Multi-strain synergistic oxygen-consuming anaerobic feed fermentation method

Abstract

The present application relates to the technical fields of microbial fermentation and feed processing, and discloses a multi-strain synergistic oxygen-consuming anaerobic feed fermentation method, which comprises the following steps: inoculating a compound fermentation inoculum containing an oxygen-consuming functional flora and an anaerobic functional flora into a feed fermentation substrate with adjusted moisture content and initial pH value at one time, and the ratio of viable bacterial count is 1:1.5-1:2.5; performing first-stage oxygen-consuming fermentation on the fermentation system, maintaining oxygen supply operation until the internal center temperature naturally rises to 40-45 DEG C; then stopping the oxygen supply operation and not performing secondary inoculation, directly applying mechanical pressure to the fermentation system to perform forced compaction and exhaust and performing physical sealing, so that the system performs second-stage anaerobic fermentation until the pH value drops to a preset acidic range, and fermented feed is prepared. The process conversion triggered by one-time inoculation and natural temperature rise avoids the introduction of external miscellaneous bacteria by segmented inoculation, and a low-oxygen high-acidity environment is constructed through microbial metabolites and physical sealing.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation and feed processing technology, specifically a method for synergistic fermentation of multi-strain aerobic anaerobic feed. Background Technology

[0002] In feed fermentation processes utilizing complex microbial communities, different metabolic types of microorganisms have varying requirements for the physicochemical environment at different stages of fermentation. Traditional multi-strain stepwise fermentation processes typically involve first inoculating aerobic functional microorganisms for the first stage of fermentation, followed by opening the fermentation vessel to add anaerobic functional microorganisms for the second stage. This process suffers from a disruption of the physical barrier during the transition between the two fermentation stages, as opening the fermentation vessel directly introduces gases and impurities from the external environment. This provides a physical pathway for external microbial contamination, leading to fluctuations in the number of harmful bacterial colonies and physicochemical indicators such as volatile basic nitrogen in the final fermented feed.

[0003] To avoid the technical problem of introducing contaminating microorganisms during secondary opening of the fermentation tank, existing technologies employ a single-stage inoculation method involving mixed fermentation strains with different metabolic types. However, in conventional single-stage inoculation processes, the fermentation system lacks clearly defined physical or chemical parameters to trigger the division of process stages. Aerobic and anaerobic microorganisms exist in a state of disordered metabolic competition within the same physical space, with the aerobic metabolism of aerobic functional groups overlapping with the acid production metabolism of anaerobic functional groups. This competition leads to a decreased survival rate of anaerobic functional groups under the high oxygen partial pressure environment at the beginning of fermentation, while aerobic functional groups are not subject to strict physical inhibition in the later stages of fermentation. A clear spatiotemporal succession pattern cannot be established within the fermentation system, failing to form a stable low oxygen partial pressure and high acidity physicochemical environment. Consequently, the target fermentation product, such as lactic acid, fails to meet quality standards, and the spore germination activity of aerobic spoilage bacteria and molds during product storage cannot be effectively suppressed. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a multi-strain synergistic aerobic anaerobic feed fermentation method, which solves the problems of easy introduction of external contaminants in the stepwise inoculation of multiple strains in existing feed fermentation processes, and easy disruption of microbial competition during fermentation due to the inoculation of different metabolic types of strains at one time.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for synergistic fermentation of multi-strain aerobic anaerobic feed, comprising the following steps:

[0007] A compound fermentation agent is inoculated into a feed fermentation substrate with adjusted moisture content and initial pH value to obtain a fermentation system. The compound fermentation agent contains aerobic functional bacteria and anaerobic functional bacteria, and the ratio of the number of live bacteria in the aerobic functional bacteria to the anaerobic functional bacteria is 1:1.5-1:2.5.

[0008] The fermentation system is subjected to the first stage of aerobic fermentation, and oxygen supply is maintained during the fermentation process until the internal core temperature of the fermentation system naturally rises to 40℃-45℃, at which point the first stage of aerobic fermentation is terminated.

[0009] After the first stage of aerobic fermentation is completed, without secondary inoculation, mechanical pressure is applied to the fermentation system to force compaction and degassing, and physical sealing is performed to allow the fermentation system to undergo the second stage of anaerobic fermentation until the pH value of the fermentation system drops to the preset acidic range, thus obtaining the finished fermented feed.

[0010] Furthermore, the aerobic functional bacterial community is composed of Bacillus, Yeast, and Mold in a live bacterial count ratio of 2.5-3.5:1.5-2.5:0.5-1.5;

[0011] The anaerobic functional bacterial community consists of homo-fermenting lactobacillus, hetero-fermenting lactobacillus, and cocci in a live count ratio of 3.5-4.5:2.5-3.5:1.5-2.5.

[0012] Furthermore, the Bacillus genus in the aerobic functional bacterial community is Bacillus subtilis, Bacillus licheniformis, or Bacillus amyloliquefaciens; the yeast genus is Saccharomyces cerevisiae, Candida utilis, or Hansenula polymorpha; and the mold genus is Aspergillus oryzae, Aspergillus niger, or Trichoderma viride.

[0013] The homofermentative lactobacilli in the anaerobic functional bacterial group are Lactobacillus plantarum, Lactobacillus acidophilus, or Lactobacillus casei; the heterofermentative lactobacilli are Lactobacillus bryonicus or Lactobacillus fermentum; and the cocci are Pediococcus pentosaceus.

[0014] Furthermore, the feed fermentation substrate comprises basic raw materials and auxiliary materials; the basic raw materials include, by mass fraction: 35.0%-45.0% corn flour, 25.0%-35.0% soybean meal, 10.0%-20.0% wheat bran, 5.0%-15.0% fiber by-products, and 3.0%-8.0% trace element and vitamin premix; the auxiliary materials include 0.5%-2.0% of readily available carbon source and 0.3%-0.8% of readily available nitrogen source, accounting for 0.5%-2.0% of the total dry matter mass of the basic raw materials.

[0015] Furthermore, the compound fermentation agent is inoculated into the feed fermentation substrate in the form of a microbial activation solution; the microbial activation solution is prepared by inoculating the compound fermentation agent into a sugar solution at 30℃-35℃ and activating it by standing for 1.0-2.0h; and the inoculation amount, based on the mass of the compound fermentation agent, is 0.3%-3.0% of the total dry matter mass of the feed fermentation substrate.

[0016] Furthermore, the total moisture content of the feed fermentation substrate, after adjusting the moisture content and initial pH value, is 55.0%-70.0%, and the initial pH value is 6.0-7.0.

[0017] Furthermore, the first stage of aerobic fermentation specifically involves: spreading the fermentation system with a material pile thickness of 5.0-10.0 cm and fermenting it for 18.0-48.0 h at an initial ambient temperature of 28℃-35℃; the oxygen supply operation involves intermittent turning or forced ventilation every 8.0-12.0 h.

[0018] Furthermore, the second stage of anaerobic fermentation specifically involves static fermentation at an ambient temperature of 25℃-32℃ for 60.0-168.0 hours.

[0019] Furthermore, in the second stage of anaerobic fermentation, the preset acidity range has a pH value of 3.8-4.5.

[0020] Furthermore, the physical sealing operation of the second stage of anaerobic fermentation is specifically carried out in a sealed fermentation bag, fermentation tank, silage pit or vertical fermentation tower equipped with a one-way exhaust valve.

[0021] The working mechanism of the technical solution of this invention is as follows:

[0022] A single-inoculation process using a compound fermentation agent resulted in a higher absolute number of viable anaerobic functional bacteria compared to aerobic functional bacteria in the initial state. During the first stage of aerobic fermentation, aerobic functional microorganisms utilized free oxygen in the substrate for aerobic metabolism by maintaining oxygen supply. During this process, the aerobic bacteria secreted extracellular enzymes to hydrolyze large molecules such as fibrous byproducts in the basic raw materials, generating small-molecule carbon sources. Simultaneously, the aerobic respiration metabolism of the microorganisms continuously released heat, leading to heat accumulation within the fermentation system. The system temperature rose non-linearly, accumulating to 40℃-45℃, and the redox potential continuously decreased from an initial positive value to a negative value. In this stage, the proliferation of anaerobic functional bacteria was limited under the oxygen-supplying and temperature-increasing physical environment, with the survival rate fluctuating within a single order of magnitude.

[0023] When the internal temperature of the fermentation system reaches the threshold condition of 40℃-45℃, the oxygen supply operation is terminated, mechanical pressure is applied to vent the gas, and the system is physically sealed to transition to the second stage of anaerobic fermentation. At this point, the oxygen partial pressure inside the fermentation system drops sharply, entering a deep reduction state. Due to the deprivation of oxygen from the environment and the limitation of physical sealing, the vegetative cells of the aerobic functional bacteria undergo lysis or enter a dormant state, resulting in a logarithmic decrease in the total number of viable bacteria. Simultaneously, the 40℃-45℃ temperature environment accumulated in the first stage activates the growth mechanism of thermotolerant homologous and heterologous lactobacilli. The anaerobic functional bacteria utilize the small molecule carbon source produced by enzymatic hydrolysis in the first stage and the established anaerobic microenvironment to undergo logarithmic proliferation, secreting organic acids such as lactic acid. The accumulation of organic acids causes the pH value of the system to decrease from above 5.9 to the range of 3.8-4.5. This method relies on the changes in the physicochemical parameters inside the fermentation system as a control trigger condition to control the alternating proliferation of the complex microbial community according to a time sequence.

[0024] This invention provides a method for synergistic fermentation of aerobic and anaerobic feed using multiple microbial strains. It offers the following beneficial effects:

[0025] 1. This invention employs a technique of inoculating aerobic functional bacteria and anaerobic functional bacteria at a live bacteria ratio of 1:1.5-1:2.5 in a single inoculation, and directly transferring to the second stage of anaerobic fermentation without secondary inoculation after the first stage of fermentation. This feature avoids the physical pathway of introducing external environmental bacteria due to the mid-stage opening of the fermentation vessel for secondary inoculation in segmented fermentation processes, thus cutting off the source of contamination from the process structure and controlling the contamination rate of the fermentation system during process switching.

[0026] 2. This invention utilizes the natural rise in the internal core temperature of the system to 40℃-45℃ during the first-stage aerobic fermentation as a physical trigger condition for terminating oxygen supply and switching processes. This feature uses the accumulation of heat generated by aerobic metabolism in the aerobic bacterial community as an objective monitoring indicator, ensuring that the aerobic bacterial community has the corresponding physiological cycle to secrete extracellular enzymes to degrade macromolecules in the substrate, and directly provides the necessary small-molecule carbon source substrate and initial temperature environment for the rapid proliferation of the second-stage thermotolerant anaerobic functional bacterial community at the physical level.

[0027] 3. This invention applies mechanical pressure directly to the fermentation system once it reaches the temperature threshold for forced compaction and degassing, followed by physical sealing. This allows the system to transition to the second stage of anaerobic fermentation until the pH value drops to 3.8-4.5. This physical degassing and sealing operation, combined with the continuous acid production by anaerobic functional bacteria, creates a dual physicochemical environment within the fermentation system characterized by low oxygen partial pressure and pre-set high acidity. This inhibits the metabolism and spore germination activity of aerobic putrefactive bacteria and molds, maintaining the stability of the physicochemical properties of the finished fermented feed during storage. Attached Figure Description

[0028] Figure 1 This is a graph showing the dynamic change of the central temperature of the system during the fermentation cycle of Example 2 of the present invention.

[0029] Figure 2 This is a graph showing the dynamic changes in redox potential (ORP) during the fermentation cycle of Example 2 of the present invention.

[0030] Figure 3 This is a dynamic change curve of the pH value of the extract during the fermentation cycle of Example 2 of the present invention;

[0031] Figure 4 This is a dynamic change curve of the effective viable number of oxygen-consuming functional bacteria during the fermentation cycle of Example 2 of the present invention.

[0032] Figure 5 This is a dynamic change curve of the effective viable number of anaerobic functional bacteria during the fermentation cycle of Example 2 of the present invention.

[0033] Figure 6 This is a comparison chart of the crude protein enhancement rate and crude fiber degradation rate of the fermentation products of each group in this invention.

[0034] Figure 7 This is a comparison chart of the lactic acid content at the fermentation endpoint of the fermented products of each group in this invention;

[0035] Figure 8 A bar chart comparing the coliform count and Salmonella detection in the fermented products of each group of the present invention;

[0036] Figure 9 Line graph showing the comparison of volatile basic nitrogen content of fermentation products from different groups in this invention;

[0037] Figure 10 This is a dynamic drift trajectory diagram of pH value of each group of fermented products of the present invention at three time points: 0 days, 90 days and 180 days of storage at room temperature.

[0038] Figure 11 This is a bar chart comparing the total number of molds in the fermented products of each group at the end of the 180-day storage period of the present invention. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Preparation Examples 1-4:

[0041] Preparation Example 1:

[0042] This preparation example provides a method for preparing a compound fermentation agent, including the following steps:

[0043] (1) Preparation of aerobic functional bacteria: Accurately weigh live Bacillus subtilis powder, live Saccharomyces cerevisiae powder, and live Aspergillus oryzae powder, and physically mix them at a live bacteria ratio of 2.5:1.5:0.5 to obtain aerobic functional bacteria. After testing, the total effective live bacteria count of the mixed aerobic functional bacteria was found to be 1.0 × 10⁻⁶. 9 CFU / g.

[0044] (2) Preparation of anaerobic functional bacteria: Accurately weigh live Lactobacillus plantarum powder, live Lactobacillus brunelli powder, and live Pediococcus pentosaceus powder, and physically mix them at a live bacteria ratio of 3.5:2.5:1.5 to obtain anaerobic functional bacteria. After testing, the total effective live bacteria count of the mixed anaerobic functional bacteria was found to be 1.0 × 10⁻⁶. 10 CFU / g.

[0045] (3) Compound fermentation agent: The aerobic functional bacteria obtained in step (1) and the anaerobic functional bacteria obtained in step (2) are mixed evenly at a ratio of 1:1.5 to obtain the whole process compound fermentation agent A.

[0046] Preparation Example 2:

[0047] This preparation example provides a method for preparing a compound fermentation agent, including the following steps:

[0048] (1) Preparation of aerobic functional bacteria: Accurately weigh Bacillus licheniformis live bacteria powder, Candida utilis live bacteria powder, and Aspergillus niger live bacteria powder, and physically mix them at a live bacteria ratio of 3.0:2.0:1.0 to obtain aerobic functional bacteria. After testing, the total effective live bacteria count of the mixed aerobic functional bacteria was found to be 2.5 × 10⁻⁶. 9 CFU / g.

[0049] (2) Preparation of anaerobic functional bacteria: Accurately weigh live Lactobacillus acidophilus powder, live Lactobacillus fermentum powder, and live Pediococcus pentosaceus powder, and physically mix them at a live bacteria ratio of 4.0:3.0:2.0 to obtain anaerobic functional bacteria. The total effective live bacteria count of the mixed anaerobic functional bacteria was found to be 2.5 × 10⁻⁶. 10 CFU / g.

[0050] (3) Compound fermentation agent: The aerobic functional bacteria obtained in step (1) and the anaerobic functional bacteria obtained in step (2) are mixed evenly at a ratio of 1:2.0 to obtain the whole process compound fermentation agent B.

[0051] Preparation Example 3:

[0052] This preparation example provides a method for preparing a compound fermentation agent, including the following steps:

[0053] (1) Preparation of aerobic functional bacteria: Accurately weigh live Bacillus amyloliquefaciens powder, live Hansenula polymorpha powder, and live Trichoderma viride powder, and physically mix them at a live bacteria ratio of 3.5:2.5:1.5 to obtain aerobic functional bacteria. The total effective live bacteria count of the mixed aerobic functional bacteria was found to be 5.0 × 10⁻⁶. 9 CFU / g.

[0054] (2) Preparation of anaerobic functional bacteria: Accurately weigh live Lactobacillus casei powder, live Lactobacillus brunelli powder, and live Pediococcus pentosaceus powder, and physically mix them at a live bacteria ratio of 4.5:3.5:2.5 to obtain anaerobic functional bacteria. The total effective live bacteria count of the mixed anaerobic functional bacteria was found to be 5.0 × 10⁻⁶. 10 CFU / g.

[0055] (3) Compound fermentation agent: The aerobic functional bacteria obtained in step (1) and the anaerobic functional bacteria obtained in step (2) are mixed evenly at a ratio of 1:2.5 to obtain the whole process compound fermentation agent C.

[0056] Preparation Example 4:

[0057] This preparation example provides a method for preparing a compound fermentation agent, including the following steps:

[0058] (1) Preparation of aerobic functional bacteria: Accurately weigh live Bacillus subtilis powder, live Bacillus licheniformis powder, live Saccharomyces cerevisiae powder, and live Aspergillus oryzae powder. The live counts of Bacillus subtilis and Bacillus licheniformis each account for half as the Bacillus genus component. The Bacillus genus component, live Saccharomyces cerevisiae powder, and live Aspergillus oryzae powder are physically mixed at a live count ratio of 3.0:2.0:1.0 to obtain the aerobic functional bacteria. The total effective live count of this aerobic functional bacteria after mixing was determined to be 3.0 × 10⁻⁶. 9 CFU / g.

[0059] (2) Preparation of anaerobic functional bacteria: Accurately weigh live Lactobacillus plantarum powder, live Lactobacillus brunelli powder, and live Pediococcus pentosaceus powder, and physically mix them at a live bacteria ratio of 4.0:3.0:2.0 to obtain anaerobic functional bacteria. The total effective live bacteria count of the mixed anaerobic functional bacteria was found to be 3.0 × 10⁻⁶. 10 CFU / g.

[0060] (3) Compound fermentation agent: The aerobic functional bacteria obtained in step (1) and the anaerobic functional bacteria obtained in step (2) are mixed evenly at a ratio of 1:2.0 to obtain the whole process compound fermentation agent D.

[0061] Examples 1-4:

[0062] Example 1:

[0063] This embodiment provides a method for synergistic fermentation of multi-strain aerobic anaerobic feed, including the following steps:

[0064] Weigh out the basic raw materials as follows by mass fraction: 45.0% corn flour, 27.0% soybean meal, 15.0% wheat bran, 5.0% miscellaneous meal, and 8.0% trace element and vitamin premix. Add 0.5% glucose and 0.3% corn steep liquor powder as auxiliary materials, and grind them together to 20 mesh to obtain the feed fermentation substrate.

[0065] The compound fermentation agent A obtained in Preparation Example 1 was inoculated into a 0.8% brown sugar aqueous solution at 30℃ and allowed to stand for 1.0 h to activate the inoculum and obtain the activated inoculum solution.

[0066] The compound fermentation agent A is inoculated into the feed fermentation substrate in the form of the activated microbial solution. The inoculation amount is 0.3% of the total dry matter mass of the feed fermentation substrate, based on the mass of the compound fermentation agent A.

[0067] The total moisture content of the feed fermentation substrate was adjusted to 55.0%, and the initial pH value was adjusted to 6.0 to obtain the fermentation system.

[0068] The fermentation system is subjected to the first stage of aerobic fermentation. The fermentation system is spread out in a 5.0cm thick material pile and fermented for 18.0h at an initial ambient temperature of 28℃. During the fermentation process, oxygen supply is maintained, specifically by intermittently turning the pile every 8.0h, until the internal core temperature of the fermentation system naturally rises to 40℃, at which point the first stage of aerobic fermentation is terminated.

[0069] After the first stage of aerobic fermentation is completed, no secondary inoculation is performed. Mechanical pressure is applied to the fermentation system to force compaction and degassing. The system is then physically sealed in a sealed fermentation bag with a one-way degassing valve to allow the fermentation system to undergo the second stage of anaerobic fermentation. The system is allowed to ferment statically at an ambient temperature of 25°C for 60.0 hours until the pH value of the fermentation system drops to the preset acidic range of 3.8, thus obtaining the finished fermented feed. This feed is then stored at room temperature, in a sealed, light-protected environment.

[0070] Example 2:

[0071] This embodiment provides a method for synergistic fermentation of multi-strain aerobic anaerobic feed, including the following steps:

[0072] Weigh out the basic raw materials as follows by mass fraction: 40.0% corn flour, 30.0% soybean meal, 15.0% wheat bran, 10.0% straw powder, and 5.0% trace element and vitamin premix. Add 1.0% brown sugar and 0.5% corn syrup powder as auxiliary materials, and pulverize them together to 30 mesh to obtain the feed fermentation substrate.

[0073] The compound fermentation agent B obtained in Preparation Example 2 was inoculated into a 1.0% (w / w) brown sugar aqueous solution at 32°C and allowed to stand for 1.5 h to activate the inoculum. The compound fermentation agent B was then inoculated into the feed fermentation substrate in the form of the activated inoculum, with the inoculation amount being 1.0% of the total dry matter mass of the feed fermentation substrate based on the mass of compound fermentation agent B. The total moisture content of the feed fermentation substrate was adjusted to 60.0%, and the initial pH was adjusted to 6.5 to obtain the fermentation system.

[0074] The fermentation system is subjected to the first stage of aerobic fermentation. The fermentation system is spread in a trough fermentation bed with a material pile thickness of 7.5 cm. Fermentation is carried out for 30.0 h at an initial ambient temperature of 30℃. During the fermentation process, oxygen supply is maintained, specifically by intermittently turning the pile every 10.0 h, until the internal core temperature of the fermentation system naturally rises to 42℃, at which point the first stage of aerobic fermentation is terminated.

[0075] After the first stage of aerobic fermentation is completed, no secondary inoculation is performed. Mechanical pressure is applied to the fermentation system to force compaction and degassing, and the system is physically sealed in a vertical fermentation tower to allow the fermentation system to undergo the second stage of anaerobic fermentation. The system is allowed to ferment at an ambient temperature of 28°C for 96.0 hours until the pH value of the fermentation system drops to the preset acidic range of 4.0, thus obtaining the finished fermented feed. The feed is then stored at room temperature, in a sealed, light-proof environment.

[0076] Example 3:

[0077] This embodiment provides a method for synergistic fermentation of multi-strain aerobic anaerobic feed, including the following steps:

[0078] Weigh out the basic raw materials as follows by mass fraction: 35.0% corn flour, 35.0% soybean meal, 15.0% wheat bran, 10.0% distillers' grains, and 5.0% trace element and vitamin premix. Add 2.0% molasses and 0.8% corn steep liquor powder as auxiliary materials, and pulverize them together to 40 mesh to obtain the feed fermentation substrate.

[0079] The compound fermentation agent C obtained in Preparation Example 3 was inoculated into a 1.2% (w / w) brown sugar aqueous solution at 35°C and allowed to stand for 2.0 h to obtain an activated inoculum solution. The compound fermentation agent C was then inoculated into the feed fermentation substrate in the form of the activated inoculum solution, with the inoculation amount being 3.0% of the total dry matter mass of the feed fermentation substrate, based on the mass of the compound fermentation agent C.

[0080] The total moisture content of the feed fermentation substrate was adjusted to 70.0%, and the initial pH value was adjusted to 7.0 to obtain the fermentation system. The fermentation system was then subjected to the first stage of aerobic fermentation. The fermentation system was spread out in a 10.0 cm thick pile and fermented at an initial ambient temperature of 35°C for 48.0 h. During the fermentation process, oxygen supply was maintained, specifically by forced ventilation every 12.0 h, until the internal core temperature of the fermentation system naturally rose to 45°C, at which point the first stage of aerobic fermentation was terminated.

[0081] After the first stage of aerobic fermentation is completed, no secondary inoculation is performed. Mechanical pressure is applied to the fermentation system to force compaction and degassing, and the fermentation tank is physically sealed to allow the fermentation system to undergo the second stage of anaerobic fermentation. The fermentation is carried out at an ambient temperature of 32℃ for 168.0 hours until the pH value of the fermentation system drops to the preset acidic range of 4.5, and the finished fermented feed is obtained. It is then stored at room temperature, in a sealed and light-proof condition.

[0082] Example 4:

[0083] This embodiment provides a method for synergistic fermentation of multi-strain aerobic anaerobic feed, including the following steps:

[0084] Weigh out the basic raw materials as follows by mass fraction: 38.0% corn flour, 32.0% soybean meal, 18.0% wheat bran, 9.0% soybean residue, and 3.0% trace element and vitamin premix. Add 1.5% glucose and 0.6% corn syrup powder as auxiliary materials, and pulverize them together to 30 mesh to obtain the feed fermentation substrate.

[0085] The compound fermentation agent D obtained in Preparation Example 4 was inoculated into a 1.0% (w / w) brown sugar aqueous solution at 33°C and allowed to stand for 1.5 h to activate the inoculum. The compound fermentation agent D was then inoculated into the feed fermentation substrate in the form of the activated inoculum, with the inoculation amount being 2.0% of the total dry matter mass of the feed fermentation substrate, based on the mass of the compound fermentation agent D. The total moisture content of the feed fermentation substrate was adjusted to 65.0%, and the initial pH was adjusted to 6.2 to obtain the fermentation system.

[0086] The fermentation system was subjected to the first stage of aerobic fermentation. The fermentation system was spread out in a material pile with a thickness of 8.0 cm and fermented for 36.0 h at an initial ambient temperature of 32℃. During the fermentation process, oxygen supply was maintained, specifically by intermittently turning the pile every 12.0 h, until the internal core temperature of the fermentation system naturally rose to 43℃, at which point the first stage of aerobic fermentation was terminated.

[0087] After the first stage of aerobic fermentation is completed, no secondary inoculation is performed. Mechanical pressure is applied to the fermentation system to force compaction and degassing, and the system is physically sealed in the silage pit to allow the fermentation system to undergo the second stage of anaerobic fermentation. The system is allowed to ferment statically at an ambient temperature of 28°C for 120.0 hours until the pH value of the fermentation system drops to the preset acidic range of 4.2, thus obtaining the finished fermented feed. The feed is then stored at room temperature, in a sealed, light-proof environment.

[0088] Comparative Examples 1-4:

[0089] Comparative Example 1:

[0090] Compared with Example 2, the difference is that only the aerobic functional bacteria obtained in step (1) of Example 2 are inoculated, and no second-stage anaerobic fermentation is carried out after inoculation. The oxygen supply operation is maintained throughout the process and the first-stage aerobic fermentation state is maintained until the total fermentation time reaches 126.0h. All other aspects are the same.

[0091] Comparative Example 2:

[0092] Compared with Example 2, the difference is that only the anaerobic functional bacteria obtained in step (2) of Example 2 were inoculated, and after the fermentation system was obtained, the first stage of aerobic fermentation was skipped. Mechanical pressure was directly applied to the fermentation system to force compaction and degassing and physical sealing was performed. The fermentation was directly placed at an ambient temperature of 28°C for 126.0 h. All other aspects were the same.

[0093] Comparative Example 3:

[0094] Compared with Example 2, the difference is that the single inoculation process was not used. Specifically, during the initial inoculation, only the aerobic functional bacteria obtained in step (1) of Preparation Example 2 were introduced and the first stage of aerobic fermentation was carried out. After the first stage of aerobic fermentation was completed, the fermentation system was opened for a second inoculation, and the anaerobic functional bacteria obtained in step (2) of Preparation Example 2 were added. Then, mechanical pressure was applied to the fermentation system to force compaction and degassing and physical sealing was carried out to enter the second stage of anaerobic fermentation. The rest were the same.

[0095] Comparative Example 4:

[0096] Compared with Example 2, the difference is that the linkage mechanism of natural temperature rise transition is missing. Specifically, after the fermentation system is prepared by inoculating compound fermentation agent B, the first stage of aerobic fermentation is skipped. No oxygen supply and turning operation is performed. Mechanical pressure is directly applied to the fermentation system to force compaction and degassing and physical sealing is performed. The system directly enters the second stage of anaerobic fermentation and is allowed to ferment statically at an ambient temperature of 28°C for 126.0 hours. All other aspects are the same.

[0097] Test Examples 1-5:

[0098] Test Example 1: Dynamic Monitoring Test of Physicochemical Environment during Fermentation

[0099] This test case uses the fermentation system of Example 2 as the object, and dynamically monitors its temperature, redox potential (ORP), and pH value throughout the two-stage fermentation process. The specific steps are as follows:

[0100] At the aerobic fermentation start point (i.e., 0h) in the first stage of Example 2, an insertable online temperature sensor and an online redox potential composite electrode were pre-embedded at the geometric center and diagonal edge of the trough fermentation bed, respectively. During fermentation, the probes remained fixed and were turned over together with the material to record real-time in-situ data at each time point and take the average value of multiple points.

[0101] pH values ​​were determined using a fixed-point, timed destructive sampling method. Starting from 0 h of fermentation, approximately 50 g of solid material was collected from multiple points in the upper, middle, and lower layers of the fermentation system at regular intervals. The samples were mixed thoroughly, and 10.0 g of the mixture was accurately weighed and placed in an Erlenmeyer flask containing 90 mL of deionized water. The mixture was shaken and extracted on a shaker at 150 r / min for 30 min, then allowed to stand. The pH value of the supernatant was measured using a precision pH meter.

[0102] During the first stage of aerobic fermentation (0h to 30.0h), data was recorded and samples were taken every 6.0h. When the internal temperature of the fermentation system reached 42℃, the stage switch was triggered, and the system was transferred to the fermentation tower to enter the second stage of anaerobic fermentation (30.0h to 126.0h). In the early stage, samples were taken every 12.0h, and in the later stage, samples were taken every 24.0h, until the end of fermentation.

[0103] The test data for dynamic monitoring of the physical and chemical environment are recorded in Table 1.

[0104] Table 1. Dynamic changes in physicochemical parameters of the fermentation system in Example 2 throughout the entire fermentation cycle.

[0105] Fermentation time (h) Fermentation stage Temperature at the center of the system (°C) ORP (mV) pH value of extract 0.0 Phase 1 30.1 +148 6.47 6.0 Phase 1 31.8 +121 6.41 12.0 Phase 1 34.2 +86 6.36 18.0 Phase 1 37.6 +52 6.28 24.0 Phase 1 40.2 +17 6.15 30.0 Phase 1 End / Switch 42.4 -15 5.92 42.0 Phase Two 40.6 -132 5.21 54.0 Phase Two 35.7 -194 4.63 78.0 Phase Two 30.4 -246 4.22 102.0 Phase Two 28.6 -273 4.08 126.0 Phase Two Ended 28.2 -282 3.98

[0106] in conclusion:

[0107] Figure 1The process of temperature rising continuously due to the metabolic heat production of aerobic microorganisms in the first stage, and then naturally dropping back after 30.0 hours when the process transitioned to the second stage of anaerobic fermentation;

[0108] Figure 2 This demonstrates the process by which residual oxygen inside the fermentation substrate is rapidly consumed, and the system gradually transitions from an initial oxidative environment to a deep reduction state, crossing the inflection point.

[0109] Figure 3 This demonstrates the process by which, after the establishment of the anaerobic microenvironment in the second stage, the explosive acid production by the lactic acid bacteria causes a rapid decrease in the pH value of the system, which eventually stabilizes within the preset acidic range.

[0110] Figure 1 , Figure 2 and Figure 3 The vertical dashed line in the figure represents the node corresponding to the fermentation time of 30.0 h, indicating the process boundary between the first stage of aerobic fermentation and the second stage of anaerobic fermentation.

[0111] According to the data in Table 1, during the first stage of aerobic fermentation (0.0 h to 30.0 h), the redox potential of the fermentation system continuously decreased from an initial +148 mV to -15 mV. The residual oxygen in the fermentation substrate was largely consumed by aerobic microorganisms such as Bacillus subtilis and Saccharomyces cerevisiae in the compound fermentation agent. During this oxygen consumption process, accompanied by intense biological metabolic heat production, the internal core temperature of the system showed a non-linear accelerating upward trend, naturally accumulating to 42.4 °C at 30.0 h. The pH value in the first stage only decreased slightly, from 6.47 to 5.92, and the lactic acid bacteria did not exhibit explosive proliferation and acid production. The data from the first stage verify that the aerobic functional bacterial community can actively alter the physicochemical microenvironment within the solid substrate through its own colonization and growth, consuming free oxygen and providing a thermal cushion.

[0112] After 30.0 hours of forced compaction, degassing, and physical sealing, followed by the transition to the second stage of anaerobic fermentation, the redox potential exhibited a precipitous drop, rapidly decreasing to -194 mV by 54.0 hours, and finally stabilizing at a highly reducing state of -282 mV. During this absolutely anaerobic process, as the temperature gradually decreased from 42.4℃ to ambient constant temperature, the previously dormant anaerobic functional lactic acid bacteria population experienced explosive proliferation, utilizing the carbon source substrate initially degraded by aerobic bacteria. Within 24 hours of entering the second stage (30.0 hours to 54.0 hours), the system pH rapidly decreased from 5.92 to 4.63, and reached the preset acidic range of 3.98 at the fermentation endpoint.

[0113] The full-cycle data in Table 1 confirms the synergistic succession mechanism among multiple microbial species in the method of this invention. The aerobic heating in the first stage not only constitutes the preliminary enzymatic hydrolysis of macromolecules but also pre-induces the anaerobic environment in the second stage. The temperature inflection point of 40℃ to 45℃ marks the peak of aerobic microbial metabolism and is also the optimal triggering condition for screening and activating thermostable homo- and hetero-type lactobacilli. This coordinated change in the physicochemical environment demonstrates the feasibility of the single-inoculation process at the spatiotemporal succession level, avoiding the cumbersome process and intermediate contamination risks caused by stepwise secondary inoculation in conventional technologies. Relying on the natural changes in temperature and oxygen partial pressure within the system, the autonomous and stable transfer of microbial dominance is achieved.

[0114] Test Example 2: Dynamic Succession Test of Microecological Community in Fermentation System

[0115] This test case uses the fermentation system of Example 2 as the object, and measures the dynamic changes in the effective viable count of each functional microbial community at three key time points: the start point of the first-stage aerobic fermentation, the end point of the first-stage aerobic fermentation, and the end point of the second-stage anaerobic fermentation. The specific steps are as follows:

[0116] Following the process flow of Example 2, samples were taken at 0.0 h after inoculation and mixing, 30.0 h at the end of the first stage fermentation switching point, and 126.0 h at the end of the second stage fermentation. The sampling method involved randomly collecting samples from multiple points in the upper, middle, and lower layers of the fermentation material pile and mixing them evenly to obtain the test samples at each time point.

[0117] Under aseptic conditions, accurately weigh 10.0 g of the sample to be tested and add it to a sterile Erlenmeyer flask containing 90 mL of physiological saline with glass beads. Shake the flask in a constant temperature shaker at 150 r / min for 30 min to allow the microorganisms in the sample to be fully released into the aqueous phase, thus preparing 10... -1 The suspension was diluted to a certain degree, and then serially diluted 10-fold to prepare 10... -2 Up to 10 -8 Sample dilutions of different gradients.

[0118] The viable count of Bacillus spp. was determined using a heat treatment screening method. Each graded dilution was placed in an 80°C water bath for 15 minutes to kill non-spore-forming vegetative cells. After cooling, 0.1 mL was spread onto nutrient agar plates and incubated aerobically at 37°C for 48 hours. Plates with colony counts between 30 and 300 were selected for counting and determining the viable count.

[0119] The total viable count of yeast and mold was determined using potato dextrose agar medium. Chloramphenicol at a final concentration of 100 mg / L was added to the medium to inhibit bacterial growth. 0.1 mL of each graded dilution was spread onto plates and incubated at 28 °C for 72 h. The number of yeast and mold colonies was counted separately based on colony morphology, and the total viable count was calculated by combining the results.

[0120] The viable count of lactic acid bacteria was determined using MRS agar medium supplemented with calcium carbonate. 0.1 mL of each graded dilution was spread onto plates, placed in an anaerobic incubator, and incubated at 37°C for 72 h. Colonies exhibiting a clear zone of calcium dissolution were selected for counting, and the viable count was calculated.

[0121] The test data of the dynamic succession of the micro-ecological community are recorded in Table 2.

[0122] Table 2. Dynamic changes in the number of viable bacteria in the microecological community at different stages of the fermentation system in Example 2.

[0123] Sampling time point (h) Fermentation stage attributes viable Bacillus spp. count (CFU / g) Total viable count of yeast and mold (CFU / g) Live lactic acid bacteria count (CFU / g) 0.0 Initial vaccination <![CDATA[1.34×10 7 ]]> <![CDATA[0.82×10 7 ]]> <![CDATA[2.15×10 8 ]]> 30.0 Phase 1 ended <![CDATA[5.86×10 8 ]]> <![CDATA[3.17×10 8 ]]> <![CDATA[4.62×10 8 ]]> 126.0 Phase Two Ended <![CDATA[1.05×10 7 ]]> <![CDATA[2.41×10 6 ]]> <![CDATA[3.94×10 9 ]]>

[0124] Summarize:

[0125] Figure 4 This demonstrates the process by which aerobic bacteria proliferate rapidly during the first stage of aerobic fermentation (0.0h to 30.0h), and then experience a significant decline in viable bacterial count after transitioning to the second stage of anaerobic fermentation (30.0h to 126.0h) due to strict physical sealing and the limitation of an oxygen-deficient environment.

[0126] Figure 5 This demonstrates the growth trend of lactic acid bacteria maintaining a stable survival state in the first stage, and then experiencing a sharp increase after entering the anaerobic environment of the second stage, taking over and dominating the growth of the later fermentation and metabolic processes.

[0127] Figure 4 and Figure 5 The vertical dashed line in the figure represents the node corresponding to the fermentation time of 30.0 h, indicating the boundary between the first stage of aerobic fermentation and the second stage of anaerobic fermentation.

[0128] According to the data in Table 2, at the initial inoculation time of 0.0 h, the number of lactic acid bacteria was higher than that of aerobic functional bacteria due to the designed compound ratio. After the first stage of aerobic fermentation (30.0 h), the viable number of Bacillus spp. increased from 1.34 × 10⁻⁶. 7 CFU / g increased to 5.86 × 10⁻⁶ 8 CFU / g, total viable count of yeast and mold decreased from 0.82×10 7 CFU / g increased to 3.17 × 10⁻⁶ 8 Both CFU / g and CFU / g showed a significant jump in order of magnitude. During the same period, the viable count of lactic acid bacteria increased from 2.15 × 10⁻⁶.8 CFU / g increased slowly to 4.62×10 8 CFU / g, no logarithmic burst was observed. Data at this stage indicate that, under the physical environment of open, shallow layering and turning for oxygen supply, aerobic functional microorganisms utilize oxygen for aerobic respiration, with a metabolic rate far exceeding that of facultative or obligate anaerobic lactic acid bacteria. While rapidly consuming free oxygen from the substrate, the aerobic bacteria multiply rapidly, achieving absolute dominance of the early-stage microecological environment of the fermentation system. There is no internal antagonistic consumption phenomenon where premature large-scale acid production by lactic acid bacteria inhibits the growth of aerobic bacteria.

[0129] At the end of the second stage of anaerobic fermentation (126.0 h), the viable count of lactic acid bacteria surged to 3.94 × 10⁻⁶. 9 CFU / g, occupying an absolute niche in the community. Aerobic bacteria were strictly confined by physical sealing and an anaerobic environment; Bacillus species mainly existed in dormant spore form, with the viable count dropping to 1.05 × 10⁻⁶. 7 The total viable count of yeast and mold decreased significantly to 2.41 × 10⁻⁶ CFU / g due to acid intolerance and lack of oxygen. 6 CFU / g. Based on the small molecule sugars produced by the enzymatic hydrolysis of macromolecules by aerobic bacteria in the first stage and the local anaerobic microenvironment, lactic acid bacteria rapidly adapt to and dominate the metabolic process in the second stage.

[0130] The microbial count data in Table 2 confirms the spatial colonization and temporal succession mechanism at the microecological level in the single-inoculation process of the compound fermentation agent. In the first stage, aerobic bacteria act as pioneer populations, consuming oxygen and raising the system temperature, thus removing environmental obstacles for subsequent anaerobic fermentation and accumulating easily fermentable carbon sources. After transitioning to physical compaction and sealing, lactic acid bacteria seamlessly take over metabolic dominance, proliferating and producing acid in large quantities. The dynamic evolution of this microecological community highly matches the changes in the physicochemical environment in Test Example 1, proving that the process design of this invention can precisely control the optimal stage for each mixed microbial community to perform its function, solving the technical problem of microbial competition disorder in traditional single-inoculation multi-species fermentation processes, and effectively improving the conversion efficiency and product uniformity of feed fermentation.

[0131] Test Example 3: Degradation, Quality Improvement, and Nutritional Index Determination of Fermentation Products

[0132] This test example uses the finished fermented feeds prepared in Examples 1 to 4 and Comparative Examples 1 to 4 as test objects. The crude fiber degradation rate, crude protein enhancement rate, and lactic acid content at the fermentation endpoint were quantitatively measured to verify the actual improvement effect of each fermentation process parameter and procedure on the physicochemical nutritional indicators of the feed substrate. The specific steps are as follows:

[0133] The finished fermented feeds from Examples 1 to 4 and Comparative Examples 1 to 4 were collected as test samples at the end of their respective specified fermentation cycles. Each group of samples was dried in a 65°C forced-air drying oven to constant weight, pulverized, and passed through a 40-mesh standard sieve to obtain dry matter test powder samples. Simultaneously, unfermented feed fermentation substrates from each group were reserved and, after the same drying and pulverizing process, served as baseline control samples.

[0134] The crude protein content was determined using the Kjeldahl method (referencing GB / T6432 standard). 0.5 g of dry matter was accurately weighed for the test sample and the baseline control sample, and placed separately in digestion tubes. A catalyst and concentrated sulfuric acid were added for high-temperature digestion. After cooling, distillation and titration were performed on an automated Kjeldahl nitrogen analyzer. The volume of standard titrating acid consumed was recorded, and the absolute crude protein content of each group of samples was calculated. Using the crude protein content of the baseline control sample as a benchmark, the crude protein enhancement rate after fermentation was calculated.

[0135] The crude fiber content was determined using the acid-base digestion method (referencing GB / T6434 standard). 2.0 g of dry matter was accurately weighed for the test sample and the baseline control sample. The samples were then digested sequentially with sulfuric acid solution and sodium hydroxide solution of specified concentrations under boiling conditions. After filtration and washing, the residue was collected, dried, weighed, and ashed in a muffle furnace at 500℃. The absolute crude fiber content was calculated based on the mass difference before and after ashing. Using the crude fiber content of the baseline control sample as a benchmark, the crude fiber degradation rate after fermentation was calculated.

[0136] The lactic acid content at the fermentation endpoint was determined by high-performance liquid chromatography (HPLC). 10.0 g of fresh, finished fermented feed sample was accurately weighed and added to 90 mL of ultrapure water. Extraction was carried out at room temperature with shaking in the dark for 2.0 h. After centrifugation at 8000 rpm for 15 min, the supernatant was collected, filtered through a 0.22 μm aqueous microporous membrane, and injected into the HPLC system. Chromatographic conditions included a C18 reversed-phase column, phosphate buffer as the mobile phase, and a detection wavelength of 210 nm. The mass fraction of lactic acid in the dry matter matrix of each sample was quantitatively calculated using the external standard method.

[0137] The test data on degradation, quality improvement, and nutritional indicators of the fermented product are recorded in Table 3.

[0138] Table 3. Comparison of Nutritional and Physicochemical Indicators of Fermented Products from Examples and Comparative Examples

[0139] Group Crude protein CP enhancement rate (%) Crude fiber CF degradation rate (%) Lactic acid content at the end of fermentation (%) Example 1 18.4 31.2 4.12 Example 2 26.7 45.8 6.85 Example 3 23.1 42.3 5.94 Example 4 24.6 40.1 6.13 Comparative Example 1 8.2 35.6 0.45 Comparative Example 2 11.5 15.4 3.21 Comparative Example 3 21.4 38.7 5.16 Comparative Example 4 14.2 18.9 3.05

[0140] in conclusion:

[0141] Figure 6 The results show that the example group is generally better than the comparative group in terms of macromolecular degradation and quality improvement of feed matrix, which intuitively reflects that the preferred process parameters of Example 2 have the best comprehensive performance in improving crude protein and degrading crude fiber.

[0142] Figure 7 The study demonstrated the differences in the final acid production capacity of fermentation systems under different process routes and parameter conditions, confirming the necessary prerequisite role of the first-stage aerobic fermentation process in triggering and promoting the efficient acid production of lactic acid bacteria in the subsequent anaerobic stage.

[0143] According to the data in Table 3, the crude fiber degradation rate of Examples 1 to 4 all reached over 31.2%, the crude protein improvement rate was over 18.4%, and the lactic acid content at the fermentation endpoint was in the high range of 4.12% to 6.85%. Example 2 performed best under the optimized process parameters, with a crude fiber degradation rate of 45.8%, a crude protein improvement rate of 26.7%, and a lactic acid content of 6.85%. Comparing the data of Example 2 with Comparative Examples 1 and 2, it can be seen that a single stage of fermentation cannot achieve comprehensive degradation and quality improvement of the feed substrate. Comparative Example 1, lacking the second stage of anaerobic fermentation, had a lactic acid content of only 0.45%, which not only failed to achieve the purpose of acidification and preservation, but also resulted in excessive oxidation and consumption of the substrate carbon source into carbon dioxide and water due to the oxygen consumption throughout the process, leading to excessive dry matter loss and a relative crude protein improvement rate of only 8.2%. Comparative Example 2, lacking the first stage of aerobic fermentation, had a crude fiber degradation rate as low as 15.4%, demonstrating that lactic acid bacteria in a single anaerobic environment lack the ability to decompose macromolecules such as cellulose and hemicellulose. Furthermore, due to the lack of easily fermentable small-molecule carbon sources generated by the initial enzymatic hydrolysis of aerobic bacteria in the substrate, the final amount of lactic acid synthesized was limited to only 3.21%.

[0144] Comparing the data from Example 2 and Comparative Example 4, it is evident that the first-stage aerobic fermentation process is a necessary prerequisite for triggering the subsequent anaerobic stage of efficient acid production. Comparative Example 4 skipped the oxygen supply and heating stage after a single inoculation with the compound microbial agent and directly underwent physical sealing; its crude fiber degradation rate was only 18.9%, and the lactic acid content at the fermentation endpoint decreased to 3.05%. This data demonstrates that the oxygen supply and accompanying natural heating process in the first stage effectively activated the extracellular enzyme secretion mechanism of Bacillus and molds, playing a decisive role in the depolymerization of the macromolecular lignocellulose structure. The absence of this stage leads to low substrate utilization of the subsequent anaerobic functional microbial community, preventing the establishment of a dominant population and the production of large amounts of acid.

[0145] Comparing the data from Example 2 and Comparative Example 3, it can be seen that the single-inoculation process used in this invention is superior to the traditional step-by-step two-inoculation process in terms of fermentation efficiency. Comparative Example 3, which uses segmented inoculation, achieved a crude fiber degradation rate of 38.7% and a lactic acid content of 5.16%, all of which were lower than those in Example 2. The data indicates that single-inoculation not only simplifies the operation process but, more importantly, directly triggers microbial succession through changes in physical state (compacted sealing), avoiding heat loss, moisture loss, and the time lag in establishing an anaerobic environment caused by secondary inoculation. Aerobic and anaerobic bacteria sequentially dominate fermentation in the same system, forming a tight substrate metabolic network and energy transfer cascade, ultimately achieving a significant improvement in feed fermentation quality.

[0146] Test Example 4: Hygienic Indicators and Anti-contamination Capacity of Fermented Products

[0147] This test example uses the fermented feed products prepared in Example 2, Comparative Examples 3 and 4 as test objects. The levels of Escherichia coli, Salmonella detection, and volatile basic nitrogen (TVB-N) content were measured to evaluate the ability of different fermentation processes to control microbial contamination and the hygienic safety of the finished products. The specific steps are as follows:

[0148] Inside a sterile operating table, 25.0 g of the deep core material from Examples 2, 3, and 4 at the end of fermentation and upon opening was collected using a sterile sampling spoon. The samples were placed in homogenization bags containing 225 mL of sterile physiological saline and homogenized using a tapping homogenizer for 2 minutes to prepare a 1:10 homogeneous solution. Subsequently, sterile pipettes were used to sequentially dilute the samples tenfold to prepare test solutions of the required dilutions.

[0149] The coliform count was determined using the crystal violet neutral red bile agar (VRBA) plate count method. Three consecutively diluted sample solutions were selected, and 1.0 mL of each was injected into a sterile Petri dish. VRBA medium, melted and incubated at 45°C, was poured over each dish, mixed thoroughly, and allowed to solidify. A further layer of VRBA medium was then placed on top. The plates were incubated upside down in a 36°C incubator for 24 hours. Colonies exhibiting typical coliform characteristics (purple-red color with a surrounding precipitate ring) were counted and converted to colony-forming units (CFU) per gram of dry matter.

[0150] Salmonella testing employed a pre-enrichment and selective isolation culture method. 25.0 g of fermented feed sample was inoculated into 225 mL of buffered peptone water (BPW) and incubated at 36°C for 18 h for pre-enrichment. Subsequently, 1.0 mL and 0.1 mL of the pre-enrichment broth were inoculated into tetrathionate brilliant green enrichment broth (TTB) and selenite cystine enrichment broth (SC), respectively, for selective enrichment. The enrichment broth was streaked onto xylose-lysine deoxycholate agar (XLD) plates and incubated. The presence of typical Salmonella colonies with a black or translucent center was observed, and the detection results were recorded.

[0151] The content of volatile basic nitrogen (TVB-N) was determined using the semi-micro Kjeldahl distillation method. 10.0 g of fermented feed sample was accurately weighed, added to 100 mL of ultrapure water, and extracted by shaking for 30 min, followed by filtration. The filtrate was placed in the distillation tube of the Kjeldahl apparatus, and magnesium oxide suspension was added to make it alkaline. Heating distillation was performed to release the volatile nitrogen compounds. The distillate was absorbed using a boric acid solution containing a methyl red-bromocresol green mixed indicator. The absorbent was titrated to the endpoint using a standard hydrochloric acid titration solution, and the number of milligrams of TVB-N per 100 g of fermented feed was calculated based on the volume of hydrochloric acid consumed.

[0152] The test data of the hygienic indicators of the fermented product are recorded in Table 4.

[0153] Table 4. Hygienic index test data of fermented products from Example 2 and the comparative example.

[0154] Group Coliform count (CFU / g) Salmonella detection rate ( / 25g) Volatile basic nitrogen TVB-N (mg / 100g) Example 2 <![CDATA[1.8×10 1 ]]> Not detected 14.6 Comparative Example 3 <![CDATA[5.2×10 3 ]]> Not detected 38.4 Comparative Example 4 <![CDATA[3.1×10 5 ]]> Detected 92.7

[0155] in conclusion:

[0156] Figure 8 The fermented product of Example 2 demonstrates a significant order-of-magnitude advantage in controlling Escherichia coli, and the Salmonella test is negative, proving that the single inoculation combined with the in-situ physical sealing process used in this scheme completely cuts off the invasion route of external environmental bacteria during fermentation and effectively inhibits the proliferation of pathogenic bacteria.

[0157] Figure 9 The results showed that the TVB-N content of the product in Example 2 was at an absolutely low level, while the comparative products that underwent a second opening or lacked an oxygen-consuming heating stage all showed obvious protein putrefaction and degradation. This confirms the decisive role of the spatiotemporal dual antibacterial barrier of the present invention in blocking putrefactive bacteria from consuming nutrients and ensuring the high hygiene and safety of the fermented product.

[0158] According to the data in Table 4, the fermented product of Example 2 had significantly lower levels of Escherichia coli and TVB-N compared to Comparative Examples 3 and 4, and no Salmonella was detected. Comparing the data of Example 2 and Comparative Example 3, Comparative Example 3, due to its stepwise two-stage inoculation process, required opening the fermentation system to introduce anaerobic bacteria after the first stage of aerobic fermentation. This operation disrupted the original physically sealed state and introduced environmental contaminants. The second opening process allowed aerobic and facultative anaerobic putrefactive bacteria to regain oxygen and access to new environments, causing their Escherichia coli count to rise to 5.2 × 10⁻⁶. 3 CFU / g, while other microorganisms decomposed the matrix proteins, producing free amines and ammonia, leading to an increase in TVB-N content to 38.4 mg / 100g. Example 2 employed a single inoculation combined with in-situ physical sealing technology, completely cutting off external contamination pathways during fermentation and maintaining the purity of the system's microenvironment.

[0159] Comparing the data of Example 2 and Comparative Example 4, Comparative Example 4 skipped the first stage of aerobic fermentation and directly entered the anaerobic state, with an Escherichia coli count as high as 3.1 × 10⁻⁶. 5 The presence of pathogenic Salmonella (CFU / g) and a sharp increase in TVB-N content to 92.7 mg / 100g indicates significant protein spoilage and degradation in the product. This result confirms that, in the absence of rapid oxygen consumption and metabolic heat generation by aerobic microorganisms in the first stage, the system failed to create an early high-temperature inhibitory physical environment (40°C to 45°C) against intestinal pathogens and putrefactive bacteria. Simultaneously, the subsequent lactic acid bacteria, lacking readily fermentable carbon sources, produced acid slowly and were unable to quickly lower the pH to the inhibition threshold (below 4.0) in the early stages of fermentation, leading to a large-scale competitive proliferation of harmful bacteria in the early fermentation phase. Data from Example 2 confirms that the accumulated heat and hypoxic environment in the aerobic stage, combined with the chemical changes from efficient acid production by lactic acid bacteria in the anaerobic stage, constitute a robust spatiotemporal dual antibacterial barrier, effectively blocking the consumption of feed nutrients by putrefactive bacteria and ensuring the hygienic quality and biosafety of the final fermented product.

[0160] Test Example 5: Stability and Shelf Life of Finished Product During Room Temperature Storage

[0161] This test example uses the fermented feed products prepared in Examples 1 to 4 and Comparative Examples 1 to 4 as test objects to measure their pH fluctuations, sensory quality, and mold growth status under normal temperature conditions, in order to evaluate the impact of each fermentation process parameter on the long-term storage stability of the finished product. The specific steps are as follows:

[0162] Freshly fermented feed products obtained at the end of their respective fermentation cycles in Examples 1 to 4 and Comparative Examples 1 to 4 were packaged into 5.0 kg bags in polyethylene bags equipped with one-way exhaust valves. The bags were then degassed and heat-sealed. All test bags were placed in a room temperature environment at 25±2℃ and stored away from light.

[0163] During storage, destructive sampling and testing were conducted at set time points of 0 days, 90 days, and 180 days. For each sampling, individual packaging bags from each group were randomly selected. After opening the bags, 50.0g of samples were taken using a multi-point mixing method, and an appropriate amount of deionized water was added for shaking and extraction. After standing, the pH value of the supernatant was measured, and the data were recorded to assess the stability of the internal acidic environment.

[0164] At the end of the 180-day storage period, sensory evaluation and total mold count were performed on each group of samples. Sensory evaluation was conducted by professional evaluators, with a maximum score of 100 points, based on the intensity of the sour aroma, color uniformity, and the presence of clumping or mold spots. Total mold count was determined using the potato dextrose agar (PDA) plate counting method. 10.0g of sample was homogenized in sterile physiological saline and serially diluted, then spread on a chloramphenicol-containing PDA plate. After incubation at 28℃ for 72 hours, colony counts were performed, and the counts were converted to the number of viable mold cells per gram of dry matter.

[0165] The test data on the stability of the finished product during room temperature storage are recorded in Table 5.

[0166] Table 5. Comparison of room temperature storage data of fermentation products from the Example and Comparative Examples

[0167] Group pH value on day 0 90-day pH value pH value over 180 days 180-day sensory rating Total mold count (CFU / g) after 180 days Example 1 4.12 4.18 4.25 87.5 <![CDATA[3.4×10 2 ]]> Example 2 3.98 4.02 4.07 94.2 1.2×10 Example 3 4.45 4.51 4.63 89.6 <![CDATA[5.1×10 2 ]]> Example 4 4.21 4.27 4.36 91.3 <![CDATA[2.7×10 2 ]]> Comparative Example 1 5.88 6.74 7.52 34.6 <![CDATA[7.8×10 6 ]]> Comparative Example 2 4.55 4.72 4.96 75.8 <![CDATA[6.3×10 3 ]]> Comparative Example 3 4.38 4.65 4.88 81.4 <![CDATA[4.9×10 4 ]]> Comparative Example 4 5.12 5.67 6.24 53.7 <![CDATA[9.2×10 5 ]]>

[0168] in conclusion:

[0169] Figure 10 The results showed that the internal acidity environment of the products in the example group was highly stable and the pH value drift was minimal. However, the test groups that lacked an effective anaerobic stage (Comparative Example 1) or failed to establish a stable first-stage oxygen consumption base (Comparative Example 4) all showed a significant pH rebound and acid barrier destruction during storage.

[0170] Figure 11 Through with Figure 10 The vertical alignment of the images visually reflects how, with the increase in local pH and the loss of the acidic antibacterial environment, the latent mold inside the fermented feed product undergoes a logarithmic explosion and proliferation. This confirms the decisive role of the low pH environment and unidirectional physical defense constructed by the process of this invention in maintaining the finished product's resistance to mold and freshness.

[0171] According to the data in Table 5, the pH values ​​of the finished products from Examples 1 to 4 fluctuated only slightly during the 180-day storage period at room temperature. Specifically, the pH value of Example 2 increased only slightly from 3.98 on day 0 to 4.07, with a sensory score maintained at 94.2 points and the total mold count suppressed to a baseline level of 1.2 × 10 CFU / g. This data indicates that the products from these examples relied on the large amount of organic acids synthesized by lactic acid bacteria during the second-stage anaerobic fermentation to create a stable low-pH antibacterial environment within the finished product. Combined with the physical isolation provided by the one-way exhaust valve packaging, this effectively inhibited the spore germination and vegetative reproduction of aerobic molds and spoilage bacteria, achieving long-term preservation at room temperature.

[0172] Comparing the data from Example 2 and Comparative Example 1, Comparative Example 1, lacking the second-stage anaerobic fermentation, did not accumulate organic acids at the fermentation endpoint, resulting in a higher initial pH value on day 0. During 180 days of room temperature and sealed storage, the remaining aerobic microorganisms and mold spores within the system rapidly metabolized using residual oxygen, consuming the substrate and releasing ammonia-like alkaline substances, causing the pH value to rebound rapidly to 7.52. With the loss of the acid barrier, the total mold count exploded to 7.8 × 10⁻⁶. 6 The CFU / g concentration dropped to a sensory score of 34.6, indicating severe clumping and mold growth in the material. This control data confirms that the products of the first-stage aerobic fermentation do not possess long-term self-stabilizing capabilities and must rely on the anaerobic acid production stage for containment and stabilization.

[0173] Comparing the data from Example 2 and Comparative Example 4, Comparative Example 4 skipped the first stage of aerobic fermentation and directly underwent anaerobic treatment, with its pH value remaining at a sub-acidic state of 5.12 on day 0. Due to the lack of enzymatic hydrolysis of macromolecular carbon sources by aerobic bacteria in the early stage of fermentation, the substrate utilization rate of lactic acid bacteria was low, and the acid production was insufficient to lower the system pH value below the safe threshold (pH value less than 4.2). During the subsequent storage process, the more acid-resistant molds overcame the slight acidity inhibition and continued to proliferate, reaching a total mold count of 9.2 × 10⁻⁶ on day 180. 5 The CFU / g concentration and pH value increased to 6.24 during the spoilage process. This indicates that the linkage mechanism in this scheme, where oxygen consumption provides substrate support for anaerobic digestion, is a prerequisite for establishing the final preservation defense line.

[0174] Comparing the data from Example 2 and Comparative Example 3, Comparative Example 3, which employed a stepwise two-stage inoculation process, achieved a total mold count of 4.9 × 10⁻⁶ at the end of its 180-day storage period. 4The CFU / g concentration was significantly higher than in Example 2, which only underwent a single inoculation. This indicates that opening the fermentation system midway for a second inoculation inevitably introduces aerobic mold spores from the environment into the deeper layers of material. Even with subsequent physical sealing, latent mold spores can still germinate due to local microenvironmental deterioration or the passage of time. Example 2, employing a single inoculation combined with a natural fermentation succession model, maintained the continuity of physical sealing throughout the entire process from aerobic high-temperature fermentation to anaerobic acid production. This blocked the infection pathways of contaminants from both physical space and microecological competition perspectives, thus extending the commercial shelf life of the fermented feed product.

Claims

1. A multi-strain synergistic aerobic anaerobic feed fermentation method, characterized in that, Includes the following steps: A compound fermentation agent is inoculated into a feed fermentation substrate with adjusted moisture content and initial pH value to obtain a fermentation system. The compound fermentation agent contains aerobic functional bacteria and anaerobic functional bacteria, and the ratio of the number of live bacteria of the aerobic functional bacteria to the number of live bacteria of the anaerobic functional bacteria is 1:1.5-1:2.

5. The fermentation system is subjected to the first stage of aerobic fermentation, and oxygen supply is maintained during the fermentation process until the internal core temperature of the fermentation system naturally rises to 40℃-45℃, at which point the first stage of aerobic fermentation is terminated. After the first stage of aerobic fermentation is completed, no secondary inoculation is performed. Mechanical pressure is applied to the fermentation system to force compaction and degassing, and physical sealing is performed to allow the fermentation system to undergo the second stage of anaerobic fermentation until the pH value of the fermentation system drops to the preset acidic range, thereby obtaining the finished fermented feed.

2. The multi-strain synergistic aerobic anaerobic feed fermentation method according to claim 1, characterized in that, The oxygen-consuming functional bacterial community consists of Bacillus, yeast, and mold in a live count ratio of 2.5-3.5:1.5-2.5:0.5-1.

5. The anaerobic functional bacterial community consists of homo-fermenting lactobacillus, hetero-fermenting lactobacillus, and cocci in a live count ratio of 3.5-4.5:2.5-3.5:1.5-2.

5.

3. The multi-strain synergistic aerobic anaerobic feed fermentation method according to claim 2, characterized in that, The Bacillus genus in the oxygen-consuming functional bacterial group is Bacillus subtilis, Bacillus licheniformis, or Bacillus amyloliquefaciens. The yeast genus is Saccharomyces cerevisiae, Candida utilis, or Hansenula polymorpha; The mold genus is Aspergillus oryzae, Aspergillus niger, or Trichoderma viride; The homofermentative lactobacilli in the anaerobic functional bacterial group are Lactobacillus plantarum, Lactobacillus acidophilus, or Lactobacillus casei. The heterologous fermenting lactobacillus is Lactobacillus bucheri or Lactobacillus fermentum. The cocci mentioned are Pediococcus pentosaceus.

4. The multi-strain synergistic aerobic anaerobic feed fermentation method according to claim 1, characterized in that, The feed fermentation substrate includes basic raw materials and auxiliary materials; The basic raw materials, by mass fraction, include: Corn flour 35.0%-45.0%, soybean meal 25.0%-35.0%, wheat bran 10.0%-20.0%, fiber by-products 5.0%-15.0%, and trace element and vitamin premix 3.0%-8.0%; The auxiliary materials include 0.5%-2.0% of the total dry matter of the basic raw materials as a readily available carbon source and 0.3%-0.8% of the readily available nitrogen source.

5. The multi-strain synergistic aerobic anaerobic feed fermentation method according to claim 1, characterized in that, The compound fermentation agent is inoculated into the feed fermentation substrate in the form of a microbial activation solution. The preparation method of the bacterial activation solution is as follows: The compound fermentation agent is prepared by inoculating it into a sugar solution at 30℃-35℃ and allowing it to stand for 1.0-2.0 hours for activation. Furthermore, based on the mass of the compound fermentation agent, the inoculation amount is 0.3%-3.0% of the total dry matter mass of the feed fermentation substrate.

6. The multi-strain synergistic aerobic anaerobic feed fermentation method according to claim 1, characterized in that, The total moisture content of the feed fermentation substrate, after adjusting the moisture content and initial pH value, is 55.0%-70.0%, and the initial pH value is 6.0-7.

0.

7. The multi-strain synergistic aerobic anaerobic feed fermentation method according to claim 1, characterized in that, The first stage of aerobic fermentation specifically involves: The fermentation system was spread out in a material pile thickness of 5.0-10.0 cm and fermented for 18.0-48.0 h at an initial ambient temperature of 28℃-35℃. The oxygen supply operation involves intermittent turning or forced ventilation every 8.0-12.0 hours.

8. The multi-strain synergistic aerobic anaerobic feed fermentation method according to claim 1, characterized in that, The second stage of anaerobic fermentation specifically involves: Static fermentation was carried out at an ambient temperature of 25℃-32℃ for 60.0-168.0 hours.

9. The multi-strain synergistic aerobic anaerobic feed fermentation method according to claim 1, characterized in that, In the second stage of anaerobic fermentation, the preset acidity range has a pH value of 3.8-4.

5.

10. The multi-strain synergistic aerobic anaerobic feed fermentation method according to claim 1, characterized in that, The physical sealing for the second stage of anaerobic fermentation is specifically as follows: It is carried out in sealed fermentation bags, fermentation tanks, silage pits or vertical fermentation towers equipped with one-way exhaust valves.

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