A live lactobacillus preparation and its preparation method

Abstract

This invention relates to the field of probiotic manufacturing technology and discloses a method for preparing a live Lactobacillus preparation, comprising the following steps: preparing a fermentation culture medium based on yeast extract, yeast extract, corn peptone or corn steep liquor powder, and a carbon source; adding a surfactant to the fermentation culture medium; inoculating the fermentation culture medium with Lactobacillus seed culture for fermentation; after fermentation, heating the fermentation broth to obtain a fermentation stock solution; centrifuging the fermentation stock solution using a disc centrifuge to collect the unwashed bacterial sludge; sequentially adding trehalose, an osmotic pressure regulator, and a composite protective agent to the bacterial sludge, and mixing to obtain a bacterial suspension. The purpose of this invention is to solve the problem that the activity retention effect of existing live Lactobacillus preparations still needs to be improved during preparation and storage.

Description

Technical Field

[0001] This invention relates to the field of probiotic manufacturing technology, specifically to a live lactobacillus preparation and its preparation method. Background Technology

[0002] Live Lactobacillus preparations are widely used in food, feed, and microecological products due to their functions in regulating the microecology, improving digestion and absorption, and maintaining bodily health. Current live Lactobacillus preparations are typically made from Lactobacillus fermentation broth through processes such as cell enrichment, mixing with preservatives, drying and powdering, and packaging. Because Lactobacillus is sensitive to the external environment, its activity is easily affected by factors such as the nutrient composition of the culture medium, fermentation state, cell collection method, drying conditions, and storage environment. Therefore, existing processes usually optimize the culture medium composition, control fermentation conditions, add desiccant agents, and use sealed packaging to obtain Lactobacillus preparations with a certain live bacteria content.

[0003] However, existing Lactobacillus preparations require continuous processing after fermentation, including cell enrichment, protective agent mixing, spray drying, and packaging. During this process, the cells are susceptible to environmental changes, heat, dehydration, and oxidation, leading to a decrease in activity and a reduction in the effective viable count of the resulting powder. This decrease continues during subsequent storage. These issues affect the potency, shelf life, and application stability of live Lactobacillus preparations, limiting their effectiveness in industrial production and long-term storage. Summary of the Invention

[0004] The purpose of this invention is to address the problem that the activity retention effect of existing live lactobacillus preparations still needs to be improved during preparation and storage, and to propose a live lactobacillus preparation and its preparation method.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A method for preparing a live lactobacillus preparation includes the following steps: S1. Prepare a fermentation medium based on yeast extract powder, yeast extract, corn peptone or corn steep liquor powder and a carbon source. Add a surfactant to the fermentation medium and inoculate the lactobacillus seed liquid into the fermentation medium for fermentation. S2. After fermentation, the fermentation liquid is heated to obtain the original fermentation liquid; S3. Centrifuge the fermentation broth using a butterfly centrifuge to collect the unwashed bacterial sludge. S4. Add trehalose, osmotic pressure regulator and composite protectant to the bacterial mud in sequence, and mix to obtain bacterial suspension; S5. Spray dry the bacterial suspension, controlling the inlet air temperature to 120-125℃ and the outlet air temperature to 78-80℃, collect the bacterial powder, and control the moisture content of the bacterial powder to 3-4%; S6. Add vitamin C to the bacterial powder, mix well, and seal and package together with the desiccant to obtain a live lactobacillus preparation.

[0006] Based on the above technical solution, the present invention can be further improved as follows.

[0007] Furthermore, in step S4, the trehalose is crystalline trehalose, and the amount added is 1.0 to 2.0% of the mass of the bacterial sludge. It is added to the bacterial sludge in 5 portions, and stirred evenly after each addition. After all the sludge is added, stirring is continued for 3 to 5 minutes. The osmotic pressure regulator is sodium chloride, and the amount added is 0.05-0.1% of the mass of the bacterial sludge. The composite protective agent includes skim milk powder, maltodextrin, and ascorbic acid, wherein the amount of skim milk powder added is 0.3-0.5% of the mass of the bacterial sludge, the amount of maltodextrin added is 0.3-0.5% of the mass of the bacterial sludge, and the amount of ascorbic acid added is 0.1-0.2% of the mass of the bacterial sludge. Sodium chloride, skim milk powder, maltodextrin, and ascorbic acid are dissolved together in water to obtain a protective agent solution. After adding crystalline trehalose to the bacterial sludge and stirring evenly, the protective agent solution is added to the bacterial sludge, and stirring is continued for 2-3 minutes to obtain the bacterial suspension.

[0008] Furthermore, the fermentation medium also includes manganese sulfate monohydrate, magnesium chloride hexahydrate, sodium chloride, dipotassium hydrogen phosphate, calcium carbonate, and an antifoaming agent; The carbon source is granulated sugar or glucose, and the surfactant is Tween-80; The fermentation medium comprises: 3.0-4.0% granulated sugar or glucose, 1.0-2.0% yeast extract, 0.3-0.6% corn peptone or corn steep liquor powder, 0.1-0.5% yeast extract, 0.02-0.05% manganese sulfate monohydrate, 0.05-1.0% magnesium chloride hexahydrate, 0.08-1.0% sodium chloride, 0.06-1.0% calcium carbonate, 0.2-0.5% dipotassium hydrogen phosphate, 0.1-0.2% Tween-80, and 0.01-0.05% defoamer.

[0009] Furthermore, the fermentation medium is sterilized at 121°C for 30 minutes before inoculation with Lactobacillus seed culture, and then cooled to 30-40°C and the initial pH is adjusted to 6.0-7.0. During fermentation, the stirring speed is 80-100 r / min. When the pH drops to 5.0-6.0, automatic alkali replenishment is activated. Fermentation ends when alkali replenishment stops.

[0010] Furthermore, in step S2, after fermentation is completed, alkali supplementation is stopped, the temperature of the fermentation liquid is raised to 40-50°C and maintained for 30 minutes, and then the obtained fermentation liquid is transported to a disc centrifuge for centrifugation. During the centrifugation process, the fermentation broth is separated into solid and liquid components, and the resulting bacterial sludge containing the centrifuged mother liquor is collected. This bacterial sludge is then used for the preparation of a subsequent bacterial suspension.

[0011] Furthermore, the preparation of the Lactobacillus seed culture includes: picking a single colony of Lactobacillus and inoculating it into 100 mL of primary culture medium, and culturing it until a primary culture medium is formed; The primary culture medium was inoculated into the secondary culture medium of a 50L fermenter at an inoculation rate of 1-5%, with a liquid volume of 60-75%, and cultured at 30-40℃ and 80-120r / min for 5-15h to obtain the secondary seed culture. The secondary seed culture is transferred into the fermentation medium under pressure using air or nitrogen gas with a purity of ≥99.999%.

[0012] Furthermore, in S3, the processing capacity of the disc centrifuge is 3-5 T / h; In step S5, the spray drying tower is preheated to an inlet air temperature of 120-125°C before spray drying. After the outlet air temperature rises to 78-80°C, clean water is first pumped in for spray drying test. After the outlet air temperature stabilizes, the bacterial suspension is pumped in, and the air hammer is turned on to collect the bacterial powder.

[0013] Furthermore, in step S6, the amount of vitamin C added is 0.1% to 0.2% of the mass of the bacterial powder; The desiccant is silica gel desiccant, with 20g of silica gel desiccant added for every 20kg of bacterial powder; The bacterial powder, vitamin C, and silica gel desiccant are vacuum-sealed and stored at 4°C.

[0014] Furthermore, the lactobacillus is one or more of the following: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus brunelli, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus johnsonii, Lactobacillus reuteri, or Lactobacillus fermentum.

[0015] A live lactobacillus preparation is prepared by the above-described preparation method.

[0016] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: This invention employs a fermentation culture system containing yeast extract, yeast paste, corn peptone or corn steep liquor powder, and a carbon source, combined with surfactants. This provides a suitable nutritional environment for the growth of Lactobacillus, allowing the cells to achieve optimal growth during the fermentation stage. After fermentation, the cells are heated to form a fermentation stock solution, and then collected using a disc centrifuge without water washing. This reduces additional environmental changes and processing damage during the enrichment and transfer process, allowing the cells to enter the subsequent formulation process in a more stable state. A bacterial suspension is prepared by sequentially adding trehalose, an osmotic pressure regulator, and a composite protectant to the bacterial sludge. This improves the environmental adaptability of the cells before and after drying, reducing the impact of the drying process on cell activity. Simultaneously, by controlling the inlet and outlet air temperatures and the moisture content of the bacterial powder during spray drying, a good balance is achieved between drying efficiency and maintaining viable cells. Finally, vitamin C is added and sealed together with a desiccant, which helps reduce the impact of the external environment on the activity of the bacterial powder during storage. Therefore, this method can improve the activity retention of Lactobacillus during preparation and preservation, and improve the product stability and application reliability of the obtained live Lactobacillus preparation, while being suitable for industrial continuous production. Attached Figure Description

[0017] Figure 1 The effect of Tween-80 dosage on the activity of live Lactobacillus preparations is shown in the figure. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the raw materials used in the following embodiments are all commercially available products in the art, the equipment used are all conventional equipment in the art, and the percentages are all mass percentages.

[0019] This invention relates to a method for preparing a live lactobacillus preparation, wherein the lactobacillus can be one or more of the following: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus brunelli, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus johnsonii, Lactobacillus reuteri, or Lactobacillus fermentum.

[0020] The preparation method of this invention includes steps such as strain activation and seed culture, preparation of fermentation medium, fermentation production, post-fermentation treatment, collection of bacterial sludge by butterfly centrifugation, addition of protective agent, spray drying, and packaging and preservation. Through systematic regulation during the fermentation stage, cell membrane regulation stage, dormancy induction stage, centrifugation stage, protective agent encapsulation stage, spray drying stage, and packaging and preservation stage, the lactobacillus is protected intracellularly, intracellularly, and extracellularly, thereby improving its activity retention during spray drying and subsequent preservation.

[0021] The fermentation medium of this invention employs a combination of rapid and slow nitrogen sources. Yeast extract powder and yeast extract serve as rapid nitrogen sources that are easily utilized by the bacteria, while corn peptone or corn steep liquor powder serves as a relatively slow-release nitrogen source. This combination of rapid and slow nitrogen sources prolongs the transition time of Lactobacillus from its rapid growth phase to its stationary phase, resulting in a more rod-like bacterial morphology. This facilitates the encapsulation of the bacteria by the preservative during subsequent spray drying, thereby reducing the loss of activity caused by high temperatures and rapid dehydration.

[0022] Add 0.1–0.2% Tween-80 to the fermentation medium. Tween-80 can increase the oleic acid content in the Lactobacillus cell membrane, improve the fatty acid composition of the cell membrane, and enhance the cell membrane's pressure resistance and stress resistance, thereby improving the cell's tolerance to centrifugation, spray drying, and storage processes. During the later stages of fermentation, add 0.01–0.03% rifampicin to the fermentation broth to induce the cells into a dormant or low-metabolic state. Before transferring the cells to the fermentation tank, raise the culture temperature by approximately 10°C (40–50°C) and maintain this temperature for 30 minutes to induce the expression of heat shock proteins, thereby improving the cells' heat resistance during the high-temperature spray drying stage.

[0023] The centrifugation stage employs butterfly centrifugation technology. Compared to tube centrifugation, butterfly centrifugation has a larger processing capacity and shorter centrifugation time, which can reduce the loss of cell activity during the waiting process. At the same time, the bacterial sludge collected by this invention is unwashed and contains some centrifugation mother liquor, which can retain some trace elements and nutrients in the yeast fermentation broth, which is beneficial for the cells to regain physiological balance after centrifugation.

[0024] First, add 1.0–2.0% crystalline trehalose to the centrifuged bacterial sludge and stir well. Crystalline trehalose can maintain cell morphology, stabilize cell membrane and intracellular protein structure, and reduce damage during rapid dehydration. Then, add 0.05–0.1% sodium chloride. Sodium chloride acts as an osmotic pressure regulator to stabilize the osmotic pressure of the bacterial sludge system and reduce damage to the cells caused by osmotic pressure mutations. Next, add 0.3–0.5% skim milk powder, 0.3–0.5% maltodextrin, and 0.1–0.2% ascorbic acid in sequence. Skim milk powder provides protective components for proteins and lactoses, maltodextrin has film-forming and encapsulating properties, and ascorbic acid has antioxidant effects. All three work together to reduce cell activity loss caused by high temperature, rapid dehydration, and oxidative stress during spray drying.

[0025] During spray drying, the inlet air temperature is controlled at 120–125℃, and the outlet air temperature is controlled at 78–80℃. Within this temperature range, the bacterial suspension can be rapidly dehydrated, causing the bacteria to enter a low-moisture, low-metabolic, or dormant state. After spray drying, the moisture content of the bacterial powder is controlled at 3–4%, which reduces the risk of decreased activity due to excessive moisture inducing cell germination or metabolic recovery, while also avoiding thermal damage to the bacteria caused by excessively high temperatures. Before spray drying, clean water is pumped in for a spray drying test. The pumping speed is controlled by adjusting the peristaltic pump speed, thereby regulating and stabilizing the outlet air temperature. Once the temperature is stable, the bacterial suspension is then pumped in for spray drying.

[0026] Add 0.1-0.2% vitamin C to the live bacteria preparation obtained by spray drying, mix thoroughly, pack into a sealed bag, and add silica gel desiccant. Add 20g of silica gel desiccant to every 20kg of bacterial preparation, and then vacuum pack. It is preferred to store in a cold storage at 4℃.

[0027] A live lactobacillus preparation is prepared by the above-described preparation method.

[0028] Example 1: Preparation of live Lactobacillus plantarum preparation After thawing the *Lactobacillus plantarum* strain preserved in glycerol tubes, streak the culture onto MRS plates or *Lactobacillus plantarum*-specific plates under aseptic conditions, and incubate at 37°C until distinct colonies appear. Single colonies are picked from the plates and streaked onto slant agar plates. Once a large amount of bacterial growth has occurred, the culture is used as a slant culture for future use.

[0029] Take vigorous slant culture, scrape one loopful of bacterial cells using a sterilized inoculation loop, and inoculate it into 100 mL of primary culture medium. Incubate at 37°C until the culture becomes turbid to obtain the primary culture broth. Inoculate the primary culture broth at a rate of 3% into the secondary culture medium of a 50 L fermenter, filling the tank to 70% capacity. Incubate at 37°C and 100 rpm for 10 h to obtain the secondary seed culture. Transfer the obtained secondary seed culture under pressure using nitrogen gas with a purity ≥99.999% into the fermentation medium of a 5 T fermenter.

[0030] The fermentation medium was prepared according to the following composition: 3.5% granulated sugar, 1.5% yeast extract, 0.5% corn peptone, 0.3% yeast extract, 0.03% manganese sulfate monohydrate, 0.08% magnesium chloride hexahydrate, 0.10% sodium chloride, 0.08% calcium carbonate, 0.3% dipotassium hydrogen phosphate, 0.15% Tween-80, 0.03% defoamer, with the balance being water. After fully dissolving the above materials in a feeding tank, the mixture was pumped into a 5T fermenter, filling it to 70% capacity. The mixture was sterilized at 121℃ for 30 minutes, then cooled to 37℃ and adjusted to an initial pH of 6.5.

[0031] The secondary seed culture was inoculated into the fermentation medium for fermentation, with the stirring speed controlled at 90 rpm during the fermentation process. When the pH dropped to 5.5, automatic alkali supplementation was activated, and fermentation ended when alkali supplementation stopped. In the later stage of fermentation, 0.02% rifampicin was added to the fermentation broth to induce the cells to enter a dormant or low metabolic state. Subsequently, the temperature of the fermentation broth was raised from 37℃ to 45℃ and maintained for 30 min to induce the expression of heat shock proteins in the cells using heat stimulation, thus obtaining the fermentation stock broth.

[0032] The fermentation broth was transferred to a storage tank in the production workshop under air pressure and centrifuged using a disc centrifuge at a capacity of 4 tons per hour. The unwashed bacterial sludge containing some of the centrifuged mother liquor was collected after centrifugation and used for subsequent preparation of the bacterial suspension.

[0033] Crystalline trehalose was added to the obtained mycelial sludge in 5 portions, with each addition being 1.5% of the sludge's mass. After each addition, the mixture was stirred thoroughly. After all the sludge was added, stirring continued for 4 minutes. Separately, sodium chloride, skim milk powder, maltodextrin, and ascorbic acid were added to an appropriate amount of water and stirred until fully dissolved to obtain a preservative solution. The sodium chloride, skim milk powder, maltodextrin, and ascorbic acid were added at 0.08% and 0.4% of the sludge's mass, respectively. The ascorbic acid was then added to the sludge containing crystalline trehalose, and stirring continued for 3 minutes to obtain a bacterial suspension.

[0034] While preparing the bacterial suspension, the spray drying tower was preheated, with the inlet air temperature adjusted to 123℃. Once the outlet air temperature reached 79℃, clean water was first pumped in for spray drying testing. The pumping speed was controlled by adjusting the peristaltic pump speed to stabilize the outlet air temperature at 79℃. After the temperature stabilized, the bacterial suspension was pumped in, and the air hammer was activated to collect the bacterial powder. During the spray drying process, the inlet air temperature was controlled at 123℃, the outlet air temperature at 79℃, and the moisture content of the bacterial powder was controlled at 3.5%.

[0035] Vitamin C was added to the obtained bacterial powder at a concentration of 0.15% of the bacterial powder mass. After thorough mixing, the mixture was placed in a sealed bag and silica gel desiccant was added. 20g of silica gel desiccant was added for every 20kg of bacterial powder. The mixture was then vacuum-packed and stored in a 4℃ cold storage to obtain a live Lactobacillus plantarum preparation.

[0036] Example 2: Preparation of live Lactobacillus rhamnosus preparation This embodiment is basically the same as Example 1, except that the lactobacillus is Lactobacillus rhamnosus. After the glycerol tube culture is thawed, it is streaked on an MRS plate or a Lactobacillus rhamnosus-specific plate for isolation, cultured at 37°C until obvious colonies grow, and then streaked on a slant to obtain the culture for preservation on a slant.

[0037] The primary culture medium was inoculated into the secondary culture medium of a 50L fermenter at a 1% inoculation rate, with a liquid volume of 60%. The culture was incubated at 30℃ and 80r / min for 15h to obtain the secondary seed culture, which was then transferred into the fermentation medium under air pressure.

[0038] The fermentation medium was prepared according to the following composition: 3.0% glucose, 1.0% yeast extract, 0.3% corn steep liquor powder, 0.1% yeast extract, 0.02% manganese sulfate monohydrate, 0.05% magnesium chloride hexahydrate, 0.08% sodium chloride, 0.06% calcium carbonate, 0.2% dipotassium hydrogen phosphate, 0.1% Tween-80, 0.01% antifoaming agent, with the remainder being water. The fermentation medium was sterilized at 121℃ for 30 min, then cooled to 30℃ and the initial pH was adjusted to 6.0. During fermentation, the stirring speed was 80 rpm. Automatic alkali supplementation was initiated when the pH dropped to 5.0, and fermentation ended when alkali supplementation stopped. In the later stages of fermentation, 0.01% rifampicin was added, and the fermentation broth temperature was then raised to 40℃ and maintained for 30 min to obtain the original fermentation broth.

[0039] The fermentation broth was centrifuged using a disc centrifuge with a processing capacity of 3 T / h, and the unwashed bacterial sludge containing some centrifugation mother liquor was collected. Crystalline trehalose was added to the bacterial sludge in five portions, at a rate of 1.0% of the bacterial sludge mass, and stirring was continued for 3 minutes after each addition. A protective agent solution was then added, comprising 0.05% sodium chloride, 0.3% skim milk powder, 0.3% maltodextrin, and 0.1% ascorbic acid, and stirring was continued for 2 minutes to obtain a bacterial suspension. During spray drying, the inlet air temperature was controlled at 120℃, the outlet air temperature at 78℃, and the moisture content of the bacterial powder at 3.0%. 0.1% vitamin C was added to the bacterial powder, mixed thoroughly, and then vacuum-sealed with silica gel desiccant and stored at 4℃ to obtain a live Lactobacillus rhamnosus preparation.

[0040] Example 3: Preparation of live Lactobacillus acidophilus preparation This embodiment is basically the same as Example 1, except that the lactobacillus is Lactobacillus acidophilus. After the glycerol tube culture is thawed, it is streaked on an MRS plate or a dedicated Lactobacillus acidophilus plate for isolation, cultured at 37°C until obvious colonies grow, and then streaked on a slant to obtain the culture for preservation on a slant.

[0041] The primary culture medium was inoculated into the secondary culture medium of a 50L fermenter at a 5% inoculation rate, with a liquid volume of 75%, and cultured at 40℃ and 120r / min for 5h to obtain the secondary seed culture.

[0042] The fermentation medium was prepared according to the following composition: 4.0% granulated sugar, 2.0% yeast extract, 0.6% corn peptone, 0.5% yeast extract, 0.05% manganese sulfate monohydrate, 1.0% magnesium chloride hexahydrate, 1.0% sodium chloride, 1.0% calcium carbonate, 0.5% dipotassium hydrogen phosphate, 0.2% Tween-80, 0.05% antifoaming agent, with the remainder being water. After sterilization, the fermentation medium was cooled to 40℃ and the initial pH was adjusted to 7.0. During fermentation, the stirring speed was 100 rpm. Automatic alkali supplementation was initiated when the pH dropped to 6.0, and fermentation ended when alkali supplementation stopped. In the later stages of fermentation, 0.03% rifampicin was added, and the fermentation broth temperature was then raised to 50℃ and maintained for 30 minutes to obtain the original fermentation broth.

[0043] The fermentation broth was centrifuged using a 5T / h disc centrifuge, and the unwashed bacterial sludge containing some centrifuged mother liquor was collected. Crystalline trehalose was added to the sludge in five portions, at a rate of 2.0% of the sludge's mass, and stirring was continued for 5 minutes after each addition. A protective agent solution was then added, comprising 0.1% sodium chloride, 0.5% skim milk powder, 0.5% maltodextrin, and 0.2% ascorbic acid, and stirring was continued for 3 minutes to obtain a bacterial suspension. During spray drying, the inlet air temperature was controlled at 125℃, the outlet air temperature at 80℃, and the moisture content of the bacterial powder at 4.0%. 0.2% vitamin C was added to the bacterial powder, mixed thoroughly, and then vacuum-sealed with silica gel desiccant. The mixture was then stored at 4℃ to obtain a live Lactobacillus acidophilus preparation.

[0044] Example 4: Preparation of a compound live bacteria preparation of Lactobacillus casei and Lactobacillus paracasei This embodiment is basically the same as Example 1, except that the lactobacillus is a compound strain of Lactobacillus casei and Lactobacillus paracasei, which are mixed at a live count ratio of 1:1 and used as the inoculum. Each strain is thawed in glycerol tubes, isolated by MRS or special plate streak, streak culture on slant, and primary seed culture, and then mixed in proportion.

[0045] The fermentation medium was prepared according to the following composition: 3.8% glucose, 1.8% yeast extract, 0.4% corn steep liquor powder, 0.4% yeast extract, 0.04% manganese sulfate monohydrate, 0.5% magnesium chloride hexahydrate, 0.5% sodium chloride, 0.5% calcium carbonate, 0.4% dipotassium hydrogen phosphate, 0.18% Tween-80, 0.04% antifoaming agent, with the remainder being water. The primary culture medium was inoculated into the secondary culture medium at a 4% inoculum rate, filling a 50L fermenter to 65% capacity. The culture was incubated at 35℃ and 100 rpm for 12 hours to obtain the secondary seed culture. During fermentation, the stirring speed was 95 rpm. Automatic alkali supplementation was initiated when the pH dropped to 5.8, and fermentation ended when alkali supplementation stopped. In the later stages of fermentation, 0.02% rifampicin was added, and the fermentation broth temperature was subsequently raised to 46℃ and maintained for 30 minutes to obtain the fermentation stock solution.

[0046] The fermentation broth was centrifuged in a 4.5 T / h disc centrifuge to collect the unwashed bacterial sludge containing some centrifugation mother liquor. Crystalline trehalose was added to the sludge in five portions, at a rate of 1.8% of the sludge's mass. A protective agent solution composed of sodium chloride, skim milk powder, maltodextrin, and ascorbic acid was also added, with sodium chloride at 0.09%, skim milk powder at 0.45%, maltodextrin at 0.45%, and ascorbic acid at 0.18% of the sludge's mass. This mixture was then used to prepare a bacterial suspension. During spray drying, the inlet air temperature was controlled at 124℃, the outlet air temperature at 79℃, and the moisture content of the bacterial powder at 3.8%. 0.18% vitamin C was added to the bacterial powder, mixed thoroughly, and then vacuum-sealed with silica gel desiccant. The mixture was then stored at 4℃ to obtain a compound live lactobacillus preparation.

[0047] Example 5: Preparation of live Lactobacillus reuteri preparation This embodiment is basically the same as Example 1, except that the lactobacillus used is Lactobacillus reuteri. After the glycerol tube culture is thawed, it is streaked on an MRS plate or a Lactobacillus reuteri-specific plate for isolation, cultured at 37°C until obvious colonies grow, and then streaked on a slant to obtain the culture for preservation on a slant.

[0048] The fermentation medium was prepared according to the following composition: 3.2% granulated sugar, 1.2% yeast extract, 0.35% corn steep liquor powder, 0.2% yeast extract, 0.025% manganese sulfate monohydrate, 0.2% magnesium chloride hexahydrate, 0.2% sodium chloride, 0.2% calcium carbonate, 0.25% dipotassium hydrogen phosphate, 0.12% Tween-80, 0.02% antifoaming agent, with the remainder being water. The primary culture medium was inoculated into the secondary culture medium in a 50L fermenter at a 2% inoculum rate, resulting in a 68% fill volume. The culture was incubated at 32℃ and 90 rpm for 13 hours to obtain the secondary seed culture. During fermentation, the stirring speed was 85 rpm. Automatic alkali supplementation was initiated when the pH dropped to 5.2, and fermentation ended when alkali supplementation stopped. In the later stages of fermentation, 0.015% rifampicin was added, and the fermentation broth temperature was subsequently raised to 42℃ and maintained for 30 minutes to obtain the fermentation stock solution.

[0049] The bacterial sludge, unwashed and containing some centrifugal mother liquor, was collected using a 3.5 T / h disc centrifuge. Crystalline trehalose was added to the sludge in five portions, at a rate of 1.2% of the sludge's mass. A protective agent solution composed of sodium chloride, skim milk powder, maltodextrin, and ascorbic acid was also added, with sodium chloride at 0.06%, skim milk powder at 0.35%, maltodextrin at 0.35%, and ascorbic acid at 0.12% of the sludge's mass. A bacterial suspension was prepared by mixing the solutions. During spray drying, the inlet air temperature was controlled at 121℃ and the outlet air temperature at 78℃, resulting in a bacterial powder moisture content of 3.2%. 0.12% vitamin C was added to the bacterial powder, mixed thoroughly, and then vacuum-sealed with silica gel desiccant. The mixture was then stored at 4℃ to obtain a live Lactobacillus reuteri preparation.

[0050] Example 6: Preparation of a compound live bacteria preparation of Lactobacillus fermentum, Lactobacillus delbrueckii, and Lactobacillus johnsonii This embodiment is basically the same as Example 1, except that the lactobacillus used is a complex strain of Lactobacillus fermentum, Lactobacillus delbrueckii, and Lactobacillus johnsonii, which are mixed at a live count ratio of 1:1:1 as the inoculum. Each strain is thawed in glycerol tubes, isolated by MRS or special plate streak, cultured on slant, and cultured as a primary seed culture before being mixed in the specified ratio.

[0051] The fermentation medium was prepared according to the following composition: 3.6% glucose, 1.6% yeast extract, 0.55% corn peptone, 0.35% yeast extract, 0.035% manganese sulfate monohydrate, 0.6% magnesium chloride hexahydrate, 0.6% sodium chloride, 0.6% calcium carbonate, 0.35% dipotassium hydrogen phosphate, 0.16% Tween-80, 0.035% antifoaming agent, with the remainder being water. The primary culture was inoculated into the secondary culture medium in a 50L fermenter at a 3.5% inoculation rate, resulting in a 72% fill volume. The culture was incubated at 38℃ and 110 rpm for 8 hours to obtain the secondary seed culture. During fermentation, the stirring speed was 90 rpm. Automatic alkali supplementation was initiated when the pH dropped to 5.6, and fermentation ended when alkali supplementation ceased. In the later stages of fermentation, 0.02% rifampicin was added, and the fermentation broth temperature was subsequently raised to 48℃ and maintained for 30 minutes to obtain the fermentation stock solution.

[0052] The fermentation broth was centrifuged using a disc centrifuge with a processing capacity of 4 T / h, and the unwashed bacterial sludge containing some centrifuged mother liquor was collected. Crystalline trehalose was added to the bacterial sludge in five portions, at a rate of 1.6% of the sludge's mass. A protective agent solution composed of sodium chloride, skim milk powder, maltodextrin, and ascorbic acid was also added, with sodium chloride at 0.085%, skim milk powder at 0.42%, maltodextrin at 0.42%, and ascorbic acid at 0.16% of the sludge's mass. A bacterial suspension was prepared by mixing the solutions. During spray drying, the inlet air temperature was controlled at 122℃, the outlet air temperature at 79℃, and the moisture content of the bacterial powder at 3.6%. 0.16% vitamin C was added to the bacterial powder, mixed thoroughly, and then vacuum-sealed with silica gel desiccant. The mixture was then stored at 4℃ to obtain a compound live lactobacillus preparation.

[0053] Comparative Example 1: Trehalose without added crystals The difference between this comparative example and Example 1 is that crystalline trehalose was not added during the preparation of the bacterial suspension; all other process conditions were the same as in Example 1. Specifically, the composition of the fermentation medium, fermentation conditions, rifampicin-induced dormancy, thermal stimulation, disc centrifugation, spray drying conditions, and packaging and storage conditions were all the same as in Example 1. After the bacterial sludge was collected by disc centrifugation, only a protective agent solution obtained by dissolving sodium chloride, skim milk powder, maltodextrin, and ascorbic acid was added. The amount of sodium chloride added was 0.08% of the bacterial sludge mass, the amount of skim milk powder added was 0.4% of the bacterial sludge mass, the amount of maltodextrin added was 0.4% of the bacterial sludge mass, and the amount of ascorbic acid added was 0.15% of the bacterial sludge mass. After stirring for 3 minutes, a bacterial suspension was obtained. Subsequently, it was spray-dried, mixed with vitamin C, vacuum-sealed with silica gel desiccant, and stored at 4°C under the same conditions as in Example 1.

[0054] Comparative Example 2: Spray drying temperature out of range Compared with Example 1, this comparative example differs in that the inlet and outlet air temperatures during spray drying both exceed the limits defined in this invention; the remaining process conditions are the same as in Example 1. Specifically, the composition of the fermentation medium, fermentation conditions, rifampicin-induced dormancy, thermal stimulation, butterfly centrifugation, bacterial suspension preparation process, and packaging and storage conditions are all the same as in Example 1. During spray drying, the spray drying tower is preheated to an inlet air temperature of 135°C, and spray drying is performed after the outlet air temperature reaches 85°C to collect the bacterial powder. Subsequently, vitamin C is added to the bacterial powder at a concentration of 0.15% of the bacterial powder mass, mixed thoroughly, and then vacuum-sealed together with silica gel desiccant. After packaging, it is stored at 4°C.

[0055] Comparative Example 3: No Vitamin C added The difference between this comparative example and Example 1 is that vitamin C was not added before packaging the bacterial powder; all other process conditions were the same as in Example 1. Specifically, fermentation medium, Lactobacillus seed liquid, fermentation stock broth, unwashed bacterial sludge containing some centrifuged mother liquor, and bacterial suspension were prepared according to the same method as in Example 1, and bacterial powder was obtained under the same spray drying conditions as in Example 1. The obtained bacterial powder was not supplemented with vitamin C and was directly vacuum-sealed with silica gel desiccant, then stored at 4°C.

[0056] To further illustrate the effects of the various technical measures of the present invention on the activity of live Lactobacillus preparations, the number of live bacteria, moisture content, number of live bacteria and survival rate after spray drying of the live bacterial preparations obtained in Examples 1-6 and Comparative Examples 1-3 were tested according to the above-mentioned live bacterial preparation activity detection method. The results are shown in Table 1.

[0057] Example 1 Lactobacillus plantarum Complete process, optimal parameters 8.5 3.5 7.2 84.7 Example 2 Lactobacillus rhamnosus Complete process, minimum parameters 8.0 3.0 6.6 82.5 Example 3 Lactobacillus acidophilus Complete process, high limit parameters 6.1 4.0 5.2 85.2 Example 4 Lactobacillus casei + Lactobacillus paracasei Compound Lactobacillus 6.8 3.8 5.7 83.8 Example 5 Lactobacillus reuteri Complete process, medium and low parameters 6.5 3.2 5.4 83.1 Example 6 Lactobacillus fermentum + Lactobacillus delbrueckii + Lactobacillus johnsonii Compound Lactobacillus 6.9 3.6 5.8 84.1 Comparative Example 1 Lactobacillus plantarum No crystalline trehalose added 4.2 3.6 2.9 69.0 Comparative Example 2 Lactobacillus plantarum Spray drying temperature out of range 3.2 2.8 2.1 65.6 Comparative Example 3 Lactobacillus plantarum No vitamin C was added before packaging. 7.6 3.5 4.8 63.2 Table 1. Performance comparison of live bacterial preparations obtained in Examples 1-6 and Comparative Examples 1-3 As shown in Table 1, Examples 1-6 all employed the complete process of this invention, including the combination of rapid and slow nitrogen sources, Tween-80 regulation of cell membrane pressure resistance, rifampicin-induced bacterial dormancy, pre-containment heating stimulation, butterfly centrifugation to collect unwashed bacterial sludge containing some centrifugal mother liquor, stepwise protection with crystalline trehalose and a composite protective agent, spray drying control at an inlet air temperature of 120-125°C and an outlet air temperature of 78-80°C, and vacuum sealing packaging using vitamin C and silica gel desiccant. The viable bacterial counts of the Lactobacillus preparations obtained in Examples 1-6 remained high after spray drying, and the survival rate remained above 80% after 6 months of storage at 4°C, indicating that the method of this invention has good applicability and stability for different Lactobacillus strains and composite strains.

[0058] Comparative Example 1, without the addition of crystalline trehalose, showed a significant decrease in both viable bacterial count and survival rate after 6 months of storage following spray drying, indicating that crystalline trehalose plays an important role in maintaining cell morphology and stabilizing cell membranes and intracellular protein structures. In Comparative Example 2, the spray drying temperature exceeded the limits specified in this invention, resulting in a significant decrease in viable bacterial count after spray drying, indicating that excessively high inlet and outlet air temperatures can cause thermal damage to the bacteria. Although Comparative Example 3 still had a relatively high viable bacterial count after spray drying, the lack of vitamin C before packaging led to a significant decrease in viable bacterial count after 6 months of storage at 4°C, demonstrating that vitamin C plays a positive role in reducing oxidative stress and improving storage stability.

[0059] Therefore, Table 1 further demonstrates that the present invention, through the synergistic effects of multiple processes including fermentation regulation, induction of dormancy, thermal stimulation, butterfly centrifugation, compounding of protective agents, spray drying temperature control, and packaging antioxidant protection, can significantly improve the spray-dried survival rate and storage stability of live Lactobacillus preparations. Examples 1-6 have significant advantages over Comparative Examples 1-3.

[0060] Methods for detecting the activity of live bacteria preparations Weigh 1.000g of live bacterial preparation and place it in a 250mL Erlenmeyer flask. The Erlenmeyer flask should be pre-filled with an appropriate amount of glass beads and sterilized and dried. In a sterile laminar flow hood, add 99mL of sterile physiological saline to the Erlenmeyer flask to dilute the sample 100 times. Then place the Erlenmeyer flask on a shaker and shake at 130r / min for 15min to ensure that the bacterial powder is fully dispersed.

[0061] Prepare eight sterilized 10mL centrifuge tubes in a sterile laminar flow hood, adding 4.5mL of sterile physiological saline to each tube. Remove the well-mixed bacterial solution from the shaker, and in the laminar flow hood, add 0.5mL of the solution from an Erlenmeyer flask to the first centrifuge tube. Shake for 15 seconds to mix thoroughly, at which point the bacterial solution is diluted to 10⁻⁶. 3 Then, take 0.5 mL of bacterial culture from the first centrifuge tube and add it to the second centrifuge tube. Shake for 15 seconds to mix. At this point, the bacterial culture is diluted to 10⁻⁶. 4 Repeat the process to dilute the bacterial culture to 10 times; 10 times.

[0062] Take sterilized MRS solid medium or strain-specific detection medium, melt it using a microwave oven, and then place it in a 50℃ water bath for later use. Take three disposable sterile agar plates, and add 1 mL of bacterial solution from the well-mixed eighth centrifuge tube to each plate. Then pour the melted medium, cooled to approximately 50℃, into each of the three plates and gently shake to evenly disperse the bacterial solution. After the medium in the plates has solidified, incubate the plates at 37℃ for 2–5 days until colonies grow. Count the number of colonies grown on the three plates with a black marker, take the average value, and multiply it by the corresponding dilution factor to obtain the number of live bacteria per gram of live bacterial preparation, expressed in CFU / g.

[0063] Tween-80 dosage adjustment experiment An experiment was conducted to adjust the amount of Tween-80 used in *Lactobacillus plantarum* CICC21809. The culture medium formula was as follows: 3.0–4.0% granulated sugar, 1.0–2.0% yeast extract, 0.3–0.6% corn steep liquor powder, 0.1–0.5% yeast extract, 0.02–0.05% manganese sulfate monohydrate, 0.05–1.0% magnesium chloride hexahydrate, 0.08–1.0% sodium chloride, 0.06–1.0% calcium carbonate, 0.2–0.5% dipotassium hydrogen phosphate, 0.1–0.2% Tween-80, 0.01–0.05% antifoaming agent, and the remainder being water. The fermentation operation was carried out according to Example 1. The activity of the live bacteria preparations corresponding to different Tween-80 contents was tested by adjusting the Tween-80 content in the fermentation medium.

[0064] Experimental results show that the use of Tween-80 can significantly enhance the activity of live Lactobacillus plantarum preparations. However, when the amount of Tween-80 added is too high, its enhancing effect gradually decreases, and it may even have a negative effect. This may be because excessively high concentrations of Tween-80 can inhibit bacterial growth. Different lactobacilli have slightly different requirements for the amount of Tween-80, so the optimal amount can be determined experimentally based on the characteristics of the strains. In this invention, the preferred amount of Tween-80 added is 0.1% to 0.2%.

[0065] Experiment on adjustment of the optimal amount of protective agent Taking Lactobacillus plantarum CICC21809 as an example, based on the fermentation and centrifugation process in Example 1, spray drying experiments with different protective agent formulations were designed to investigate the effects of the dosage of trehalose, skim milk powder, maltodextrin and ascorbic acid on the activity of the live bacteria preparation. The results are shown in Table 2.

[0066] control group 0 0 0 0 0.5 Group 1 1.0 0.3 0.3 0.1 3.2 Group 2 1.5 0.4 0.4 0.15 6.8 Group 3 2.0 0.5 0.5 0.2 8.5 Group 4 2.5 0.6 0.6 0.25 6.5 Table 2. Effects of different dosages of preservative on the activity of live Lactobacillus plantarum preparations. Table 2 shows that without the addition of a preservative, the viable bacterial count after spray drying was only 0.5 × 10¹⁰ CFU / g; after adding the preservative, the viable bacterial count increased significantly. Among them, group 3, with 2.0% trehalose, 0.5% skim milk powder, 0.5% maltodextrin, and 0.2% ascorbic acid, had the highest viable bacterial count, reaching 8.5 × 10¹⁰ CFU / g. In group 4, the viable bacterial count decreased after excessive addition of the preservative, possibly due to increased viscosity of the bacterial sludge, resulting in poor flowability during spray drying and affecting atomization and drying uniformity. Therefore, the amounts of trehalose, skim milk powder, maltodextrin, and ascorbic acid should be controlled within appropriate ranges.

[0067] Experiment on the effect of spray drying temperature on bacterial activity Using Lactobacillus plantarum CICC21809 as the experimental strain, and with the optimal formulation of the fixative protectant as shown in Table 2, the effects of different combinations of inlet and outlet air temperatures on the activity of the live bacteria preparation were further investigated. The results are shown in Table 3.

[0068] control group 100 60 2.1 5.2 Group A 110 70 4.5 4.8 Group B 120 78 8.5 3.5 Group C 125 80 7.9 3.2 Group D 130 85 3.2 2.8 Table 3 Effect of spray drying temperature on microbial activity Table 3 shows that when the temperature is too low, the bacterial powder is not completely dehydrated, resulting in a high moisture content, which is not conducive to subsequent preservation. When the temperature is too high, although the moisture content decreases, the high temperature causes a large number of bacteria to become inactive. Group B, with an inlet air temperature of 120℃ and an outlet air temperature of 78℃, has the highest viable bacterial count, reaching 8.5×10^10 cfu / g, and a moisture content of 3.5%. Group C, with an inlet air temperature of 125℃ and an outlet air temperature of 80℃, still maintains a relatively high viable bacterial count, with a moisture content of 3.2%. Therefore, the inlet air temperature for spray drying is preferably controlled at 120–125℃, the outlet air temperature is preferably controlled at 78–80℃, and the moisture content of the bacterial powder is preferably controlled at 3–4%.

[0069] Adaptability test of different lactobacillus strains Four common lactobacilli—Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus paracasei—were selected and spray-dried using the technical solution of this invention, namely the basic process of Example 1, the optimized protective agent formulation, and the optimized spray drying temperature conditions. The adaptability of different strains was compared, and the results are shown in Table 4.

[0070] Lactobacillus plantarum 6.8 8.5 82.4 Lactobacillus acidophilus 5.2 6.1 85.7 Lactobacillus rhamnosus 7.1 8.0 83.1 Lactobacillus paracasei 5.9 6.8 86.4 Table 4. Adaptability experiments of different Lactobacillus strains As shown in Table 4, the technical solution of this invention is applicable to various types of Lactobacillus. All strains maintained a high viable count after spray drying, with a survival rate of no less than 82%, indicating that the method of this invention has good strain adaptability.

[0071] Stability test of live bacterial preparation The *Lactobacillus plantarum* live bacteria preparation prepared in Example 1 was stored under refrigeration at 4°C, room temperature at 25°C, and accelerated aging conditions at 37°C, respectively. The viable bacterial count was tested periodically, and the results are shown in Table 5. The viable bacterial count in Table 5 is in units of ×10^10 cfu / g.

[0072] Refrigerate at 4℃ 8.5 8.1 7.2 6.5 25℃ normal temperature 8.5 6.8 5.0 2.1 37℃ acceleration 8.5 4.2 1.5 0.3 Table 5. Experiment on the preservation stability of Lactobacillus plantarum Table 5 shows that after 6 months of storage at 4℃, the viable count of the *Lactobacillus plantarum* live bacteria preparation was 7.2 × 10^10 cfu / g, and the survival rate was: (7.2 / 8.5)×100%≈84.7% After 12 months of storage, the viable bacterial count remained at 6.5 × 10^10 cfu / g, indicating that the Lactobacillus live bacteria preparation prepared in this invention has good storage stability under refrigeration at 4°C. The viable bacterial count decreased rapidly under ambient temperature (25°C) and accelerated storage (37°C). Therefore, the data from ambient temperature storage can be used as a comparison for stability evaluation. The product of this invention is preferably stored under refrigeration at 4°C.

[0073] Based on the above embodiments, comparative examples, and experimental results, it can be seen that this invention protects Lactobacillus simultaneously intracellularly, intracellularly, and extracellularly through measures such as the combination of rapid and slow nitrogen sources, Tween-80 regulation of cell membrane composition, rifampicin-induced bacterial dormancy, temperature-induced heat stress protein expression, rapid collection of unwashed bacterial sludge containing some centrifugation mother liquor via butterfly centrifugation, stepwise addition of composite protective agents, appropriate spray drying temperature control, and vacuum sealing packaging with vitamin C and silica gel desiccant. These measures ensure that Lactobacillus maintains its activity as much as possible during spray drying and storage. The viable Lactobacillus preparation obtained by spray drying can achieve an activity of 1.0 × 10^10 to 1.0 × 10^11 cfu / g, and the survival rate after 6 months of storage at 4°C can reach over 70%, preferably over 80%.

[0074] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, equivalent substitutions or conventional adjustments can be made to the types of lactobacillus, culture medium components, fermentation conditions, rifampicin dosage, protectant dosage, spray drying parameters, and packaging and storage methods without departing from the concept of the present invention. All such equivalent substitutions or conventional adjustments should fall within the protection scope of the present invention.

Claims

1. A method for preparing a live lactobacillus preparation, characterized in that, Includes the following steps: S1. Prepare a fermentation medium based on yeast extract powder, yeast extract, corn peptone or corn steep liquor powder and a carbon source. Add a surfactant to the fermentation medium and inoculate the lactobacillus seed liquid into the fermentation medium for fermentation. S2. After fermentation, the fermentation liquid is heated to obtain the original fermentation liquid; S3. Centrifuge the fermentation broth using a butterfly centrifuge to collect the unwashed bacterial sludge. S4. Add trehalose, osmotic pressure regulator and composite protectant to the bacterial mud in sequence, and mix to obtain bacterial suspension; S5. Spray dry the bacterial suspension, controlling the inlet air temperature to 120-125℃ and the outlet air temperature to 78-80℃, collect the bacterial powder, and control the moisture content of the bacterial powder to 3-4%; S6. Add vitamin C to the bacterial powder, mix well, and seal and package together with the desiccant to obtain a live lactobacillus preparation.

2. The method for preparing a live lactobacillus preparation according to claim 1, characterized in that, In step S4, the trehalose is crystalline trehalose, and the amount added is 1.0-2.0% of the mass of the bacterial sludge. It is added to the bacterial sludge in 5 portions, and stirred evenly after each addition. After all the sludge is added, stirring is continued for 3-5 minutes. The osmotic pressure regulator is sodium chloride, and the amount added is 0.05-0.1% of the mass of the bacterial sludge. The composite protective agent includes skim milk powder, maltodextrin, and ascorbic acid, wherein the amount of skim milk powder added is 0.3-0.5% of the mass of the bacterial sludge, the amount of maltodextrin added is 0.3-0.5% of the mass of the bacterial sludge, and the amount of ascorbic acid added is 0.1-0.2% of the mass of the bacterial sludge. Sodium chloride, skim milk powder, maltodextrin, and ascorbic acid are dissolved together in water to obtain a protective agent solution. After adding crystalline trehalose to the bacterial sludge and stirring evenly, the protective agent solution is added to the bacterial sludge, and stirring is continued for 2-3 minutes to obtain the bacterial suspension.

3. The method for preparing a live lactobacillus preparation according to claim 1, characterized in that, The fermentation medium also includes manganese sulfate monohydrate, magnesium chloride hexahydrate, sodium chloride, dipotassium hydrogen phosphate, calcium carbonate, and an antifoaming agent; The carbon source is granulated sugar or glucose, and the surfactant is Tween-80; The fermentation medium comprises: 3.0-4.0% granulated sugar or glucose, 1.0-2.0% yeast extract, 0.3-0.6% corn peptone or corn steep liquor powder, 0.1-0.5% yeast extract, 0.02-0.05% manganese sulfate monohydrate, 0.05-1.0% magnesium chloride hexahydrate, 0.08-1.0% sodium chloride, 0.06-1.0% calcium carbonate, 0.2-0.5% dipotassium hydrogen phosphate, 0.1-0.2% Tween-80, and 0.01-0.05% defoamer.

4. The method for preparing a live lactobacillus preparation according to claim 3, characterized in that, The fermentation medium was sterilized at 121°C for 30 min before inoculation with Lactobacillus seed culture, and then cooled to 30-40°C and the initial pH was adjusted to 6.0-7.

0. During fermentation, the stirring speed is 80-100 r / min. When the pH drops to 5.0-6.0, automatic alkali replenishment is activated. Fermentation ends when alkali replenishment stops.

5. The method for preparing a live lactobacillus preparation according to claim 4, characterized in that, In step S2, after fermentation is completed, alkali addition is stopped, the temperature of the fermentation liquid is raised to 40-50°C and maintained for 30 minutes, and then the obtained fermentation liquid is transported to a disc centrifuge for centrifugation. During the centrifugation process, the fermentation broth is separated into solid and liquid components, and the resulting bacterial sludge containing the centrifuged mother liquor is collected. This bacterial sludge is then used for the preparation of a subsequent bacterial suspension.

6. The method for preparing a live lactobacillus preparation according to claim 1, characterized in that, The preparation of the Lactobacillus seed culture includes: picking a single colony of Lactobacillus and inoculating it into 100 mL of primary culture medium, and culturing it until a primary culture medium is formed; The primary culture medium was inoculated into the secondary culture medium of a 50L fermenter at an inoculation rate of 1-5%, with a liquid volume of 60-75%, and cultured at 30-40℃ and 80-120r / min for 5-15h to obtain the secondary seed culture. The secondary seed culture is transferred into the fermentation medium under pressure using air or nitrogen gas with a purity of ≥99.999%.

7. The method for preparing a live lactobacillus preparation according to claim 1, characterized in that, In S3, the processing capacity of the disc centrifuge is 3-5 T / h; In step S5, the spray drying tower is preheated to an inlet air temperature of 120-125°C before spray drying. After the outlet air temperature rises to 78-80°C, clean water is first pumped in for spray drying test. After the outlet air temperature stabilizes, the bacterial suspension is pumped in, and the air hammer is turned on to collect the bacterial powder.

8. The method for preparing a live lactobacillus preparation according to claim 1, characterized in that, In step S6, the amount of vitamin C added is 0.1% to 0.2% of the mass of the bacterial powder; The desiccant is silica gel desiccant, with 20g of silica gel desiccant added for every 20kg of bacterial powder; The bacterial powder, vitamin C, and silica gel desiccant are vacuum-sealed and stored at 4°C.

9. The method for preparing a live lactobacillus preparation according to claim 1, characterized in that, The lactobacillus is one or more of the following: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus brunelli, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus johnsonii, Lactobacillus reuteri, or Lactobacillus fermentum.

10. The live lactobacillus preparation obtained by the preparation method according to any one of claims 1 to 9.

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