A feed lactic acid bacteria additive and a preparation method thereof

Abstract

The present application relates to the technical field of microbial feed additive, and discloses a feed lactic acid bacteria additive and a preparation method thereof.The preparation method comprises the following steps: taking Lactobacillus plantarum and / or Enterococcus faecalis as a bacterial strain to activate and expand culture, taking agricultural and sideline product waste as a substrate to obtain mature fermentation liquor through deep liquid fermentation; mixing the mature fermentation liquor with a composite protective agent containing double essential oil, chitosan, theabrownin and porous silicon dioxide to obtain a semi-dry granule core through granulation and low-temperature pre-drying; and coating the semi-dry granule core with a κ-carrageenan / konjac glucomannan / plant sterol ester composite gel, and drying and packaging to obtain a finished product.The double essential oil, chitosan and theabrownin jointly form a full-spectrum active antibacterial system; the elastic composite gel outer layer realizes dual protection of liquid water and water vapor; the porous silicon dioxide actively maintains low water activity of the granule to prevent the lactic acid bacteria from recovering in advance; and the product has high viable count, strong heat and acid resistance, and excellent storage stability.

Description

Technical Field

[0001] This invention relates to the field of microbial feed additive technology, and more specifically, to a feed lactic acid bacteria additive and its preparation method. Background Technology

[0002] With the global ban on antibiotics in animal feed, probiotic feed additives have become an important antibiotic alternative in the livestock industry. Lactic acid bacteria, with their ability to regulate intestinal microecology, inhibit pathogens, and enhance immunity, are widely used and are among the most promising feed microbial additives. Lactic acid bacteria additives are typically produced by liquid fermentation to obtain high concentrations of live bacteria, followed by microencapsulation or carrier adsorption to create solid granule products, thus protecting the live bacteria during storage and feeding. Currently, commercially available products usually use inert physical protective components such as skim milk powder and starch as protective agents, with water-soluble materials as the outer coating, and are produced through liquid fermentation using specialized commercial culture media such as MRS.

[0003] However, existing feed lactic acid bacteria additives still have significant shortcomings in actual production applications: existing protectant formulations do not contain active antibacterial components, and the products are easily contaminated by exogenous bacteria during storage, leading to a continuous decline in the number of viable bacteria; existing water-soluble outer layers dissolve and fall off rapidly in feeding environments such as water accumulation in farm feed troughs, high humidity climates, and alternating wet and dry conditions, or develop micro-cracks due to repeated expansion and contraction, causing a significant reduction in lactic acid bacteria before they are ingested by animals; in addition, traditional liquid fermentation relies on specialized commercial culture media such as MRS, which results in high production costs and is not conducive to large-scale industrial production. Summary of the Invention

[0004] This invention provides a feed lactic acid bacteria additive and its preparation method, solving the technical problems of existing feed lactic acid bacteria additives lacking active antibacterial function, protective layer failure in humid farm environments, and high production costs.

[0005] This invention provides a method for preparing a feed lactic acid bacteria additive, comprising the following steps:

[0006] Step 1: Inoculate lactic acid bacteria into liquid culture medium for activation culture to obtain primary seed culture;

[0007] Step 2: Prepare a liquid fermentation culture medium using agricultural and sideline product waste as carbon and nitrogen sources, and then sterilize it.

[0008] Step 3: Inoculate the primary seed culture into the liquid fermentation medium and ferment in a sealed container until the viable cell count is not less than [amount missing]. CFU / mL was used to obtain a mature fermentation broth;

[0009] Step 4: Mix porous starch, skim milk powder, trehalose, monosodium glutamate, natural plant essential oils, food-grade chitosan, black tea theaflavins, and food-grade porous silica to obtain a composite protective agent.

[0010] Step 5: Mix the mature fermentation broth and the compound protective agent at a mass ratio of 1:(1.5-2.5) and granulate to obtain wet granules;

[0011] Step 6: Dry the wet particles at 30-40℃ until the moisture content is 8%-12% to obtain semi-dry particle cores;

[0012] Step 7: Prepare a coating solution by mixing κ-carrageenan, konjac glucomannan and phytosterol esters, spray it onto the core surface of the semi-dry granules, cool and solidify it, and then dry it until the moisture content is less than 8% to obtain the feed lactic acid bacteria additive.

[0013] Preferably, the lactic acid bacteria in step one are at least one of Lactobacillus plantarum and Enterococcus faecalis; the liquid culture medium is MRS liquid culture medium; and the activation culture conditions are static culture at 35-38°C for 18-24 hours.

[0014] Preferably, the agricultural by-product waste mentioned in step two is one or more of the following: water chestnut starch processing wastewater, cassava residue aqueous extract, or wheat bran aqueous extract; the cassava residue aqueous extract is extracted at a solid-liquid ratio of 1:10 to 1:15 (w / v) at 60 to 80°C for 1 to 2 hours, and then filtered to obtain the supernatant; the wheat bran aqueous extract is extracted at a solid-liquid ratio of 1:8 to 1:12 (w / v) at 60 to 80°C for 1 to 2 hours, and then filtered to obtain the supernatant; the fermentation culture medium is sterilized at 115°C for 30 minutes.

[0015] Preferably, the composite protective agent in step four comprises, by weight, 25-40 parts porous starch (oil absorption not less than 80%, w / w), 20-30 parts skim milk powder, 10-20 parts trehalose, 5-10 parts monosodium glutamate, 5-10 parts natural plant essential oil, 5-10 parts food-grade chitosan, 5-8 parts theaflavins from black tea, and 3-8 parts food-grade porous silica (specific surface area not less than 150 m²). 2 / g).

[0016] Preferably, the natural plant essential oil in step four is a mixture of thymol / carvacrol essential oil and clove essential oil, with a mass ratio of 1 to 2:1; the total content of thymol and carvacrol in the thymol / carvacrol essential oil is not less than 80% (w / w), of which the thymol content accounts for 50% to 70% (w / w) of the total content; the eugenol content in the clove essential oil is not less than 75% (w / w).

[0017] Preferably, the degree of deacetylation of the food-grade chitosan in step four is not less than 85%, and the viscosity-average molecular weight is 100,000 to 200,000 Da; the food-grade porous silica is amorphous silica, food-grade or feed-grade, with a specific surface area of ​​not less than 150 m². 2 / g.

[0018] Preferably, the natural plant essential oils in step four are introduced by pre-adsorption: after mixing thymol / carvacrol essential oil with clove essential oil, it is added to 40% to 60% of the total amount of porous starch used in step four, and stirred for 15 to 30 minutes until the mixture appears as a free-flowing dry powder to obtain porous starch containing essential oils, which is then mixed evenly with the remaining components in step four.

[0019] Preferably, the coating solution in step seven comprises, by weight: 3-5 parts κ-carrageenan, 1-2 parts konjac glucomannan, 1-2 parts phytosterol esters, and 0.3-0.8 parts feed-grade potassium chloride; the total concentration of κ-carrageenan and konjac glucomannan in the coating solution is 3%-6% (w / v).

[0020] Preferably, the preparation steps of the coating solution in step seven are as follows: κ-carrageenan, konjac glucomannan, and potassium chloride are added to water and heated to 70-80°C until completely dissolved to obtain a polymer solution; phytosterol esters are preheated separately to 70-80°C until completely melted and then added to the polymer solution; the mixture is stirred at 3000-5000 r / min for 10-15 minutes at 70-80°C; the resulting coating solution is kept at 60-65°C for later use; the coating is applied using a fluidized bed coating machine, with the coating amount increasing by 5%-12% of the total particle mass; after coating, the mixture is cooled to below 35°C to solidify the composite gel.

[0021] The beneficial effects of this invention are as follows:

[0022] (1) The three antibacterial components in the compound protectant, namely, dual essential oil (penetrating type), food-grade chitosan (contact type) and black tea theaflavins (film-forming type), complement each other in terms of antibacterial pathway and coverage spectrum of miscellaneous bacteria. When used together, they show a significant synergistic effect. They have a continuous active inhibition ability against exogenous miscellaneous bacteria throughout the storage and circulation process, effectively preventing the decline in viable bacteria count caused by miscellaneous bacteria contamination.

[0023] (2) The outer layer of the κ-carrageenan / konjac glucomannan composite gel has excellent elasticity and water stability. It does not dissolve or crack under conditions of water accumulation in the farm feed trough and repeated wet and dry conditions. After the hydrophobic microcrystalline domains of plant sterol esters are embedded in the gel layer, the water vapor permeability is further reduced, so that the outer layer can achieve dual protection against liquid water impact and water vapor penetration at the same time, which fundamentally solves the problem of failure of the existing water-soluble outer layer in the humid feeding environment.

[0024] (3) The food-grade porous silica inside the particles continuously adsorbs the water that seeps into the particles, keeping the water activity inside the particles below the deep dormancy threshold of lactic acid bacteria for a long time. This prevents the lactic acid bacteria from prematurely reviving and metabolizing due to water seepage before entering the animal digestive tract, and the number of live bacteria in the finished product can be kept stable during long-term storage.

[0025] (4) The three-layer progressive physical protection consists of a skim milk powder-trehalose-monosodium glutamate protein-sugar matrix inner layer, a chitosan and porous starch dense particle matrix middle layer, and a κ-carrageenan / KGM composite gel outer layer, which significantly improves the ability of lactic acid bacteria to withstand the high temperature of feed pelleting and the acidic environment of animal stomach, ensuring that the effective number of live bacteria can reach the animal intestine smoothly and complete colonization.

[0026] (5) Using agricultural by-products such as water chestnut starch processing wastewater, cassava residue and bran water extract as fermentation substrates instead of dedicated commercial culture media can significantly reduce production costs and realize the resource utilization of agricultural waste, thereby reducing industrial wastewater discharge. Attached Figure Description

[0027] Figure 1 This is a bar chart comparing the E. coli colony counts of each treatment group in Experiment 1 of this invention on day 30.

[0028] Figure 2 These are SEM microstructure comparison images of the coated sections of samples F and H in Experiment 2 of this invention;

[0029] Figure 3 This is a bar chart comparing the water vapor transmission rate (WVP) of each coating treatment group in Experiment 2 of this invention;

[0030] Figure 4 This is a line graph showing the changes in particle water activity and viable bacteria count over time for two groups of samples in Experiment 3 of this invention under storage conditions at 37℃.

[0031] Figure 5 This is a bar chart comparing the resistance of each sample group in Experiment 4 of this invention to high temperatures during feed pelleting and to animal stomach acid. Detailed Implementation

[0032] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0033] Example 1

[0034] In this embodiment, a method for preparing a feed lactic acid bacteria additive using water chestnut starch processing wastewater as a fermentation substrate and Lactobacillus plantarum as a single strain is disclosed. The method includes the following steps:

[0035] Step 1: Activation and scale-up of bacterial strains

[0036] Lactobacillus plantarum was selected, inoculated into MRS liquid medium, and cultured statically at 37°C for 20 hours to obtain primary seed culture of Lactobacillus plantarum.

[0037] Step 2: Preparation of liquid fermentation culture medium

[0038] Wastewater from water chestnut starch processing was collected, filtered through a 200-mesh filter to remove large particulate impurities, and the pH was adjusted to 6.5 with food-grade lactic acid. The mixture was then sterilized at 115°C for 30 minutes and cooled to room temperature to obtain a liquid fermentation medium.

[0039] Step 3: Liquid Deep Fermentation

[0040] The primary seed culture obtained in step one was inoculated into the liquid fermentation medium obtained in step two at an inoculation rate of 5%. Fermentation was carried out in a sealed container at 35°C for 65 hours, until the pH of the fermentation broth dropped to 3.7 and the viable cell count reached a certain level. Once both CFU / mL and other indicators meet the target, fermentation is stopped to obtain a mature fermentation broth.

[0041] Step 4: Preparation of the composite protective agent

[0042] Weigh the following raw materials by weight: 30 parts porous starch (85% oil absorption, w / w), 25 parts skim milk powder, 15 parts trehalose, 7 parts monosodium glutamate, 6 parts natural plant essential oil, 6 parts food-grade chitosan powder (88% deacetylation, viscosity-average molecular weight 120,000 Da), 6 parts black tea theaflavin powder, and 5 parts food-grade porous silica (amorphous, specific surface area 220 m²). 2 / g).

[0043] The natural plant essential oils used consist of 3 parts thymol / carvacrol essential oil and 3 parts clove essential oil, with a mass ratio of 1:1. The total content of thymol and carvacrol in the thymol / carvacrol essential oil is 85% (w / w), of which thymol accounts for 55% (w / w) of the total content. The eugenol content in the clove essential oil is 80% (w / w).

[0044] Essential oil pre-adsorption: Mix 3 parts of thymol / carvacrol essential oil and 3 parts of clove essential oil evenly, then add 60% (i.e. 18 parts) of the total amount of porous starch (30 parts) to the porous starch, stir for 20 minutes until the mixture appears as a free-flowing dry powder, and obtain porous starch containing essential oil (18 parts).

[0045] Mixing of the composite protective agent: 18 parts of the above-mentioned porous starch containing essential oil, 12 parts of the remaining porous starch (i.e., 40% of the total amount of porous starch), 25 parts of skim milk powder, 15 parts of trehalose, 7 parts of monosodium glutamate, 6 parts of food-grade chitosan powder, 6 parts of black tea theaflavin powder, and 5 parts of food-grade porous silica are added sequentially to a high-speed mixer and mixed at 1000 r / min for 20 minutes until all components are evenly dispersed and there are no visible lumps, to obtain 100 parts of composite protective agent powder.

[0046] Step 5: Liquid-solid adsorption granulation

[0047] The mature fermentation broth obtained in step three and the composite protective agent powder obtained in step four are added to a mixing granulator at a mass ratio of 1:1.8. The mixture is stirred for 15 minutes to allow the fermentation broth to be fully adsorbed by the solid carrier. The soft material is then extruded and granulated through an 18-mesh sieve to obtain wet granules containing live bacteria with a particle size of 1.0–1.2 mm.

[0048] Step Six: Low-Temperature Pre-Drying

[0049] The wet granules containing live bacteria obtained in step five are placed in a fluidized bed dryer, and the material temperature is controlled at 35°C (air inlet temperature at 50°C) and dried until the moisture content of the granules drops to 10%, thus obtaining semi-dried granule cores.

[0050] Step 7: Coating the outer layer of the composite gel

[0051] Preparation of coating solution: Weigh 3 parts κ-carrageenan, 1 part konjac glucomannan (molecular weight approximately 1.5 million Da), 1 part phytosterol ester, and 0.4 parts feed-grade potassium chloride. Add an appropriate amount of water to prepare a total concentration of κ-carrageenan and KGM of 4.0% (w / v). Heat to 75°C and stir continuously until completely dissolved to obtain a polymer solution. Preheat the phytosterol ester separately to 75°C until completely melted, then add it to the above polymer hot solution. Stir at 4000 r / min for 12 minutes at 75°C to uniformly disperse the phytosterol ester in fine droplets to obtain a coating solution. Keep warm at 62°C for later use.

[0052] Coating process: The coating solution, kept at 62℃, was uniformly coated onto the core surface of the semi-dry granules obtained in step six using a fluidized bed coating machine, resulting in a 7% weight gain. After coating, heating was stopped, and the granules were cooled to below 30℃, allowing the κ-carrageenan / KGM composite gel to solidify into an elastic gel film layer, with phytosterol esters simultaneously crystallizing and embedding into the gel network. Subsequently, the product was dried at 32℃ (material temperature) until the total moisture content reached 6.5%, passed through a 30-mesh sieve, and sealed in packaging to obtain the finished feed lactic acid bacteria additive.

[0053] Product performance test results:

[0054]

[0055] Example 2

[0056] In this embodiment, a method for preparing a feed lactic acid bacteria additive using cassava residue aqueous extract as a fermentation substrate and a compound strain of Lactobacillus plantarum and Enterococcus faecalis is disclosed. The method includes the following steps:

[0057] Step 1: Activation and scale-up of bacterial strains

[0058] Lactobacillus plantarum and Enterococcus faecalis were separately inoculated into MRS liquid medium and incubated at 37°C for 20 hours to obtain primary seed cultures of Lactobacillus plantarum and Enterococcus faecalis, respectively. These were then mixed at a 1:1 volume ratio before inoculation in step three to obtain a composite primary seed culture.

[0059] Step 2: Preparation of liquid fermentation culture medium

[0060] Add water to cassava residue at a solid-liquid ratio of 1:12 (w / v), extract at 70℃ for 1.5 hours, filter through a 200-mesh filter to obtain the supernatant, adjust the pH to 6.5 with food-grade lactic acid, sterilize at 115℃ for 30 minutes, and cool to room temperature to obtain the liquid fermentation medium.

[0061] Step 3: Liquid Deep Fermentation

[0062] The composite primary seed culture obtained in step one was inoculated into the liquid fermentation medium obtained in step two at an inoculation rate of 7%. Fermentation was carried out in a sealed environment at 37°C for 60 hours, until the pH of the fermentation broth dropped to 3.6 and the viable cell count reached a certain level. When the concentration of CFU / mL is reached, fermentation is stopped, and a mature fermentation broth is obtained.

[0063] Step 4: Preparation of the composite protective agent

[0064] Weigh the following raw materials by weight: 35 parts porous starch (oil absorption 88%, w / w), 22 parts skim milk powder, 12 parts trehalose, 6 parts monosodium glutamate, 8 parts natural plant essential oil, 8 parts food-grade chitosan powder (degree of deacetylation 90%, viscosity-average molecular weight 150,000 Da), 7 parts black tea theaflavin powder, and 4 parts food-grade porous silica (amorphous, specific surface area 180 m²). 2 / g).

[0065] The natural plant essential oils used consist of 4.8 parts of thymol / carvacrol essential oil and 3.2 parts of clove essential oil, with a mass ratio of 1.5:1. The total content of thymol and carvacrol in the thymol / carvacrol essential oil is 85% (w / w), of which thymol accounts for 62% (w / w) of the total content. The eugenol content in the clove essential oil is 82% (w / w).

[0066] Essential oil pre-adsorption: Mix 4.8 parts of thymol / carvacrol essential oil and 3.2 parts of clove essential oil evenly, then add 50% (i.e. 17.5 parts) of the total amount of porous starch (35 parts) to the porous starch, stir for 20 minutes until the mixture appears as a free-flowing dry powder, and obtain porous starch containing essential oil (17.5 parts).

[0067] Mixing of the composite protective agent: 17.5 parts of the above-mentioned porous starch containing essential oil, 17.5 parts of the remaining porous starch (i.e., 50% of the total amount of porous starch), 22 parts of skim milk powder, 12 parts of trehalose, 6 parts of monosodium glutamate, 8 parts of food-grade chitosan powder, 7 parts of black tea theaflavin powder, and 4 parts of food-grade porous silica were added sequentially to a high-speed mixer and mixed at 1000 r / min for 20 minutes until all components were evenly dispersed and there were no visible lumps, to obtain 102 parts of composite protective agent powder.

[0068] Step 5: Liquid-solid adsorption granulation

[0069] The mature fermentation broth obtained in step three and the composite protective agent powder obtained in step four are added to a mixing granulator at a mass ratio of 1:2.0. The mixture is stirred for 15 minutes to allow the fermentation broth to be fully adsorbed by the solid carrier. The soft material is then extruded and granulated through an 18-mesh sieve to obtain wet granules containing live bacteria with a particle size of 1.0–1.2 mm.

[0070] Step Six: Low-Temperature Pre-Drying

[0071] The wet granules containing live bacteria obtained in step five are placed in a fluidized bed dryer, and the material temperature is controlled at 35°C (air inlet temperature at 50°C) and dried until the moisture content of the granules drops to 10%, thus obtaining semi-dried granule cores.

[0072] Step 7: Coating the outer layer of the composite gel

[0073] Preparation of coating solution: Weigh 4 parts κ-carrageenan, 1.5 parts konjac glucomannan (molecular weight approximately 1.8 million Da), 1.5 parts phytosterol ester, and 0.5 parts feed-grade potassium chloride. Add an appropriate amount of water to prepare a total concentration of κ-carrageenan and KGM of 4.5% (w / v). Heat to 75°C and stir continuously until completely dissolved to obtain a polymer solution. Preheat the phytosterol ester separately to 75°C until completely melted, then add it to the above polymer hot solution. Stir at 4000 r / min for 12 minutes at 75°C to uniformly disperse the phytosterol ester in fine droplets to obtain a coating solution. Keep warm at 62°C for later use.

[0074] Coating process: The coating solution, kept at 62℃, was uniformly coated onto the core surface of the semi-dry granules obtained in step six using a fluidized bed coating machine, resulting in an 8% weight gain. After coating, heating was stopped, and the granules were cooled to below 30℃, allowing the κ-carrageenan / KGM composite gel to solidify into an elastic gel film layer, with phytosterol esters simultaneously crystallizing and embedding into the gel network. Subsequently, the product was dried at 32℃ (material temperature) until the total moisture content reached 6.8%, passed through a 30-mesh sieve, and sealed in packaging to obtain the finished feed lactic acid bacteria additive.

[0075] Product performance test results:

[0076]

[0077] Example 3

[0078] In this embodiment, a method for preparing a feed lactic acid bacteria additive using wheat bran aqueous extract as a fermentation substrate and a compound strain of Lactobacillus plantarum and Enterococcus faecalis is disclosed. The method includes the following steps:

[0079] Step 1: Activation and scale-up of bacterial strains

[0080] Lactobacillus plantarum and Enterococcus faecalis were separately inoculated into MRS liquid medium and incubated at 38°C for 18 hours to obtain their respective primary seed solutions. These were then mixed at a 1:1 volume ratio before inoculation in step three to obtain a composite primary seed solution.

[0081] Step 2: Preparation of liquid fermentation culture medium

[0082] Add water to wheat bran at a solid-liquid ratio of 1:10 (w / v), extract at 65°C for 2 hours, filter through a 200-mesh filter to obtain the supernatant, adjust the pH to 6.8 with food-grade sodium hydroxide solution, sterilize at 115°C for 30 minutes, and cool to room temperature to obtain the liquid fermentation medium.

[0083] Step 3: Liquid Deep Fermentation

[0084] The composite primary seed culture obtained in step one was inoculated into the liquid fermentation medium obtained in step two at an inoculation rate of 9%. Fermentation was carried out in a sealed environment at 39°C for 55 hours, until the pH of the fermentation broth dropped to 3.5 and the viable cell count reached a certain level. When the concentration of CFU / mL is reached, fermentation is stopped, and a mature fermentation broth is obtained.

[0085] Step 4: Preparation of the composite protective agent

[0086] Weigh the following raw materials by weight: 38 parts porous starch (90% oil absorption, w / w), 28 parts skim milk powder, 18 parts trehalose, 9 parts monosodium glutamate, 9 parts natural plant essential oil, 9 parts food-grade chitosan powder (92% deacetylation, viscosity-average molecular weight 180,000 Da), 7.5 parts black tea theaflavin powder, and 7 parts food-grade porous silica (amorphous, specific surface area 250 m²). 2 / g).

[0087] The natural plant essential oils used consist of 6 parts thymol / carvacrol essential oil and 3 parts clove essential oil, with a mass ratio of 2:1. The total content of thymol and carvacrol in the thymol / carvacrol essential oil is 88% (w / w), of which thymol accounts for 68% (w / w) of the total content. The eugenol content in the clove essential oil is 78% (w / w).

[0088] Essential oil pre-adsorption: Mix 6 parts of the above thymol / carvacrol essential oil with 3 parts of clove essential oil evenly, then add 40% (i.e. 15.2 parts) of the total amount of porous starch (38 parts) to porous starch, stir for 25 minutes until the mixture appears as a free-flowing dry powder, and obtain porous starch containing essential oil (15.2 parts).

[0089] Composite Protectant Mixing: 15.2 parts of the above-mentioned porous starch containing essential oil, 22.8 parts of the remaining porous starch (i.e., 60% of the total amount of porous starch), 28 parts of skim milk powder, 18 parts of trehalose, 9 parts of monosodium glutamate, 9 parts of food-grade chitosan powder, 7.5 parts of black tea theaflavin powder, and 7 parts of food-grade porous silica were added sequentially to a high-speed mixer and mixed at 1000 r / min for 20 minutes until all components were evenly dispersed and no visible lumps were found, resulting in 126 parts of composite protective agent powder.

[0090] Step 5: Liquid-solid adsorption granulation

[0091] The mature fermentation broth obtained in step three and the composite protective agent powder obtained in step four are added to a mixing granulator at a mass ratio of 1:2.3. The mixture is stirred for 18 minutes to allow the fermentation broth to be fully adsorbed by the solid carrier. The soft material is then extruded and granulated through a 16-mesh sieve to obtain wet granules containing live bacteria with a particle size of 1.1–1.4 mm.

[0092] Step Six: Low-Temperature Pre-Drying

[0093] The wet granules containing live bacteria obtained in step five are placed in a fluidized bed dryer, and the material temperature is controlled at 38°C (air inlet temperature 53°C) and dried until the moisture content of the granules drops to 11%, thus obtaining semi-dried granule cores.

[0094] Step 7: Coating the outer layer of the composite gel

[0095] Preparation of coating solution: Weigh 5 parts κ-carrageenan, 2 parts konjac glucomannan (molecular weight approximately 1.2 million Da), 2 parts phytosterol ester, and 0.7 parts feed-grade potassium chloride. Add an appropriate amount of water to prepare a total concentration of κ-carrageenan and KGM of 5.5% (w / v). Heat to 78°C and stir continuously until completely dissolved to obtain a polymer solution. Preheat the phytosterol ester separately to 78°C until completely melted, then add it to the above polymer hot solution. Stir at 4500 r / min for 15 minutes at 78°C to uniformly disperse the phytosterol ester in fine droplets to obtain a coating solution. Keep warm at 64°C for later use.

[0096] Coating process: The coating solution, kept at 64℃, is uniformly coated onto the core surface of the semi-dry granules obtained in step six using a fluidized bed coating machine, resulting in a 10% weight gain. After coating, heating is stopped, and the granules are cooled to below 30℃, allowing the κ-carrageenan / KGM composite gel to solidify into an elastic gel film layer, with phytosterol esters simultaneously crystallizing and embedding into the gel network. Subsequently, the product is dried at 36℃ (material temperature) until the total moisture content reaches 7.2%, passed through a 30-mesh sieve, and sealed in packaging to obtain the finished feed lactic acid bacteria additive.

[0097] Product performance test results:

[0098]

[0099] Example 4

[0100] In this embodiment, a method for preparing a feed lactic acid bacteria additive using water chestnut starch processing wastewater as a fermentation substrate and a compound strain of Lactobacillus plantarum and Enterococcus faecalis is disclosed. The method includes the following steps:

[0101] Step 1: Activation and scale-up of bacterial strains

[0102] Lactobacillus plantarum and Enterococcus faecalis were selected and inoculated separately into MRS liquid medium. They were then statically cultured at 37°C for 20 hours to allow the strains to recover from their preservative state to a vigorous growth state, yielding primary seed cultures for Lactobacillus plantarum and Enterococcus faecalis, respectively. These were then mixed at a 1:1 volume ratio before inoculation in step three to obtain a composite primary seed culture for later use. When using both Lactobacillus plantarum and Enterococcus faecalis strains simultaneously, each strain must be inoculated separately into MRS liquid medium and independently activated under the same temperature and time conditions to obtain primary seed cultures for Lactobacillus plantarum and Enterococcus faecalis, respectively. These primary seed cultures were then mixed at a 1:1 volume ratio before inoculation in step three.

[0103] This step is a routine bacterial activation procedure in the field, aimed at restoring bacterial viability and ensuring that the bacterial solution used for inoculation in step three has a sufficient concentration of live bacteria. The procedure is well known to those skilled in the art.

[0104] Step 2: Preparation of liquid fermentation culture medium

[0105] Wastewater from water chestnut starch processing was collected, filtered through a 200-mesh screen to remove large particulate impurities, and the pH was adjusted to 6.5 with food-grade lactic acid. The mixture was then sterilized at 115℃ for 30 minutes to obtain a liquid fermentation medium for later use. This wastewater does not require a solid-liquid extraction step and can be used directly after filtration. The wastewater naturally contains a high concentration of soluble carbohydrates and amino acids, making it rich in nutrients and ensuring stable fermentation.

[0106] Compared with existing technologies that use specialized commercial culture media such as MRS, this step utilizes the fermentable sugars, free amino acids and minerals naturally present in water chestnut starch processing wastewater, which can meet the nutritional requirements of deep liquid fermentation of lactic acid bacteria without the need to add additional commercial culture media components, thus significantly reducing the cost of fermentation substrate.

[0107] Step 3: Liquid Deep Fermentation

[0108] The composite primary seed culture obtained in step one was inoculated into the liquid fermentation medium obtained in step two at an inoculation rate of 8%. The mixture was then subjected to closed-loop deep liquid fermentation at 37°C for 60 hours, until the pH of the fermentation broth dropped below 3.7 and the viable cell count reached at least [amount missing]. When the concentration of CFU / mL reaches a certain level, fermentation is stopped to obtain a mature fermentation broth for later use. The actual fermentation endpoint is determined by the simultaneous achievement of both pH and viable cell count targets. The specific time may vary depending on the substrate batch and the state of the microbial strain, and the test results should be used as the reference.

[0109] This step is a standard liquid deep fermentation procedure in this field. The fermentation broth pH is lowered to below 3.7 while the viable cell count remains at a minimum. CFU / mL is used as an indicator to determine the fermentation endpoint, ensuring that the number of viable bacteria in the mature fermentation broth meets the requirements of the subsequent granulation process.

[0110] Step 4: Preparation of the composite protective agent

[0111] Compared with existing protective agent formulations that only use inert physical protective components such as skim milk powder, trehalose, monosodium glutamate, and porous starch, this step introduces three additional components with active antibacterial functions (double essential oils, food-grade chitosan, and black tea theaflavins) into the aforementioned inert protective matrix, as well as food-grade porous silica that continuously adsorbs moisture inside the particles. This allows the composite protective agent to provide physical protection for lactic acid bacteria while also having a continuous active inhibitory effect on exogenous bacteria, and to maintain the water activity inside the particles at the low level required for lactic acid bacteria dormancy.

[0112] Weigh the following components by weight: 30 parts porous starch (oil absorption not less than 80%, w / w), 25 parts skim milk powder, 15 parts trehalose, 7 parts monosodium glutamate, 6 parts natural plant essential oil (mixture of two essential oils), 6 parts food-grade chitosan powder, 6 parts black tea theaflavin powder, and 5 parts food-grade porous silica.

[0113] (I) Preparation and pre-adsorption of natural plant essential oils (double essential oils)

[0114] The natural plant essential oil is a mixture of thymol / carvacrol essential oil and clove essential oil in a 1:1 mass ratio. The thymol / carvacrol essential oil is a plant-derived essential oil with a total content of thymol and carvacrol of not less than 80% (w / w), wherein the thymol content accounts for 55% (w / w) of the total content of thymol and carvacrol; the clove essential oil is a plant-derived essential oil with a eugenol content of not less than 75% (w / w). Thymol / carvacrol essential oil (terpene phenolic compounds) and clove essential oil (whose main active ingredient is eugenol, belonging to the phenylpropanoid phenolic compounds) belong to different categories of natural phenolic antibacterial substances in terms of chemical structure: terpene phenols (thymol, carvacrol) inhibit bacteria by altering the permeability of the phospholipid bilayer of the bacterial cell membrane, causing leakage of cell contents; phenylpropanoids (eugenol) inhibit bacteria by occupying different binding sites in the cell membrane, disrupting the conformation and activity of functional proteins bound to the cell membrane. Because the two types of phenolic substances have different sites of action, their disruption of the bacterial cell membrane is complementary, exhibiting a synergistic antibacterial effect at a 1:1 mass ratio—the total concentration required to achieve the same antibacterial effect when used together is lower than the sum of their effective concentrations when used alone, thus achieving broader spectrum of bacterial inhibition coverage with a lower total essential oil dosage. Meanwhile, the lipids in the cell membranes of lactic acid bacteria contain a high proportion of unsaturated fatty acids and cyclopropane fatty acids, which makes them significantly more tolerant to the two types of essential oils than Gram-negative bacteria and molds. Introducing the two types of essential oils into the same protectant can selectively inhibit bacteria without significantly reducing the viable count of the target lactic acid bacteria.

[0115] Since natural plant essential oils are liquid, they cannot be directly mixed with other dry powder components (direct mixing will result in uneven distribution of the essential oil and powder clumping). Pre-adsorption treatment is necessary: ​​Mix 3 parts thymol / carvacrol essential oil and 3 parts clove essential oil in a 1:1 mass ratio until homogeneous. Then add this mixture to 50% (15 parts) of the total porous starch required in step four. Stir at 1000 r / min using a high-speed mixer for 20 minutes until the essential oil is completely adsorbed by the porous starch pores and the mixture appears as a free-flowing dry powder, yielding 15 parts of porous starch containing essential oil. This step transforms the liquid essential oil into a solid powder form, facilitating subsequent homogeneous mixing with other dry powder components and ensuring uniform dispersion of the essential oil within the composite protective agent particles.

[0116] (II) Introduction of food-grade chitosan

[0117] Food-grade chitosan powder (degree of deacetylation not less than 85%, viscosity-average molecular weight 100,000–200,000 Da) is added to the formulation system of the composite protective agent. Food-grade chitosan is a natural cationic polysaccharide whose molecular chain contains a large number of free amino groups (…). In weakly acidic to neutral aqueous solutions, it is protonated to form a positively charged amino group ( In step five, when the mature fermentation broth is mixed with the composite protective agent powder, the acidic environment provided by the fermentation broth allows the chitosan amino groups to be fully protonated. The positively charged chitosan segments can electrostatically adsorb onto the anionic groups carried on the surface of the bacterial cell walls (phosphate groups in the teichoic acid of Gram-positive bacteria cell walls and anionic groups in the lipopolysaccharide of Gram-negative bacteria outer membranes), directly binding to the surface of the bacterial cell walls. This achieves contact-type antibacterial action by disrupting cell wall integrity and cell membrane permeability. This contact-type solid-phase antibacterial action complements the permeation-type gas / liquid-phase antibacterial action of essential oils in terms of pathway: essential oils act on the bacteria around the particle surface through volatilization or liquid-phase diffusion, while chitosan directly acts on the bacteria on the contact particle surface. The two have different coverage areas and are synergistically complementary.

[0118] (III) Introduction of theabrownins in dark tea

[0119] Theabrownin powder from dark tea (a powdered extract obtained through water extraction, filtration, concentration, and spray drying of dark tea) was added to the compound protective agent formulation system. Theabrownin from dark tea is a high-molecular-weight polymeric polyphenol mixture formed by a series of oxidative condensation reactions of catechin monomers in tea leaves during the microbial post-fermentation process. As a water-soluble polymeric polyphenol, theabrownin, after being introduced into the compound protective agent powder, dissolves in the aqueous phase provided by the fermentation broth during the liquid-solid adsorption granulation process in step five. During the low-temperature pre-drying process in step six, as the moisture in the particles evaporates, the dissolved theabrownin migrates to the particle surface with the capillary water flow and accumulates, forming a continuous polymeric polyphenol film containing a large number of phenolic hydroxyl groups on the particle surface after drying. This membrane has two functions: First, the numerous phenolic hydroxyl groups in the polymeric polyphenols can form multi-point hydrogen bonds with the amino and hydroxyl groups in the cell wall proteins of bacteria and fungi, disrupting the normal conformation and function of the cell wall proteins, and exhibiting inhibitory activity against Gram-positive bacteria, Gram-negative bacteria, and various types of fungi; Second, the tea brown pigment membrane, together with chitosan contact-type antibacterial and essential oil penetration-type antibacterial, constitutes a full-spectrum active antibacterial combination with three complementary antibacterial pathways. The three components complement each other in terms of coverage of miscellaneous bacteria and antibacterial targets, jointly reducing the risk of miscellaneous bacteria contaminating the target lactic acid bacteria during storage.

[0120] (iv) Introduction of food-grade porous silica

[0121] Food-grade porous silica (amorphous silica, food-grade or feed-grade, with a specific surface area of ​​not less than 150 m²) is used. 2 / g) is added to the compound protective agent formulation system. This component is not an antibacterial agent, but an active hygroscopic agent placed in the internal matrix of the granules. When a small amount of moisture from the outer gel coating slowly seeps into the granules during storage or feeding, food-grade porous silica, with its high specific surface area microporous network structure, preferentially and continuously physically adsorbs this seeped moisture, reducing the water activity ( / g) inside the granules. The water activity level is maintained below 0.3 for an extended period. Under this water activity condition, the trehalose inside the granules forms an amorphous vitrified state, which solidifies and maintains the lactic acid bacteria cells. The metabolic activity of the lactic acid bacteria is inhibited, and the lactic acid bacteria remain in a dormant state, without consuming the protective nutrients (trehalose, monosodium glutamate, etc.) inside the granules. As a result, the number of live bacteria in the finished product is maintained stably during long-term storage.

[0122] (v) Mixing of composite protective agents

[0123] Add 15 parts of the porous starch containing essential oil obtained in step four (a), 15 parts of the remaining porous starch (i.e., 50% of the total amount of porous starch), 25 parts of skim milk powder, 15 parts of trehalose, 7 parts of monosodium glutamate, 6 parts of food-grade chitosan powder, 6 parts of black tea theaflavin powder, and 5 parts of food-grade porous silica to a high-speed mixer in sequence. Mix at 1000 r / min for 20 minutes until each component is uniformly dispersed visually and by touch, with no visible lumps, to obtain the composite protective agent powder for later use.

[0124] Step 5: Liquid-solid adsorption granulation

[0125] The mature fermentation broth obtained in step three and the composite protective agent powder obtained in step four are added to a mixing granulator at a mass ratio of 1:2.0. The mixture is stirred for 15 minutes to allow the live bacteria cells and nutrient solution in the fermentation broth to be fully adsorbed by the solid carriers such as porous starch. The soft material is then extruded and granulated through an 18-mesh sieve to obtain wet granules containing live bacteria (particle size 1.0-1.2 mm, wet granules with porous starch as the carrier and live bacteria evenly distributed in the granule matrix).

[0126] This step is a conventional liquid-solid adsorption granulation operation, which utilizes the porous structure of porous starch to transform the live bacteria in the mature fermentation broth from a liquid state into solid particles, providing shaped particles for subsequent drying and coating steps.

[0127] Step Six: Low-Temperature Pre-Drying

[0128] The wet granules containing live bacteria obtained in step five were placed in a fluidized bed dryer, and the material temperature was controlled at 35°C (corresponding to an inlet air temperature of approximately 50°C). Drying was carried out under these low-temperature conditions until the moisture content of the granules decreased to 10%, resulting in semi-dried granule cores. This step uses low-temperature drying to protect the activity of the lactic acid bacteria and avoid inactivation due to high temperatures. Controlling the moisture content to 10%, rather than the final target value (below 8%), is to retain an appropriate moisture content on the granule surface before coating in step seven, which is beneficial for the spreading and adhesion of the coating solution to form a film on the granule surface.

[0129] Step 7: Coating the outer layer of the composite gel

[0130] This step applies a κ-carrageenan / konjac glucomannan (KGM) composite gel coating to the semi-dried particle core obtained in step six, addressing the technical problem of the outer layer of existing products dissolving and detaching upon contact with water. Compared to existing technologies that use water-soluble skim milk powder, starch, or other materials as a protective outer layer, this step replaces the soluble outer layer with an insoluble elastic gel. Simultaneously, it introduces hydrophobic microcrystalline domains of phytosterol esters into the gel, enabling the outer coating to protect against liquid water impact and water vapor permeation.

[0131] (a) Preparation of coating solution

[0132] Weigh out 4 parts κ-carrageenan, 1.5 parts konjac glucomannan (KGM), 1.5 parts phytosterol ester, and 0.5 parts feed-grade potassium chloride. Add an appropriate amount of water to prepare a total concentration of κ-carrageenan and KGM of 4.5% (w / v). Preparation steps: Add κ-carrageenan, konjac glucomannan, and potassium chloride to water, heat to 75°C, and continuously stir until κ-carrageenan and KGM are completely dissolved to obtain a polymer solution. Preheat the phytosterol ester (melting point approximately 50–60°C) separately to 75°C until completely melted, then add it to the above-dissolved polymer solution. Stir continuously at 4000 r / min for 12 minutes at 75°C to uniformly disperse the molten phytosterol ester in the polymer solution as fine droplets, obtaining a coating solution. Keep the coating solution at 62°C for later use.

[0133] Regarding the roles of each component: κ-carrageenan (sulfated polysaccharide) forms a gel upon cooling, but when used alone, the gel is brittle and hard, easily cracking due to repeated swelling and contraction in alternating wet and dry environments, failing to meet the long-term protection requirements of farm environments. Konjac glucomannan (KGM, β-1,4-glucomannan) cannot form a gel on its own, but when KGM and κ-carrageenan are mixed in the above proportion, the KGM polymer chains and κ-carrageenan chains intertwine, forming a more compact and continuous three-dimensional network in the composite gel. The elongation at break (elasticity) of the composite gel is significantly improved, noticeably superior to pure κ-carrageenan gel. It does not undergo brittle fracture under external pressure or stretching but can elastically recover, thus preventing cracks caused by particle volume changes due to alternating wet and dry conditions. Potassium chloride ( source): This process promotes the aggregation of κ-carrageenan chains, improves the gel strength of the composite gel, and enables the gel to solidify into a film more quickly and completely during the coating cooling process. Phytosterol esters (phytosterol fatty acid esters) are hydrophobic crystalline solids (melting point approximately 50–60°C), uniformly dispersed as molten droplets in the κ-carrageenan / KGM polymer solution during coating preparation. Upon cooling after coating, the suspended phytosterol ester droplets crystallize simultaneously with the solidification of the κ-carrageenan / KGM gel network, uniformly embedding themselves into the gel network as solid hydrophobic microcrystalline domains. Because these sterol ester microcrystalline domains are hydrophobic, water (including liquid water and water vapor) must bypass these domains when passing through the gel layer. This significantly increases the tortuosity (winding) of the water transport path in the gel layer, thus significantly reducing the water vapor permeability (WVP) of the gel layer. This allows the outer coating to simultaneously provide protection against both liquid water (which is insoluble in elastic gels) and water vapor (which is impeded by the sterol ester microcrystalline domains).

[0134] (II) Implementation of the coat system

[0135] The coating solution, kept at 62℃, was uniformly applied to the core surface of the semi-dried granules obtained in step six using a fluidized bed coating machine. The coating amount was based on an 8% increase in the total granule mass. After coating, heating was stopped, and the granules were cooled to below 30℃. During cooling, the outer κ-carrageenan / KGM composite gel solidified into an elastic gel film, while phytosterol esters crystallized and embedded in the gel network, forming an elastic composite gel protective layer containing hydrophobic phytosterol ester microcrystalline domains on the granule surface. Drying continued at 32℃ (material temperature) until the total moisture content of the product was below 7%. The drying temperature was controlled at 32℃ during this stage to ensure that the material temperature remained below the lower limit of the gelation temperature of the κ-carrageenan / KGM composite gel (approximately 40℃) throughout the drying process. This prevented the solidified gel film from softening locally due to temperature approaching the phase transition point, ensuring the integrity of the outer coating structure. The product was then passed through a 30-mesh sieve, sealed, and packaged to obtain the finished feed lactic acid bacteria additive.

[0136] Product performance test results:

[0137]

[0138] Experimental verification

[0139] Experiment 1: Verification of the synergistic antibacterial effect of three types of active antibacterial components in a compound protectant.

[0140] 1. Experimental objective: To verify the inhibitory effects of three active antibacterial components in the composite protectant—double essential oils (penetrating type), food-grade chitosan (contact type), and black tea theaflavins (film-forming type)—on Gram-negative bacteria (Escherichia coli) and molds (Aspergillus niger), as well as their selective protection of target lactic acid bacteria (Lactobacillus plantarum), when used in combination, and to compare with the effects of using each component alone and a blank control.

[0141] 2. Sample preparation: Semi-dry granular cores (particle size 1.0–1.2 mm, moisture content 10%) were prepared according to steps one through six of Example 1. The following five treatments were set up on the composite protective agent formulation (the outer coating conditions in step seven were the same, with a weight gain of 7%):

[0142] (1) Sample A (complete formula, positive control): formulated according to step four of Example 1, containing 6 parts of double essential oil, 6 parts of food-grade chitosan powder, 6 parts of black tea theaflavins powder, and the remaining components remain unchanged.

[0143] (2) Sample B (containing only dual essential oils): Based on the formulation in step four of Example 1, retain 6 parts of dual essential oils, remove chitosan powder and tea brown powder, and supplement with an equal amount of porous starch.

[0144] (3) Sample C (containing only chitosan): Based on the formulation in step four of Example 1, retain 6 parts of chitosan powder, remove the double essential oil and theabrownin powder, and supplement with an equal amount of porous starch.

[0145] (4) Sample D (containing only theabrownin): Based on the formula in step four of Example 1, retain 6 parts of black tea theabrownin powder, remove the double essential oil and chitosan powder, and supplement with an equal amount of porous starch.

[0146] (5) Sample E (blank control, no antibacterial components): Based on the formulation in step four of Example 1, remove the double essential oil, chitosan powder and tea brown powder, and make up the difference with an equal amount of porous starch.

[0147] 3. Experimental conditions: Each group of samples (5 g each) was placed at 25℃ and 90% relative humidity (saturated barium chloride solution in a sealed container, maintaining approximately 90% RH, simulating a farm feed trough with accumulated water or a continuously high-humidity feeding environment) for 30 days. Samples were taken every 10 days (days 0, 10, 20, and 30) and plate counts were performed to detect Escherichia coli (ATCC 25922). (CFU / g level, after artificial contamination of each group of samples and storage) Colony count, Aspergillus niger (ATCC 6275), and other indicators. The number of colonies (artificial contamination level CFU / g) and the number of viable *Lactobacillus plantarum* (target strain) were recorded. Three replicates were set for each group, and the average value was taken. 90% RH ( ≈0.90) instead of 75% RH ( The minimum water activity (MDI) is approximately 0.75, because the minimum water activity for *E. coli* growth is approximately 0.95, and for *Aspergillus niger* it is approximately 0.75–0.80. Under 75% RH conditions, *E. coli* cannot proliferate, making it impossible to effectively verify the necessity of the antibacterial component. 90% RH conditions simulate the most unfavorable continuous high-humidity environment on a farm. Under these conditions, *E. coli* can slowly proliferate on the particle surface (near-saturation humidity produces a localized micro-water film), and *Aspergillus niger* can also grow actively, consistent with the actual high-humidity contamination risk on a farm.

[0148] 4. Experimental steps:

[0149] (1) Artificial contamination: E. coli solution ( CFU / mL) and Aspergillus niger spore suspension ( 0.5 mL of each of the dried particle samples (100 g each) was sprayed evenly onto the surface of each group of dried particle samples. After mixing, the samples were stored at 25℃ / 90%RH (in a sealed container of saturated barium chloride solution).

[0150] (2) Sampling and detection: 5 g of sample was taken at each time point and shaken to dissolve in 45 mL of sterile phosphate buffer (pH 7.0). Escherichia coli colonies were counted on VRBA selective medium (37℃, 24 h), Aspergillus niger colonies were counted on potato dextrose agar medium (25℃, 72 h), and Lactobacillus plantarum viable counts were counted on MRS agar medium (37℃, 48 h, anaerobic).

[0151] (3) Calculation of results: Based on the bacterial count of the sample on day 0, calculate the inhibition rate (%) of Escherichia coli and Aspergillus niger and the viable bacterial retention rate (%) of Lactobacillus plantarum in each group of samples on day 30.

[0152] 5. Experimental Results:

[0153] Table 1. Number of contaminating bacteria and retention rate of target lactic acid bacteria in samples from each treatment group on day 30.

[0154]

[0155] Figure 1 Bar chart comparing the number of E. coli in each sample on day 30.

[0156] 6. Results Analysis:

[0157] After being stored at 25℃ / 90%RH for 30 days, the number of Escherichia coli and Aspergillus niger in Sample A (complete Class III antibacterial formula) was below the detection limit (<10 CFU / g), and the viable bacteria retention rate of Lactobacillus plantarum reached 93.1%.

[0158] In contrast, no single antibacterial component could achieve the same full-spectrum antibacterial effect; the number of E. coli in the blank control group was as high as [missing information]. The CFU / g retention rate of *Lactobacillus plantarum* was only 36.2%. The antibacterial mechanisms (penetration type / contact type / film-forming type) of the three types of components are complementary. The combined use of these components has a significantly better overall antibacterial effect than the sum of the effects of using each component alone, which verifies the synergistic effect of the three types of active antibacterial systems of this invention and their selective protection of the target lactic acid bacteria.

[0159] Experiment 2: Verification of the moisture-proof stability and water vapor barrier performance of κ-carrageenan / KGM composite gel coating

[0160] 1. Experimental objective: To verify the structural stability of the outer coating of κ-carrageenan / konjac glucomannan (KGM) composite gel (containing hydrophobic microcrystalline domains of phytosterol esters) under humid farm conditions (simulating water immersion in feed troughs and high humidity climate), and to compare the moisture barrier performance of the coating of the present invention with that of the traditional skim milk powder outer coating by measuring water vapor transmission rate (WVP). At the same time, the microstructure of the coating cross section was characterized by scanning electron microscopy (SEM).

[0161] 2. Sample Preparation: Semi-dried particle cores of the same batch were prepared according to steps one through six of Example 2, and the following three outer coating treatments were applied respectively:

[0162] (1) Sample F (Coating of the present invention): The κ-carrageenan / KGM / phytosterol ester composite gel coating was applied according to the formulation in step seven of Example 2, with a weight gain of 8%. After cooling and curing, it was dried at 32°C to a moisture content of 6.8%.

[0163] (2) Sample G (no phytosterol ester control): The coating solution formula is the same as that of sample F, but phytosterol ester (1.5 parts) is removed and made up with an equal mass of water. The other coating conditions are the same, and the weight gain is 8%.

[0164] (3) Sample H (conventional skim milk powder outer layer control): 15% (w / v) skim milk powder aqueous solution was used as coating liquid and applied to the same particle core surface by spraying with a fluidized bed coating machine. The weight gain was 8%, and it was dried at 35°C to a moisture content of 6.8%.

[0165] 3. Experimental conditions and procedures:

[0166] (1) Water vapor transmission rate (WVP) determination: Referring to GB / T 1037-2021 standard, each group of coating liquids were cast onto a polytetrafluoroethylene template (60 mm in diameter, flat). After cooling and solidification according to the actual coating process, the templates were dried at 25℃ / 20%RH for 48 h to prepare flat films. The film thickness was measured at 5 different locations with a micrometer and the average value was taken (the thickness was controlled to be 0.15-0.20 mm). A circular sample with a diameter of 57 mm was cut and placed in a permeation cup (Note: The WVP test film was prepared by casting the coating liquid and was used to characterize the intrinsic water vapor barrier performance of the coating material; the actual coating thickness of the particles was about 12-15 μm, calculated from the 8% weight gain and particle size, which is different from the WVP test film thickness. The test film thickness was used to ensure measurement accuracy). The permeability per unit area per unit time was measured at 25℃ and relative humidity gradient (0% / 75%RH), expressed as g·mm / (m 2 ·d) represents water vapor transmission rate (WVP), with 3 parallel membrane samples in each group and the average value taken.

[0167] (2) Water stability test: Each group of samples (5 g each) was immersed in deionized water at 25℃. Every 5 minutes, the appearance of the coating layer (dissolution, peeling, or remaining intact) and the changes in particle texture were observed and recorded. The time required for the coating layer to undergo obvious dissolution and peeling (swelling failure time, min) was recorded.

[0168] (3) Dry and wet cycle stability: After each group of samples was cycled 10 times alternately at 25℃ / 90%RH (24 h) and 25℃ / 20%RH (24 h), the surface of the particles was observed with a stereomicroscope (×20) to see if there were any cracks, and the microstructure of the coating cross section was characterized by SEM.

[0169] (4) Viable count determination after 30 days of storage: After the above samples completed 10 wet-dry cycles, they were stored at 25℃ / 75%RH for 30 days, and the viable count of Lactobacillus plantarum was determined by plate count method.

[0170] 4. Experimental Results:

[0171] Table 2 Comparison of moisture-proof performance and number of viable bacteria in storage for each coating treatment group

[0172]

[0173] Figure 2 Microstructure characterization of the cross-section using SEM;

[0174] Figure 3 : Bar chart comparing WVP of different coating types;

[0175] 5. Results Analysis:

[0176] The water vapor transmission rate of sample F (κ-carrageenan / KGM / phytosterol ester composite gel coating of the present invention) was 2.3 g·mm / m. 2 ·d) This is only the outer layer of traditional skim milk powder (sample H, 18.6 g·mm / m 2 The WVP was 12.4% of that of sample F (d); the introduction of hydrophobic microcrystalline domains of phytosterol esters (sample F vs. sample G) further reduced WVP to 29.5% of that of sample G, confirming that the tortuosity barrier effect of phytosterol ester microcrystalline domains on water vapor was significant.

[0177] Samples F and G maintained their outer layers intact (no dissolution or detachment) after immersion in water for 120 minutes, while sample H showed significant dissolution and detachment after approximately 8 minutes of immersion, demonstrating the excellent liquid water stability of the κ-carrageenan / KGM gel. After 10 wet-dry cycles, samples F and G showed no surface cracks (elastic gel absorption volume change), while sample H exhibited extensive cracking, resulting in a significant decrease in viable bacterial count on day 30. CFU / g (decreased by approximately 98% from the initial value), while sample F remained unchanged. CFU / g. The above results fully verify the structural stability and moisture-proof barrier effect of the outer coating of the present invention under actual humid conditions on a farm.

[0178] Experiment 3: Verification of the effect of food-grade porous silica in actively regulating the water activity within particles and preventing premature resuscitation of lactic acid bacteria in vitro.

[0179] 1. Experimental objective:

[0180] Verification of the water activity inside the particles after adding food-grade porous silica to the composite protective agent ( The variation pattern of ) during storage, and the maintenance of low water activity within the particles ( The effect of ≤0.3% on preventing premature recovery and metabolism of lactic acid bacteria in vitro and maintaining a high number of viable bacteria was quantitatively compared with the control group without porous silica.

[0181] 2. Sample preparation:

[0182] The finished product (complete formula, containing 5 parts of food-grade porous silica) was prepared according to steps one through seven of Example 1 and designated as Sample I (containing silica). A control sample J (without silica) was also prepared. The remaining formula and process were identical to those of Example 1, except that the 5 parts of food-grade porous silica were removed and replaced with an equal amount of porous starch. All other parameters remained unchanged. The moisture content of the finished products in both groups was controlled at 6.5% (±0.2%).

[0183] 3. Experimental conditions:

[0184] Two groups of finished samples (50 g each) were placed in open weighing dishes (Φ90 mm glass petri dishes, without lids) and then placed in sealed desiccators containing saturated sodium chloride solution (maintaining relative humidity of 75% RH). The samples were stored in a constant temperature oven at 37°C to simulate the high temperature and high humidity storage and transportation conditions on a farm. Water vapor in the external 75% RH environment can slowly permeate into the particle interior through the outer coating, thereby causing the water activity inside the particles of the two groups of samples to change gradually over time.

[0185] The reason for choosing open containers instead of sealed bags is that the total moisture content is constant in a sealed system, and the water activity within the particles cannot increase; this experiment aims to verify the active hygroscopic capacity of porous silica when external moisture slowly penetrates, requiring a continuous external moisture driving force (75% RH external environment). Every 7 days, 5 g samples were taken and the particle water activity was measured using a water activity meter (Rotronic HC2-AW, equilibrated at 25℃ for 30 minutes). Simultaneously, the viable count of *Lactobacillus plantarum* was determined using the MRS agar plate counting method (37℃, 48 h, anaerobic), and monitored continuously for 30 days. Each group had 3 replicates, and the average value was taken.

[0186] 4. Experimental steps:

[0187] (1) Sampling: At 0, 7, 14, 21 and 30 days, weighing dishes were taken out of the desiccator at 37℃. 5 g of particles were taken from each group of samples and the water activity and viable bacteria were immediately measured. The weighing dishes were then put back into the desiccator for continued storage.

[0188] (2) Water activity determination: Take about 2 g of particles and place them in the sample cell of the water activity meter. After equilibration at 25°C for 30 minutes, read the value. value.

[0189] (3) Viable bacteria count: Dissolve the remaining 3 g of sample in 30 mL of sterile phosphate buffer (PBS, pH 7.0), vortex for 3 minutes to fully dissolve the particles, serially dilute and spread on MRS agar plates, anaerobic incubate at 37℃ for 48 h and count the colonies, converting them to CFU / g sample.

[0190] 5. Experimental Results:

[0191] Table 3. Changes in particle water activity and viable bacteria count over time during storage at 37℃ for the two sample groups.

[0192]

[0193] Figure 4 Line graphs showing the changes in water activity and viable bacteria count over time for two groups of samples;

[0194] 6. Results Analysis:

[0195] Sample I, containing food-grade porous silica, underwent open storage at 37℃ / 75%RH for 30 days. External moisture continuously permeated into the particles through the outer coating, but the internal water activity remained consistently between 0.21 and 0.25 (below the lactic acid bacteria resuscitation threshold of 0.30). The number of viable lactic acid bacteria decreased from [previous value]. CFU / g only decreased to CFU / g, with a 30-day viable bacteria retention rate of 91.4%.

[0196] In control sample J, which does not contain porous silica, the water activity within the particles continuously increased to 0.62 over time (day 30) under the same storage conditions. The lactic acid bacteria prematurely resumed metabolism and consumed internal protective nutrients due to the water activity exceeding the dormancy threshold, resulting in a significant decrease in the number of viable bacteria. The CFU / g count retained only 14.7% of the initial viable count, a decrease that was 6.2 times greater than that of Sample I. These results confirm that food-grade porous silica, by continuously capturing water that permeates into the particles, maintains the water activity within the particles below the deep dormancy threshold for lactic acid bacteria, effectively preventing premature reactivation and metabolic decline of lactic acid bacteria before they enter the animal digestive tract due to water infiltration.

[0197] Experiment 4: Verification of the product's ability to withstand high temperatures during feed pelleting and the acidic environment of animal stomachs.

[0198] 1. Experimental objective: To verify the ability of the product prepared by the method of the present invention to have a significantly higher survival rate of live Lactobacillus plantarum than conventional products under simulated feed pelleting high temperature (85℃, 5 minutes) and simulated animal gastric acid environment (pH 2.5 artificial gastric juice, 2 hours), and to compare and analyze the contribution of the multi-layer progressive physical protective structure to heat resistance and acid resistance.

[0199] 2. Sample Preparation: Three sets of product samples are prepared as follows:

[0200] (1) Sample K (fully protected by this invention, corresponding to Example 2): prepared according to all steps of Example 2 (steps one to seven), containing dual essential oils, chitosan, theaflavins, porous silica (inner protective layer) and κ-carrageenan / KGM / phytosterol ester outer coating, with a viable count of the finished product. CFU / g.

[0201] (2) Sample L (without outer gel coating): Prepare a semi-dry particle core (containing all components of the composite protective agent inner layer) according to steps one to six of Example 2, omitting the outer coating in step seven, and directly dry at 32°C to a moisture content of 6.8%, with the viable count of the finished product being... CFU / g.

[0202] (3) Sample M (conventional process control): prepared using conventional bran adsorption process. Take the same batch of mature fermentation broth as in Example 2 (viable cell count...). The fermentation broth (CFU / mL) and wheat bran (passed through a 40-mesh sieve, moisture content approximately 12%) were added to a mixing granulator at a 1:1 mass ratio. The mixture was stirred for 15 minutes to ensure the fermentation broth was fully absorbed by the wheat bran. After granulation through a 20-mesh sieve, the granules were dried in a fluidized bed at 35°C until the moisture content was below 8%. No compound protective agent was prepared, no outer coating was applied, and the mixture was sieved and sealed in packaging to obtain a control sample for the conventional process. The viable count of the finished product was... CFU / g.

[0203] High temperature resistance test: Each group of samples (1 g each) was placed in a sealed aluminum foil bag and placed in a preheated oven at 85°C for 5 minutes. After removal, the samples were immediately cooled to room temperature. The number of viable bacteria after treatment was detected by MRS agar plate counting method, and the survival rate (%) was calculated.

[0204] Gastric acid resistance test: Prepare artificial gastric solution (pH 2.5, containing 3 g / L pepsin, pH adjusted with hydrochloric acid). Add each group of samples (1 g each) to 20 mL of artificial gastric solution, and treat with shaking in a water bath at 37°C for 2 hours. Immediately after treatment, detect the viable bacterial count using the MRS agar plate counting method and calculate the survival rate (%). Each group has 3 replicates, and the average value is taken.

[0205] 3. Experimental Results:

[0206] Table 4 Comparison of viable bacterial survival rates after high-temperature and gastric acid resistance treatments in different sample groups

[0207]

[0208] Figure 5 Bar chart comparing the survival rates of each sample group in terms of high temperature resistance and gastric acid resistance.

[0209] 5. Results Analysis: The survival rate of sample K (fully protected structure of this invention) was 76% after treatment at 85℃ for 5 minutes and 85% after treatment with artificial gastric fluid at pH 2.5 for 2 hours, which is significantly higher than that of sample L (without outer gel coating) (64% / 71%) and sample M (conventional bran adsorption) (23% / 28%).

[0210] The improved heat / acid resistance of sample K compared to sample L (76% vs. 64%; 85% vs. 71%) mainly stemmed from the additional physical isolation and protection provided by the outer layer of the κ-carrageenan / KGM composite gel to lactic acid bacteria in high-temperature / acidic environments. The significant improvement in the resistance of sample L compared to sample M (64% vs. 23%; 71% vs. 28%) was primarily due to the direct chemical protection of the lactic acid bacteria cells by the inner layer of the composite protective agent, which consists of skim milk powder, trehalose, monosodium glutamate protein, and glycobase. Together, these two components constitute the three-layer progressive physical protection system of this invention, enabling the finished product to exhibit significantly higher heat and stomach acid resistance than conventional products during feed processing and animal digestion.

[0211] The embodiments of the present invention have been described above. However, the embodiments are not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make more equivalent embodiments under the guidance of the present embodiments, and all of them are within the protection scope of the present embodiments.

Claims

1. A method for preparing a feed lactic acid bacteria additive, characterized in that, Includes the following steps: Step 1: Inoculate lactic acid bacteria into liquid culture medium for activation culture to obtain primary seed culture; Step 2: Prepare a liquid fermentation culture medium using agricultural and sideline product waste as carbon and nitrogen sources, and then sterilize it. Step 3: Inoculate the primary seed culture into the liquid fermentation medium and ferment in a sealed container until the viable cell count is not less than [amount missing]. CFU / mL was used to obtain a mature fermentation broth; Step 4: Mix porous starch, skim milk powder, trehalose, monosodium glutamate, natural plant essential oils, food-grade chitosan, black tea theaflavins, and food-grade porous silica to obtain a composite protective agent. The composite protective agent contains, by weight, 25-40 parts of porous starch, 20-30 parts of skim milk powder, 10-20 parts of trehalose, 5-10 parts of monosodium glutamate, 5-10 parts of natural plant essential oil, 5-10 parts of food-grade chitosan, 5-8 parts of black tea theaflavins, and 3-8 parts of food-grade porous silica. The natural plant essential oil is a mixture of thymol / carvacrol essential oil and clove essential oil, with a mass ratio of 1 to 2:

1. Natural plant essential oils are introduced using a pre-adsorption method: after mixing thymol / carvacrol essential oil with clove essential oil, add it to 40% to 60% of the total amount of porous starch used in step four, stir for 15 to 30 minutes until the mixture appears as a free-flowing dry powder, and obtain porous starch containing essential oils, and then mix it evenly with the remaining components in step four. Step 5: Mix the mature fermentation broth and the compound protective agent at a mass ratio of 1:1.5 to 2.5 and granulate to obtain wet granules; Step 6: Dry the wet particles at 30-40℃ until the moisture content is 8%-12% to obtain semi-dry particle cores; Step 7: Prepare a coating solution by mixing κ-carrageenan, konjac glucomannan, phytosterol esters and feed-grade potassium chloride, spray it onto the core surface of the semi-dry granules, cool and solidify it, and then dry it until the moisture content is less than 8% to obtain the feed lactic acid bacteria additive. The coating solution contains, by weight, 3-5 parts of κ-carrageenan, 1-2 parts of konjac glucomannan, 1-2 parts of phytosterol esters, and 0.3-0.8 parts of feed-grade potassium chloride; the total concentration of κ-carrageenan and konjac glucomannan in the coating solution is 3%-6%.

2. The preparation method according to claim 1, characterized in that, The lactic acid bacteria mentioned in step one are at least one of Lactobacillus plantarum and Enterococcus faecalis; the liquid culture medium is MRS liquid culture medium; the activation culture conditions are static culture at 35-38℃ for 18-24 hours.

3. The preparation method according to claim 1, characterized in that, The agricultural waste mentioned in step two is one or more of the following: water chestnut starch processing wastewater, cassava residue aqueous extract, or wheat bran aqueous extract; Cassava residue aqueous extract was extracted at 60-80℃ for 1-2 hours with a solid-liquid ratio of 1:10 to 1:15 and then filtered to obtain the supernatant. The bran aqueous extract was extracted at 60-80℃ for 1-2 hours at a solid-liquid ratio of 1:8 to 1:12, and then filtered to obtain the supernatant. The fermentation medium was sterilized at 115°C for 30 minutes.

4. The preparation method according to claim 1, characterized in that, The total content of thymol and carvacrol in the thymol / carvacrol essential oil is not less than 80%, of which thymol accounts for 50% to 70% of the total content; the eugenol content in the clove essential oil is not less than 75%.

5. The preparation method according to claim 1, characterized in that, The food-grade chitosan mentioned in step four has a degree of deacetylation of not less than 85% and a viscosity-average molecular weight of 100,000 to 200,000 Da; the food-grade porous silica is amorphous silica with a specific surface area of ​​not less than 150 m². 2 / g.

6. The preparation method according to claim 1, characterized in that, The preparation steps of the coating solution in step seven are as follows: add κ-carrageenan, konjac glucomannan and potassium chloride to water and heat to 70-80℃ to completely dissolve them to obtain a polymer solution; preheat the phytosterol ester separately to 70-80℃ to completely melt it and then add it to the above polymer solution. Stir at 3000-5000 r / min for 10-15 minutes at 70-80℃. Keep the resulting coating solution at 60-65℃ for later use; spray it with a fluidized bed coating machine. The amount of coating solution should be based on an increase of 5%-12% in the total mass of the particles. After coating, cool to below 35℃ to solidify the composite gel.

7. A feed lactic acid bacteria additive, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.

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