Penicillin bottle packaged thallus preparation and preparation method thereof

Abstract

The invention discloses a thallus preparation packaged by a penicillin bottle and a preparation method of the thallus preparation, and belongs to the technical field of probiotics. The thallus preparation packaged by the penicillin bottle comprises the penicillin bottle serving as a packaging container and content located in the penicillin bottle, the content comprises solid particles and powder, at least one of the solid particles and the powder contains an edible thallus preparation, and the thallus preparation is selected from one or more of freeze-dried bacteria, inactivated bacteria and thallus metabolite. The consumer-oriented thallus preparation product disclosed by the invention can effectively play a physiological role. In terms of selection of packaging materials, penicillin bottles made of glass are adopted, and plastic materials are not involved. By means of the design, the risk that an eater intakes the micro-plastic particles is effectively avoided, and health and safety of consumers are guaranteed when the consumers use the probiotic products.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology, and in particular relates to a vial-packaged bacterial preparation and its preparation method. Background Technology

[0002] Probiotics are live microorganisms that, when ingested in sufficient quantities, produce beneficial effects on human health. A vast and complex microbial community exists in the human gut. Probiotics compete with harmful bacteria, inhibiting the colonization of harmful bacteria by occupying adhesion sites on the intestinal mucosa, thereby maintaining the balance of the gut microbiota. For example, probiotics such as Bifidobacterium and Lactobacillus acidophilus can produce organic acids such as lactic acid and acetic acid, lowering the intestinal pH and creating an environment unfavorable to the growth of harmful bacteria. Furthermore, probiotics play a crucial role in the development and regulation of the immune system. They can stimulate the development and maturation of the intestinal immune system, activating immune cells such as macrophages and T lymphocytes, and enhancing their phagocytic capacity and activity. For example, lactic acid bacteria can produce immunomodulatory factors, such as cytokines and chemokines, which can promote interactions between immune cells and regulate immune responses. Studies have shown that probiotic supplementation helps improve the body's resistance to pathogens and reduce the occurrence of infectious diseases, such as respiratory and intestinal infections.

[0003] As people pay increasing attention to health, the market demand for probiotic products is also growing steadily. Consumers' awareness and acceptance of probiotic products are gradually increasing, and more and more people are choosing probiotic products as a daily health maintenance option. There are many types of probiotic products, including probiotic supplements, foods and beverages containing probiotics, etc. These products provide consumers with a convenient way to ingest probiotics by adding specific strains of probiotics, thereby obtaining their various health benefits. In the future, with the continuous advancement of scientific research and further improvement in consumers' health awareness, probiotic products are expected to play an even greater role in the health field.

[0004] Most commercially available powdered probiotics currently use plastic packaging materials. However, plastic packaging poses certain health risks. During the plastic production process, microplastic particles may be released. These tiny particles can enter the human body through the food chain, and long-term accumulation may have adverse effects on human health. Studies have shown that microplastics may cause intestinal flora imbalance, cytotoxicity, reproductive toxicity, neurotoxicity, and may even induce chronic diseases such as intestinal disorders, cardiovascular disease, obesity, and diabetes. Therefore, finding safer and more environmentally friendly packaging materials to reduce microplastic release and improve the stability of probiotics has become an important research direction in the field of probiotic packaging. Summary of the Invention

[0005] To address the aforementioned problems, this invention proposes a vial-packaged microbial preparation and its preparation method. The consumer-oriented microbial preparation product of this invention includes one or more of freeze-dried bacteria, inactivated bacteria, and microbial metabolites, which can effectively exert physiological effects. Its packaging material is a glass vial, which does not involve plastic materials, effectively preventing consumers from ingesting microplastic particles.

[0006] A first aspect of the present invention discloses a vial-packaged microbial preparation comprising a vial as a packaging container and contents located within the vial, the contents comprising solid particles and powder, at least one of the solid particles and powder containing an edible microbial preparation, the microbial preparation being selected from one or more of freeze-dried bacteria, inactivated bacteria, and microbial metabolites.

[0007] In some embodiments of the present invention, the bacterial preparation is freeze-dried bacteria to maintain the active state of the microorganisms.

[0008] In some embodiments of the present invention, the bacterial preparation is an inactivated bacterium, which is obtained by heat inactivation, ultraviolet inactivation or irradiation inactivation treatment.

[0009] In some embodiments of the present invention, the bacterial preparation is a bacterial metabolite, which includes one or more of short-chain fatty acids, bacteriocins, extracellular polysaccharides, organic acids, and bioactive peptides.

[0010] In some embodiments of the present invention, the prohibited ingredients in the probiotic food are one or more of L-cysteine ​​hydrochloride, mannitol, dimethyl sulfoxide, manganese sulfate, and ribitol. While these ingredients can protect the bacterial cells and reduce the inactivation rate of the strains during freeze-drying, due to the strict restrictions on freeze-drying protectant systems stipulated in GB 2760-2025 (National Food Safety Standard for the Use of Food Additives), they cannot be added to food or the amount added is limited, thus failing to achieve the freeze-drying protection effect.

[0011] In some embodiments of the present invention, the vial is filled with nitrogen, the purpose of which is to improve the stability of the contents.

[0012] In some embodiments of the present invention, the solid particles are regular or irregular spheres or cylinders.

[0013] In some embodiments of the present invention, the lyophilized bacteria packaged in vials do not contain allergenic ingredients, such as lactose.

[0014] In some embodiments of the present invention, the bacterial preparation comprises one or more units of an effective dose that exerts physiological effects in the human body, which can be used once or multiple times to ensure sufficient bacterial quantity each time it is used. Preferably, the effective dose of one unit of the freeze-dried bacteria to exert physiological effects in the human body is 10. 7 -10 11 TFU.

[0015] In some embodiments of the present invention, the bacterial preparation is derived from edible bacteria and / or fungi, including but not limited to one or more of Lactobacillus, Bifidobacterium, Lactococcus, Leuconostoc mesenteroides, Pediococcus, Propionibacterium, Bacillus, Akermania, Zoococcus calf, Staphylococcus xylose, Staphylococcus carinatum, and yeast.

[0016] In some embodiments of the present invention, the contents further include an adjuvant, which includes a lyophilization protectant. The lyophilization protectant includes one or more of skim milk powder, trehalose, lactose, maltodextrin, porous dextrin, and vitamin C, but does not include L-cysteine ​​hydrochloride.

[0017] In some embodiments of the present invention, the raw materials for preparing the freeze-drying protectant include the following components: 5-50 parts by weight of skim milk powder, 1-10 parts by weight of trehalose, 1-10 parts by weight of maltodextrin, and 0.1-2 parts by weight of vitamin C.

[0018] In some embodiments of the present invention, the raw materials for preparing the freeze-drying protectant include the following components: 10 parts by weight of skim milk powder, 5 parts by weight of trehalose, 5 parts by weight of maltodextrin, and 0.5 parts by weight of vitamin C.

[0019] In some embodiments of the present invention, the adjuvant further includes a flavor improver, which includes one or more of sweeteners, acidulants, colorants, and flavorings.

[0020] In some embodiments of the present invention, the contents further include one or more of prebiotics and plant extracts.

[0021] In some embodiments of the present invention, the contents also include fruit and vegetable powders.

[0022] The second aspect of this invention discloses a method for preparing the lyophilized bacteria packaged in vials as described in the first aspect, comprising the following steps: S01, vial preparation: including cleaning, sterilization, and drying of the vials; S02, Preparation of bacterial preparations; S03. Under aseptic conditions, the vials obtained in step S01 and the bacterial powder obtained in step S02 are packaged and then vacuum-sealed or filled with nitrogen.

[0023] In some embodiments of the present invention, in S02, when the bacterial preparation is lyophilized bacteria, the bacterial cells are mixed with a sterile lyophilization protectant and then lyophilized to obtain lyophilized bacterial powder. When the bacterial preparation is an inactivated bacterium, the bacterial cells are dried after inactivation treatment to obtain inactivated bacterial powder; When the bacterial preparation is a bacterial metabolite, the bacterial metabolite is collected and dried to obtain a bacterial metabolite powder.

[0024] In some embodiments of the present invention, in S02, when the bacterial preparation is lyophilized bacteria, the bacterial strain is activated by passage on a solid culture medium, then inoculated into a sterilized liquid culture medium and cultured in a constant temperature incubator at the optimal growth temperature, thus serving as the seed culture; the above seed culture is inoculated into a sterile fermentation medium, the corresponding process parameters are adjusted, and fermentation is carried out until the late logarithmic growth phase ends; the bacterial cells are collected by centrifugation, weighed, and then a sterile lyophilization protectant is added in proportion, quickly mixed evenly, and the prepared mixture is pre-frozen until it is completely frozen, and then the frozen sample is quickly transferred to a vacuum freeze dryer for freeze-drying to obtain lyophilized bacterial cells, which are then pulverized and sieved, and if necessary, flavor improvers are added to obtain the bacterial preparation.

[0025] In some embodiments of the present invention, in S02, when the bacterial product is an inactivated bacterial product, the bacterial cells are directly dried or freeze-dried after being treated by heat inactivation, ultraviolet inactivation or irradiation inactivation to obtain inactivated bacterial powder. If necessary, a flavor improver is added to obtain the bacterial preparation.

[0026] In some embodiments of the present invention, in S02, when the bacterial product is a bacterial cell metabolite, the bacterial cell fermentation supernatant or fermentation product is collected, concentrated, dried or freeze-dried to obtain a metabolite powder, and if necessary, a flavor improver is added to obtain the bacterial cell preparation.

[0027] A third aspect of the present invention discloses a vial-packaged food, comprising a vial as a packaging container and the contents of the vial-packaged bacterial preparation described in the first aspect located within the vial.

[0028] The beneficial effects of this invention are: This invention provides a consumer-oriented probiotic preparation product, comprising freeze-dried bacteria, inactivated bacteria, and bacterial metabolites. It can be administered in a single dose (one unit) or in doses that can be divided into multiple administrations (multiple units), ensuring that each dose effectively exerts its physiological effect. Regarding packaging materials, this invention uses glass vials, avoiding the use of plastic materials. This design effectively avoids the risk of consumers ingesting microplastic particles, ensuring their health and safety when using probiotic products. Glass vials offer excellent sealing and stability, effectively protecting the activity of probiotics and extending the product's shelf life. Attached Figure Description

[0029] Figure 1 The effect of different lyophilization protectants on the stability of BO1; Figure 2 The effect of different lyophilization protectants on the stability of BO2; Figure 3 The effect of different lyophilization protectants on the stability of BO3; Figure 4 The effect of different lyophilization protectants on the stability of L01; Figure 5 The effect of different lyophilization protectants on the stability of L02; Figure 6 The effect of different lyophilization protectants on the stability of L03; Figure 7 The effect of different lyophilization protectants on the stability of K01; Figure 8 The effect of different lyophilization protectants on the stability of K02. Detailed Implementation

[0030] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0031] Unless otherwise specified, the examples and comparative examples are parallel experiments with the same components, component content, preparation steps, and preparation parameters.

[0032] Example 1: A vial-packaged freeze-dried bacteria and its preparation method, comprising the following steps: (1) Preparation of vials: including cleaning, sterilization and drying of the vials; (2) Preparation of mycelium powder; The probiotic is Bifidobacterium animalis. After activation by passage on a solid culture medium, the bacterial strain is inoculated into sterilized liquid culture medium and cultured in a constant temperature incubator at the optimal growth temperature (37℃) for 18 hours, serving as the seed culture. This seed culture is then inoculated at a rate of 5% (V / V) into a tank with a liquid volume (sterile fermentation medium) of 70%. The corresponding process parameters are adjusted: stirring speed is 30 r / min; 12.5% ​​ammonia is automatically added to maintain a constant pH of 5.6; nitrogen is intermittently purged to maintain a certain anaerobic environment; fermentation is carried out at 37℃ for approximately 12-14 hours until the late logarithmic growth phase. The bacterial cells are collected by centrifugation, washed, and then collected again by centrifugation. The bacterial sludge is weighed and mixed rapidly with a sterile freeze-drying protectant at a 1:1 ratio. The prepared mixture is then transferred to a vacuum freeze dryer, pre-frozen at -40℃ for 4 hours, and then freeze-dried for 48 hours to obtain freeze-dried bacterial cells. After pulverization and sieving, the bacterial powder of the desired mesh size is obtained.

[0033] The specific components of the freeze-drying protectant are: 10g of skim milk powder, 5g of trehalose, 5g of maltodextrin, and 1g of vitamin.

[0034] (3) Package the vials obtained in step (1) and the bacterial powder obtained in step (2) under sterile conditions.

[0035] The number of probiotics added is 10. 6 -10 11 AFU.

[0036] The above preparation process is applicable to anaerobic or facultative anaerobic bacteria, including Bifidobacterium, Lactobacillus, Lactobacillus, Streptococcus salivarius, Lactococcus lactis, Leuconostoc mesenteroides, Propionibacterium, Bacillus, Akkermansia, etc., only the composition of the culture medium used in the fermentation process is different.

[0037] When preparing aerobic bacteria, sterile air simply needs to be introduced during the fermentation process, such as with yeast.

[0038] Example 2: Effects of different lyophilization protectants on the stability of Bifidobacterium.

[0039] Table 1. Lyophilizing agent formulation Three strains of Bifidobacterium were selected: Bifidobacterium animalis subsp. lactis B01, Bifidobacterium longum B02, and Bifidobacterium adolescentis B03. The bacterial sludge was obtained by fermentation in Example 1 and mixed with the freeze-drying agent formulations in Table 1 (the “control” is the commonly used freeze-drying formulation for bacterial strains). The mixture was then freeze-dried and its stability was tested under ambient temperature and accelerated conditions at 37°C.

[0040] like Figures 1-3As shown, the freeze-dried bacteria using freeze-drying protectants No. 3, 4, and 5 had a higher viable count after being accelerated at 37°C for 6 months. Furthermore, the components of freeze-drying protectants No. 3, 4, and 5 do not contain ingredients that cannot be added to probiotic foods (L-cysteine ​​hydrochloride) or allergenic ingredients (lactose). Considering both effectiveness and cost, freeze-drying protectant No. 3 is preferred, which consists of 10g of skim milk powder, 5g of trehalose, 5g of maltodextrin, and 1g of vitamin.

[0041] Example 3: Effects of different lyophilization protectants on the stability of Lactobacillus and Bacillus.

[0042] Table 2. Lyophilizing agent formulation

[0043] Three strains of bacteria were selected: Lactobacillus fermentum L01, Lactobacillus reuteri L02, and Clostridium butyricum L03. The bacterial sludge was obtained by fermentation using the method described in Example 1 and mixed with the freeze dryer formulas in Table 1 (the “control” is the commonly used freeze dryer formula for bacterial strains). The mixture was then freeze-dried and its stability was tested under ambient temperature and accelerated conditions at 37°C.

[0044] like Figure 4-6 As shown, the freeze-dried bacteria using freeze-drying protectants No. 3, 4, and 5 had a higher viable count after being accelerated at 37°C for 6 months. Furthermore, the components of freeze-drying protectants No. 3, 4, and 5 do not contain ingredients that cannot be added to probiotic foods (L-cysteine ​​hydrochloride) or allergenic ingredients (lactose). Considering both effectiveness and cost, freeze-drying protectant No. 3 is preferred, which consists of 10g of skim milk powder, 5g of trehalose, 5g of maltodextrin, and 1g of vitamin.

[0045] Example 4: Effect of different lyophilization protectants on the stability of AKK bacteria.

[0046] Table 3. Lyophilizing agent formulation Two strains of Akk bacteria, K01 and K02, were selected and fermented using the method described in Example 1 to obtain bacterial sludge. This sludge was then mixed with the freeze-drying formulations listed in Table 1 (the "control" is a commonly used freeze-drying formulation for bacterial strains), freeze-dried, and its stability was tested under both ambient temperature and accelerated conditions at 37°C. Akk bacteria, also known as Akkermansia muciniphila, are myxotrophic Akkermansia muciniphila.

[0047] like Figure 7 , Figure 8As shown, for K01, the lyophilized bacteria using freeze-drying protectants No. 3, 4, and 5 had a higher viable count after 6 months of accelerated drying at 37°C. For K02, there was no significant difference in the effect of different formulations on the stability of the strains. The components of freeze-drying protectants No. 3, 4, and 5 do not contain ingredients that cannot be added to probiotic foods (L-cysteine ​​hydrochloride) or allergenic ingredients (lactose). Considering both effectiveness and cost, freeze-drying protectant No. 3 is preferred, which consists of 10g of skim milk powder, 5g of trehalose, 5g of maltodextrin, and 1g of vitamin.

[0048] Comparative Example 1: The effect of vial packaging and other probiotic packaging on strain activity.

[0049] The prepared bacterial powder was packaged in five different ways: vials filled with bacterial powder and then directly sealed (air), vacuum sealed, and nitrogen-filled sealed; vials filled with bacterial powder and then directly sealed (air) and nitrogen-filled sealed. The bacterial powder was stored at room temperature and away from light, and the viable count (AFU / g) was tested at 0 months, 3 months, and 6 months.

[0050] Table 4. Effects of different packaging methods on strain activity * Vacuum refers to evacuating to an internal pressure of 1 Pa; nitrogen purging refers to first evacuating to a vacuum and then purging with nitrogen until the internal pressure reaches 1 × 10⁻⁶ Pa. 5 Pa.

[0051] As shown in the table above, both vial-packaged and commercially available strip-packaged probiotic powders exhibited higher live bacteria counts at 30 and 60 days in accelerated testing, with nitrogen-filled or vacuum-packed packaging demonstrating a positive effect on extending shelf life. When filled with nitrogen or air, there was no significant difference between vial-packaged and strip-packaged products. Therefore, vial-packaged probiotic powder ensures higher probiotic viability throughout its shelf life, with vacuum or nitrogen-filled packaging showing even better results.

[0052] The preferred embodiments and examples of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments and examples. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the concept of the present invention.

Claims

1. A bacterial preparation packaged in a vial, characterized in that, The invention includes a vial serving as a packaging container and contents located within the vial, the contents comprising solid particles and powders, at least one of which contains an edible microbial preparation, the microbial preparation being selected from one or more of freeze-dried bacteria, inactivated bacteria, and microbial metabolites.

2. The vial-packaged bacterial preparation according to claim 1, characterized in that, The vials are filled with nitrogen.

3. The vial-packaged bacterial preparation according to claim 1, characterized in that, Solid particles are regular or irregular spheres or cylinders.

4. The vial-packaged bacterial preparation according to claim 1, characterized in that, The bacterial preparation comprises one or more units of an effective dose that exerts a physiological effect in the human body; Preferably, the effective dose of one unit of the freeze-dried bacteria to exert physiological effects in the human body is 10. 7 -10 11 TFU.

5. The lyophilized bacteria packaged in vials according to claim 1, characterized in that, The bacterial preparation is derived from edible bacteria and / or fungi.

6. The vial-packaged bacterial preparation according to claim 1, characterized in that, The contents also include adjuvants, including lyophilization protectants, which include one or more of skim milk powder, trehalose, lactose, maltodextrin, porous dextrin, and vitamin C, but do not include L-cysteine ​​hydrochloride.

7. The vial-packaged bacterial preparation according to claim 6, characterized in that, The raw materials for preparing the freeze-drying protectant include the following components: 5-50 parts by weight of skim milk powder, 1-10 parts by weight of trehalose, 1-10 parts by weight of maltodextrin, and 0.1-2 parts by weight of vitamin C.

8. The vial-packaged bacterial preparation according to claim 7, characterized in that, The raw materials for preparing the freeze-drying protectant include the following components: 10 parts by weight of skim milk powder, 5 parts by weight of trehalose, 5 parts by weight of maltodextrin, and 0.5 parts by weight of vitamin C.

9. The vial-packaged bacterial preparation according to claim 6, characterized in that, The additives also include flavor improvers, which include one or more of sweeteners, acidulants, colorings, and flavorings.

10. The vial-packaged bacterial preparation according to claim 1, characterized in that, The contents also include one or more of prebiotics and plant extracts.

11. The vial-packaged bacterial preparation according to claim 1, characterized in that, The contents also include fruit and vegetable powders.

12. A method for preparing lyophilized bacteria packaged in vials according to any one of claims 1-11, characterized in that, Includes the following steps: S01, vial preparation: including cleaning, sterilization, and drying of the vials; S02, Preparation of bacterial preparations; S03, under sterile conditions, the vials obtained in step S01 and the bacterial preparations obtained in step S02 are packaged, preferably under vacuum or filled with nitrogen.

13. The method for preparing lyophilized bacteria packaged in vials according to claim 12, characterized in that, In S02, when the bacterial preparation is lyophilized bacteria, the bacterial cells are mixed with a sterile lyophilization protectant and then lyophilized to obtain lyophilized bacterial powder. When the bacterial preparation is an inactivated bacterium, the bacterial cells are dried after inactivation treatment to obtain inactivated bacterial powder; When the bacterial preparation is a bacterial metabolite, the bacterial metabolite is collected and dried to obtain a bacterial metabolite powder.

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