A high-purity multifunctional powder of probiotics and a preparation method thereof

Abstract

This invention discloses a high-purity, multifunctional probiotic powder and its preparation method. Based on the different characteristics of lactic acid bacteria, Bacillus subtilis, and actinomycetes, this invention employs different methods for their respective proliferation. This not only avoids potential mutual interference and contamination during their individual proliferation processes but also achieves high viable cell counts while maintaining high purity, and the high viable cell count is maintained even after mixing. This invention mixes the proliferated bacterial powders in a reasonable ratio, allowing lactic acid bacteria to be the dominant strain while fully utilizing the functional characteristics of each of the three bacteria: lactic acid bacteria are anaerobic, while Bacillus subtilis and actinomycetes are aerobic. The latter can absorb oxygen released by the former, ensuring that in the anaerobic environment of the human intestine, Bacillus subtilis and actinomycetes remain active rather than dormant, generating resistant endospores through synergistic action with lactic acid bacteria.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, and in particular to a high-purity, multifunctional probiotic powder and its preparation method. Background Technology

[0002] Traditionally, dried powder containing high-purity lactic acid bacteria (lactic acid bacteria dried product) is known to be used as a probiotic in health foods or livestock feed. For example, one method for manufacturing lactic acid bacteria dried product includes: mixing 1.5% pure culture of *Lactobacillus fermentum*, 4% skim milk powder, 0.5%–1.5% natural salt, 1% molasses, 0.5% monosodium glutamate, 2%–6% potato starch, 3%–9% defatted soybean flour, and 100% pure water; after a specific period of proliferation, drying in a spray dryer with an inlet temperature of 150°C–180°C and an outlet temperature of 75°C–87°C, ultimately yielding a viable count of 1.1 × 10⁻⁶. 9 ~1.2×10¹ 8 CFU / g, average particle size 1~9 μm powdered lactic acid bacteria dried product (see Japanese invention patent JP4067474).

[0003] The dried lactic acid bacteria in the aforementioned Japanese patent is a mixed bacteria (hybrid bacteria: a process in which three types of bacteria are cultured separately and then mixed) formed by mixing lactic acid bacteria with other types of bacteria. However, in the preparation of lactic acid bacteria, because multiple types of bacteria are mixed, there is a risk that the functions of each type of bacteria may be limited, thereby affecting its efficacy. Summary of the Invention

[0004] Therefore, based on the above background, the present invention provides a high-purity multifunctional probiotic powder and its preparation method. By avoiding contamination during the proliferation process of the three bacteria, high purity is maintained while achieving high viable count proliferation, and high purity and high viable count are still maintained after mixing.

[0005] The technical solution provided by this invention is as follows: A high-purity, multifunctional probiotic powder, by weight, is made from the following raw materials: Lactic acid bacteria dry powder 50%-70%, Bacillus subtilis dry powder 15%-25%, and actinomycete dry powder 15%-25%.

[0006] Preferably, it is made from the following raw materials by weight: The formula contains 60% lactic acid bacteria powder, 20% Bacillus subtilis powder, and 15%-25% actinomycete powder.

[0007] Preferably, the lactobacillus powder is selected from fermented lactobacillus; The Bacillus subtilis powder is made from Bacillus natto.

[0008] Based on the same inventive concept, the present invention also provides a method for preparing high-purity multifunctional probiotic powder, comprising the following steps: 1) Lactic acid bacteria, Bacillus subtilis and actinomycetes were separately cultured and pulverized to prepare dry powders of lactic acid bacteria, Bacillus subtilis and actinomycetes respectively; 2) Mix the lactic acid bacteria powder, Bacillus subtilis powder and actinomycete powder according to the mass ratio.

[0009] Furthermore, the preparation of the lactic acid bacteria powder includes: Lactobacillus strains were cultured in broth at 35-40℃ until the viable count reached 1.0 × 10⁻⁶. 2 CFU / g or higher; The proliferated bacterial solution was then dried and pulverized.

[0010] Preferably, the raw materials for preparing the culture medium include yeast, glucose, peptone, and purified water.

[0011] Preferably, the preparation of the Bacillus subtilis dry powder includes: Boiled, cooled, and drained soybeans were used as a culture medium to proliferate Bacillus subtilis until the viable bacterial count reached 1.0 × 10⁻⁶. 12 After reaching a CFU / g level, the product undergoes drying and pulverization.

[0012] Preferably, the preparation of the actinomycete powder includes: After sun-drying, the pineapple peels were crushed, soaked in purified water, and chitosan was added as a culture medium. Then, actinomycetes were cultured at 35-40℃ until the viable count reached 1.0 × 10¹. 0 CFU / g or higher; The bacterial cell solution was obtained by filtration, and a protectant and water were added to the solution and mixed. The mixed materials are then dried and pulverized.

[0013] Preferably, the protective agent is composed of trehalose and inulin, wherein the mass ratio of trehalose to inulin is (2-3):(1-2). The mass ratio of the bacterial solution and the added protective agent to water is (5-15):(30-45):(40-55).

[0014] The beneficial effects achieved by this invention are as follows: Based on the different characteristics of lactic acid bacteria, Bacillus subtilis and actinomycetes, this invention adopts different methods for their respective proliferation. This not only avoids the mutual influence and contamination that may occur during their respective proliferation processes, but also achieves high viable count proliferation while maintaining high purity, and the high viable count can still be maintained after mixing.

[0015] This invention mixes lactic acid bacteria powder, Bacillus subtilis powder, and actinomycete powder prepared after their respective proliferation in a reasonable ratio. This allows lactic acid bacteria to be the main strain while also maximizing the functional characteristics of each of the three strains. Since lactic acid bacteria are anaerobic, while Bacillus subtilis and actinomycetes are aerobic, the latter can absorb the oxygen released by the former. This allows the probiotic powder to maintain the activity of Bacillus subtilis and actinomycetes in the anaerobic environment of the human intestine rather than entering dormancy. Through synergistic action with lactic acid bacteria, they generate resistant endospores. Attached Figure Description

[0016] Appendix Figure 1 A schematic diagram of the process for preparing lactic acid bacteria powder in the embodiments is shown.

[0017] Appendix Figure 2 A schematic diagram of the process for preparing Bacillus subtilis powder in the embodiments is shown.

[0018] Appendix Figure 3 A schematic diagram of the process for preparing actinomycete powder in the embodiments is shown.

[0019] Appendix Figure 4 A schematic diagram illustrating the overall preparation process of the high-purity multifunctional probiotic powder in the embodiments is shown.

[0020] Appendix Figure 5 An exemplary structural schematic diagram of the stirring device used in the embodiments is shown.

[0021] Appendix Figure 6 An exemplary structural schematic diagram of another stirring device used in the embodiments is shown. Attached Figure

[0022] 1. Mixer; 2. Feeding section; 3. Hopper; 4. Mixing section; 4a. Inner cylinder; 41. Spiral blade; 42. Drive wheel; 43. Support section; 5. Second hopper; 6. Conveying section; 7. Second conveying section; 8. Belt; 9. Motor; 10. Auxiliary wheel. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The technical solution of the present invention: A method for preparing a high-purity multifunctional probiotic powder, which produces a mixture of three bacteria: lactic acid bacteria, Bacillus subtilis, and actinomycetes, and maintains high purity and high viable count of each type of bacteria during the preparation process.

[0025] Specifically, three types of bacteria—lactic acid bacteria powder, Bacillus subtilis powder, and actinomycete powder—prepared separately through propagation and pulverization, are mixed in a specific ratio. This avoids contamination during the individual propagation process of each bacteria, achieves high viable counts while maintaining high purity, and retains these high viable counts even after mixing.

[0026] Specifically, by weight, the formula consists of 60% lactic acid bacteria, 20% Bacillus subtilis, and 20% actinomycetes. This formulation ensures that lactic acid bacteria are the dominant bacteria while also allowing each of the three bacteria to fully utilize its unique functional characteristics.

[0027] Specifically, the preparation method of lactic acid bacteria dry powder includes: culturing Lactobacillus fermentum at a constant temperature, adding a culture medium composed of yeast, glucose, peptone, and purified water to the strain, and pulverizing the bacteria when the viable count reaches 1.0 × 10² CFU / g or higher to obtain the lactic acid bacteria dry powder. This method ensures that a sufficient quantity of lactic acid bacteria dry powder is obtained for probiotic powder production.

[0028] In practice, Lactobacillus fermentation can be cultured at a constant temperature of 36°C for 24 hours. An example of culture medium preparation is as follows: Mix 5g of yeast, 5g of glucose, and 5g of peptone in 1000ml of purified water to prepare the additive solution.

[0029] The preparation method of Bacillus subtilis dry powder includes: using boiled, cooled, and drained soybeans as a culture medium to proliferate Bacillus subtilis until the viable cell count reaches 1.0 × 10⁻⁶. 12 When the CFU / g is above a certain level, the particles are pulverized to a particle size of 0.1~0.2 mm under the condition of a moisture content ≤8% to obtain dried Bacillus subtilis. This method can effectively ensure the mass production needs of dried Bacillus subtilis.

[0030] The preparation method of actinomycete powder includes: pulverizing sun-dried pineapple peel, soaking it in purified water, adding chitosan, and incubating at a warm temperature until the viable count is ≥1.0×10¹. 0 After filtering the bacterial solution (CFU / g), a protective agent was added at a mass ratio of 10 (solution):25 (trehalose):15 (inulin):50 (water). The product was then dried at low temperature for 72 hours to obtain the final product. Pineapple peel can be pre-treated by sun-drying for up to 3 years. Specific parameters are as follows: 200g of crushed pineapple peel is soaked in 1000ml of purified water, 20g of chitosan is added, and the mixture is kept at 35-40℃ for 72 hours. Subsequent protective agent addition and drying processes are performed according to the aforementioned ratio. This method can efficiently obtain dried actinomycetes.

[0031] In this invention, a high-purity, multifunctional probiotic powder is formulated using the following ratio: 60% powdered lactic acid bacteria, 20% Bacillus subtilis, and 20% actinomycetes by mass. This ratio ensures that all types of bacteria maintain a high viable count and high purity. Since lactic acid bacteria are anaerobic, while Bacillus subtilis and actinomycetes are aerobic, the latter can absorb the oxygen released by the former. This allows the probiotic powder to maintain its activity rather than enter dormancy in anaerobic environments such as the human gut, where Bacillus subtilis and actinomycetes can synergistically generate resistant endospores.

[0032] Example 1: Combination Figure 1 The flowchart shown illustrates the preparation process of lactobacillus powder, providing a detailed explanation of the preparation process.

[0033] The specific preparation includes the following steps: S101: Culture of Lactobacillus fermentum strains (accession numbers NBRC3071 and NBRC3072).

[0034] To detect lactic acid bacteria and determine their count, melted agar was poured into petri dishes (round glass containers) to form BCP plate counting agar medium. The *Lactobacillus fermentum* strain was then spread into a 2 mm thick layer using a platinum ring. The inoculated petri dishes were then placed in a constant temperature incubator and incubated at 36°C for 24 hours.

[0035] S102: Preparation of culture medium The culture medium was prepared as follows: 1000 ml of purified water, 5 g of yeast, 5 g of glucose, and 5 g of peptone were added to a rigid heat-resistant container. The mixture was then placed in an autoclave and sterilized at 120°C for 20 minutes, followed by cooling to below 40°C.

[0036] S103: Inoculate the colonies formed in the petri dish in S101 into the culture medium in step S102.

[0037] S104: Proliferation and Culture of Lactic Acid Bacteria The specific procedure is as follows: Place the culture medium inoculated with the Lactobacillus strain in step S103 into an incubator and carry out proliferation culture at 35-40℃ for 48 hours.

[0038] The number of lactic acid bacteria was increased to 1.0 × 10⁻⁶. 12 CFU / g or higher. Indicators for confirming proliferation include: the culture solution is turbid, and a vortex can be observed when the container is tilted; if pH is measured, the value should be between pH 3.2 and 3.5.

[0039] S105: The solution after S104 proliferation culture is dried and then the lactic acid bacteria are powdered.

[0040] Example 2: Combination Figure 2 The flowchart shown illustrates the preparation process of Bacillus subtilis powder, providing a detailed explanation of the preparation process.

[0041] This embodiment illustrates a specific example of manufacturing Bacillus subtilis powder using soybeans as a culture medium.

[0042] It is worth noting that Bacillus subtilis (Natto Bacillus), a spore-bearing bacterium of soil bacteria, proliferates by attaching to dead leaves in the form of spores. Therefore, achieving pure culture technology for this spore-bearing bacterium is of crucial significance.

[0043] The specific preparation includes the following steps: S106: Culture Medium Preparation Soybeans are soaked, boiled, cooled, and drained. These soybeans will then be used as a culture medium for Bacillus subtilis.

[0044] S107: Inoculate soybeans with Bacillus subtilis. In this example, Bacillus subtilis was selected as Bacillus natto.

[0045] S108: Proliferation culture of Bacillus subtilis Specifically, soybeans inoculated with Bacillus subtilis in S107 were placed in an incubator and cultured at a constant temperature of 37°C for 24 hours. During this time, the soybean surface was covered with spore-like or cotton-like Bacillus subtilis spores, emitting an ammonia odor. The Bacillus subtilis count was then increased to 1.0 × 10⁻⁶. 12 CFU / g or higher.

[0046] S109: After the Bacillus subtilis cultured in S108 is dried at low temperature to a moisture content of less than 8%, it is pulverized to achieve a particle size of 0.1mm~0.2mm. By controlling the moisture content to below 8% during pulverization, the ammonia odor generated in the S108 process can be eliminated.

[0047] Example 3: Combination Figure 2 The flowchart shown illustrates the preparation process of actinomycete powder, providing a detailed explanation of the preparation process.

[0048] This embodiment illustrates a specific example of manufacturing actinomycete powder using pineapple peel as raw material.

[0049] S110: Preparation of Culture Medium Raw Materials Pineapple peels that have undergone three years of sun-drying were used as the culture medium raw material. S111: Preparation of Culture Medium The sun-dried pineapple peel of S110 was crushed using a mixer. 200g of the crushed pineapple peel dust was placed in a 2L container and soaked in 1000ml of purified water. 20g of chitosan was added as a nutrient for actinomycetes to prepare a culture medium. S112: After inoculating the actinomycetes into the culture medium prepared by S111, place them in an incubator and incubate at a constant temperature of 35~40℃ for 72 hours; S113: By stirring the solution (because the aerobic bacteria need oxygen supplementation for cultivation due to the operation of the stirrer), filamentous and sticky actinomycetes are generated. The actinomycetes are then filtered through a cloth to obtain a bacterial solution. At this point, the number of actinomycetes should reach 1.0 × 10⁻⁶. 10 CFU / g or higher, preferably 1.0×10⁻⁶. 11 CFU / g, more preferably, proliferation to 1.0 × 10⁻⁶. 12 CFU / g or higher.

[0050] S114: Mix inulin and trehalose into the bacterial solution according to a mass ratio of 10:25:15:50 for the bacterial solution, trehalose, inulin and water.

[0051] S115: The mixed materials are dehumidified and dried at low temperature; S116: Powder the dried mixture.

[0052] Finally, S117: The lactic acid bacteria powder prepared in Example 1, the Bacillus subtilis powder prepared in Example 2, and the actinomycete powder prepared in Example 3 are mixed using a mixer (see...). Figure 5 , Figure 6 The mixture is stirred and blended to produce a high-purity, multifunctional probiotic powder.

[0053] Lactic acid bacteria powder, Bacillus subtilis powder, and actinomycete powder are mixed in a weight ratio of 60%, 20%, and 20%, respectively. In other words, the main strain in this high-purity, multifunctional probiotic powder is lactic acid bacteria.

[0054] Lactic acid bacteria, Bacillus subtilis, and actinomycetes are all positive-positive bacteria (beneficial bacteria), and can produce good results even when taken alone. For example, taking lactic acid bacteria alone can improve intestinal health. When taking a high-purity, multifunctional probiotic powder made from a mixture of lactic acid bacteria, Bacillus subtilis, and actinomycetes, as mentioned earlier, the synergistic effect with the lactic acid bacteria can generate endospores and form resistant spores. This resistance can achieve effects such as inhibiting cancer cells.

[0055] Furthermore, if the lactic acid bacteria, Bacillus subtilis, and actinomycetes are mixed before being dried and pulverized separately, there is a risk of hydrolysis of each type of bacteria. In this embodiment, by drying each type of bacteria separately to a moisture content of less than 8% before pulverizing, such hydrolysis can be effectively suppressed.

[0056] Figure 5 and Figure 6 This is an example of a mixer 1 used in this invention to manufacture high-purity, multifunctional probiotic powder. Schematic diagram. The following is combined with... Figure 5 and Figure 6 This describes the structure of a mixer 1 used for mixing lactic acid bacteria powder, Bacillus subtilis powder, and actinomycete powder.

[0057] exist Figure 5 In section A, lactic acid bacteria powder, Bacillus subtilis powder, and actinomycete powder (hereinafter referred to as...) are first fed through the feed section 2. The "three-bacterial mixed powder" is fed in. A conveyor belt is installed inside the feeding section 2, which transfers the three-bacterial mixed powder to the hopper 3. The hopper 3 is connected to the mixing section 4. The mixing section 4 includes an inner cylinder 4a with internal spiral blades 41, i.e. Figure 5 As shown in Figure A, the mixing section 4 adopts a double-layer cylindrical structure.

[0058] Figure 5 B is Figure 5 A cross-sectional schematic diagram of the mixing section 4 along section XX in section A (the helical blade 41 is not shown). The mixing section 4 has a double circular cross-section structure. Furthermore, the inner cylinder 4a of the mixing section 4 is connected to a drive wheel 42, while the outer cylinder of the mixing section 4 is connected to two larger diameter support sections 43. Through the rotation of the two drive wheels 42, the inner cylinder 4a and the helical blade 41 inside the mixing section 4 rotate synchronously. The support sections 43 are used to fix the mixing section 4, while the outer cylinder and the support sections 43 themselves remain stationary. In addition, after the helical blade 41 rotates continuously at a rate of 10 revolutions per minute along the powder propulsion direction for 30 minutes, it will rotate in the opposite direction once. This single reverse rotation effectively removes powder adhering to the helical blade 41 (e.g., agglomerated portions).

[0059] Back Figure 5 Explanation of A. The tri-bacterial mixed powder, pushed out by the rotation of the spiral blade 41, will be transferred to the second hopper 5. During this process, if the preset number of cycles is not exceeded, the tri-bacterial mixed powder will be fed back into the feed section 2 after entering the second hopper 5, and the above process will be repeated. The path structure for the return from the hopper 5 to the feed section 2 will be explained later. Figure 6 It will be displayed in China.

[0060] Figure 6 A is a plan view of the mixer 1 showing the flow of the three-bacterial mixture powder after it enters the conveyor section 6 from the second hopper 5. The conveyor section 6 also has a conveyor belt inside, which transfers the three-bacterial mixture powder to the second conveyor section 7. After passing through the second conveyor section 7, the powder returns... Figure 5 The feed section 2 shown in Figure A. It should be noted that... Figure 6 In diagram A, the conveying section 6 and the second conveying section 7 are drawn as a continuous straight line structure, but they can also be designed in a staggered manner, that is, allowing a drop in the powder when it is transferred from the conveying section 6 to the second conveying section 7. Figure 6 B is Figure 6 A. Side view along the direction of the arrow. Since the feed section 2 is positioned higher than the conveying section 6 and the second conveying section 7, they can be designed to be inclined towards the mixer 1 as a whole and raised towards the feed section 2. Figure 6 C is Figure 6 A. Cross-sectional view of the second conveying section 7 along the YY section. The three-bacterial mixed powder is conveyed by the belt 8 of the second conveying section 7 (the conveying section 6 is also equipped with this belt), and the belt 8 is driven by the motor 9 and the auxiliary wheel 10.

[0061] As described above, the operation of mixer 1 can achieve efficient stirring and mixing of the three-strain mixed powder, and the high purity of each of the three strains can still be maintained after mixing.

[0062] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A high-purity, multifunctional probiotic powder, characterized in that, By weight, it is made from the following raw materials: Lactic acid bacteria dry powder 50%-70%, Bacillus subtilis dry powder 15%-25%, and actinomycete dry powder 15%-25%.

2. The high-purity, multifunctional probiotic powder according to claim 1, characterized in that, By weight, it is made from the following raw materials: The formula contains 60% lactic acid bacteria powder, 20% Bacillus subtilis powder, and 15%-25% actinomycete powder.

3. The high-purity multifunctional probiotic powder according to claim 1 or 2, characterized in that, The Lactobacillus powder is made from fermented Lactobacillus. The Bacillus subtilis powder is made from Bacillus natto.

4. A method for preparing a high-purity, multifunctional probiotic powder according to any one of claims 1 to 3, characterized in that, Includes the following steps: 1) Lactic acid bacteria, Bacillus subtilis and actinomycetes were separately cultured and pulverized to prepare dry powders of lactic acid bacteria, Bacillus subtilis and actinomycetes respectively; 2) Mix the lactic acid bacteria powder, Bacillus subtilis powder and actinomycete powder according to the mass ratio.

5. The method for preparing a high-purity multifunctional probiotic powder according to claim 4, characterized in that, The preparation of the lactic acid bacteria dry powder includes: The lactobacillus species is proliferated in the culture solution at 35-40°C until the viable cell count reaches 1.0 x 10 2 CFU / g or more; The proliferated bacterial solution was then dried and pulverized.

6. The method for preparing a high-purity multifunctional probiotic powder according to claim 5, characterized in that, The raw materials for preparing the culture medium include yeast, glucose, peptone, and purified water.

7. The method for preparing a high-purity multifunctional probiotic powder according to claim 4, characterized in that, The preparation of the Bacillus subtilis dry powder includes: The boiled and cooled soybean, after draining water, is used as a culture medium to proliferate Bacillus subtilis, and when the viable cell count reaches 1.0 x 10 12 CFU / g or more, drying and powdering processes are performed.

8. The method for preparing a high-purity multifunctional probiotic powder according to claim 4, characterized in that, The preparation of the actinomycete dry powder includes: After sun-drying, the pineapple peels were crushed, soaked in purified water, and chitosan was added as a culture medium. Then, actinomycetes were cultured at 35-40℃ until the viable count reached 1.0 × 10⁻⁶. 10 CFU / g or higher; The bacterial cell solution was obtained by filtration, and a protectant and water were added to the solution and mixed. The mixed materials are then dried and pulverized.

9. The method for preparing a high-purity multifunctional probiotic powder according to claim 8, characterized in that, The protective agent is composed of trehalose and inulin, wherein the mass ratio of trehalose to inulin is (2-3):(1-2). The mass ratio of the bacterial solution and the added protective agent to water is (5-15):(30-45):(40-55).

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