A process for preparing a high-activity lactic acid bacterial inoculant using granular biochar

Abstract

The present application belongs to the technical field of lactic acid bacteria agent preparation, and particularly relates to a process for preparing high-activity lactic acid bacteria agent by using granular biochar. In view of the influence of heat drying on the survival rate of lactic acid bacteria in the preparation process of lactic acid bacteria agent, the present application first provides a heat-resistant protective agent, and the modified inulin and corn oil are used as the components of the heat-resistant protective agent. The heat-resistant protective agent can not only improve the survival rate of lactic acid bacteria, but also increase the probiotic function of the bacterial powder. In addition, the granular biochar is used as a lactic acid bacteria adsorbent to prepare high-activity bacterial agent, and is applied to the feed industry. The granular biochar can not only play a role in adsorbing and fixing lactic acid bacteria, but also effectively reduce the accumulation of intestinal toxins of animals, and has important industrial value and environmental protection significance.

Description

Technical Field

[0001] This invention belongs to the field of lactic acid bacteria inoculant preparation technology, specifically relating to a process for preparing highly active lactic acid bacteria inoculants using granular biochar. Background Technology

[0002] Lactic acid bacteria, as an important probiotic additive in feed, have significant effects on improving animal gut health, increasing feed utilization, and promoting growth. The preparation process involves heat drying, but lactic acid bacteria are not heat-resistant, and the drying process easily causes them to die.

[0003] Currently, commonly used lactic acid bacteria protection technologies in the feed industry mainly include microencapsulation, enteric coating, and conventional freeze-drying protectants. For example, patent CN120665717A discloses a composite system composed of glycerol, skim milk powder, trehalose, and inulin, but it uses freeze-drying technology, resulting in excessively high production costs. Another example is patent CN110623066A, which proposes a double-layer encapsulation technology for lactic acid bacteria. While this improves stomach acid tolerance, the process is complex and difficult to apply on a large scale in the feed industry.

[0004] Therefore, the main problems with existing technologies are: complex processes, difficulty in large-scale production, unsuitable selection of protective agents for large-scale applications in the feed industry, or insufficient protection against the high temperature and high pressure conditions unique to feed pelleting. Summary of the Invention

[0005] To address the problems mentioned in the background art, the present invention provides a process for preparing highly active lactic acid bacteria agents using granular biochar.

[0006] The technical solution of the present invention is as follows: This invention provides a process for preparing highly active lactic acid bacteria inoculants using granular biochar, comprising: (1) Prepare the heat-resistant protective agent according to the following mass percentages: Skim milk powder 15-20%; Modified inulin 6-10%; Trehalose 4-8%; Corn oil 2-5%; Glycerin 3-9%; Monosodium glutamate 1-2%; The remainder is water; The modified inulin is obtained by enzymatic hydrolysis of inulin. (2) The lactic acid bacteria cells are mixed with a heat-resistant protectant to obtain a bacterial slurry; the bacterial slurry is mixed with granular biochar and then granulated and subjected to boiling drying.

[0007] Based on the above-described process for preparing highly active lactic acid bacteria agents using granular biochar, in step (1), enzymatic hydrolysis is performed using endoinulinase.

[0008] Based on the above-described process for preparing highly active lactic acid bacteria agents using granular biochar, step (1) includes the preparation of the heat-protecting agent: First, dissolve the modified inulin, trehalose, glycerin, and monosodium glutamate in a portion of the water and stir until homogeneous. Then, disperse the skim milk powder and corn oil evenly in the remaining water. Finally, mix the two mixtures together.

[0009] Based on the above-described process for preparing highly active lactic acid bacteria using granular biochar, in step (2), the lactic acid bacteria cells and the heat protection agent are mixed at a mass ratio of 1:2 to 1:6.

[0010] Based on the above-described process for preparing highly active lactic acid bacteria using granular biochar, in step (2), the viable count of the bacterial slurry is 1.0 × 10⁻⁶. 11 -1.0×10 12 cfu / g.

[0011] Based on the above-described process for preparing highly active lactic acid bacteria agents using granular biochar, in step (2), the bacterial slurry and granular biochar are mixed at a mass ratio of 1:1 to 1:2.

[0012] Furthermore, the granular biochar is coconut shell biochar, walnut shell biochar, apricot shell biochar, or bamboo biochar.

[0013] Based on the above-described process for preparing highly active lactic acid bacteria agents using granular biochar, in step (2), during the boiling drying process, the air inlet temperature is 48-50℃, and the moisture content of the dried bacterial powder is controlled at 5%-10%.

[0014] The present invention also provides a highly active lactic acid bacteria agent prepared by the process described above, wherein the lactic acid bacteria survival rate is not less than 90% after 60 days of storage at room temperature, the lactic acid bacteria survival rate is not less than 70% after granulation, and the bacterial cell recovery rate is not less than 80%.

[0015] The present invention also provides the application of the highly active lactic acid bacteria agent prepared by the above process in animal feed additives.

[0016] Beneficial effects To address the impact of heat drying on the survival rate of lactic acid bacteria during the preparation of lactic acid bacteria inoculants, this invention first provides a heat-resistant protectant. Modified inulin and corn oil are used together as components of this heat-resistant protectant, which not only improves the survival rate of lactic acid bacteria but also enhances the probiotic function of the inoculant powder. Furthermore, this invention uses granular biochar as an adsorbent to prepare a highly active inoculant for the feed industry. This not only adsorbs and immobilizes lactic acid bacteria but also effectively reduces the accumulation of intestinal toxins in animals, demonstrating significant industrial value and environmental significance. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of a process for preparing highly active lactic acid bacteria agents using granular biochar according to the present invention. Detailed Implementation

[0018] The following examples are intended to illustrate the present invention, and not to further limit the invention.

[0019] This invention provides a process for preparing highly active lactic acid bacteria agents using granular biochar, such as... Figure 1 ,include: (1) Prepare the heat-resistant protective agent according to the following mass percentages: Skim milk powder 15-20%; Modified inulin 6-10%; Trehalose 4-8%; Corn oil 2-5%; Glycerin 3-9%; Monosodium glutamate 1-2%; The balance is water, and the sum of the mass percentages of all components is 100%.

[0020] The modified inulin is obtained by enzymatic hydrolysis of inulin, preferably using endoinulinase.

[0021] Furthermore, based on the mass of inulin, the amount of endoinulinase added is 7-12 U / g.

[0022] The specific processing procedure is as follows.

[0023] First, weigh out a certain amount of inulin and add it to an acetate-sodium acetate buffer solution with a pH of 4.5-5.0 at 10 times the amount.

[0024] Then, add the above mixture to a mixer and stir until it is fully dissolved and pre-equilibrated.

[0025] Next, add endoinulinase to the solution and keep the solution temperature at 50-60℃, stirring at this constant temperature for 12 hours.

[0026] After the enzymatic hydrolysis reaction is complete, place the mixture in a boiling water bath and heat for 5-10 minutes to completely inactivate the enzyme and terminate the reaction.

[0027] Finally, the inactivated solution is centrifuged, the supernatant is collected, and the supernatant is spray-dried to obtain the modified inulin powder.

[0028] Preferably, the modified inulin contains more than 65% fructooligosaccharides.

[0029] Inulin, as a natural plant material, possesses excellent water-holding and water-absorbing properties and is rich in polysaccharides, which can regulate the intestinal tract. However, due to its large molecular weight and low hydroxyl content, directly adding inulin during the preparation of heat-resistant protectants makes it difficult for its large molecules to adhere to the phospholipids of lactic acid bacteria cell membranes, and it cannot form a dense heat-insulating protective layer during high-temperature processing. Therefore, this invention modifies inulin, breaking down long-chain inulin into short-chain inulin, thereby enhancing the adsorption of inulin to lactic acid bacteria and its heat-insulating protective properties.

[0030] This invention uses modified inulin and corn oil together as heat-protecting agents, which not only improves the survival rate of lactic acid bacteria, but also increases the probiotic function of the bacterial powder.

[0031] Based on the raw material components involved in the formulation, preferably, the formulation of the heat-resistant protective agent includes: Modified inulin, trehalose, glycerin, and monosodium glutamate were first dissolved in a portion of water and stirred until homogeneous. Skim milk powder and corn oil were then dispersed evenly in the remaining water. Finally, the two mixtures were combined to obtain a liquid heat protectant.

[0032] The heat-resistant protective agent provided by this invention has a simple preparation process and is easy to use for large-scale production of lactic acid bacteria powder. Because of its good heat resistance, its storage time at room temperature and survival rate are higher than those of conventional freeze-dried bacterial powder and other bacterial powders.

[0033] (2) The lactic acid bacteria cells are mixed with a heat-resistant protectant to obtain a bacterial slurry; the bacterial slurry is mixed with granular biochar and then granulated and subjected to boiling drying.

[0034] Specifically, the first step is to culture and collect the bacteria. Lactic acid bacteria (such as Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus plantarum) are inoculated into the culture medium and gradually expanded. After culturing to the early stationary phase, the bacteria are collected by centrifugation to obtain wet bacterial sludge.

[0035] Then, the lactic acid bacteria cells are mixed with a heat protectant at a mass ratio of 1:2 to 1:6 to form a uniform bacterial slurry.

[0036] Preferably, the viable count of the mixed bacterial slurry is 1.0 × 10⁻⁶. 11 -1.0×10 12 cfu / g.

[0037] Next, the bacterial slurry and granular biochar are mixed at a mass ratio of 1:1 to 1:2. After being mixed evenly, the mixture is poured into a vibrating granulator for granulation.

[0038] The granular biochar is made from coconut shell, walnut shell, apricot shell, or bamboo.

[0039] In the feed industry, the immobilization and drying of lactic acid bacteria mainly relies on inorganic adsorbents such as diatomaceous earth, zeolite powder, and montmorillonite. Although these materials have good adsorption properties, they have the following disadvantages: high cost; limited nutritional value; potential safety hazards such as carrying heavy metals; and limitations on their addition to feed.

[0040] This invention uses granular biochar as an adsorbent for lactic acid bacteria to prepare a highly active bacterial agent, which can be applied to the feed industry. It can not only adsorb and fix lactic acid bacteria, but also effectively reduce the accumulation of intestinal toxins in animals, which has important industrial value and environmental significance.

[0041] Finally, the granulated wet bacterial powder is subjected to fluidized bed drying. During fluidized bed drying, the inlet air temperature is 48-50℃, and the moisture content of the dried bacterial powder is controlled at 5%-10%.

[0042] The present invention also provides a process for preparing a highly active lactic acid bacteria agent using granular biochar. The agent has a lactic acid bacteria survival rate of not less than 90% after 60 days of storage at room temperature, a lactic acid bacteria survival rate of not less than 70% after granulation, and a bacterial cell recovery rate of not less than 80%.

[0043] The present invention also provides an application of a highly active lactic acid bacteria agent prepared by a process for preparing highly active lactic acid bacteria agent using granular biochar in animal feed additives. Specifically, the agent is used in animal feed additives that improve average daily weight gain, reduce feed conversion ratio and diarrhea rate, and have good gastrointestinal fluid tolerance.

[0044] To address the impact of heat drying on the survival rate of lactic acid bacteria during the preparation of lactic acid bacteria inoculants, this invention first provides a heat-resistant protectant. Modified inulin and corn oil are used together as components of this heat-resistant protectant, which not only improves the survival rate of lactic acid bacteria but also enhances the probiotic function of the inoculant powder. Furthermore, this invention uses granular biochar as an adsorbent to prepare a highly active inoculant for the feed industry. This not only adsorbs and immobilizes lactic acid bacteria but also effectively reduces the accumulation of intestinal toxins in animals, demonstrating significant industrial value and environmental significance.

[0045] Example 1 This embodiment provides a process for preparing highly active lactic acid bacteria agents using granular biochar, including: (1) Prepare the heat-resistant protective agent according to the following mass percentages: Skim milk powder 16%; Modified inulin 8%; Trehalose 4%; Corn oil 3%; Glycerin 6%; Monosodium glutamate 1.5%; The balance is water, and the sum of the mass percentages of all components is 100%.

[0046] The inulin modification process is as follows.

[0047] First, weigh out a certain amount of inulin and add it to an acetate-sodium acetate buffer solution with a pH of 4.5-5.0 at 10 times the amount.

[0048] Then, add the above mixture to a mixer and stir until it is fully dissolved and pre-equilibrated.

[0049] Next, endoinulinase was added to the solution, and the solution temperature was maintained at 50-60℃ with constant stirring for 12 hours. The amount of endoinulinase added was 10 U / g based on the mass of inulin.

[0050] After the enzymatic hydrolysis reaction is complete, the mixture is placed in a boiling water bath and heated for 8 minutes to completely inactivate the enzyme and terminate the reaction.

[0051] Finally, the inactivated solution is centrifuged, the supernatant is collected, and the supernatant is spray-dried to obtain the modified inulin powder.

[0052] Modified inulin, trehalose, glycerin, and monosodium glutamate were first dissolved in a portion of water and stirred until homogeneous. Skim milk powder and corn oil were then dispersed evenly in the remaining water. Finally, the two mixtures were combined to obtain a liquid heat protectant.

[0053] (2) Lactobacillus casei was inoculated into MRS liquid medium and gradually expanded. The culture was maintained at 37°C until the early stationary phase. The bacterial cells were collected by centrifugation to obtain wet bacterial slurry. The collected wet bacterial cells were thoroughly mixed with the prepared heat protectant at a ratio of 1:2 (mass ratio) to form a homogeneous bacterial slurry. The viable count of the obtained slurry was 1.0 × 10⁻⁶. 11 —1.0×10 12 cfu / g.

[0054] The bacterial slurry and granulated biochar coconut shell charcoal were further mixed and adsorbed at a mass ratio of 1:2. After stirring evenly, the mixture was poured into a vibrating granulator for granulation. The mass of the wet bacterial powder and the number of live bacteria in the wet bacterial powder after granulation were recorded.

[0055] The granulated wet bacterial powder was subjected to boiling drying with an inlet air temperature of 48-50℃. The moisture content of the dried bacterial powder was controlled at 5%-10%. The quality of the dried bacterial powder and the number of live bacteria in the dried bacterial powder were recorded.

[0056] Comparative Example 1 Lactobacillus casei was inoculated into MRS liquid medium and scaled up stepwise. The culture was maintained at 37°C until the early stationary phase. The bacterial cells were collected by centrifugation to obtain a wet bacterial slurry. The collected wet bacterial cells were mixed thoroughly with an appropriate amount of physiological saline to form a homogeneous bacterial slurry. The viable count of the obtained slurry was 1.0 × 10⁻⁶. 11 —1.0×10 12 cfu / g.

[0057] The bacterial slurry and granulated biochar coconut shell charcoal were further mixed and adsorbed at a mass ratio of 1:2. After stirring evenly, the mixture was poured into a vibrating granulator for granulation. The mass of the wet bacterial powder and the number of live bacteria in the wet bacterial powder after granulation were recorded.

[0058] The granulated wet bacterial powder was subjected to boiling drying with an inlet air temperature of 48-50℃. The moisture content of the dried bacterial powder was controlled at 5%-10%. The quality of the dried bacterial powder and the number of live bacteria in the dried bacterial powder were recorded.

[0059] Comparative Example 2 (1) Prepare the heat-resistant protective agent according to the following mass percentages: Skim milk powder 16%; Inulin 8%; Trehalose 4%; Corn oil 3%; Glycerin 6%; Monosodium glutamate 1.5%; The balance is water, and the sum of the mass percentages of all components is 100%.

[0060] First, dissolve inulin, trehalose, glycerin, and monosodium glutamate in a portion of water and stir until homogeneous. Then, disperse skim milk powder and corn oil evenly in the remaining water. Finally, mix the two mixtures to obtain a liquid heat protectant.

[0061] (2) Lactobacillus casei was inoculated into MRS liquid medium and gradually expanded. The culture was maintained at 37°C until the early stationary phase. The bacterial cells were collected by centrifugation to obtain wet bacterial slurry. The collected wet bacterial cells were thoroughly mixed with the prepared heat protectant at a ratio of 1:2 (mass ratio) to form a homogeneous bacterial slurry. The viable count of the obtained slurry was 1.0 × 10⁻⁶. 11 —1.0×10 12 cfu / g.

[0062] The bacterial slurry and granulated biochar coconut shell charcoal were further mixed and adsorbed at a mass ratio of 1:2. After stirring evenly, the mixture was poured into a vibrating granulator for granulation. The mass of the wet bacterial powder and the number of live bacteria in the wet bacterial powder after granulation were recorded.

[0063] The granulated wet bacterial powder was subjected to boiling drying with an inlet air temperature of 48-50℃. The moisture content of the dried bacterial powder was controlled at 5%-10%. The quality of the dried bacterial powder and the number of live bacteria in the dried bacterial powder were recorded.

[0064] Comparative Example 3 (1) Prepare the heat-resistant protective agent according to the following mass percentages: Skim milk powder 16%; Modified inulin 8%; Trehalose 4%; Corn oil 10%; Glycerin 6%; Monosodium glutamate 1.5%; The balance is water, and the sum of the mass percentages of all components is 100%.

[0065] The inulin modification process is as follows.

[0066] First, weigh out a certain amount of inulin and add it to an acetate-sodium acetate buffer solution with a pH of 4.5-5.0 at 10 times the amount.

[0067] Then, add the above mixture to a mixer and stir until it is fully dissolved and pre-equilibrated.

[0068] Next, endoinulinase was added to the solution, and the solution temperature was maintained at 50-60℃ with constant stirring for 12 hours. The amount of endoinulinase added was 10 U / g based on the mass of inulin.

[0069] After the enzymatic hydrolysis reaction is complete, the mixture is placed in a boiling water bath and heated for 8 minutes to completely inactivate the enzyme and terminate the reaction.

[0070] Finally, the inactivated solution is centrifuged, the supernatant is collected, and the supernatant is spray-dried to obtain the modified inulin powder.

[0071] Modified inulin, trehalose, glycerin, and monosodium glutamate were first dissolved in a portion of water and stirred until homogeneous. Skim milk powder and corn oil were then dispersed evenly in the remaining water. Finally, the two mixtures were combined to obtain a liquid heat protectant.

[0072] (2) Lactobacillus casei was inoculated into MRS liquid medium and gradually expanded. The culture was maintained at 37°C until the early stationary phase. The bacterial cells were collected by centrifugation to obtain wet bacterial slurry. The collected wet bacterial cells were thoroughly mixed with the prepared heat protectant at a ratio of 1:2 (mass ratio) to form a homogeneous bacterial slurry. The viable count of the obtained slurry was 1.0 × 10⁻⁶. 11 —1.0×10 12 cfu / g.

[0073] The bacterial slurry and granulated biochar coconut shell charcoal were further mixed and adsorbed at a mass ratio of 1:2. After stirring evenly, the mixture was poured into a vibrating granulator for granulation. The mass of the wet bacterial powder and the number of live bacteria in the wet bacterial powder after granulation were recorded.

[0074] The granulated wet bacterial powder was subjected to boiling drying with an inlet air temperature of 48-50℃. The moisture content of the dried bacterial powder was controlled at 5%-10%. The quality of the dried bacterial powder and the number of live bacteria in the dried bacterial powder were recorded.

[0075] Comparative Example 4 (1) Prepare the heat-resistant protective agent according to the following mass percentages: Skim milk powder 16%; Modified inulin 8%; Trehalose 4%; Corn oil 3%; Glycerin 6%; Monosodium glutamate 1.5%; The balance is water, and the sum of the mass percentages of all components is 100%.

[0076] The inulin modification process is as follows.

[0077] First, weigh out a certain amount of inulin and add it to an acetate-sodium acetate buffer solution with a pH of 4.5-5.0 at 10 times the amount.

[0078] Then, add the above mixture to a mixer and stir until it is fully dissolved and pre-equilibrated.

[0079] Next, endoinulinase was added to the solution, and the solution temperature was maintained at 50-60℃ with constant stirring for 12 hours. The amount of endoinulinase added was 10 U / g based on the mass of inulin.

[0080] After the enzymatic hydrolysis reaction is complete, the mixture is placed in a boiling water bath and heated for 8 minutes to completely inactivate the enzyme and terminate the reaction.

[0081] Finally, the inactivated solution is centrifuged, the supernatant is collected, and the supernatant is spray-dried to obtain the modified inulin powder.

[0082] Modified inulin, trehalose, glycerin, and monosodium glutamate were first dissolved in a portion of water and stirred until homogeneous. Skim milk powder and corn oil were then dispersed evenly in the remaining water. Finally, the two mixtures were combined to obtain a liquid heat protectant.

[0083] (2) Lactobacillus casei was inoculated into MRS liquid medium and gradually expanded. The culture was maintained at 37°C until the early stationary phase. The bacterial cells were collected by centrifugation to obtain wet bacterial slurry. The collected wet bacterial cells were thoroughly mixed with the prepared heat protectant at a ratio of 1:2 (mass ratio) to form a homogeneous bacterial slurry. The viable count of the obtained slurry was 1.0 × 10⁻⁶. 11 —1.0×10 12 cfu / g.

[0084] The mycelium slurry and powdered biochar coconut shell charcoal were further mixed and adsorbed at a mass ratio of 1:2. After stirring evenly, the mixture was poured into a vibrating granulator for granulation. The mass of the wet mycelium powder and the number of live bacteria in the wet mycelium powder after granulation were recorded.

[0085] The granulated wet bacterial powder was subjected to boiling drying with an inlet air temperature of 48-50℃. The moisture content of the dried bacterial powder was controlled at 5%-10%. The quality of the dried bacterial powder and the number of live bacteria in the dried bacterial powder were recorded.

[0086] Comparative Example 5 (1) Prepare the heat-resistant protective agent according to the following mass percentages: Skim milk powder 16%; Modified inulin 8%; Trehalose 4%; Corn oil 3%; Glycerin 6%; Monosodium glutamate 1.5%; The balance is water, and the sum of the mass percentages of all components is 100%.

[0087] The inulin modification process is as follows.

[0088] First, weigh out a certain amount of inulin and add it to an acetate-sodium acetate buffer solution with a pH of 4.5-5.0 at 10 times the amount.

[0089] Then, add the above mixture to a mixer and stir until it is fully dissolved and pre-equilibrated.

[0090] Next, endoinulinase was added to the solution, and the solution temperature was maintained at 50-60℃ with constant stirring for 12 hours. The amount of endoinulinase added was 10 U / g based on the mass of inulin.

[0091] After the enzymatic hydrolysis reaction is complete, the mixture is placed in a boiling water bath and heated for 8 minutes to completely inactivate the enzyme and terminate the reaction.

[0092] Finally, the inactivated solution is centrifuged, the supernatant is collected, and the supernatant is spray-dried to obtain the modified inulin powder.

[0093] Modified inulin, trehalose, glycerin, and monosodium glutamate were first dissolved in a portion of water and stirred until homogeneous. Skim milk powder and corn oil were then dispersed evenly in the remaining water. Finally, the two mixtures were combined to obtain a liquid heat protectant.

[0094] (2) Lactobacillus casei was inoculated into MRS liquid medium and gradually expanded. The culture was maintained at 37°C until the early stationary phase. The bacterial cells were collected by centrifugation to obtain wet bacterial slurry. The collected wet bacterial cells were thoroughly mixed with the prepared heat protectant at a ratio of 1:2 (mass ratio) to form a homogeneous bacterial slurry. The viable count of the obtained slurry was 1.0 × 10⁻⁶. 11—1.0×10 12 cfu / g.

[0095] The bacterial slurry and diatomaceous earth were further mixed and adsorbed at a mass ratio of 1:2. After stirring evenly, the mixture was poured into a swing granulator for granulation. The mass of the wet bacterial powder and the number of live bacteria in the wet bacterial powder after granulation were recorded.

[0096] The granulated wet bacterial powder was subjected to boiling drying with an inlet air temperature of 48-50℃. The moisture content of the dried bacterial powder was controlled at 5%-10%. The quality of the dried bacterial powder and the number of live bacteria in the dried bacterial powder were recorded.

[0097] Experimental results 1. Detection of bacterial cell recovery rate and survival rate Example 1 and Comparative Examples 1-5 were used to test the bacterial cell recovery rate and survival rate.

[0098] Bacterial cell recovery rate (%) = ×100%.

[0099] Bacterial cell survival rate (%) = ×100%.

[0100] Feed pellet survival rate (%) = ×100%.

[0101] The results are shown in Table 1.

[0102] Table 1. Results of bacterial cell recovery and survival rate under different treatments. 2. Gastrointestinal fluid tolerance test To verify the protective effect of the process provided by this invention on the bacteria, a gastrointestinal fluid tolerance test was conducted on the bacterial powder. The experimental method was as follows: 1.00g of the bacterial powder prepared in Example 1 and Comparative Examples 1-5 was weighed and mixed in test tubes containing 8ml of simulated gastric fluid and simulated intestinal fluid. The mixture was treated in a shaker at 37℃ and 60r / min for 60min, 90min, and 120min, respectively. The viable bacteria were counted in the treated liquids, and the survival rate was calculated. The results are shown in Tables 2 and 3.

[0103] Table 2. Results of artificial gastric fluid tolerance under different treatments Table 3. Results of artificial intestinal fluid tolerance under different treatments The results showed that the bacterial powder of Example 1 exhibited higher tolerance to artificial gastric and intestinal fluids than the comparative examples. This indicates that the process provided by the present invention can improve the survival rate of lactic acid bacteria in complex environments, demonstrating strong stress resistance.

[0104] 3. Animal testing The lactic acid bacteria preparations obtained in Example 1 and Comparative Examples 1-5 were added to the diet of growing pigs at a ratio of 0.15% for a 42-day feeding trial.

[0105] The experimental method was as follows: Healthy pigs of similar weight (40-50kg) were randomly divided into 6 groups: Example 1 group and Comparative Examples 1-5 groups, with 10 piglets in each group. All groups received the same basal diet, with each experimental group additionally supplemented with a corresponding lactic acid bacteria preparation at a concentration of 0.15% (percentage of diet weight). Feeding continued for 42 days. Routine immunization and cleaning were performed daily, and the incidence of diarrhea and feed intake were recorded.

[0106] Average daily weight gain (grams / day) = .

[0107] Feed conversion ratio (%) = ×100%.

[0108] Diarrhea rate (%) = ×100%.

[0109] The results are shown in Table 4.

[0110] Table 4 Results of animal experiments The results showed that the average daily weight gain in Example 1 was significantly higher than that in the control groups, the diarrhea rate was significantly reduced, and the feed conversion ratio was lower than that in the other groups.

Claims

1. A process for preparing highly active lactic acid bacteria inoculants using granular biochar, characterized in that, include: (1) Prepare the heat-resistant protective agent according to the following mass percentages: Skim milk powder 15-20%; Modified inulin 6-10%; Trehalose 4-8%; Corn oil 2-5%; Glycerin 3-9%; Monosodium glutamate 1-2%; The remainder is water; The modified inulin is obtained by enzymatic hydrolysis of inulin. (2) The lactic acid bacteria cells are mixed with a heat-resistant protectant to obtain a bacterial slurry; the bacterial slurry is mixed with granular biochar and then granulated and subjected to boiling drying.

2. The process for preparing highly active lactic acid bacteria inoculants using granular biochar according to claim 1, characterized in that, In step (1), enzymatic hydrolysis is performed using endoinulinase.

3. The process for preparing highly active lactic acid bacteria agents using granular biochar according to claim 1, characterized in that, In step (1), the preparation of the heat-resistant protective agent includes: First, dissolve the modified inulin, trehalose, glycerin, and monosodium glutamate in a portion of the water and stir until homogeneous. Then, disperse the skim milk powder and corn oil evenly in the remaining water. Finally, mix the two mixtures together.

4. The process for preparing highly active lactic acid bacteria inoculants using granular biochar according to claim 1, characterized in that, In step (2), the lactic acid bacteria cells and the heat protectant are mixed at a mass ratio of 1:2 to 1:

6.

5. The process for preparing highly active lactic acid bacteria agents using granular biochar according to claim 1, characterized in that, In step (2), the viable count of the mycelium slurry is 1.0 × 10⁻⁶. 11 -1.0×10 12 cfu / g.

6. The process for preparing highly active lactic acid bacteria inoculants using granular biochar according to claim 1, characterized in that, In step (2), the bacterial slurry and granular biochar are mixed at a mass ratio of 1:1 to 1:

2.

7. The process for preparing highly active lactic acid bacteria inoculants using granular biochar according to claim 6, characterized in that, The granular biochar is coconut shell biochar, walnut shell biochar, apricot shell biochar, or bamboo biochar.

8. The process for preparing highly active lactic acid bacteria agents using granular biochar according to claim 1, characterized in that, In step (2), during the boiling drying process, the air inlet temperature is 48-50℃, and the moisture content of the dried bacterial powder is controlled at 5%-10%.

9. A highly active lactic acid bacteria inoculant prepared by the process described in claim 1, characterized in that, The survival rate of lactic acid bacteria after 60 days of storage at room temperature is not less than 90%, the survival rate of lactic acid bacteria after granulation is not less than 70%, and the bacterial cell recovery rate is not less than 80%.

10. The application of a highly active lactic acid bacteria agent prepared by the process described in claim 1 in animal feed additives.

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