differ. Therefore, how to match the decomposition of plant tissues and the release of substances with the current conversion capacity of the fermentation system is a problem that still needs to be solved in existing edible grass compound fermentation processes. Summary of the Invention

[0007] The purpose of this invention is to provide a compound fermentation product of leafy grass enzymes, its preparation method and application, in order to solve the problem that in the existing compound fermentation process of leafy grass, the deconstruction of plant tissues and the release of substances are difficult to match with the current conversion capacity of the fermentation system, and the release load after concentrated crushing of plant raw materials is too large, which can easily affect the stability of subsequent anaerobic fermentation.

[0008] This invention provides a method for preparing a compound fermentation product of edible clover enzymes, comprising the following steps: coarsely crushing plant raw materials including edible clover and artemisia, mixing with fermentation bacteria for aerobic pre-fermentation, and then performing coarse solid-liquid separation to obtain a liquid phase containing fermenting microorganisms and a wet solid phase retaining coarse particle structure; placing the liquid phase containing microorganisms in a main fermentation vessel for anaerobic acidification, temporarily storing the wet solid phase in a sealed container, and using all materials currently participating in fermentation in the main fermentation vessel as yeast feed; in each round of processing, sampling is performed on the mixed yeast feed and the remaining wet solid phase, and test groups are configured according to multiple candidate mass ratios of wet solid phase to mother material. A portion of the mother material sample from each test group is used as a wet grinding medium to wet grind the wet solid phase sample in that group, and the resulting slurry is then combined with the remaining mother material sample in that group; pH is measured during and after each round of testing, and changes in pH and titratable acidity are measured during continued anaerobic fermentation. A candidate mass ratio that simultaneously meets pH control conditions and acidification recovery conditions is selected, and the selected mass ratio is then used to further refine the mixture. The wet solid phase processing volume for this round is determined by selecting the mass ratio and the actual mass of all yeast material in the main fermentation vessel. According to the corresponding test group's mother material usage ratio, wet milling endpoint, temperature conditions, and round method, under closed low-oxygen conditions, a portion of the yeast material taken from the main fermentation vessel is used as the wet milling medium to wet mill the wet solid phase for this round. The resulting slurry is then returned to the main fermentation vessel, mixed with the remaining yeast material, and anaerobic fermentation continues. After the yeast material meets the acidification recovery conditions after the round, samples are resampled to determine the wet solid phase processing volume for the next round. Wet milling, rounds, and anaerobic fermentation are repeated until all wet solid phase processing is completed and returned. Anaerobic fermentation continues, and post-processing yields the composite fermentation product. The pH control condition is that the pH detection value during the round and within a preset initial time period after the round does not exceed a preset control upper limit. The acidification recovery condition is that the pH detection value within the preset recovery time period reaches below the preset recovery upper limit, and the titration acidity increases to a preset range compared to the initial titration acidity after the round.

[0009] Furthermore, all plant materials and fermentation strains are added before the start of aerobic pre-fermentation, and no additional sugar sources, acidifiers, alkaline neutralizers, or other batches of mature fermentation broth are added during subsequent processing; the wet solid phase is temporarily stored in a sealed container at 4–8°C, and the liquid precipitated during the temporary storage period is mixed with the corresponding wet solid phase and then sampled, measured, and processed together.

[0010] Furthermore, the testing groups are configured with 10, 20, 30, and 40 parts of wet solid phase for every 100 parts of masterbatch. Each testing group takes out a portion of masterbatch equivalent to twice the mass of the wet solid phase from its own group as wet grinding media, and the remaining masterbatch is used to receive the grinding slurry of its own group. The masterbatch samples of subsequent rounds retain the fine plant tissue returned from the previous rounds.

[0011] Furthermore, the pH control conditions are that the pH values ​​detected during the round and at 5, 15, and 30 minutes after the round are not higher than 4.5; the acidification recovery conditions are that the last two pH values ​​at 30-minute intervals during the 4-hour observation period after the round are not higher than 4.2, and the titration acidity 4 hours after the round is not less than 10% higher than the titration acidity 30 minutes after the round is completed; the round completion refers to the complete addition of the corresponding remaining masterbatch to the pulp and the completion of mixing.

[0012] Furthermore, the pH value is the measured value after the sample is adjusted to 25°C; the titration acidity is determined by the following method: weigh 10.00 g of the mixed sample, add 90 g of analytical water to remove carbon dioxide, stir at 25°C for 10 minutes, titrate with a standardized 0.1 mol / L sodium hydroxide solution to pH 8.2 and maintain for 30 seconds, and express the titration acidity per 10.00 g of sample as the volume of alkali consumed after deducting the analytical water blank.

[0013] Furthermore, after determining the amount of wet solid phase treatment in this round and before performing this round of wet milling, the yeast feed is remixed and its pH and titratable acidity are checked. If the pH changes by more than 0.1 or the titratable acidity changes by more than 10% compared with the masterbatch sampled in the corresponding test group, the sample is resampled for candidate mass ratio determination, and the previously determined treatment amount is not directly used.

[0014] Furthermore, each round of wet milling is carried out in a local loop consisting of an independent, sealed feeding container, wet milling equipment, and connecting pipelines. The remaining masterbatch in the main fermentation container does not enter the wet milling loop of this round. The outlet temperature of the wet mill is controlled to not exceed 35°C, and the wet milling endpoint is defined as the particle size of 90% of the particles in the wet particle size distribution not exceeding 0.8 mm. The resulting slurry is returned from below the liquid surface of the main fermentation container through a sealed pipeline, and the returned material does not re-enter the wet milling loop of this round.

[0015] Furthermore, if the remaining wet solid phase is insufficient to meet the processing capacity of the measured treatment level, a final round of testing is conducted based on the actual mass of the remaining wet solid phase and the mass ratio of the current total yeast feed. After passing the test, wet milling and reprocessing are performed. After all the wet solid phase is recovered and the acidification recovery conditions are met, anaerobic fermentation continues until the pH is not higher than 4.2 and the change is not more than 0.1 in two consecutive tests at 6-hour intervals, and the change in titration acidity is not more than 5%. Then, the temperature is lowered and the solid-liquid mixture is collected, or the liquid product is collected through solid-liquid separation.

[0016] Furthermore, the present invention also provides a compound fermentation product of leafy grass enzymes, wherein the compound fermentation product is a liquid product or a solid-liquid mixture obtained by the above preparation method.

[0017] Furthermore, the present invention also provides the application of the compound fermentation product of the leafy grass enzyme in the preparation of fermented feed or drinking water for farmed animals.

[0018] The beneficial effects achieved by the present invention using the above solution are as follows:

[0019] First, by dividing the material after aerobic pre-fermentation into a liquid phase containing bacteria and a wet solid phase retaining coarse particle structure, the liquid phase containing bacteria is first subjected to anaerobic acidification. Then, the amount of material to be processed in each round is determined according to the actual decomposition load of the wet solid phase on the current yeast material. This changes the release of substances inside the plant tissue from concentrated release to a sequential release that is adapted to the current transformation state of the fermentation system, reducing the possibility that the material release load caused by one-time deep crushing will not match the subsequent acidification process.

[0020] Second, by using the same batch of yeast feed and the remaining wet solid phase to set different candidate mass ratios, and by combining pH changes and titration acidity changes after actual wet milling and rounds to determine the amount of wet solid phase to be processed in each round, the amount of wet solid phase to be decomposed can be adjusted according to the state of the raw materials and the fermentation process, reducing the deviation caused by switching stages based solely on fixed time, fixed feed ratio or a single pH value.

[0021] Third, by using the current yeast feed as the wet grinding medium in the wet solid phase and directly returning the ground material to the original main fermentation vessel, the fermentable substances released after the plant tissue is decomposed can directly enter the ongoing anaerobic fermentation system. At the same time, there is no need to add water, sugar source, acidifier or other batches of mature fermentation liquid outside the formula, thus maintaining the continuity of the raw material composition and fermentation system of the same batch.

[0022] Fourth, by limiting each round of wet milling to an independent local loop, the yeast material that has not been extracted from the main fermentation vessel does not pass through the wet milling equipment, and the material that has been returned is restricted from re-entering the wet milling loop in this round. While completing the further deconstruction of the wet solid phase, the impact of repeated mechanical shearing and temperature rise caused by wet milling on the entire batch of fermentation material is reduced.

[0023] Fifth, by determining the amount of wet solid phase treatment in the next round after each round of deconstruction and acidification recovery conditions are met, the fermentable substances released in the previous round can participate in subsequent anaerobic transformation, and the fermented state after transformation is used as the basis for the next round of treatment. This forms a preparation process in which plant tissue is deconstructed in rounds, substances are released gradually, and the fermentation system is continuously transformed. Attached Figure Description

[0024] Figure 1 This is a flowchart of the preparation method of the compound fermentation product of leafy grass enzyme of the present invention. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] This embodiment uses *Artemisia argyi* as the main plant material, combined with *Artemisia argyi* and other plant materials. Through aerobic pre-fermentation, phase separation of the same batch of materials, initial acidification of the bacterial liquid phase, successive rounds of wet-solid phase decomposition, and subsequent anaerobic fermentation, a complex fermentation product of *Artemisia argyi* enzymes is prepared. The *Artemisia argyi* enzyme complex fermentation product is a liquid or solid-liquid mixture formed from plant materials through microbial transformation, and is not limited to a single enzyme preparation.

[0027] This implementation method maintains the same composition, proportion, microbial composition, and total inoculum amount for each plant material in the same batch. All plant materials and microbial strains are added before the start of aerobic pre-fermentation. No plant material is selectively discarded during subsequent phase separation and round-by-round treatment, and no additional sugar sources, acidifiers, alkaline neutralizers, or mature fermentation broth from other batches are added. Reagents used for testing are not introduced into the production system, and the protective gas is used only to replace air and maintain a low-oxygen environment.

[0028] The combination of microbial strains used should be able to complete aerobic pre-fermentation in the mixed plant materials and continue to utilize the fermentable substrates for acid production after the oxygen supply is stopped. This embodiment determines whether the current raw materials and microbial system meet the conditions for sequential deconstruction treatment by first acidifying the liquid phase containing bacteria and then measuring the actual material carried over. Not all edible fungi strains are considered capable of completing the process.

[0029] Weigh each plant material according to the same measurement standard, remove mud, sand, rotten tissue and foreign matter, and wash and drain the surface water. When using fresh weight measurement, use the weight of the washed and drained raw materials as the basis for feeding; when using dry matter measurement, determine the feeding weight based on the moisture content of the raw materials. Record the source, part used, feeding weight and moisture content of each raw material to avoid mixing fresh weight ratios and dry matter ratios.

[0030] like Figure 1 As shown, the plant materials are coarsely crushed using cutting or low-speed shredding methods, forming coarse particles of 5-10 mm in length for the main plant tissues, while retaining identifiable leaf, vein, and stem segments. After coarse crushing, the plant materials are mixed evenly, and the inoculum and process water from the original formula are added. All materials are ground into a uniform fine slurry before inoculation.

[0031] The mixture was fed into a fermentation vessel equipped with aeration, low-speed stirring, and temperature control functions. The aerobic pre-fermentation temperature was set at 32°C, and the treatment time was 18 hours. Filtered air and intermittent stirring ensured that different parts of the material were exposed to air. Stirring aimed to agitate the material and reduce localized accumulation, preventing premature breakup of coarse particles due to continuous high-speed shearing. During fermentation, the material temperature was controlled via a jacketed heat exchanger, and adjustments were made when the temperature deviated from the set value.

[0032] After aerobic pre-fermentation, coarse solid-liquid separation is performed using a sieve with a pore size of 1–2 mm. The flowing liquid phase is first collected by natural drainage, supplemented by low-intensity pressing if necessary. During the separation process, the coarse particle state of the wet solid phase is retained, without the aim of completely crushing the plant tissue. The resulting liquid phase retains the fermenting microorganisms, soluble components, and fine suspended matter, and is not subjected to sterilization filtration, serving as the bacterial liquid phase; the plant tissue and its attached liquid retained on the sieve are considered the wet solid phase.

[0033] Record the mass of the bacterial liquid phase and the wet solid phase separately, and maintain the same batch label. Gently stir the wet solid phase to disperse coarse particles from different plant sources, then place it in a sealed temporary storage container and store it at 4–8°C. During temporary storage, the precipitated liquid is retained along with the corresponding wet solid phase, and gently mixed again before subsequent sampling and processing. Do not pour the precipitated liquid separately into the main fermentation vessel.

[0034] This implementation sets 48 hours as the upper limit of the cumulative time for completing each round of wet solid-phase processing. This time is calculated from the end of the coarse solid-liquid separation and includes the time spent on initial liquid phase acidification, load measurement, acidification recovery, and temperature adjustment before wet milling. Transferring containers or repackaging does not restart the calculation. This upper limit is a process management condition and is not an independent basis for judging the safety of wet solid-phase storage. If the wet solid-phase exhibits abnormal spontaneous temperature rise, visible mold growth, or other quality abnormalities, it should be discontinued from the normal processing flow.

[0035] The bacterial-containing liquid phase is introduced into the main fermentation vessel, maintained at 32°C, and air supply is stopped. The material is fed in and out through a closed pipeline. Nitrogen is used to replace the air at the top of the vessel, reducing the gaseous oxygen content to below 1%. Operating conditions are then maintained to prevent air ingress. The main fermentation vessel is equipped with an exhaust structure and pressure relief protection to prevent backflow of air, allowing the gases produced during fermentation to escape. The gaseous oxygen content is used to confirm the state of air replacement at the top and is not the sole criterion for determining whether the liquid phase has been completely deoxygenated.

[0036] During the initial acidification in the liquid phase, the pH was measured every hour, and the titration acidity was measured every two hours. When the pH of the bacterial-containing liquid phase reached below 4.2, the wet solid phase decomposition loading test was initiated. This pH is only used as a condition for initiating the test; whether the addition of wet solid phase decomposition material is permitted is still determined based on the actual test results with the substrate. If the bacterial-containing liquid phase fails to establish the required acidification state, no temporary acid supplementation, addition of sugar sources, or replacement of the bacterial strain is used to meet the treatment conditions.

[0037] In this embodiment, all materials currently participating in fermentation in the main fermentation vessel are referred to as fermentation yeast stock. Before the first round of treatment, the fermentation yeast stock mainly consists of a pre-acidified bacterial liquid phase; before subsequent rounds of treatment, the fermentation yeast stock also includes the small plant tissues and their fermentation products returned from previous rounds. The wet solid phase treatment volume for each round is based on the actual mass of all fermentation yeast stock in the main vessel, not on the amount of wet grinding media extracted in this round.

[0038] The acidification tolerance refers to the processing capacity of the pre-fermented yeast feed, under specified temperature, low-oxygen conditions, wet milling degree, and reprocessing method, to meet the specified initial acidity changes and subsequent acidification recovery conditions after receiving a certain amount of wet solid phase decomposed material. This capacity is determined by actual material measurements of the same batch of masterbatch and the wet solid phase to be treated, and is not only determined based on the current pH or titratable acidity of the masterbatch.

[0039] All pH measurements were performed using electrodes suitable for liquids and suspensions. Before measurement, the samples were calibrated using the appropriate standard buffer solution, brought to 25°C, gently mixed, and then a stable value was read. The pH thresholds described below are based on this temperature and measurement method. When online pH monitoring is used during production, the online readings are used for trend observation; formal determinations are based on sampling and measurement results under the same conditions, or on a pre-established correlation between online readings and sampling and measurement results.

[0040] The acidity titration was performed using a fixed sample volume and a fixed titration endpoint. Specifically, 10.00 g of the mixed sample was weighed, 90 g of analytical water (with carbon dioxide removed) was added, and the mixture was stirred at 25°C for 10 minutes. The solution was then titrated with a standardized 0.1 mol / L sodium hydroxide solution until the pH reached 8.2, and maintained for 30 seconds. The volume of alkali consumed after deducting the analytical water blank was recorded. The process titration acidity is expressed as the volume of alkali consumed per 10 g of original sample. The same sampling, pretreatment, and titration methods were used for each assay.

[0041] Titration acidity determination is performed using parallel samples for verification, and the analyzed samples are not returned to the production system. The titration acidity is a process control indicator and is not directly equivalent to the content of lactic acid, acetic acid, or other specific organic acids, nor is it used to directly calculate acid production.

[0042] Before each load test, the masterbatch in the main container was mixed at a low speed, and samples were taken from different depths. If obvious stratification was observed, mixing was continued and samples were taken again. For subsequent rounds of masterbatch samples, the existing fine plant tissues were retained, and only the upper clear liquid was taken. At least five subsamples were taken from different locations of the remaining wet solid phase, gently mixed to form a representative solid sample, and its temperature was kept close to that of the masterbatch sample.

[0043] Multiple candidate treatment amounts were prepared by configuring 10, 20, 30, and 40 parts by weight of wet solid phase for every 100 parts by weight of masterbatch. Each candidate treatment amount was prepared using a sample of the same masterbatch after a single mixing process and a sample of the same representative wet solid phase, and the measurements were performed in parallel. The actual configuration amount of the small sample was determined according to the needs of subsequent sampling and testing, and the mass relationship of each part was kept unchanged when scaling up or down the small sample.

[0044] For each candidate throughput, a portion of the masterbatch equivalent to twice the mass of the wet solid phase is taken from that masterbatch group as the wet grinding media, and the remaining masterbatch is retained in the receiving container of that group. For example, when 100 grams of masterbatch is used to prepare 20 grams of wet solid phase, 40 grams are taken from the masterbatch group for wet grinding, and the remaining 60 grams are used to receive the material after wet grinding. The wet grinding media is taken from the existing masterbatch in that group; no additional masterbatch or water is added.

[0045] The wet solid phase and the extracted masterbatch were mixed under closed conditions and subjected to secondary decomposition using a rotor-stator wet mill. The rotor-stator gap was set to 0.5–1.0 mm, and coarse particles were further refined through a single pass or a limited number of cycles. During the treatment, the outlet temperature of the wet mill was controlled to not exceed 35°C. Small-sample measurements and formal treatments used the same decomposition endpoint and temperature control conditions, and the correspondence between the two was determined not only by whether the equipment rotation speed was the same.

[0046] When using wet particle size analysis, particles with a cumulative volume of 90% having a diameter no greater than 0.8 mm are considered the proposed decomposition endpoint in this embodiment. Since subsequent batches of masterbatch already contain fine particles formed in previous batches, samples must be taken from the front, middle, and rear sections of the wet mill output. After mixing, these samples are passed through a 0.8 mm sieve for auxiliary inspection to observe whether intact leaves, vein clusters, or stem segments close to the initial coarsely broken size remain on the sieve. If such tissues are present, a limited number of processing cycles are performed in the local loop of this batch, adhering to temperature control conditions. Particle size and sieving results are used to define the degree of tissue refinement and are not directly used as proof of complete cell rupture.

[0047] The small sample of wet-milled material is fed back into the remaining masterbatch in the same batch according to the prescribed method, and is gently mixed during the feeding back. When continuous feeding is used in formal production, the small sample is added continuously in a quantitative manner or in small amounts in multiple batches, and the feeding back duration corresponds to the formal processing. In this embodiment, the planned completion time for a single round of wet milling and feeding back is set to within 15 minutes, and the specific time used is consistent between the small sample round and the formal processing.

[0048] The acidification recovery time was calculated from the moment all wet milled material was recombined and fully mixed. pH was measured at 5, 15, and 30 minutes after the first round, and then every hour thereafter, with an additional measurement in the last 30 minutes of the 4-hour observation period. The titratable acidity 30 minutes after the first round was used as the initial acidity for this candidate treatment and compared with the titratable acidity after 4 hours of continued fermentation. The comparisons were made with materials after the addition of the same wet solid phase; recovery to the acidity before the addition of the wet solid phase was not required.

[0049] Candidate treatments are deemed to have passed this round of load testing if they simultaneously meet the following conditions: The pH value is not higher than 4.5 at each detection point during the return process and in the first 30 minutes after the round; the pH values ​​at the last two 30-minute intervals during the 4-hour observation period are not higher than 4.2; and the titratable acidity after 4 hours of continued fermentation increases by at least 10% compared to the initial titratable acidity 30 minutes after the round, confirmed by parallel measurements. Candidate treatments that only meet the initial pH conditions but not the subsequent recovery conditions are not considered qualified treatments.

[0050] From all the candidate treatment quantities that passed the initial assessment, select one level for the formal treatment. If a higher single-round treatment quantity is needed, select the higher level that has already passed the assessment; if a treatment margin needs to be maintained, select the lower level that has also already passed the assessment. Without prior assessment, it is not assumed that a lower or higher treatment quantity will necessarily meet the requirements. If all candidate treatment quantities fail, no formal secondary deconstruction will be performed in this round. Fermentation can continue within the allowed waiting time, followed by resampling, or other candidate treatment quantities can be added for assessment.

[0051] Before formal processing, mix again and verify the current state of the masterbatch. If the pH of the masterbatch changes by more than 0.1, the titratable acidity changes by more than 10%, or there is significant sedimentation, floating, or other changes in state compared to the sampling at the time of load testing, the previous test results should not be directly used. Even if the pH of the masterbatch decreases further, the wet solid phase treatment volume should not be temporarily increased accordingly.

[0052] The wet solid phase used for formal processing should correspond to the representative sample taken for the load test. If significant leaching, localized temperature rise, or further tissue disintegration occurs in the wet solid phase during the test, the corresponding precipitate should be remixed and resampled. In each round, only the wet solid phase of a certain processing volume is removed from refrigeration conditions, while the remaining wet solid phase continues to be temporarily stored to avoid repeated heating and cooling of the entire wet solid phase.

[0053] Based on the actual mass of the current masterbatch, the mass of the determined sample is mapped to the processing volume for this round. For example, if the acceptable level is 20 parts by mass of wet solid phase per 100 parts by mass of masterbatch, and there are 100 kg of masterbatch in the main container, then 20 kg of wet solid phase will be processed in this round, with 40 kg taken from the masterbatch as wet grinding media, leaving 60 kg of masterbatch in the main container. After all the wet grinding media is returned, the material in the main container will be approximately 120 kg. The actual mass is based on the weighing result after deducting the sampling and equipment residue. This example is for illustrative purposes only.

[0054] The masterbatch extracted in this round is fed into a separate, sealed wet mill feed container and mixed with the wet solid phase from this round. The feed temperature is brought into contact with the small sample testing conditions through jacket heat exchange, followed by secondary decomposition. When repeated passage through the wet mill is required, the circulation range is limited to the feed container, the wet mill, and its connecting pipelines; the remaining masterbatch in the main container does not enter the wet mill cycle.

[0055] After the material reaches the decomposition endpoint in this round of grinding, it is returned to the main fermentation vessel through a closed pipeline. The return port is located below the liquid surface to reduce air entrainment during the return process. The returned material is not continuously extracted from the main vessel for repeated wet grinding. When it is necessary to recover pipeline residue, the same batch of masterbatch is used for flushing, and the masterbatch used for flushing is included in the current round's medium volume. No additional water is added to change the mixing relationship.

[0056] Record the outlet temperature, reflow time, and material state in the main container during wet grinding and reflow. If the temperature exceeds the limit, the equipment cannot deliver material stably, or the degree of decomposition is inconsistent with the sample, suspend operation and investigate. If it is necessary to change the wet grinding medium volume, decomposition endpoint, or reflow duration, re-measure the load according to the changed conditions, and continue feeding directly without making temporary adjustments.

[0057] After all the wet milled material has been returned, the material in the main container will be mixed at a low speed until homogeneous, and fermentation will continue under 32°C and low-oxygen conditions. Acidification recovery will be observed using the same testing time points and judgment conditions as the small sample. If the pH exceeds the upper limit of control during the return process, wet solid phase feeding and milled material return will be suspended. Milled material not yet returned will be temporarily stored in a separate, sealed receiving container. Any abnormalities during the return process will be recorded separately and will not be ignored even if the pH returns to the required level after all material has been returned.

[0058] Once the recovery conditions for this round are met, the current masterbatch mass is re-weighed or verified. Samples are then taken from the current masterbatch and the remaining wet solid phase to determine the processing volume for the next round. The remaining wet solid phase remains in a coarse particle state until the corresponding processing round, and is not pre-ground before being added. The processing volumes and intervals for each round can be different; it is not required that the processing volumes of subsequent rounds gradually increase, nor is it necessary to use the processing volume of the first round until all materials have been processed.

[0059] If the remaining wet solid phase is insufficient for a complete processing cycle, a candidate processing relationship for the final round is established based on its actual quality and the current masterbatch quality. Measurements are then performed before wet milling and reprocessing; the decision is not omitted due to a small remaining quantity. After the final round, it is still necessary to confirm that the material from that round meets the acidification recovery conditions before proceeding to subsequent heat preservation fermentation.

[0060] The wet solid phase storage time limit is managed in conjunction with the sequential processing rounds. Small samples of different candidate processing volumes are measured in parallel, but each formal processing round is conducted sequentially according to the current state of the masterbatch. If the remaining time is insufficient to complete the next round of measurement and processing, or if the recovery time after a formal round exceeds the set value, the reception of wet solid phase according to the normal process is stopped, and the remaining material is not crushed and fed in all at once. Batches that cannot be fully returned are recorded separately, and products that are not recorded as having completed sequential processing of all formulation materials are not recorded.

[0061] After all wet solid phase has been decomposed and returned, anaerobic fermentation continues, and the entire batch of masterbatch is no longer subjected to repeated high-intensity wet milling. In this implementation method, the pH value is no higher than 4.2 and the change between the two measurements is no more than 0.1, and the titratable acidity change is no more than 5%, in two consecutive measurements taken 6 hours apart, as reference conditions for ending the continued heat preservation process. This endpoint determination is set separately from the load recovery determination for each round, and it is not required that the titratable acidity continue to increase by 10% during the endpoint observation period.

[0062] Once the process values ​​stabilize, post-processing arrangements are determined in conjunction with product quality testing. Product qualification is not solely determined by the cessation of pH or titratable acidity changes. The pH, titratable acidity, and observation time mentioned are used to control the decomposition rounds and fermentation process, and do not replace the judgment of raw material quality, microbiological hygiene, or product suitability.

[0063] After fermentation, the material is cooled and post-processed. When preparing a solid-liquid mixture, the fermentation broth and small plant tissues are retained, mixed at low speed, and then packaged. When preparing a liquid product, the fermentation material is coarsely filtered or subjected to solid-liquid separation, and the liquid phase is collected. The separated liquid product and the unseparated solid-liquid mixture are sampled and tested separately; the composition of the whole material before separation is not directly used as the composition of the liquid product.

[0064] For products intended to retain live bacteria, the post-processing stage will not employ terminal treatments aimed at killing bacteria. Instead, refrigeration and packaging conditions that maintain the corresponding quality requirements will be used. Packaging should consider the possibility of continued gas production, avoiding the direct loading of continuously gas-producing materials into containers lacking appropriate pressurization or venting capabilities. The specific shelf life will be determined based on product stability; the wet-solid phase storage period will not be used as the finished product shelf life.

[0065] Before sampling, the product should be mixed at a low speed, and samples should be taken from different locations and combined to form a representative sample. The product can be characterized by indicators such as water-extracted soluble nitrogen, free amino acids, major organic acids, plant active ingredients, and the corresponding fermentation bacteria count, and the sampling status, pretreatment method, and unit of measurement for each indicator should be clearly defined.

[0066] In a detection setup for water-extractable soluble nitrogen, 10 grams of a well-mixed sample is weighed and added to 90 grams of analytical water. The mixture is stirred at 25°C for 10 minutes, centrifuged at 6000 times the force of gravity for 10 minutes, and the supernatant is collected to determine the nitrogen content, which is then converted to the original sample volume. This indicator is called water-extractable soluble nitrogen under specified conditions and is not directly equivalent to the content of soluble proteins or small peptides. When detecting free amino acids, small peptides, and components such as lactic acid and acetic acid, detection methods that can distinguish the corresponding substances are used respectively, and the actual types and contents detected are reported.

[0067] The resulting compound fermentation product can be used as an additive in the preparation of fermented feed. It is added to the feed to be fermented by spraying or mixing to ensure uniform distribution of liquid and fine solids, and then the subsequent fermentation is completed according to the corresponding feed processing conditions. When adding liquid product, the moisture it introduces is included in the total water consumption of the feed. This application is not based on the premise that this product directly replaces a fixed proportion of soybean meal or other protein raw materials.

[0068] When using liquid products to prepare drinking water for farmed animals, select liquid products that are compatible with the filtration and delivery conditions of the water supply equipment, and determine the addition method and dosage based on the animal species, the actual composition of the product, and the usage conditions. This application is limited to farmed animals and is not extended to nutrient solutions for human consumption, nor is the achievement of acidity conditions during the process directly used as a basis for determining whether the solution is potable.

[0069] Through the above preparation process, an acidification state is first established using the same batch of bacterial-containing liquid phase. Then, the processing volume for each round is determined based on the actual material load caused by the wet solid phase under specified decomposition conditions, and the current masterbatch is used as the wet grinding medium for the corresponding wet solid phase. After each round of decomposition, the material is returned for further fermentation. Once the recovery conditions are met, the next round of wet solid phase is processed, thus linking the release sequence of substances within the plant tissue with the processing state of the same batch of fermentation system. At the same time, the coarse particle state of the unprocessed wet solid phase is preserved, limiting the repeated passage of the returned masterbatch through the wet grinding equipment.

[0070] The above are merely specific embodiments of the present invention and are not intended to limit the invention. For those skilled in the art, any modifications, equivalent substitutions, or improvements made to the above embodiments without departing from the inventive concept should fall within the protection scope of the present invention. The protection scope of the present invention is defined by the claims, and the specification and drawings can be used to interpret the claims.

Claims

1. A method for preparing a compound fermentation product of leafy green enzymes, characterized in that, Includes the following steps: Plant materials, including leafy grass and mugwort, are coarsely crushed, mixed with fermentation bacteria for aerobic pre-fermentation, and then subjected to coarse solid-liquid separation to obtain a liquid phase containing fermentation microorganisms and a wet solid phase containing coarse particle structure. The bacterial liquid phase is placed in the main fermentation vessel for anaerobic acidification, the wet solid phase is temporarily stored in a sealed container, and all the materials currently participating in fermentation in the main fermentation vessel are used as yeast feed. In each round of processing, samples were taken from the mixed yeast feed and the remaining wet solid phase. Test groups were configured according to multiple candidate mass ratios of wet solid phase to masterbatch. A portion of the masterbatch sample in each test group was used as wet grinding medium to wet grind the wet solid phase sample in that group. The resulting slurry was then combined with the remaining masterbatch sample in that group. The pH was measured during and after each round of testing, and the changes in pH and titrated acidity were measured during the continued anaerobic fermentation. Candidate mass ratios that simultaneously met the pH control conditions and acidification recovery conditions were selected. Based on the selected candidate mass ratios and the actual mass of all yeast material in the main fermentation vessel, the wet solid phase treatment amount for this round was determined. According to the proportion of masterbatch used in the corresponding test group, wet milling endpoint, temperature conditions and round method, under closed low oxygen conditions, a portion of the yeast material taken from the main fermentation container is used as the wet milling medium to wet mill the wet solid phase in this round, and the resulting slurry is sent back to the main fermentation container, mixed with the yeast material retained therein and then anaerobic fermentation continues. After the fermentation material meets the acidification recovery conditions after the round, the sample is resampled to determine the amount of wet solid phase to be processed in the next round. The wet milling, round and anaerobic fermentation are repeated until the wet solid phase is completely processed and returned. Then, anaerobic fermentation is continued and post-processing is performed to obtain the compound fermentation product. The pH control condition is that the pH detection value during the round and the preset initial time period after the round does not exceed the preset control upper limit, and the acidification recovery condition is that the pH detection value during the preset recovery time period reaches below the preset recovery upper limit, and the titration acidity increases to a preset range compared to the initial titration acidity after the round.

2. The method for preparing the compound fermentation product of leafy green enzymes according to claim 1, characterized in that, All plant materials and fermentation strains are added before the start of aerobic pre-fermentation. No additional sugar sources, acidifiers, alkaline neutralizers, or other batches of mature fermentation broth are added during subsequent processing. The wet solid phase is temporarily stored in a sealed container at 4–8°C. The liquid precipitated during the temporary storage period is mixed with the corresponding wet solid phase and then sampled, measured, and processed together.

3. The method for preparing the compound fermentation product of leafy green enzymes according to claim 1, characterized in that, The testing groups were configured with 10, 20, 30, and 40 parts of wet solid phase for every 100 parts of masterbatch. Each testing group took out a portion of masterbatch equivalent to twice the mass of the wet solid phase from its own group as wet grinding media, and the remaining masterbatch was used to receive the grinding slurry from its own group. The masterbatch samples from subsequent rounds retained the fine plant tissue returned from the previous rounds.

4. The method for preparing the compound fermentation product of leafy green enzymes according to claim 1, characterized in that, The pH control condition is that the pH values ​​detected during the round and at 5, 15, and 30 minutes after the round are not higher than 4.5; the acidification recovery condition is that the last two pH values ​​at 30-minute intervals during the 4-hour observation period after the round are not higher than 4.2, and the titration acidity 4 hours after the round is not less than 10% higher than the titration acidity 30 minutes after the round is completed; the round completion means that all the pulped material has been added to the corresponding remaining masterbatch and the mixing is completed.

5. The method for preparing the compound fermentation product of leafy green enzymes according to claim 4, characterized in that, The pH value is the measured value after the sample is adjusted to 25°C; the titration acidity is determined by the following method: weigh 10.00 g of the mixed sample, add 90 g of analytical water to remove carbon dioxide, stir at 25°C for 10 minutes, titrate with a standardized 0.1 mol / L sodium hydroxide solution to pH 8.2 and maintain for 30 seconds, and express the titration acidity per 10.00 g of sample as the volume of alkali consumed after deducting the analytical water blank.

6. The method for preparing the compound fermentation product of leafy green enzymes according to claim 1, characterized in that, After determining the wet solid phase treatment volume for this round and before performing this round of wet milling, the yeast feed is remixed and its pH and titratable acidity are checked. If the pH changes by more than 0.1 or the titratable acidity changes by more than 10% compared with the masterbatch sampled in the corresponding test group, the sample is resampled for candidate mass ratio determination, and the previously determined treatment volume is not directly used.

7. The method for preparing the compound fermentation product of leafy green enzymes according to claim 1, characterized in that, Each round of wet milling is carried out in a local loop consisting of an independent, sealed feeding container, wet milling equipment, and connecting pipelines. The remaining masterbatch in the main fermentation container does not enter the wet milling loop of this round. The outlet temperature of the wet mill is controlled to not exceed 35°C, and the wet milling endpoint is defined as the particle size of 90% of the particles in the wet particle size distribution not exceeding 0.8 mm. The resulting slurry is returned from below the liquid surface of the main fermentation container through a sealed pipeline, and the returned material does not re-enter the wet milling loop of this round.

8. The method for preparing the compound fermentation product of leafy green enzymes according to claim 1, characterized in that, When the remaining wet solid phase is insufficient to meet the processing capacity of the measured treatment level, the final test is performed according to the ratio of the actual mass of the remaining wet solid phase to the mass of all the current yeast feed. After passing the test, wet milling and reprocessing are carried out. After all the wet solid phase is recovered and the acidification recovery conditions are met, anaerobic fermentation continues until the pH is not higher than 4.2 and the change is not more than 0.1 in two consecutive tests with a 6-hour interval, and the change in titration acidity is not more than 5%. Then, the temperature is lowered and the solid-liquid mixture is collected, or the liquid product is collected through solid-liquid separation.

9. A compound fermentation product of leafy green enzymes, characterized in that, The composite fermentation product is a liquid product or a solid-liquid mixture obtained by the preparation method according to any one of claims 1 to 8.

10. The application of the compound fermentation product of leafy grass enzymes as described in claim 9 in the preparation of fermented feed or drinking water for farmed animals.

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