A probiotic formula suitable for highland population and a preparation method thereof

Abstract

The present application relates to the technical field of probiotic preparation, in particular to a probiotic formula suitable for plateau population and a preparation method thereof, which formula comprises the following components by weight: Lactobacillus plantarum 3-5 parts, Bifidobacterium 2-4 parts, Lactobacillus acidophilus 1.5-3 parts, Lactobacillus rhamnosus 1-2.5 parts and Bacillus coagulans 0.5-1.5 parts. The method comprises the following steps: step one, strain activation and expansion; step two, strain fermentation; step three, strain collection; step four, strain treatment and packaging. The present application forms a double barrier by Lactobacillus plantarum and Lactobacillus rhamnosus, which can reduce the risk of intestinal infection of the user, and at the same time, by adding Bacillus coagulans in the formula, it provides a growth environment for other strains, further ensuring the stability of the formula.

Description

Technical Field

[0001] This invention relates to the field of probiotic preparation technology, specifically to a probiotic formula and preparation method suitable for people living in high-altitude areas. Background Technology

[0002] Probiotics are a class of live microorganisms that are beneficial to the host's health. They typically colonize the host's gut and reproductive system, producing definite health benefits by regulating the host's mucosal and systemic immune functions or adjusting the balance of gut microbiota. They are usually available in forms such as probiotic powders, tablets, capsules, or yak milk powder containing probiotics, and are suitable for people with weak gut function, low immunity, sleep disorders, or those needing to recover their energy. Probiotics suitable for people living at high altitudes are live microorganisms specifically selected to adapt to the extreme environment of high altitudes. They aim to help alleviate intestinal damage caused by hypoxia at high altitudes by strengthening the intestinal barrier, regulating immune responses, and maintaining metabolic homeostasis, while also improving oxygen utilization efficiency and shortening the adaptation period, helping the body quickly adapt to the high-altitude environment.

[0003] Existing probiotic formulas, such as Culturelle, use Lactobacillus rhamnosus LGG as the core strain. Lactobacillus rhamnosus LGG's strong acid resistance and bile salt tolerance can successfully pass through the acidic environment of the stomach to reach the intestines and colonize. At the same time, it can also inhibit the growth of harmful bacteria and the secretion of antibacterial substances, thereby directly weakening pathogens, enhancing the intestinal barrier function, reducing the possibility of pathogen invasion, and thus achieving the effect of immune regulation.

[0004] While the above formula can achieve immunomodulatory effects, its core strain contains only *Lactobacillus rhamnosus* LGG, which primarily stimulates local immunity and may be less effective in regulating systemic immunity. Therefore, it is necessary to propose a probiotic formula and preparation method suitable for people living in high-altitude areas. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a probiotic formula and preparation method suitable for high-altitude populations. This formula utilizes Lactobacillus rhamnosus and Bifidobacterium to simultaneously activate T cells and B cells, thereby inhibiting excessive inflammatory responses while enhancing the neutralizing capacity of B cells against pathogens. This ensures the effectiveness of systemic immune regulation.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: A probiotic formula suitable for people living in high-altitude areas, comprising the following components by weight: 3-5 parts of Lactobacillus plantarum, 2-4 parts of Bifidobacterium, 1.5-3 parts of Lactobacillus acidophilus, 1-2.5 parts of Lactobacillus rhamnosus, and 0.5-1.5 parts of Bacillus coagulans.

[0007] Furthermore, a probiotic formula suitable for people living in high-altitude areas comprises the following components by weight: 3 parts Lactobacillus plantarum, 2 parts Bifidobacterium, 1.5 parts Lactobacillus acidophilus, 1 part Lactobacillus rhamnosus, and 0.5 parts Bacillus coagulans.

[0008] Furthermore, a probiotic formula suitable for people living in high-altitude areas comprises the following components by weight: 4 parts Lactobacillus plantarum, 3 parts Bifidobacterium, 2 parts Lactobacillus acidophilus, 1.5 parts Lactobacillus rhamnosus, and 1 part Bacillus coagulans.

[0009] Furthermore, a probiotic formula suitable for people living in high-altitude areas comprises the following components by weight: 5 parts Lactobacillus plantarum, 4 parts Bifidobacterium, 3 parts Lactobacillus acidophilus, 2.5 parts Lactobacillus rhamnosus, and 1.5 parts Bacillus coagulans.

[0010] Furthermore, a method for preparing probiotics suitable for people living in high-altitude areas includes the following steps: Step 1, strain activation and expansion: Lactobacillus plantarum, Bifidobacterium, Lactobacillus acidophilus and Lactobacillus rhamnosus were inoculated into MRS broth medium for anaerobic culture and activated 2-3 times using multi-stage expansion and activation technology; at the same time, Bacillus coagulans was inoculated into nutrient broth medium for aerobic culture and activated twice using multi-stage expansion and activation technology.

[0011] Step 2, Fermentation: Carbon and nitrogen sources are added to the MRS broth medium to obtain the fermentation medium, which is then sterilized. The activated anaerobic bacteria are inoculated into the fermentation medium at 3%-5% of their respective weight for anaerobic fermentation. The viable cell count is monitored using the plate count method. Once the viable cell count reaches 10⁻⁶, fermentation continues. 9 Anaerobic fermentation is completed when the concentration of CFU / mL is above a certain level. The fermentation broth is collected to obtain the anaerobic fermentation broth of each anaerobic strain. Among them, one or more of glucose and sucrose are selected as carbon sources, and one or more of yeast powder and beef extract are selected as nitrogen sources.

[0012] The activated Bacillus coagulans was inoculated into the fermentation medium for aerobic fermentation. The inoculation amount was the sum of the inoculation amounts of each anaerobic strain during anaerobic fermentation. After more than 70% of the fermentation medium was converted into Bacillus coagulans spores, the aerobic fermentation was completed. The fermentation broth was collected to obtain the aerobic fermentation broth of Bacillus coagulans.

[0013] Step 3, bacterial collection: The aerobic fermentation broth and anaerobic fermentation broth were distinguished by the type of bacteria and placed in a centrifuge. At a temperature of 4°C, the anaerobic fermentation broth and aerobic fermentation broth of different bacterial species were centrifuged at 8000 rpm for 15 minutes. After centrifugation, bacterial sludge of each anaerobic bacterial species and bacterial sludge of Bacillus coagulans were obtained.

[0014] Step 4, Inoculum Processing and Packaging: The anaerobic bacterial sludge and Bacillus coagulans sludge are placed in a mixing device and mixed using the mixing components to obtain a mixed bacterial sludge. Simultaneously, three times the mass of a freeze-drying protectant is added to the mixed bacterial sludge through the feeding components in the mixing device and stirred to obtain a mixture. After mixing, the mixture is removed from the mixing device and pre-frozen at -40℃ for 4 hours. It is then transferred to a freeze dryer, where the temperature of the freeze dryer's baffles is gradually increased from -20℃ to 0℃, and the mixture is dried for 20 hours under a vacuum of <10 Pa to complete the initial drying. The temperature of the freeze dryer's baffles is then increased from 20℃ to 25℃, and the mixture is dried again for 4 hours under a vacuum of <10 Pa to complete the freeze-drying operation and obtain freeze-dried bacterial powder. Finally, the freeze-dried bacterial powder is packaged in nitrogen-filled aluminum foil bags to complete the preparation of the compound probiotic preparation.

[0015] Furthermore, in step one, during anaerobic culture, each anaerobic strain is anaerobic cultured at 37°C for 18-24 hours; during aerobic culture, Bacillus coagulans is aerobic cultured at 37°C for 24 hours.

[0016] Furthermore, in step two, during anaerobic fermentation, the fermentation temperature of each anaerobic strain is controlled at 37℃, and the pH value range is 5.5-6.0; during aerobic fermentation, the fermentation temperature of Bacillus coagulans is controlled at 37℃, and it is placed in a constant temperature shaking incubator and shaken at a frequency of 200 rpm.

[0017] Furthermore, in step four, when packaging the freeze-dried bacterial powder, the temperature is controlled below 15℃ and the relative humidity is below 20%.

[0018] Furthermore, the mixing device includes a controller and a housing. The top of the housing has a feed inlet with a detachable rubber plug. The lower side wall of the housing has a discharge outlet with a hinged door. A fixed frame and a drive unit are fixedly connected to the inner side wall of the housing. The controller controls the opening and closing of the drive unit. A fixed cylinder is fixedly connected to the side of the fixed frame away from the inner side wall of the housing. The top of the housing has a first through hole and a storage tank is fixedly connected thereto. The storage tank is filled with freeze-drying preservative. A feeding pipe is connected to the bottom of the storage tank. One end of the feeding pipe is connected to the inside of the storage tank through the first through hole. A second through hole is opened on one side of the fixed cylinder, and the other end of the feeding pipe is connected to the inside of the fixed cylinder through the second through hole. The fixed cylinder is equipped with a feeding component for batching the freeze-drying preservative into the housing. A guide platform is fixedly connected to the bottom of the housing.

[0019] The output shaft of the drive unit extends through the fixed cylinder and is fixedly connected to a first bevel gear on one side of the fixed cylinder. A rotating rod is rotatably fitted on the top wall of the outer shell, and the bottom end of the rotating rod is rotatably fitted with the bottom wall of the outer shell. A mixing component is provided on the rotating rod. The mixing component includes a second bevel gear fixedly connected to the rotating rod. The first bevel gear meshes with the second bevel gear. A stirring rod is fixedly connected to the lower circumference of the rotating rod.

[0020] Furthermore, the feeding assembly includes a feeding wheel fixedly connected to the output shaft of the drive component. The feeding wheel is rotatably engaged with the inner wall of the fixed cylinder. Several feeding grooves are opened at equal angles on the feeding wheel. Each feeding groove has a baffle hinged to its inner wall. The length of each baffle is the same as the opening length of the feeding groove. Each baffle is rotatably engaged with the inner wall of the fixed cylinder. A discharge port is opened at the bottom of the fixed cylinder. The discharge port is located in the movement path of all the baffles.

[0021] The technical principles of the above solution are as follows: Workers added the anaerobic bacterial sludge and Bacillus coagulans sludge to the mixing device through the feed inlet. Then, the controller activated the drive unit, causing the output shaft to sequentially rotate the first bevel gear, the second bevel gear, and the rotating rod. The rotating rod then rotated the stirring rod, mixing the anaerobic bacterial sludge and the Bacillus coagulans sludge. During this process, the drive unit's output shaft also activated the feeding component, which added the freeze-drying preservative, located in the storage tank, in batches to the anaerobic bacterial sludge and the Bacillus coagulans sludge, mixing them together.

[0022] The above approach has the following beneficial effects: 1. This invention activates T cells and B cells simultaneously using Lactobacillus rhamnosus and Bifidobacterium, which can enhance the neutralizing ability of B cells against pathogens while inhibiting excessive inflammatory response, thereby ensuring the effectiveness of immune regulation during systemic immune regulation.

[0023] 2. This invention improves the growth efficiency of each strain by culturing anaerobic bacteria and Bacillus coagulans separately, thereby increasing the activity rate of the strains and thus improving the effect of the probiotic formula.

[0024] 3. The present invention delivers the freeze-drying protectant in batches through the feeding component, ensuring that it is uniformly mixed with the fungal sludge of each strain, thereby forming a uniform protective film in the fungal sludge and reducing the possibility of cell membrane rupture or protein denaturation in the fungal sludge.

[0025] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0026] Figure 1This is a schematic diagram of the method steps in an embodiment of the method for preparing probiotics suitable for people in high-altitude areas according to the present invention; Figure 2 This is an isometric view of the mixing device in an embodiment of the method for preparing probiotics suitable for high-altitude populations according to the present invention; Figure 3 This is an isometric view of the mixing component in an embodiment of the method for preparing probiotics suitable for high-altitude populations according to the present invention; Figure 4 This is an isometric view of the feeding component in an embodiment of the method for preparing probiotics suitable for high-altitude populations according to the present invention.

[0027] The reference numerals in the accompanying drawings of the instruction manual include: 1. outer casing; 2. fixing frame; 3. fixing cylinder; 4. storage box; 5. feeding pipe; 6. guide table; 7. stepper motor; 8. feeding wheel; 9. baffle; 10. first bevel gear; 11. rotating rod; 12. second bevel gear; 13. stirring rod. Detailed Implementation

[0028] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0030] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0031] The following detailed description illustrates the specific implementation method: Example 1:

[0032] As attached Figure 1 As shown: A probiotic formula suitable for people living in high-altitude areas, comprising the following components by weight: 3 parts Lactobacillus plantarum, 2 parts Bifidobacterium, 1.5 parts Lactobacillus acidophilus, 1 part Lactobacillus rhamnosus, and 0.5 parts Bacillus coagulans.

[0033] Its production method is as follows Figure 1 As shown, it includes the following steps: Step 1, Strain Activation and Expansion: First, *Lactobacillus plantarum*, *Bifidobacterium*, *Lactobacillus acidophilus*, and *Lactobacillus rhamnosus* were inoculated separately into MRS broth medium as anaerobic strains and cultured anaerobically at 37°C for 18-24 hours. Then, a multi-stage expansion and activation technique was used to activate each strain 2-3 times. Simultaneously, *Bacillus coagulans* was inoculated into nutrient broth medium and cultured aerobically at 37°C for 24 hours. This was followed by two activation stages using a multi-stage expansion and activation technique, completing the strain activation and expansion process.

[0034] Step two, microbial fermentation: Workers add carbon and nitrogen sources to the MRS broth culture medium to obtain the fermentation medium, which is then sterilized. In this embodiment, high-pressure steam sterilization is used to sterilize the fermentation medium.

[0035] After sterilizing the fermentation medium, the activated anaerobic bacteria were inoculated into the fermentation medium at 4% of their respective weights. The fermentation temperature for each anaerobic bacteria was controlled at 37°C, and the pH range was 5.5-6.0. In this example, the pH was controlled at 5.7. Anaerobic fermentation was carried out separately, and the viable cell count was monitored during the fermentation process using the plate count method. Once the viable cell count reached 10... 9 Anaerobic fermentation is completed when the concentration of CFU / mL reaches a certain level. The fermentation broth is then collected to obtain the anaerobic fermentation broth for each anaerobic strain. The carbon source is selected from one or more of glucose and sucrose; glucose is selected in this embodiment. The nitrogen source is selected from one or more of yeast powder and beef extract; yeast powder and beef extract are selected in this embodiment.

[0036] While conducting anaerobic fermentation, the staff also inoculated activated Bacillus coagulans into the fermentation medium, controlled the fermentation temperature at 37℃, and placed it in a constant temperature shaking incubator, shaking it at a frequency of 200 rpm to carry out aerobic fermentation. The inoculation amount was the sum of the inoculation amounts of each anaerobic strain during anaerobic fermentation. After more than 70% of the fermentation medium was converted into Bacillus coagulans spores, the aerobic fermentation was completed, and the fermentation broth was collected to obtain the aerobic fermentation broth of Bacillus coagulans.

[0037] Step 3, bacterial collection: The staff distinguished the aerobic fermentation broth and anaerobic fermentation broth according to the type of bacteria, and placed them into centrifuges. At a temperature of 4°C, the anaerobic fermentation broth and aerobic fermentation broth of different bacterial species were centrifuged at 8000 rpm for 15 minutes. After centrifugation, bacterial sludge of each anaerobic bacterial species and bacterial sludge of Bacillus coagulans were obtained.

[0038] Step 4, Microbial Processing and Packaging: After obtaining the anaerobic bacterial sludge and Bacillus coagulans sludge, the workers placed them into a mixing device and mixed them using the mixing components to obtain a mixed sludge. The mixing ratio was the same as the weight component ratio in this embodiment. Simultaneously, the workers added three times the mass of a freeze-drying protectant to the mixed sludge using the feeding component in the mixing device and stirred to obtain a mixture. After mixing, the mixture was removed from the mixing device and pre-frozen at -40°C for 4 hours. It was then transferred to a freeze dryer, where the temperature of the freeze dryer's baffles was gradually increased from -20°C to 0°C, and the mixture was dried for 20 hours under a vacuum of <10 Pa to complete the initial drying. Subsequently, the temperature of the freeze dryer's baffles was increased from 20°C to 25°C, and the mixture was dried again for 4 hours under a vacuum of <10 Pa to complete the freeze-drying operation and obtain freeze-dried bacterial powder.

[0039] Finally, the staff took out the freeze-dried bacterial powder and, under temperature conditions below 15°C and relative humidity conditions below 20%, used aluminum foil bags to fill the freeze-dried bacterial powder with nitrogen, thus completing the preparation of the compound probiotic preparation.

[0040] Specifically, the mixing device in this embodiment is as follows: Figure 2 and Figure 3 As shown, it includes a controller and a housing 1. The top of the housing 1 has a feed inlet, and a rubber plug is detachably engaged at the feed inlet. The lower part of the side wall of the housing 1 has a discharge outlet, and an opening and closing door is hinged at the discharge outlet.

[0041] A fixing bracket 2 and a drive component are fixedly connected to the inner wall of the outer casing 1 by screws. A controller is used to control the opening and closing of the drive component. A fixing cylinder 3 is welded to the side of the fixing bracket 2 away from the inner wall of the outer casing 1. A first through hole is opened on the top of the outer casing 1 and a storage box 4 is welded thereon. The storage box 4 is filled with freeze-drying preservative. A feeding pipe 5 is connected to the bottom of the storage box 4. One end of the feeding pipe 5 is connected to the inside of the storage box 4 through the first through hole. A second through hole is opened on one side of the fixing cylinder 3. The other end of the feeding pipe 5 is connected to the inside of the fixing cylinder 3 through the second through hole. The feeding component is located inside the fixing cylinder 3. A guide platform 6 is integrally formed on the bottom of the outer casing 1. In this embodiment, a stepper motor 7 is selected as the drive component.

[0042] like Figure 4As shown, the feeding assembly includes a feeding wheel 8 fixedly connected to the output shaft of the stepper motor 7 with screws. The feeding wheel 8 is rotatably engaged with the inner wall of the fixed cylinder 3. Several feeding grooves are opened at equal angles on the feeding wheel 8. Each feeding groove has a baffle 9 hinged to its inner wall. The length of each baffle 9 is the same as the opening length of the feeding groove. Each baffle 9 is rotatably engaged with the inner wall of the fixed cylinder 3. A discharge port is opened at the bottom of the fixed cylinder 3. The discharge port is located in the movement path of all the baffles 9.

[0043] Specifically, workers place the anaerobic bacterial sludge and Bacillus coagulans sludge into the mixing device through the inlet. Then, the controller rotates the output shaft of the stepper motor 7, causing it to drive the feeding wheel 8, which is screwed to it, to rotate. The feeding wheel 8 then drives the feeding trough and the baffle 9 hinged to the inner wall of the feeding trough to rotate. During this process, since the two ends of the feeding pipe 5 are connected to the inside of the storage tank 4 and the fixed cylinder 3 respectively, the freeze-drying preservative located in the storage tank 4 can enter the fixed cylinder 3 through the feeding pipe 5, allowing it to enter different feeding troughs.

[0044] During this process, as the output shaft of the stepper motor 7 continues to rotate, since the discharge port is located in the movement path of all the baffles 9, when a baffle 9 moves to the discharge port, it can swing under its own gravity, thereby releasing the freeze-drying protectant located in the feeding tank in batches onto the guide table 6, and mixing it with the bacterial mud of various anaerobic bacteria and Bacillus coagulans under the guidance of the guide table 6.

[0045] The output shaft of the stepper motor 7 extends through the fixed cylinder 3 and is fixedly engaged with the first bevel gear 10 on one side of the fixed cylinder 3. The inner top wall of the outer shell 1 is rotatably engaged with the rotating rod 11. The bottom end of the rotating rod 11 is rotatably engaged with the inner bottom wall of the outer shell 1. The mixing component is located on the rotating rod 11. The mixing component includes a second bevel gear 12 fixedly engaged with the rotating rod 11. The first bevel gear 10 and the second bevel gear 12 mesh. The rotating rod 11 has a stirring rod 13 integrally formed along its lower circumference.

[0046] Specifically, when the output shaft of the stepper motor 7 rotates, it can drive the first bevel gear 10, which is fixedly engaged with it, to rotate. The first bevel gear 10 drives the second bevel gear 12, which meshes with it, to rotate. The second bevel gear 12 drives the rotating rod 11 to rotate, which in turn drives the stirring rod 13 to rotate, thereby stirring and mixing the anaerobic bacterial mud and Bacillus coagulans bacterial mud inside the outer shell 1.

[0047] This invention activates both T cells and B cells simultaneously using Lactobacillus rhamnosus and Bifidobacterium, which can enhance the neutralizing ability of B cells against pathogens while suppressing excessive inflammatory responses, thereby ensuring the effectiveness of immune regulation during systemic immune modulation.

[0048] Example 2:

[0049] As attached Figure 1 As shown, the difference from Example 1 is that a probiotic formula suitable for people in high-altitude areas includes the following components by weight: 4 parts of Lactobacillus plantarum, 3 parts of Bifidobacterium, 2 parts of Lactobacillus acidophilus, 1.5 parts of Lactobacillus rhamnosus and 1 part of Bacillus coagulans.

[0050] The preparation method is the same as in Example 1.

[0051] Example 3:

[0052] As attached Figure 1 As shown, the difference from Example 2 is that a probiotic formula suitable for people in high-altitude areas includes the following components by weight: 5 parts of Lactobacillus plantarum, 4 parts of Bifidobacterium, 3 parts of Lactobacillus acidophilus, 2.5 parts of Lactobacillus rhamnosus and 1.5 parts of Bacillus coagulans.

[0053] The preparation method is the same as in Example 1.

[0054] The probiotic formulations from Examples 1, 2, and 3 were used as experimental groups 1, 2, and 3, respectively, while Culturelle from the prior art was used as the control group. The results are shown in the table below: Table 1. Comparison of Experimental Data Experimental group 1 Experimental group 2 Experimental group 3 control group Strain survival rate (%) 57.31±0.16 56.12±0.11 55.85±0.12 50.64±0.25 SOD activity (U / mg) 28±2 25±2 22±1 20±2 As shown in Table 1, the survival rate and SOD activity of the strains in experimental groups 1, 2 and 3 were higher than those in the control group. It can be seen that, with the weight ratio of Examples 1, 2 and 3, the probiotics prepared by the method of Example 1 have higher survival and activity than existing products. Therefore, it can be concluded that the probiotics in Examples 1, 2 and 3 are all superior to existing products, and Example 1 is the best.

[0055] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A probiotic formula suitable for people living in high-altitude areas, characterized in that, It consists of the following components by weight: 3-5 parts of Lactobacillus plantarum, 2-4 parts of Bifidobacterium, 1.5-3 parts of Lactobacillus acidophilus, 1-2.5 parts of Lactobacillus rhamnosus, and 0.5-1.5 parts of Bacillus coagulans.

2. The probiotic formula for high-altitude populations according to claim 1, characterized in that, It consists of the following components by weight: 3 parts Lactobacillus plantarum, 2 parts Bifidobacterium, 1.5 parts Lactobacillus acidophilus, 1 part Lactobacillus rhamnosus, and 0.5 parts Bacillus coagulans.

3. The probiotic formula suitable for high-altitude populations according to claim 2, characterized in that, It consists of the following components by weight: 4 parts Lactobacillus plantarum, 3 parts Bifidobacterium, 2 parts Lactobacillus acidophilus, 1.5 parts Lactobacillus rhamnosus and 1 part Bacillus coagulans.

4. The probiotic formula suitable for high-altitude populations according to claim 3, characterized in that, It consists of the following components by weight: 5 parts of Lactobacillus plantarum, 4 parts of Bifidobacterium, 3 parts of Lactobacillus acidophilus, 2.5 parts of Lactobacillus rhamnosus, and 1.5 parts of Bacillus coagulans.

5. A method for preparing probiotics suitable for high-altitude populations, used to prepare the probiotic formula for high-altitude populations according to any one of claims 1-4, characterized in that, Includes the following steps: Step 1, strain activation and expansion: Lactobacillus plantarum, Bifidobacterium, Lactobacillus acidophilus, and Lactobacillus rhamnosus were inoculated into MRS broth medium for anaerobic culture, and activated 2-3 times using multi-stage expansion and activation technology; at the same time, Bacillus coagulans was inoculated into nutrient broth medium for aerobic culture, and activated twice using multi-stage expansion and activation technology. Step 2, Fermentation: Carbon and nitrogen sources are added to the MRS broth medium to obtain the fermentation medium, which is then sterilized. Activated anaerobic bacteria are inoculated into the fermentation medium at 3%-5% of their respective mass for anaerobic fermentation. The viable cell count is monitored during fermentation using the plate count method. Anaerobic fermentation is complete when the viable cell count reaches 10⁹ CFU / mL or higher. The fermentation broth is collected to obtain the anaerobic fermentation broth for each anaerobic bacteria. The carbon source is selected from one or more of glucose and sucrose, and the nitrogen source is selected from one or more of yeast powder and beef extract. The activated Bacillus coagulans was inoculated into the fermentation medium for aerobic fermentation. The inoculation amount was the sum of the inoculation amounts of each anaerobic strain during anaerobic fermentation. After more than 70% of the fermentation medium was converted into Bacillus coagulans spores, the aerobic fermentation was completed. The fermentation broth was collected to obtain the aerobic fermentation broth of Bacillus coagulans. Step 3, bacterial collection: The aerobic fermentation broth and anaerobic fermentation broth were distinguished by the type of bacteria and placed in a centrifuge. At a temperature of 4°C, the anaerobic fermentation broth and aerobic fermentation broth of different bacterial species were centrifuged at 8000 rpm for 15 minutes. After centrifugation, bacterial sludge of each anaerobic bacterial species and bacterial sludge of Bacillus coagulans were obtained. Step 4, Inoculum Processing and Packaging: The anaerobic bacterial sludge and Bacillus coagulans sludge are placed in a mixing device and mixed using the mixing components to obtain a mixed bacterial sludge. Simultaneously, three times the mass of a freeze-drying protectant is added to the mixed bacterial sludge through the feeding components in the mixing device and stirred to obtain a mixture. After mixing, the mixture is removed from the mixing device and pre-frozen at -40℃ for 4 hours. It is then transferred to a freeze dryer, where the temperature of the freeze dryer's baffles is gradually increased from -20℃ to 0℃, and the mixture is dried for 20 hours under a vacuum of <10 Pa to complete the initial drying. The temperature of the freeze dryer's baffles is then increased from 20℃ to 25℃, and the mixture is dried again for 4 hours under a vacuum of <10 Pa to complete the freeze-drying operation and obtain freeze-dried bacterial powder. Finally, the freeze-dried bacterial powder is packaged in nitrogen-filled aluminum foil bags to complete the preparation of the compound probiotic preparation.

6. The method for preparing probiotics suitable for high-altitude populations according to claim 5, characterized in that, In step one, during anaerobic culture, each anaerobic strain is cultured anaerobicly at 37°C for 18-24 hours; during aerobic culture, Bacillus coagulans is cultured aerobically at 37°C for 24 hours.

7. The method for preparing probiotics suitable for high-altitude populations according to claim 6, characterized in that, In step two, during anaerobic fermentation, the fermentation temperature of each anaerobic strain is controlled at 37℃ and the pH range is 5.5-6.0; during aerobic fermentation, the fermentation temperature of Bacillus coagulans is controlled at 37℃, and it is placed in a constant temperature shaking incubator and shaken at a frequency of 200 rpm.

8. The method for preparing probiotics suitable for high-altitude populations according to claim 7, characterized in that, In step four, when packaging the freeze-dried bacterial powder, the temperature should be controlled below 15℃ and the relative humidity below 20%.

9. The method for preparing probiotics suitable for high-altitude populations according to claim 8, characterized in that, The mixing device includes a controller and a housing (1). The top of the housing (1) has a feed inlet with a detachable rubber plug. The lower side wall of the housing (1) has a discharge outlet with a hinged door. The inner side wall of the housing (1) is fixedly connected to a mounting bracket (2) and a drive unit. The controller is used to control the opening and closing of the drive unit. A mounting cylinder (3) is fixedly connected to the side of the mounting bracket (2) away from the inner side wall of the housing (1). The top of the housing (1) has a first through hole and a storage box is fixedly connected thereto. (4) The storage box (4) is filled with freeze-drying protectant. The bottom of the storage box (4) is connected to a feeding pipe (5). One end of the feeding pipe (5) is connected to the inside of the storage box (4) through the first through hole. A second through hole is opened on one side of the fixed cylinder (3). The other end of the feeding pipe (5) is connected to the inside of the fixed cylinder (3) through the second through hole. The fixed cylinder (3) is equipped with a feeding component for conveying the freeze-drying protectant to the inside of the outer shell (1) in batches. A guide platform (6) is fixedly connected to the bottom of the outer shell (1). The output shaft of the drive unit extends through the fixed cylinder (3) and is fixedly connected to the first bevel gear (10) on one side of the fixed cylinder (3). The inner top wall of the outer shell (1) is rotatably fitted with a rotating rod (11). The bottom end of the rotating rod (11) is rotatably fitted with the inner bottom wall of the outer shell (1). A mixing component is provided on the rotating rod (11). The mixing component includes a second bevel gear (12) fixedly connected to the rotating rod (11). The first bevel gear (10) meshes with the second bevel gear (12). A stirring rod (13) is fixedly connected to the lower circumference of the rotating rod (11).

10. The method for preparing probiotics suitable for high-altitude populations according to claim 9, characterized in that, The feeding assembly includes a feeding wheel (8) fixedly connected to the output shaft of the drive component. The feeding wheel (8) is rotatably engaged with the inner wall of the fixed cylinder (3). The feeding wheel (8) has several feeding grooves at equal angles. Each feeding groove has a baffle (9) hinged to its inner wall. The length of each baffle (9) is the same as the opening length of the feeding groove. Each baffle (9) is rotatably engaged with the inner wall of the fixed cylinder (3). The bottom of the fixed cylinder (3) has a discharge port, which is located in the movement path of all the baffles (9).

Images