A compound probiotic fermented feed additive and a preparation method thereof

Abstract

The present application relates to the field of feed additive, and discloses a kind of compound probiotic fermented feed additive and preparation method thereof;The additive is granular structure, including fermentation core, calcium source transition zone in the outer edge aperture of fermentation core and the crosslinking fixed layer covering the outer surface of fermentation core and partially embedded in the outer edge aperture.Its preparation method includes: with protein-containing feed raw material, pectin-containing plant raw material and fermentation auxiliary material to make granular fermentation matrix, inoculate bacillus and yeast to carry out first stage aeration fermentation;The pectin on the outside of fermentation core is de-esterified, and the slightly soluble calcium source is loaded inside the low esterification pectin zone using pressure difference;Inoculate lactic acid-producing probiotics to carry out second stage closed fermentation, so that lactic acid promotes calcium ion release of calcium source and crosslinking with low esterification pectin, and form the crosslinking fixed layer of fixed lactic acid-producing probiotics.The method can take into account feed substrate hydrolysis, probiotic spatial zoning and viable bacteria stability.

Description

Technical Field

[0001] This invention relates to the field of feed additives, specifically to a compound probiotic fermented feed additive and its preparation method. Background Technology

[0002] Compound probiotic fermented feed typically uses soybean meal, wheat bran, corn flour, and plant by-products as main raw materials. It undergoes solid-state or semi-solid-state fermentation by inoculating microorganisms such as Bacillus, yeast, and lactic acid bacteria to degrade some macromolecular proteins, starches, and anti-nutritional factors, while retaining a certain number of beneficial microorganisms and their metabolites in the fermentation products. Current technologies often employ simultaneous inoculation of multiple microbial strains, or follow aerobic fermentation followed by anaerobic fermentation based on the aerobic characteristics of the strains, controlling moisture content, temperature, fermentation time, and inoculation ratio to obtain the fermented feed. While these processes can improve the palatability and nutrient utilization of the raw materials to some extent, different microorganisms vary in growth conditions, enzyme production characteristics, and acid tolerance. Controlling these factors solely through inoculation sequence and conventional fermentation parameters makes it difficult to simultaneously ensure the degree of substrate hydrolysis, viable cell stability, and product structural integrity.

[0003] In existing fermented feeds, after the first stage of protein and carbohydrate degradation, lactic acid bacteria are typically directly mixed into the fermentation material for further fermentation. The lactic acid bacteria are randomly distributed inside and outside the particles, making them susceptible to the effects of temperature, moisture changes, and mechanical shearing during subsequent drying, transportation, and storage. Some technologies use alginate, pectin, starch, or protein-based wall materials to microencapsulate or granulate probiotics; however, this type of protective treatment is usually carried out independently after cell culture or feed fermentation, requiring additional wall materials, cross-linking agents, and encapsulation equipment. The external coating layer mainly adheres to the surface of the fermentation particles, and the coating layer is prone to cracking or delamination during drying, impact, or moisture absorption. Furthermore, the coating material may block the original pores of the fermentation particles, affecting the release of fermentation products and subsequent digestibility and utilization.

[0004] Furthermore, existing technologies for immobilizing probiotics using calcium ion cross-linked polysaccharides often involve pre-mixing soluble or slightly soluble calcium sources with polysaccharide materials, followed by the addition of acid or a cross-linking agent to form a gel. However, the spatial position of the calcium source within the material is difficult to control. When the calcium source is uniformly distributed throughout the fermentation particle, the cross-linking reaction may penetrate the particle interior, causing core pore shrinkage and restricting mass transfer. Conversely, when the calcium source remains only on the outermost surface of the particle, it easily forms a surface gel that separates rapidly from the core. Current technologies struggle to maintain the protein hydrolysis and pore structure of the fermentation core while simultaneously ensuring stable accumulation of lactic acid bacteria on the outer side of the particle, forming a continuous and non-detachable fixed structure connected to the fermentation core. Consequently, issues such as unstable fermentation control, disordered spatial distribution of probiotics, and insufficient product storage stability persist. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a compound probiotic fermented feed additive and its preparation method, thereby solving the technical problems existing in the prior art.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution: A compound probiotic fermented feed additive, wherein the compound probiotic fermented feed additive is a granular structure formed from protein-containing feed raw materials and pectin-containing plant raw materials. The granular structure includes a fermentation core, a calcium source transition zone formed in the pores at the outer edge of the fermentation core, and a cross-linking fixation layer covering the outer surface of the fermentation core and at least partially embedded in the pores at the outer edge of the fermentation core. The calcium source transition zone is located inside the cross-linking fixation layer. The fermented core contains protein hydrolysates, live Bacillus bacteria, live yeast, and core pectin derived from pectin-containing plant materials. The cross-linked immobilization layer contains low-esterified pectin formed by demethylation of the outer part of the core pectin. The low-esterified pectin is continuously connected to the core pectin and forms an ionic cross-linking network through cross-linked calcium. Lactic acid-producing probiotics are immobilized in the ionic cross-linking network. The calcium source transition zone forms a calcium enrichment area derived from a slightly soluble calcium source. According to the calcium element line scan detection of the granular structure cross section, the calcium element signal peak is located between the outer edge of the fermentation core and the inner side of the cross-linked fixation layer. After separating the cross-linked immobilization layer and the fermentation core, based on the dry weight of each separated part, the number of viable lactic acid-producing probiotics in the cross-linked immobilization layer was higher than that in the fermentation core, and the number of viable Bacillus in the fermentation core was higher than that in the cross-linked immobilization layer.

[0007] Preferably, based on the dry matter mass of each raw material at the time of feeding, the raw materials forming the fermentation core include 45-65 parts of protein-containing feed ingredients, 15-30 parts of pectin-containing plant ingredients, 8-20 parts of fermentable carbohydrate ingredients, and 5-15 parts of wheat bran; the protein-containing feed ingredients include soybean meal, and the pectin-containing plant ingredients include at least one of citrus pomace, apple pomace, and beet pomace, with an equivalent particle size of 2-8 mm and a moisture content of 8%-12%.

[0008] Preferably, the low-esterified pectin in the cross-linked immobilization layer is formed by the demethylation and esterification of the core pectin on the outside of the fermentation core. The degree of esterification of the low-esterified pectin is less than 50%, the degree of esterification of the core pectin inside the fermentation core is not less than 50%, and the difference in degree of esterification between the low-esterified pectin and the core pectin inside the fermentation core is not less than 10 percentage points.

[0009] Preferably, the slightly soluble calcium source is calcium carbonate, and the circumferential coverage of the calcium source transition zone on the cross-section of the granular structure is not less than 70%; the cross-linked fixing layer extends along the outer surface of the fermentation core, and the portion of the cross-linked fixing layer embedded in the outer edge pores of the fermentation core is continuously connected to the portion located on the outer surface of the fermentation core.

[0010] Preferably, the Bacillus is Bacillus subtilis, the yeast is Saccharomyces cerevisiae, and the lactic acid-producing probiotic is Lactobacillus plantarum; based on the dry weight of each separated part, the ratio of the number of viable Lactobacillus plantarum in the cross-linked immobilization layer to the number of viable Lactobacillus plantarum in the fermentation core is not less than 3, and the ratio of the number of viable Bacillus subtilis in the fermentation core to the number of viable Bacillus subtilis in the cross-linked immobilization layer is not less than 2.

[0011] A method for preparing a compound probiotic fermented feed additive includes the following steps: S1: Mix protein-containing feed ingredients, pectin-containing plant ingredients and fermentation additives, adjust the moisture content of the mixture and make it into a granular fermentation substrate, inoculate the granular fermentation substrate with a first probiotic including Bacillus and yeast, and carry out the first stage of fermentation under aeration conditions to hydrolyze the protein-containing feed ingredients and form a fermentation core containing protein hydrolysate, live Bacillus, live yeast and core pectin; S2: After the first stage of fermentation is completed, pectin methyl esterase solution is sprayed onto the outer surface of the fermentation core to cause the pectin on the outside of the fermentation core to undergo demethylation and esterification, forming a low-esterified pectin zone on the outside of the fermentation core, and making the esterification degree of pectin in the low-esterified pectin zone lower than that of the pectin in the inside of the fermentation core. S3: While maintaining the particle shape of the fermentation core, the pressure difference is used to allow the suspension containing a slightly soluble calcium source to enter the outer pores of the fermentation core, and the slightly soluble calcium source is controlled to accumulate on the inner side of the low-esterified pectin zone, and the free suspension attached to the outer surface of the low-esterified pectin zone is removed, so as to form a calcium source transition zone on the outer edge of the fermentation core. S4: Inoculate lactic acid-producing probiotics on the outside of the low-esterified pectin zone and carry out the second stage of fermentation under closed conditions. This allows the lactic acid produced by the lactic acid-producing probiotics to migrate from the low-esterified pectin zone to the calcium source transition zone. The lactic acid dissolves the slightly soluble calcium source and releases calcium ions. S5: Causes calcium ions to migrate from the calcium source transition region to the low-esterified pectin region and undergo ionic cross-linking with the low-esterified pectin in the low-esterified pectin region, transforming the low-esterified pectin region into a cross-linked immobilization layer that covers the outer surface of the fermentation core and is at least partially embedded in the pores of the outer edge of the fermentation core, while immobilizing at least some lactic acid-producing probiotics in the cross-linked immobilization layer. S6: Dry the granules after the second stage of fermentation to obtain a compound probiotic fermented feed additive.

[0012] Preferably, based on the dry matter mass of each raw material at the time of feeding, 45-65 parts of protein-containing feed raw materials, 15-30 parts of pectin-containing plant raw materials, 8-20 parts of fermentable carbohydrate raw materials and 5-15 parts of wheat bran are mixed, the moisture content of the resulting mixture is adjusted to 42%-50%, and then a granular fermentation substrate with an equivalent particle size of 2-8 mm is prepared. Protein-containing feed ingredients include soybean meal; pectin-containing plant ingredients include at least one of citrus pomace, apple pomace, and beet pomace; the primary probiotics include Bacillus subtilis and Saccharomyces cerevisiae, with an inoculum size of 110 g of Bacillus subtilis based on the dry matter mass of the granular fermentation substrate. 6 ~110 8 CFU / g, the inoculum size of *Saccharomyces cerevisiae* was 110. 5 ~110 7 CFU / g.

[0013] Preferably, the temperature of the first stage of fermentation is 32-36℃. During the fermentation, the granular fermentation substrate is intermittently aerated and turned, and the carbon dioxide concentration in the fermentation tail gas is continuously collected. The amount of carbon dioxide released per unit dry matter granular fermentation substrate per unit time is determined based on the fermentation tail gas flow rate and carbon dioxide concentration. When the carbon dioxide release rate drops to 40%–70% of the peak value after reaching its peak, and the relative change rate of acid-soluble protein content between two fermentation core samples obtained at a 2-hour interval is no greater than 5%, the first stage of fermentation ends. The amount of pectin methyl esterase solution sprayed is 2% to 8% of the dry weight of the fermentation core, and the amount of pectin methyl esterase added is 50 to 500 U per 100g of dry matter in the fermentation core. After spraying, keep it at 25 to 35℃ for 20 to 90 minutes to ensure that the degree of esterification of pectin in the low-esterification pectin zone is less than 50%, and that the difference in the degree of esterification between pectin in the low-esterification pectin zone and the pectin in the core of the fermentation core is not less than 10 percentage points.

[0014] Preferably, the fermentation core with the low-esterified pectin zone is placed under an absolute pressure of 20-60 kPa for 2-10 minutes, and the fermentation core is brought into contact with a calcium carbonate suspension with a mass fraction of 1%-5% under the absolute pressure. Then the pressure is restored to 90-105 kPa, so that the calcium carbonate suspension enters the outer pores of the fermentation core. After the pressure is restored, the calcium carbonate suspension that did not enter the fermentation core is discharged, and the fermentation core is subjected to rolling drainage and surface air supply to remove the calcium carbonate suspension attached to the outer surface of the low-esterified pectin zone, while retaining the calcium carbonate that entered the outer edge pores of the fermentation core, so that the peak value of the calcium element signal obtained by the calcium element line scan detection through the cross section of the fermentation core is located inside the low-esterified pectin zone.

[0015] Preferably, the lactic acid-producing probiotic is *Lactobacillus plantarum*, and the inoculum quantity of *Lactobacillus plantarum* is 110 g / L based on the dry matter mass of the fermentation core. 7 ~110 9 CFU / g; After inoculation, the moisture content of the particles was adjusted to 52%–60%, and the second stage of fermentation was carried out under sealed conditions at 30–34℃; The second stage of fermentation ends when the pH of the fermentation system drops to 3.8–4.2 and the relative change rate of bound calcium content between two cross-linked immobilized layer samples obtained at a 2-hour interval is no greater than 5%. After the second stage of fermentation, vacuum drying or fluidized bed drying is carried out under the condition that the particle temperature does not exceed 40℃ until the moisture content of the resulting compound probiotic fermented feed additive is 8% to 12%.

[0016] In summary, the present invention has the following main beneficial effects: By first using Bacillus subtilis and Saccharomyces cerevisiae to conduct a first-stage aerated fermentation of the granular fermentation substrate, the macromolecular proteins in soybean meal are hydrolyzed, and a fermentation core containing protein hydrolysis products and outer pores is formed while maintaining the granular shape. At the same time, the endpoint of the first-stage fermentation is determined by the changes in carbon dioxide release rate and acid-soluble protein content. This achieves the effect of reducing the impact of different batches of raw materials and the fluctuation of bacterial activity on the degree of fermentation, avoiding under-fermentation or over-fermentation, and maintaining the structural integrity of the fermentation core.

[0017] By performing localized demethylation and esterification of the pectin derived from pectin-containing plant materials on the outer side of the fermentation core, a low-esterified pectin zone is formed on the outer side of the particle. A slightly soluble calcium source is then loaded into the pores at the outer edge of the fermentation core within this low-esterified pectin zone using a pressure difference. This achieves the effect of creating a gradient in pectin esterification and a localized distribution of calcium within the same particle. This maintains material continuity between the low-esterified pectin zone and the fermentation core, avoiding the use of externally added commercial low-esterified pectin to form an easily separable coating from the fermentation core. It also prevents the slightly soluble calcium source from uniformly entering the entire fermentation core, which could lead to excessive cross-linking and pore blockage within the core.

[0018] By inoculating *Lactobacillus plantarum* on the outer side of the low-esterified pectin zone, it continuously produces lactic acid during the second-stage closed fermentation. This promotes the migration of lactic acid from the outside to the inside, releasing calcium ions from the calcium source transition zone. The calcium ions then migrate from the inside to the outside and undergo ionic cross-linking with the low-esterified pectin, achieving the effect of forming a continuous cross-linked immobilization layer in situ on the outer surface of the fermentation core and in its outer edge pores. This cross-linked immobilization layer can fix *Lactobacillus plantarum* on the outer side of the particle, while keeping *Bacillus subtilis* mainly within the fermentation core, thus forming a spatial partition of the complex probiotics and reducing the impact of subsequent drying, storage, and acidic environments on the activity of *Lactobacillus plantarum*. Attached Figure Description

[0019] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0020] 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.

[0021] refer to Figure 1 This application discloses a compound probiotic fermented feed additive and its preparation method. The compound probiotic fermented feed additive described herein does not involve simultaneously adding multiple probiotics to feed ingredients for ordinary mixed fermentation, nor does it involve independently coating lactic acid bacteria with an external wall material after fermentation. Instead, it first forms a fermentation core with protein hydrolysis products and outer pores. Then, the pectin from pectin-containing plant materials on the outside of the fermentation core undergoes localized demethylation and esterification. Subsequently, a slightly soluble calcium source is loaded into the outer pores of the fermentation core inside the low-esterified pectin zone. Finally, *Lactobacillus plantarum* located outside the low-esterified pectin zone continuously produces acid, causing lactic acid to migrate inward and release calcium ions. The released calcium ions migrate outward and cross-link with the low-esterified pectin, thereby forming a cross-linked fixed layer continuously connected to the fermentation core.

[0022] During implementation, the mass fraction of each raw material is based on the dry matter mass at the time of feeding; the inoculation amount is expressed as the number of viable bacteria per gram of granular fermentation substrate dry matter or per gram of fermentation core dry matter. When there are differences in moisture content, pectin content, initial pectin esterification degree, and water absorption capacity among different batches of raw materials, the amount of water added and the amount of pectin methyl esterase added are calculated based on the actual test results, and the wet mass of the raw materials is not directly used to replace the dry matter mass.

[0023] Soybean meal is used as the protein feed ingredient. Before use, the moisture and crude protein content of the soybean meal are tested, and it is ground to a size that can pass through a 2mm sieve.

[0024] Pectin-containing plant materials include citrus pomace, apple pomace, or beet pomace. Before use, the moisture content, pectin content, and initial esterification degree of these materials are tested, and the material is pulverized to pass through a 2mm sieve. When changing pectin-containing plant materials, the pectin methyl esterase treatment time is re-determined based on the actual measured pectin content and initial esterification degree of that batch of material; the treatment time of other materials is not directly adopted.

[0025] The fermentable carbohydrate raw material is corn flour, and the bran is wheat bran. Corn flour is used to provide the available carbon source required for the first and second stages of fermentation, while wheat bran is used to regulate the water-holding capacity, pellet formation properties, and pore structure of the fermentation core.

[0026] The Bacillus strain used is Bacillus subtilis, the yeast strain is Saccharomyces cerevisiae, and the lactic acid-producing probiotic strain is Lactobacillus plantarum. The strains used are inoculum agents or seed liquids with strain identification data and viable cell count data.

[0027] When using inoculum powder, first verify the plate count of the inoculum powder, and then calculate the added mass based on the measured number of viable bacteria. When using liquid seed liquid, first determine the concentration of viable bacteria in the seed liquid, and then calculate the inoculation volume based on the dry matter mass of the granular fermentation substrate or fermentation core.

[0028] Bacillus subtilis was cultured in a liquid medium suitable for Bacillus growth at 35–37°C and 160–200 rpm for 12–16 h; Saccharomyces cerevisiae was cultured in yeast extract peptone glucose medium at 28–30°C and 120–180 rpm for 16–20 h; and Lactobacillus plantarum was cultured statically in MRS medium at 35–37°C for 16–20 h. The viable cell count in the seed culture was determined before inoculation.

[0029] Pectin methyl esterase is used in food-grade or feed-grade pectin methyl esterase. One unit of pectin methyl esterase activity is defined as the amount of enzyme required to catalyze the production of 1 mol of titratable carboxyl groups per minute using a 1% (w / w) high-ester pectin solution as a substrate at 30°C and pH 7.0. When using commercial enzyme preparations, the enzyme activity labeled on the product should be converted according to the above definition.

[0030] The slightly soluble calcium source used is feed-grade calcium carbonate. The particle size distribution of the calcium carbonate is tested before use, and at least recorded. The selected calcium carbonate should be able to enter the pores at the outer edge of the fermentation core with the suspension, but should not penetrate to the central region of the fermentation core with the suspension. The calcium carbonate suspension should be continuously and slowly stirred after preparation to prevent the calcium carbonate particles from settling during loading.

[0031] Mix protein-containing feed ingredients, pectin-containing plant ingredients, fermentable carbohydrate ingredients and wheat bran. Calculate the amount of water to add based on the measured moisture content of each ingredient, and adjust the moisture content of the mixture to 42%–50%.

[0032] The conditioned mixture is granulated into wet granules through extrusion granulation, disc granulation, or balling granulation. The granules do not need to be perfectly spherical; the equivalent particle size is calculated based on the projected area of ​​the granules in the vertical projection direction. At least 30 granules from each batch are randomly selected for image measurement, and granules with an equivalent particle size of 2–8 mm are retained.

[0033] When the equivalent particle size is less than 2 mm, pectin methyl esterase solution and calcium carbonate suspension easily enter the center of the particle, which is not conducive to the formation of a low-esterified pectin zone and a calcium source transition zone. When the equivalent particle size is greater than 8 mm, the internal aeration, heat dissipation, and mass transfer of the particle are affected. Therefore, the particle size is determined by the common criteria of uniform fermentation in the first stage, pectin methyl esterase mainly acting on the outer side of the particle, and calcium carbonate being able to accumulate in the outer edge pores.

[0034] Bacillus subtilis and Saccharomyces cerevisiae were inoculated into the granular fermentation substrate, and the first stage of fermentation was carried out under aeration conditions of 32–36°C. During fermentation, intermittent aeration and slow turning were used. The aeration rate was adjusted according to the material temperature to ensure that the internal temperature of the particles did not exceed the upper limit of the set fermentation temperature, and the particles were kept intact during the turning process.

[0035] The first-stage fermentation vessel is equipped with an inlet gas flow meter, an exhaust gas flow meter, and an exhaust carbon dioxide detector. The carbon dioxide release rate is determined based on the carbon dioxide content and flow rate in the inlet and exhaust gases. The carbon dioxide release rate is calculated using the following formula: ; In the formula, The amount of carbon dioxide released per unit dry matter granular fermentation substrate per unit time. To convert the exhaust gas flow rate to standard conditions; To convert the intake airflow to standard conditions; This represents the volume fraction of carbon dioxide in the exhaust gas. This represents the volume fraction of carbon dioxide in the intake air. This refers to the molar volume of the gas under standard conditions. This refers to the dry matter mass of the fermentation material.

[0036] The average value of the data collected over a continuous 1-hour period was used as the carbon dioxide release rate for that period, and the maximum 1-hour average value that occurred during the first stage of fermentation was used as the peak value.

[0037] After the carbon dioxide release rate reached its peak, fermentation core samples were collected every 2 hours, and the acid-soluble protein content was determined using the trichloroacetic acid extraction method. The percentage obtained by dividing the absolute value of the difference between the current measurement and the previous measurement by the previous measurement was taken as the relative change rate of acid-soluble protein content.

[0038] The first stage of fermentation ends when the carbon dioxide release rate drops to 40%–70% of the peak value and the relative change rate of acid-soluble protein content between two samples obtained 2 hours apart is no more than 5%.

[0039] The carbon dioxide release rate reflects the overall metabolic state of Bacillus subtilis and Saccharomyces cerevisiae, while the acid-soluble protein content reflects the degree of hydrolysis of soybean meal protein. If neither condition is met simultaneously, the first stage of fermentation continues, and the endpoint is not directly determined by a fixed fermentation time.

[0040] After the first stage of fermentation, a fermented core that retains its granular shape is obtained. The fermented core contains acid-soluble protein components formed from the hydrolysis of soybean meal protein, live Bacillus subtilis, live Saccharomyces cerevisiae, and core pectin derived from pectin-containing plant materials.

[0041] After the first stage of fermentation is completed, the fermentation core is not crushed. The fermentation core is then sprayed with pectin methyl esterase solution while rolling. The amount of pectin methyl esterase solution sprayed is 2% to 8% of the dry weight of the fermentation core, and the amount of pectin methyl esterase added is 50 to 500 U per 100g of dry matter in the fermentation core.

[0042] During the spraying process, the enzyme solution covers the outer surface of the fermentation core, but the fermentation core is not completely immersed in the pectin methyl esterase solution. After spraying, maintain the temperature at 25–35°C for 20–90 minutes to allow the pectin on the outer side of the fermentation core to preferentially undergo demethylation.

[0043] After processing, the particles are frozen and fixed, and then cut along a plane passing through the geometric center of the particles. The portion extending from the outer surface of the particles inwards, not exceeding 15% of the particle's equivalent radius, is designated as the outer sampling area; the portion centered on the particle's geometric center, not exceeding 40% of the particle's equivalent radius, is designated as the core sampling area.

[0044] Pectin was extracted from the two regions separately, and the degree of pectin esterification was determined by titration. When the degree of esterification of the outer pectin was less than 50%, the degree of esterification of the pectin inside the core was not less than 50%, and the difference between the two degrees of esterification was not less than 10 percentage points, a low-esterified pectin zone was determined to have formed on the outer side of the fermentation core.

[0045] If the degree of esterification of the outer pectin layer does not meet the above conditions, the treatment should be extended by 10-15 minutes each time, and then retested, but the total treatment time should not exceed 90 minutes. If the target esterification degree difference still cannot be achieved after a total treatment time of 90 minutes, the initial degree of esterification of the pectin-containing plant material, the equivalent particle size, the activity of pectin methyl esterase, and the uniformity of spraying should be rechecked, and the material should not proceed directly to the calcium source loading step.

[0046] The low-esterified pectin zone is formed by localized demethylation and esterification of the core pectin in the fermentation core itself, rather than by an independent coating layer formed by spraying commercial low-ester pectin onto the outer surface of the fermentation core.

[0047] The fermentation core that forms the low-esterified pectin zone is placed in a vacuum container, and the absolute pressure of the container is reduced to 20-60 kPa and maintained for 2-10 minutes to allow some of the gas in the pores at the outer edge of the fermentation core to be discharged.

[0048] Under negative pressure, the fermentation core is brought into contact with a calcium carbonate suspension with a mass fraction of 1%–5%, and then the container pressure is restored to 90–105 kPa. During the pressure restoration process, the calcium carbonate suspension enters the pores at the outer edge of the fermentation core under the action of the pressure difference.

[0049] Immediately after the pressure is restored, the calcium carbonate suspension that did not enter the fermentation core is discharged. The particles are then placed in a rolling draining device and slowly rolled while surface air is supplied to remove the free suspension adhering to the outer surface of the low-esterified pectin zone.

[0050] The obtained particles were weighed twice at 1-minute intervals. When the particle mass change rate was no greater than 0.2% and there was no continuous liquid film on the outer surface of the particles, the rolling draining and surface aeration were stopped. This treatment retains calcium carbonate that enters the pores at the outer edge of the fermentation core while reducing calcium carbonate residue on the outermost surface of the particles.

[0051] No fewer than five particles were randomly selected from each batch of particles, and after resin embedding, they were cut along a plane passing through the geometric center of the particles. The calcium element was then scanned by line and surface scanning using a scanning electron microscope energy dispersive spectroscopy or a micro-area X-ray fluorescence analyzer.

[0052] When the peak value of the calcium element signal is located between the outer edge of the fermentation core and the inner side of the low-esterified pectin zone, rather than at the geometric center of the particle or the outermost surface, it is determined that a calcium source transition zone has been formed.

[0053] The arc length of the calcium enrichment region along the outer edge of the fermentation core was determined by scanning surface images of calcium. When the circumferential coverage of the calcium enrichment region on the particle cross-section was not less than 70%, the batch of particles entered the second stage of fermentation.

[0054] When the calcium signal peak is located on the outermost surface of the particles, extend the rolling drainage and surface air supply time; when the calcium signal peak enters the central region of the particles, shorten the vacuum holding time, increase the absolute pressure of the container, or increase the particle size of calcium carbonate particles; when the circumferential coverage is insufficient, adjust the contact time of the suspension after back pressure without allowing the calcium source to enter the center of the particles.

[0055] Lactobacillus plantarum was inoculated onto the outer side of the low-esterified pectin zone. The inoculation amount of Lactobacillus plantarum was 110 g / L based on the dry matter mass of the fermentation core. 7 ~110 9 CFU / g. After inoculation, the moisture content of the particles was adjusted to 52%–60%, and the second stage of fermentation was carried out under sealed conditions at 30–34℃.

[0056] Lactobacillus plantarum grows on the outer side of the particle and continuously produces lactic acid. Lactic acid migrates from the low-esterified pectin region to the calcium source transition region, causing calcium carbonate in the calcium source transition region to gradually release calcium ions. The released calcium ions migrate from the calcium source transition region to the low-esterified pectin region and undergo ionic cross-linking with the carboxyl groups in the low-esterified pectin, gradually transforming the low-esterified pectin region into a cross-linked fixed layer.

[0057] Because calcium carbonate is located inside the low-esterification pectin region, the cross-linking reaction proceeds from the inside of the low-esterification pectin region outwards. The formed cross-linked immobilization layer covers the outer surface of the fermentation core and enters the outer edge pores of the fermentation core, thus continuously connecting the cross-linked structures on the outer surface with the cross-linked structures that enter the outer edge pores.

[0058] During the second stage of fermentation, the pH value of the fermentation system was continuously monitored. After the pH value decreased to 3.8–4.2, particle samples were obtained every 2 hours. The free calcium salts on the outer surface of the particles were quickly washed off to obtain cross-linked immobilized layer samples and the bound calcium content was determined.

[0059] The absolute value of the difference between the current bound calcium content and the previous bound calcium content, divided by the previous bound calcium content, is used as the percentage to represent the relative change rate of bound calcium content. The second stage of fermentation ends when the pH value is 3.8–4.2 and the relative change rate of bound calcium content between two samples taken 2 hours apart is no greater than 5%.

[0060] If the pH condition is met but the calcium content continues to increase, the second stage of fermentation continues to avoid prematurely ending the fermentation before the cross-linked fixed layer has been stably formed.

[0061] After the second stage of fermentation, vacuum drying or fluidized bed drying is used to reduce the moisture content of the particles. During the drying process, a temperature sensor inserted into the particle layer is used to monitor the material temperature, ensuring that the particle temperature does not exceed 40℃.

[0062] The control here refers to the actual temperature of the pellets, not just the air inlet temperature of the equipment. Drying is stopped when the moisture content of the compound probiotic fermented feed additive reaches 8%–12%, then it is cooled to room temperature and sealed for storage.

[0063] The dried, intact particles were frozen and fixed, and a cross-section passing through the geometric center of the particles was prepared. The cross-section was then observed using a scanning electron microscope, a confocal microscope, or a staining method that can distinguish between the pectin cross-linked structure and the fermentation core.

[0064] The cross-linked immobilization layer and the fermentation core are considered to form a continuous connection structure when the following conditions are met: The cross-linked fixation layer covers the outer surface of the fermentation core; the cross-linked fixation layer at least partially enters the original pores at the outer edge of the fermentation core; the cross-linked structure entering the outer edge pores is continuous with the cross-linked structure located on the outer surface of the particle; there is no continuous separation gap between the cross-linked fixation layer and the fermentation core extending along the entire observation section.

[0065] At least 5 particles should be randomly tested in each batch, and at least two mutually perpendicular central cross sections should be observed for each particle. The formation of the cross-linked fixation layer cannot be inferred solely from the particle's appearance, surface hardness, or water absorption.

[0066] Take the dried granules and rinse them three times quickly with sterile physiological saline at a ratio of 1:10 between the mass of the granules and the volume of physiological saline, shaking for 30 seconds each time, to remove free bacteria that are not fixed to the cross-linked fixation layer.

[0067] The rinsed particles were added to a 1% (w / w) sterile sodium citrate solution at a particle mass to sodium citrate solution volume ratio of 1:10. The mixture was shaken at 4℃ and 60 rpm for 10 min to de-crosslink the pectin calcium ion crosslinking network. The de-crosslinking solution and its solids were collected as the crosslinking fixation layer sample.

[0068] The remaining particles after removing the cross-linking fixation layer were quickly rinsed once with sterile physiological saline and aseptically ground to obtain the fermentation core sample. The dry matter mass of the cross-linking fixation layer and the fermentation core, as well as the viable cell count of each bacterial species, were determined separately.

[0069] Lactobacillus plantarum was cultured in a lactic acid bacteria selective medium at 37°C for 48 hours under anaerobic conditions before counting; Bacillus subtilis was cultured in a selective medium that had been verified to distinguish Bacillus subtilis for 24–36 hours under aerobic conditions at 37°C before counting; and Saccharomyces cerevisiae was cultured in a yeast selective medium at 28–30°C for 48–72 hours before counting.

[0070] The ratio of the cross-linked immobilized layer of *Lactobacillus plantarum* to the viable cell count in the fermentation core was calculated as follows: ; In the formula, The ratio of the cross-linked immobilization layer of *Lactobacillus plantarum* to the number of viable bacteria in the fermentation core; The number of viable Lactobacillus plantarum bacteria in the cross-linked immobilization layer; This represents the number of viable Lactobacillus plantarum bacteria in the fermentation core.

[0071] The ratio of viable bacteria in the Bacillus subtilis fermentation core to the cross-linked immobilization layer was as follows: ; In the formula, The ratio of viable bacteria in the Bacillus subtilis fermentation core to the cross-linked immobilization layer; The number of viable Bacillus subtilis cells in the fermentation core; This represents the number of viable Bacillus subtilis cells in the cross-linked immobilization layer. Not less than 3, and When the value is not less than 2, it is determined that the compound probiotic fermented feed additive forms a spatial distribution structure in which Lactobacillus plantarum is mainly distributed in the cross-linked fixed layer and Bacillus subtilis is mainly distributed in the fermentation core.

[0072] The viable cell count of the brewing yeast was performed at the end of the first stage of fermentation, the end of the second stage of fermentation, and after drying to confirm the presence of live brewing yeast in the final product.

[0073] Example 1 Weigh out 55 parts soybean meal, 20 parts citrus pomace, 15 parts corn flour, and 10 parts wheat bran based on the dry matter mass of each raw material at the time of feeding. Calculate the amount of water to add based on the measured moisture content of each raw material, and adjust the moisture content of the mixture to 46% to prepare a granular fermentation substrate with an equivalent particle size of 3-6 mm.

[0074] Based on the dry matter weight of the granular fermentation substrate, inoculate with Bacillus subtilis up to 110... 7 CFU / g, inoculated with Saccharomyces cerevisiae to 110 6 CFU / g. The first stage of fermentation was carried out at 34℃, with aeration for 10 minutes every 30 minutes and slow turning of the material every 4 hours.

[0075] The carbon dioxide release rate was continuously monitored. After the carbon dioxide release rate reached its peak, the acid-soluble protein content was measured every 2 hours. The first stage of fermentation ended when the carbon dioxide release rate dropped to 55% of the peak value and the relative change rate of the acid-soluble protein content was no more than 5%.

[0076] Spray pectin methyl esterase solution at 5% of the dry weight of the fermentation core. The amount of pectin methyl esterase added is 200U per 100g of dry fermentation core. Treat at 30℃ for 45min.

[0077] After processing, the esterification degree of pectin in the outer layer and the pectin esterification degree in the core of the particles were measured separately. Calcium source loading was carried out only after the conditions were met that the esterification degree of pectin in the outer layer was less than 50%, the esterification degree of pectin in the core was not less than 50%, and the difference between the two was not less than 10 percentage points.

[0078] The fermentation core was placed in a vacuum container, and the absolute pressure was reduced to 40 kPa and maintained for 5 minutes. Under this pressure, the fermentation core was brought into contact with a 3% (w / w) calcium carbonate suspension, and then the pressure was restored to 100 kPa.

[0079] Drain the calcium carbonate suspension that did not enter the fermentation core, slowly roll the particles for 3 minutes and perform surface air blowing until there is no continuous liquid film on the particle surface, and the change rate of particle mass obtained by weighing twice at 1 minute intervals is not greater than 0.2%.

[0080] Linear and surface scans of calcium were performed on the particle cross-section. The second stage of fermentation proceeded only after the calcium signal peak was located inside the low-esterified pectin region and the circumferential coverage of the calcium source transition zone was no less than 70%.

[0081] Based on the dry matter mass of the fermentation core, *Lactobacillus plantarum* was inoculated onto the outer side of the low-esterified pectin zone up to 110 μL. 8 The particle moisture content was adjusted to 56% by CFU / g, and the second stage of fermentation was carried out under sealed conditions at 32℃.

[0082] The second stage of fermentation ends when the pH of the fermentation system decreases to 3.8–4.2 and the relative change rate of bound calcium content between two cross-linked immobilized layer samples obtained at a 2-hour interval is no greater than 5%.

[0083] Vacuum drying was used to dry the particles, ensuring the particle temperature did not exceed 40℃. After drying to a particle moisture content of 8%–12%, the particles were cooled and sealed for storage, yielding a compound probiotic fermented feed additive.

[0084] The obtained additives were subjected to particle cross-section observation, calcium element scanning, and stratified viable bacteria counting to confirm that the cross-linked fixation layer and the fermentation core were continuously connected, that the calcium element signal peak was located between the outer edge of the fermentation core and the inner side of the cross-linked fixation layer, and to confirm the spatial distribution relationship of Lactobacillus plantarum and Bacillus subtilis.

[0085] Example 2 Weigh out 45 parts soybean meal, 30 parts citrus pomace, 20 parts corn flour, and 5 parts wheat bran based on the dry matter mass of each raw material at the time of feeding. Adjust the moisture content of the mixture to 42% to prepare a granular fermentation substrate with an equivalent particle size of 2-5 mm.

[0086] Based on the dry matter weight of the granular fermentation substrate, inoculate with Bacillus subtilis up to 110... 6 CFU / g, inoculated with Saccharomyces cerevisiae to 110 5 The first stage of fermentation was carried out at 32°C with CFU / g.

[0087] The first stage of fermentation ends when the carbon dioxide release rate drops to 70% of its peak and the relative change rate of acid-soluble protein content is no more than 5%.

[0088] Spray pectin methyl esterase solution at 2% of the dry weight of the fermentation core. The amount of pectin methyl esterase added is 50U per 100g of dry matter in the fermentation core. Treat at 25℃ for 20min and confirm the low esterification pectin zone based on the actual measured pectin esterification degree.

[0089] The absolute pressure was reduced to 60 kPa and maintained for 2 minutes to bring the fermentation core into contact with a 1% (w / w) calcium carbonate suspension. The pressure was then restored to 90–105 kPa. The free suspension on the outer surface of the particles was removed, and the calcium source transition zone was identified by the peak position of the calcium element signal and the circumferential coverage.

[0090] Based on the dry matter weight of the fermentation core, inoculate with Lactobacillus plantarum to a concentration of 110. 7 The particle moisture content was adjusted to 52% by adjusting the CFU / g, and the second stage of fermentation was carried out under sealed conditions at 30℃.

[0091] After the second stage of fermentation reaches the dual endpoints of pH value and relative change rate of bound calcium content, the particles are dried to a moisture content of 8% to 12% under conditions where the particle temperature does not exceed 40℃.

[0092] Example 3 Weigh out 65 parts soybean meal, 15 parts apple pomace, 8 parts corn flour, and 12 parts wheat bran based on the dry matter mass of each raw material at the time of feeding. Adjust the moisture content of the mixture to 50% to prepare granular fermentation substrate with an equivalent particle size of 5-8 mm.

[0093] Based on the dry matter weight of the granular fermentation substrate, inoculate with Bacillus subtilis up to 110... 8 CFU / g, inoculated with Saccharomyces cerevisiae to 110 7 The first stage of fermentation was carried out at 36°C with CFU / g.

[0094] The first stage of fermentation ends when the carbon dioxide release rate drops to 40% of the peak value and the relative change rate of acid-soluble protein content is no more than 5%.

[0095] Spray pectin methyl esterase solution at 8% of the dry weight of the fermentation core. The amount of pectin methyl esterase added is 500U per 100g of dry fermentation core. Treat at 35℃ for 90min and measure the degree of pectin esterification in the outer layer of the particles and the inside of the core.

[0096] The absolute pressure was reduced to 20 kPa and maintained for 10 minutes to bring the fermentation core into contact with a 5% (w / w) calcium carbonate suspension. The pressure was then restored to 90–105 kPa. The free suspension on the particle surface was removed, and cross-sections of the particle center were prepared from different directions to confirm that the calcium signal peak did not enter the particle center region.

[0097] Based on the dry matter weight of the fermentation core, inoculate with Lactobacillus plantarum to a concentration of 110. 9 The particle moisture content was adjusted to 60% and the second stage of fermentation was carried out under sealed conditions at 34℃.

[0098] After the second stage of fermentation reaches the dual endpoints of pH value and relative change rate of bound calcium content, the particles are dried to a moisture content of 8% to 12% under conditions where the particle temperature does not exceed 40℃.

[0099] Example 4 In this embodiment, beet pulp is used as a pectin-containing plant material.

[0100] Weigh out 55 parts soybean meal, 20 parts beet pulp, 15 parts corn flour, and 10 parts wheat bran based on the dry matter mass of each raw material at the time of feeding. Before use, determine the moisture content, pectin content, and initial pectin esterification degree of the beet pulp, adjust the moisture content of the mixture to 46%, and prepare granular fermentation substrate with an equivalent particle size of 3-6 mm.

[0101] The inoculation amounts of Bacillus subtilis and Saccharomyces cerevisiae, the first-stage fermentation conditions, the calcium source loading conditions, the inoculation amount of Lactobacillus plantarum, the second-stage fermentation conditions, and the drying conditions were the same as in Example 1.

[0102] Pectin methyl esterase treatment was initiated with an initial addition of 200U per 100g of fermented core dry matter, and the treatment time was adjusted based on the actual esterification degree of pectin from beet pulp. Calcium source loading was only carried out after the esterification degree of pectin on the outer layer of the particles was less than 50%, the esterification degree of pectin inside the core was not less than 50%, and the difference between the two was not less than 10 percentage points.

[0103] This embodiment illustrates that when changing pectin-containing plant raw materials, the actual measured results of pectin esterification degree should be used as the basis for unified process judgment, and it is not appropriate to directly assume that pectin from different sources has reached the same degree of demethylation and esterification based on the same treatment time.

[0104] Comparative Example 1 A granular fermentation substrate was prepared according to the raw material composition and moisture content of Example 1. Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum were inoculated simultaneously without distinguishing between the first stage fermentation and the second stage fermentation, and without limiting the inoculation position of Lactobacillus plantarum.

[0105] Comparative Example 1 was used to compare the effects of simultaneous inoculation and staged localized inoculation on the spatial distribution of Lactobacillus plantarum and Bacillus subtilis.

[0106] Comparative Example 2 The first stage of fermentation was completed according to Example 1, but without spraying pectin methyl esterase solution, and the second stage of fermentation of calcium carbonate and Lactobacillus plantarum was carried out directly.

[0107] Comparative Example 2 was used to confirm the effect of pectin localized demethylation on the outside of the fermentation core on the formation of the cross-linked fixed layer.

[0108] Comparative Example 3 The first stage of fermentation was completed according to Example 1. Instead of localized demethylation of the pectin in the fermentation core itself, a commercial low-ester pectin solution was sprayed onto the outer surface of the fermentation core, followed by calcium source loading and the second stage of fermentation.

[0109] Comparative Example 3 was used to compare the interfacial connection between the independent coating formed by the addition of low-ester pectin and the cross-linked fixation layer formed by the continuous extension of the core pectin in this application.

[0110] Comparative Example 4 In preparing the granular fermentation substrate, calcium carbonate is directly and evenly mixed with soybean meal, citrus pomace, corn flour and wheat bran, without using pressure differential loading.

[0111] Comparative Example 4 was used to compare the peak position and cross-linking region of calcium element signal when calcium carbonate was uniformly distributed throughout the fermentation core and when calcium carbonate was localized in the outer pores.

[0112] Comparative Example 5 The first stage of fermentation and pectin methyl esterase treatment were completed according to Example 1. Instead of using pressure difference to allow calcium carbonate to enter the outer pores of the fermentation core, the calcium carbonate suspension was directly sprayed onto the outer surface of the low-esterified pectin zone, and rolling drainage and surface air supply were omitted.

[0113] Comparative Example 5 was used to confirm whether a surface cross-linking layer that is easily separated from the fermentation core is formed when calcium carbonate is located on the outermost surface of the particles.

[0114] Comparative Example 6 The first stage of fermentation, pectin methyl esterase treatment, and calcium source loading were completed according to Example 1. Instead of inoculating with Lactobacillus plantarum, lactic acid was added to the outer surface of the particles in one go.

[0115] The amount of lactic acid added was determined based on the actual amount of total organic acids formed during the second stage of fermentation in Example 1. Comparative Example 6 was used to compare the effects of single-time external lactic acid addition and continuous fermentation of Lactobacillus plantarum on the location of calcium ion release, cross-linking process, and continuity of the cross-linked fixation layer.

[0116] The range of raw material ratios is determined based on the following criteria: the particles can be stably shaped, the first stage of fermentation can form protein hydrolysates, and the pectin-containing plant raw materials can provide continuous core pectin.

[0117] The equivalent particle size of 2–8 mm was determined by three factors: mass transfer during the first stage of fermentation, the external localization effect of pectin methyl esterase, and the loading of calcium carbonate on the outer edge.

[0118] The first stage of fermentation is based on the following criteria: fermentation temperature is 32-36℃, carbon dioxide release rate drops to 40%-70% of peak value, and relative change rate of acid-soluble protein content is no more than 5%. The protein-containing feed ingredients are judged to have reached a stable hydrolysis state without significant over-fermentation.

[0119] The amount of pectin methyl esterase added, the amount of spraying, the treatment temperature, and the treatment time are determined based on the formation of a gradient of pectin esterification degree between the outer layer of the particles and the inside of the core, and are not determined solely by the enzyme treatment time.

[0120] Vacuum pressure, holding time, calcium carbonate suspension concentration, and calcium carbonate particle size were determined based on the fact that the peak value of the calcium element signal was located inside the low-esterified pectin region and the circumferential coverage of the calcium source transition region was not less than 70%.

[0121] The changes in pH value and bound calcium content during the second stage of fermentation were determined based on the completion of acid production by Lactobacillus plantarum, the release of calcium ions from the slightly soluble calcium source, and the formation of a stable cross-linked fixed layer.

[0122] The drying temperature and final moisture content were determined based on minimizing the impact of the drying process on the activity of probiotics and maintaining the storage stability of the granules. In formal experiments, the actual test results of each example and comparative example should be based on the original test records, and theoretical predictions should not be used to replace measured values.

[0123] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound probiotic fermented feed additive, characterized in that, The compound probiotic fermented feed additive is a granular structure formed from protein-containing feed ingredients and pectin-containing plant ingredients. The granular structure includes a fermentation core, a calcium source transition zone formed in the pores at the outer edge of the fermentation core, and a cross-linking fixation layer covering the outer surface of the fermentation core and at least partially embedded in the pores at the outer edge of the fermentation core. The calcium source transition zone is located inside the cross-linking fixation layer. The fermented core contains protein hydrolysates, live Bacillus bacteria, live yeast, and core pectin derived from pectin-containing plant materials. The cross-linked immobilization layer contains low-esterified pectin formed by demethylation of the outer part of the core pectin. The low-esterified pectin is continuously connected to the core pectin and forms an ionic cross-linking network through cross-linked calcium. Lactic acid-producing probiotics are immobilized in the ionic cross-linking network. The calcium source transition zone forms a calcium enrichment area derived from a slightly soluble calcium source. According to the calcium element line scan detection of the granular structure cross section, the calcium element signal peak is located between the outer edge of the fermentation core and the inner side of the cross-linked fixation layer. After separating the cross-linked immobilization layer and the fermentation core, based on the dry weight of each separated part, the number of viable lactic acid-producing probiotics in the cross-linked immobilization layer was higher than that in the fermentation core, and the number of viable Bacillus in the fermentation core was higher than that in the cross-linked immobilization layer.

2. The compound probiotic fermented feed additive according to claim 1, characterized in that, Based on the dry matter mass of each raw material at the time of feeding, the raw materials forming the fermentation core include 45-65 parts of protein-containing feed ingredients, 15-30 parts of pectin-containing plant ingredients, 8-20 parts of fermentable carbohydrate ingredients, and 5-15 parts of wheat bran; the protein-containing feed ingredients include soybean meal, and the pectin-containing plant ingredients include at least one of citrus pomace, apple pomace, and beet pomace, with an equivalent particle size of 2-8 mm and a moisture content of 8%-12%.

3. The compound probiotic fermented feed additive according to claim 2, characterized in that, The low-esterified pectin in the cross-linked immobilization layer is formed by the demethylation and esterification of the core pectin on the outside of the fermentation core. The degree of esterification of the low-esterified pectin is less than 50%, the degree of esterification of the core pectin inside the fermentation core is not less than 50%, and the difference in degree of esterification between the low-esterified pectin and the core pectin inside the fermentation core is not less than 10 percentage points.

4. The compound probiotic fermented feed additive according to claim 3, characterized in that, The slightly soluble calcium source is calcium carbonate, and the circumferential coverage of the calcium source transition zone on the cross-section of the granular structure is not less than 70%. The cross-linked fixation layer extends along the outer surface of the fermentation core, and the part of the cross-linked fixation layer embedded in the outer edge pores of the fermentation core is continuously connected with the part located on the outer surface of the fermentation core.

5. The compound probiotic fermented feed additive according to claim 4, characterized in that, The Bacillus is Bacillus subtilis, the yeast is Saccharomyces cerevisiae, and the lactic acid-producing probiotic is Lactobacillus plantarum. Based on the dry weight of each separated part, the ratio of the number of viable Lactobacillus plantarum in the cross-linked immobilization layer to the number of viable Lactobacillus plantarum in the fermentation core is not less than 3, and the ratio of the number of viable Bacillus subtilis in the fermentation core to the number of viable Bacillus subtilis in the cross-linked immobilization layer is not less than 2.

6. A method for preparing a compound probiotic fermented feed additive, applicable to the compound probiotic fermented feed additive described in any one of claims 1-5, characterized in that, Includes the following steps: S1: Mix protein-containing feed ingredients, pectin-containing plant ingredients and fermentation additives, adjust the moisture content of the mixture and make it into a granular fermentation substrate, inoculate the granular fermentation substrate with a first probiotic including Bacillus and yeast, and carry out the first stage of fermentation under aeration conditions to hydrolyze the protein-containing feed ingredients and form a fermentation core containing protein hydrolysate, live Bacillus, live yeast and core pectin; S2: After the first stage of fermentation is completed, pectin methyl esterase solution is sprayed onto the outer surface of the fermentation core to cause the pectin on the outside of the fermentation core to undergo demethylation and esterification, forming a low-esterified pectin zone on the outside of the fermentation core, and making the esterification degree of pectin in the low-esterified pectin zone lower than that of the pectin in the inside of the fermentation core. S3: While maintaining the particle shape of the fermentation core, the pressure difference is used to allow the suspension containing a slightly soluble calcium source to enter the outer pores of the fermentation core, and the slightly soluble calcium source is controlled to accumulate on the inner side of the low-esterified pectin zone, and the free suspension attached to the outer surface of the low-esterified pectin zone is removed, so as to form a calcium source transition zone on the outer edge of the fermentation core. S4: Inoculate lactic acid-producing probiotics on the outside of the low-esterified pectin zone and carry out the second stage of fermentation under closed conditions. This allows the lactic acid produced by the lactic acid-producing probiotics to migrate from the low-esterified pectin zone to the calcium source transition zone. The lactic acid dissolves the slightly soluble calcium source and releases calcium ions. S5: Causes calcium ions to migrate from the calcium source transition region to the low-esterified pectin region and undergo ionic cross-linking with the low-esterified pectin in the low-esterified pectin region, transforming the low-esterified pectin region into a cross-linked immobilization layer that covers the outer surface of the fermentation core and is at least partially embedded in the pores of the outer edge of the fermentation core, while immobilizing at least some lactic acid-producing probiotics in the cross-linked immobilization layer. S6: Dry the granules after the second stage of fermentation to obtain a compound probiotic fermented feed additive.

7. The preparation method of a compound probiotic fermented feed additive according to claim 6, characterized in that, Based on the dry matter mass of each raw material at the time of feeding, mix 45-65 parts of protein-containing feed raw materials, 15-30 parts of pectin-containing plant raw materials, 8-20 parts of fermentable carbohydrate raw materials and 5-15 parts of wheat bran, adjust the moisture content of the resulting mixture to 42%-50%, and then make it into granular fermentation substrate with an equivalent particle size of 2-8 mm. Protein-containing feed ingredients include soybean meal; pectin-containing plant ingredients include at least one of citrus pomace, apple pomace, and beet pomace; the primary probiotics include Bacillus subtilis and Saccharomyces cerevisiae, with an inoculum size of 110 g of Bacillus subtilis based on the dry matter mass of the granular fermentation substrate. 6 ~110 8 CFU / g, the inoculum size of *Saccharomyces cerevisiae* was 110. 5 ~110 7 CFU / g.

8. The preparation method of a compound probiotic fermented feed additive according to claim 7, characterized in that, The temperature of the first stage of fermentation is 32-36℃. During the fermentation, the granular fermentation substrate is intermittently aerated and turned, and the carbon dioxide concentration in the fermentation tail gas is continuously collected. The amount of carbon dioxide released per unit dry matter granular fermentation substrate per unit time is determined based on the fermentation tail gas flow rate and carbon dioxide concentration. When the carbon dioxide release rate drops to 40%–70% of the peak value after reaching its peak, and the relative change rate of acid-soluble protein content between two fermentation core samples obtained at a 2-hour interval is no greater than 5%, the first stage of fermentation ends. The amount of pectin methyl esterase solution sprayed is 2% to 8% of the dry weight of the fermentation core, and the amount of pectin methyl esterase added is 50 to 500 U per 100g of dry matter in the fermentation core. After spraying, keep it at 25 to 35℃ for 20 to 90 minutes to ensure that the degree of esterification of pectin in the low-esterification pectin zone is less than 50%, and that the difference in the degree of esterification between pectin in the low-esterification pectin zone and the pectin in the core of the fermentation core is not less than 10 percentage points.

9. The preparation method of a compound probiotic fermented feed additive according to claim 8, characterized in that, The fermentation core with the low-esterified pectin zone is placed under an absolute pressure of 20-60 kPa for 2-10 minutes. Under the absolute pressure, the fermentation core is brought into contact with a calcium carbonate suspension with a mass fraction of 1%-5%. The pressure is then restored to 90-105 kPa, allowing the calcium carbonate suspension to enter the outer pores of the fermentation core. After the pressure is restored, the calcium carbonate suspension that did not enter the fermentation core is discharged, and the fermentation core is subjected to rolling drainage and surface air supply to remove the calcium carbonate suspension attached to the outer surface of the low-esterified pectin zone, while retaining the calcium carbonate that entered the outer edge pores of the fermentation core, so that the peak value of the calcium element signal obtained by the calcium element line scan detection through the cross section of the fermentation core is located inside the low-esterified pectin zone.

10. The preparation method of a compound probiotic fermented feed additive according to claim 9, characterized in that, The lactic acid-producing probiotic is *Lactobacillus plantarum*. Based on the dry matter mass of the fermentation core, the inoculum quantity of *Lactobacillus plantarum* is 110... 7 ~110 9 CFU / g; After inoculation, the moisture content of the particles was adjusted to 52%–60%, and the second stage of fermentation was carried out under sealed conditions at 30–34℃; The second stage of fermentation ends when the pH of the fermentation system drops to 3.8–4.2 and the relative change rate of bound calcium content between two cross-linked immobilized layer samples obtained at a 2-hour interval is no greater than 5%. After the second stage of fermentation, vacuum drying or fluidized bed drying is carried out under the condition that the particle temperature does not exceed 40℃ until the moisture content of the resulting compound probiotic fermented feed additive is 8% to 12%.

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