A probiotic drying protectant, its preparation method and application

Abstract

The application discloses a probiotic dry protection agent and a preparation method and application thereof, relates to the technical field of biological medicine, and the preparation method is that Bacillus subtilis is cultured in a culture medium by adding calcium ions, spores are generated, the spores are extracted and broken to nanoscale, calcium-loaded spore nanocrushing products are obtained, and the calcium-loaded spore nanocrushing products are the probiotic dry protection agent. The application also discloses the probiotic dry protection agent prepared by the method, application of the probiotic dry protection agent in preparation of probiotic preparations and related probiotic preparations. The calcium-loaded spore nanocrushing products are used as the probiotic dry protection agent, and the survival rate of the probiotic after dry treatment is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a probiotic desiccant, its preparation method, and its application. Background Technology

[0002] Probiotics are a collective term for microorganisms that have beneficial effects on human health. They are widely distributed in the human gut and participate in physiological processes such as food digestion, nutrient absorption, and immune regulation through metabolic activities, secreting beneficial metabolites. Studies have shown that specific probiotics and gut microbiota can effectively inhibit the colonization and growth of pathogenic bacteria in the gut. Oral administration of sufficient amounts of live probiotics can help alleviate acute gastroenteritis, treat diarrhea, improve lactose intolerance, and show potential in the adjunctive treatment of neurodevelopmental disorders and metabolic syndrome. Related live probiotic preparations have already been used in clinical treatment. However, the industrialization of probiotic preparations faces a core challenge: live bacteria are easily affected by environmental stresses (such as temperature, humidity, and oxygen) during processing, storage, and transportation, leading to a significant decrease in survival rate, which severely restricts their efficacy and commercialization process.

[0003] To preserve the activity of probiotics for extended periods and facilitate transportation and storage, the pharmaceutical industry typically uses drying methods to prepare solid probiotic formulations. The main preparation processes include vacuum freeze-drying, spray drying, fluidized bed drying, and microwave vacuum drying. Among these, spray drying has become the preferred process for industrial production due to its advantages such as high efficiency and continuous production (drying time of only 5-30 seconds), low cost (6-50 times lower than freeze-drying), and controllable particle uniformity. However, a fatal flaw of this technology is that the bacteria must be directly exposed to a high-temperature airflow (inlet temperature is typically 120-200℃), causing triple lethal damage: ① Thermodynamic damage: High temperatures disrupt cell membrane integrity, leading to the loss of intracellular enzymes (such as Na+). + / K + The following factors contribute to the failure of probiotics to maintain their activity during spray drying: ① Inactivation of ATPase and DNA strand breaks; ② Osmotic imbalance: rapid dehydration leads to protein denaturation and failure of cytoplasmic glass transition; ③ Oxidative stress: high temperature accelerates the generation of free radicals, attacking the lipid bilayer and mitochondrial function.

[0004] Adding spores to protect probiotics is a feasible method. CN202411121487.9 discloses adding Bacillus subtilis spore shell liquid and a protectant to Lactobacillus plantarum, followed by spray drying to obtain Lactobacillus plantarum powder. However, this method involves complex and inefficient preparation of the spore shell liquid. Furthermore, the Bacillus subtilis fermentation broth used is a mixture of active bacteria and spores, containing various harmful metabolites related to the active bacteria. The proportion of spores is difficult to control and cannot be accurately quantified. CN202011426215.1 discloses a composition of spore shell nanomaterials and probiotics. A spore solution is placed in a filter membrane extruder, filtered, and spore shell nanomaterials are obtained. These nanomaterials are then co-incubated with a probiotic solution to prepare the composition. The spore shell nanomaterials act as a protective shell against stomach acid for probiotics, while also promoting their proliferation and colonization. However, the effectiveness of spore materials in protecting probiotics during high-temperature drying processes requires further improvement and optimization. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a probiotic desiccant, its preparation method, and its application. The present invention uses calcium-loaded spore nanoparticles as a probiotic desiccant, which significantly improves the survival rate of probiotics after drying.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a method for preparing a probiotic desiccant is provided, wherein calcium ions are added to a culture medium to culture spore-producing probiotics to produce spores, and then the spores are extracted and crushed to obtain calcium-loaded spore nanoparticles, which are the probiotic desiccant.

[0007] Furthermore, the concentration of calcium ions is 0.01-2 mg / mL. Calcium ions are provided using calcium chloride, or other soluble salts of calcium may be used.

[0008] In some embodiments, the soluble calcium salt is selected from inorganic calcium salts and organic calcium salts; preferably, the inorganic calcium salt is selected from one or more of calcium chloride, calcium nitrate, calcium bromide, and calcium iodide; the organic calcium salt is selected from one or more of calcium acetate, calcium lactate, calcium formate, calcium gluconate, and calcium propionate.

[0009] Furthermore, the spore-forming probiotic is at least one of Bacillus subtilis, Clostridium butyricum, Bacillus coagulans, and Bacillus licheniformis. Other spore-forming probiotics may also be included.

[0010] In some embodiments, the particle size of the calcium-loaded spore nanoparticles is 50-200 nm.

[0011] In some implementations, the preparation method of the probiotic desiccant includes the following steps in sequence:

[0012] (1) Prepare liquid culture medium by adding calcium ions to the liquid culture medium;

[0013] (2) Inoculate the spore-producing probiotics into liquid culture medium, carry out subculture under constant temperature conditions, then transfer to an ice-water bath and stand, then raise the temperature to 23-27℃, and finally transfer to 70-90℃ for 20-40 min to kill the vegetative cells, and centrifuge to collect the spores to obtain calcium-loaded spores.

[0014] (3) The calcium-loaded spores obtained in step (2) are crushed to obtain calcium-loaded spore nanoparticles, which are probiotic desiccant.

[0015] In one embodiment, in step (1), the liquid culture medium comprises 4-6 g / L peptone, 2-4 g / L beef extract, 4-6 g / L sodium chloride, and water.

[0016] In one embodiment, inoculation is performed after the spore-forming probiotics have been cultured to the logarithmic growth phase.

[0017] In one embodiment, in step (2), the culture is carried out at a constant temperature of 36-38°C and 120-180 rpm.

[0018] In one embodiment, in step (2), the mixture is left to stand in an ice-water bath at 0-4°C for 2-4 hours.

[0019] In one embodiment, the temperature is increased to 23-27°C at a rate of 0.5°C / min.

[0020] In one embodiment, the crushing process in step (3) is as follows: the calcium-loaded spores are diluted with sterile deionized water to a concentration of 1×10⁻⁶. 10 ~1×10 11 Transfer CFU / mL to a grinding tube, then add grinding beads with a diameter of 0.1-0.5 mm, and grind at 5-7 m / s for 5-10 min.

[0021] In this invention, the calcium-loaded spore nanoparticles have been sterilized and purified to remove live bacteria and other harmful impurities, allowing for accurate quantification of the spore concentration in the raw material, "diluted to a concentration of 1×10⁻⁶". 10 ~1×10 11 "CFU / mL" refers to the spore concentration, which does not involve live bacteria and does not contain impurities related to live bacteria. It is more stable and can effectively exert the metagenic function and protective effect of the spore shell, thereby reducing the risks to human health.

[0022] In this invention, the technical solution utilizes a laboratory-grade grinder for automated and efficient crushing of spore shells. Simply place a sample tube containing spore fluid and grinding beads into the instrument before grinding, grind to the desired particle size, and then remove the sample. The specialized grinding tubes used in the preparation process have excellent sealing properties, preventing spore waste. Furthermore, the large single-batch processing capacity, minimal manual operation, and low-cost consumables significantly reduce preparation costs, facilitating industrial-scale experiments and actual production.

[0023] As used in this invention, the term "probiotic" is defined as any non-pathogenic microorganism that, when administered to a host in sufficient quantities in live form, can have a beneficial effect on the host's health.

[0024] In one aspect, the present invention also provides a probiotic desiccant prepared by the above-described method for preparing the probiotic desiccant.

[0025] In one aspect, the present invention also provides the use of the above-mentioned probiotic desiccant in the preparation of probiotic formulations. The probiotic desiccant functions as an excipient in the probiotic formulation.

[0026] In some embodiments, the present invention provides a probiotic preparation comprising the aforementioned probiotic desiccant and probiotics.

[0027] In some embodiments, the mass ratio of the probiotics to the probiotic desiccant is 10:1 to 10. After protection with the probiotic desiccant, the survival rate of lactic acid bacteria can reach 95%-99% after spray drying.

[0028] Protected probiotics include, but are not limited to, commonly used probiotics such as Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium, and yeast.

[0029] In some embodiments, the protected probiotics are selected from Lactobacillus, Bifidobacterium, Streptococcus, Lactobacillus, Propionibacterium, Pediococcus, Staphylococcus, Kluyveromyces, or combinations thereof.

[0030] The present invention has the following beneficial effects:

[0031] 1. The preparation method of this invention uses low-temperature ion permeation coupled gradient temperature recovery technology to prepare spore shells containing calcium ions, and then reduces their particle size to the nanoscale (50-200nm) through efficient "one-step grinding", providing an efficient method for preparing spore nanofragments, laying the foundation for industrial-scale production.

[0032] 2. This invention combines the heat-enhancing function of calcium ions with the protective function of spore nanoparticles in the field of probiotic spray drying protection. Calcium-loaded spore nanoparticles are anchored on the surface of the bacteria through natural affinity, forming a natural protective barrier and encapsulating the bacteria. Through the dual effects of chemical bonding and physical barrier, the protective efficacy against probiotics is significantly improved. Attached Figure Description

[0033] Figure 1 This is a staining observation diagram of Bacillus subtilis sporulation process in Example 1;

[0034] Figure 2 Transmission electron microscopy image of calcium-loaded spore nanoparticles prepared in Example 1;

[0035] Figure 3 This is a comparison chart of the calcium content in the spore fragments obtained in Example 1 and Comparative Example 1.

[0036] Figure 4 The graph shows the survival rate of Streptococcus thermophilus under different protective agents.

[0037] Figure 5 The graph shows the survival rate of Streptococcus thermophilus under different addition amounts. Detailed Implementation

[0038] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0039] Example 1

[0040] A probiotic desiccant (CLSF) is prepared by adding calcium ions to a culture medium to culture Bacillus subtilis to induce spore production, then extracting and breaking the spores to obtain calcium-loaded spore nanoparticles, which are the probiotic desiccant.

[0041] The preparation method includes the following steps in sequence:

[0042] (1) Prepare liquid culture medium. The components of the liquid culture medium include 5 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride and water. Add 1 mg / mL calcium chloride to the liquid culture medium.

[0043] (2) Bacillus subtilis was inoculated into liquid culture medium for subculture. It was cultured at 37℃ and 120-180 rpm until a distinct odor could be detected. Then it was transferred to an ice-water bath at 4℃ and left to stand for 3 hours. The temperature was then increased to 25℃ at 0.5℃ / min. Finally, it was transferred to 80℃ and incubated for 30 minutes to kill the vegetative cells. The spores were collected by centrifugation to obtain calcium-loaded spores.

[0044] (3) Dilute the calcium-loaded spores with sterile deionized water to a concentration of 5×10⁻⁶. 10 The concentration of CFU / mL was transferred to a grinding tube, and then grinding beads with a diameter of 0.1 mm were added. The mixture was ground at 6 m / s for 8 min, centrifuged, filtered, and dried to obtain calcium-loaded spore nanoparticles, which are the probiotic desiccant.

[0045] Example 2

[0046] A probiotic desiccant is prepared by adding calcium ions to a culture medium to culture Clostridium butyricum to induce spore production, then extracting and breaking the spores to obtain calcium-loaded spore nanoparticles, which are the probiotic desiccant.

[0047] The preparation method includes the following steps in sequence:

[0048] (1) Prepare liquid culture medium. The components of the liquid culture medium include 4 g / L peptone, 2 g / L beef extract, 4 g / L sodium chloride and water. Add 0.01 mg / mL calcium chloride to the liquid culture medium.

[0049] (2) Clostridium butyricum was inoculated into liquid culture medium for subculture, and cultured at 36℃ and 120 rpm. Then it was transferred to an ice water bath at 0℃ and left to stand for 2 hours. The temperature was then raised to 23℃ and finally transferred to 70℃ for 40 minutes to kill the vegetative cells. The spores were collected by centrifugation to obtain calcium-loaded spores.

[0050] (3) Dilute the calcium-loaded spores with sterile deionized water to a concentration of 1×10⁻⁶. 10 The concentration of CFU / mL was transferred to a grinding tube, and then grinding beads with a diameter of 0.5 mm were added. The mixture was ground at 5 m / s for 10 min, centrifuged, filtered, and dried to obtain calcium-loaded spore nanoparticles, which are the probiotic desiccant.

[0051] Example 3

[0052] A probiotic desiccant is prepared by adding calcium ions to a culture medium to culture Bacillus licheniformis, causing it to produce spores, then extracting and breaking the spores to obtain calcium-loaded spore nanoparticles, which are the probiotic desiccant.

[0053] The preparation method includes the following steps in sequence:

[0054] (1) Prepare liquid culture medium. The components of the liquid culture medium include 6 g / L peptone, 4 g / L beef extract, 6 g / L sodium chloride and water. Add 2 mg / mL calcium chloride to the liquid culture medium.

[0055] (2) Bacillus licheniformis was inoculated into liquid culture medium for subculture, and cultured at 38℃ and 180 rpm. Then it was transferred to an ice-water bath at 2℃ and left to stand for 4 hours. The temperature was then raised to 27℃ and finally transferred to 90℃ for 20 minutes to kill the vegetative cells. The spores were collected by centrifugation to obtain calcium-loaded spores.

[0056] (3) Dilute the calcium-loaded spores with sterile deionized water to a concentration of 1×10⁻⁶. 11 The concentration of CFU / mL was transferred to a grinding tube, and then grinding beads with a diameter of 0.3 mm were added. The mixture was ground at 7 m / s for 5 min, centrifuged, filtered, and dried to obtain calcium-loaded spore nanoparticles, which are the probiotic desiccant.

[0057] Comparative Example 1

[0058] A spore fragment, the preparation method of which differs from that of Example 1, is that calcium chloride is not added to the culture medium.

[0059] Experimental Example 1

[0060] In Example 1, during the sporulation process of Bacillus subtilis culture, malachite green staining was used for observation, and the results were as follows: Figure 1 As shown in the figure, the results indicate that the cultured Bacillus subtilis is growing well, producing sporulation normally, and the extracted spores are morphologically complete, oval, and 1-2 μm in size.

[0061] Experimental Example 2

[0062] The calcium-loaded spore nanoparticles prepared in Example 1 were observed by scanning transmission electron microscopy, and the results are as follows: Figure 2 As shown, the results indicate that the spore shell is fragmented, with uniform fragment size and a particle size of 50-200 nm.

[0063] Experimental Example 3

[0064] The calcium content in the spore fragments obtained in Example 1 and Comparative Example 1 was detected separately, and the results were compared as follows: Figure 3 As shown in the figure, the results indicate that the calcium ion content in the prepared spore nanoparticles increased significantly after calcium ions were added to the culture medium.

[0065] Test Example 4

[0066] To investigate the protective effect of the probiotic desiccant in Example 1 on probiotics, widely used lactic acid bacteria in actual production were selected for spray drying. Streptococcus thermophilus, a common lactic acid bacteria species, is an important industrial lactic acid bacteria widely used in the production of some important fermented dairy products. The lactic acid it produces can regulate intestinal flora and enhance the body's immunity. However, it does not produce spores, has poor resistance, and is easily inactivated at high temperatures. Therefore, by adding a certain amount of desiccant, the lactic acid bacteria were protected from inactivation under high temperature and dehydration stress during the spray drying process. The protective effect of the desiccant was tested by measuring the survival rate of Streptococcus thermophilus. The steps are as follows:

[0067] (1) Cultivation of lactic acid bacteria

[0068] First, accurately weigh 4.83 g of MRS medium (Lactobacillus delbrueckii medium) into a 250 mL Erlenmeyer flask, add 100 mL of distilled water, and sonicate to dissolve the bacteria to obtain a liquid culture medium for Streptococcus thermophilus. Then, inoculate the activated Streptococcus thermophilus into this liquid culture medium at a 4% inoculation rate and incubate at 150 rpm for 9 hours in a 37°C constant temperature shaker until the logarithmic growth phase. Finally, centrifuge the bacterial suspension in the logarithmic growth phase at 5000 rpm for 10 minutes at 4°C, and wash three times with physiological saline to obtain fresh bacterial sludge.

[0069] (2) Preparation of active probiotic preparations by spray drying

[0070] A certain amount of fresh Streptococcus thermophilus bacterial sludge was weighed and mixed with different preservatives, then 100 mL of sterile water was added and mixed thoroughly. The experimental groups were: Group A, 5% bacterial sludge; Group B, 5% bacterial sludge + 5% skim milk powder (SMP) + 10% maltodextrin (MD); Group C, 5% bacterial sludge + 5% SMP + 10% MD + 2% CLSF, with preservatives expressed as mass fractions. The spray drying instrument parameters were set as follows: feed flow rate 120 mL / h, needle frequency 6 times / min, outlet temperature 75℃, inlet temperature 140℃, and air flow rate 415 L / h. Bacterial solutions were prepared according to the above groups, thoroughly mixed, and then spray-dried to obtain the corresponding probiotic powders.

[0071] Separately, a certain amount of fresh Streptococcus thermophilus sludge was taken and mixed with different preservatives, followed by the addition of 100 mL of sterile water and thorough mixing. The experimental groups were: blank group, 5% SMP, 10% MD, 0.5% CLSF, 1% CLSF, 2% CLSF, and 5% CLSF, with preservatives expressed as mass fractions. The spray drying instrument parameters and other processing steps are as described above.

[0072] (3) Calculation of viable cell count and cell survival rate

[0073] Accurately weigh 1.0 g of the spray-dried bacterial powder and calculate the viable count using the spread dilution method. Record the number of colonies (CEU) on the plate, expressed as CFU / g. Calculate the cell viability using the following formula: Cell viability (%) = Total viable count after spray drying (CFU / g) / Total viable count before spray drying (CFU / g) × 100.

[0074] The survival rate of Streptococcus thermophilus under different protective agent conditions is as follows: Figure 4 As shown in the figure, the results indicate that after adding the probiotic desiccant from Example 1, the survival rate of Streptococcus thermophilus after spray drying was significantly improved, reaching 95-99%.

[0075] The survival rate of Streptococcus thermophilus under different dosage conditions is as follows: Figure 5 As shown, the results indicate that the probiotic desiccant of Example 1, compared to existing desiccant, significantly improves the survival rate of lactic acid bacteria during spray drying with a smaller feed amount.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a probiotic desiccant, characterized in that, Calcium ions are added to the culture medium to culture spore-producing probiotics, causing them to produce spores. The spores are then extracted and broken down to obtain calcium-loaded spore nanoparticles, which are the probiotic desiccant.

2. The preparation method of the probiotic desiccant as described in claim 1, characterized in that, The concentration of the calcium ions is 0.01-2 mg / mL; preferably, the calcium ions are derived from soluble salts of calcium; more preferably, the calcium ions are provided by calcium chloride.

3. The preparation method of the probiotic desiccant as described in claim 1, characterized in that, The spore-producing probiotic is at least one of Bacillus subtilis, Clostridium butyricum, Bacillus coagulans, and Bacillus licheniformis.

4. The preparation method of the probiotic desiccant as described in claim 1, characterized in that, The steps are as follows: (1) Prepare a liquid culture medium and add calcium ions to the liquid culture medium; (2) Inoculate the spore-producing probiotics into the liquid culture medium, carry out subculture under constant temperature conditions, then transfer to an ice water bath and stand for 2-4 hours, then raise the temperature to 23-27℃, and finally transfer to 70-90℃ for 20-40 minutes to kill the vegetative cells, and centrifuge to collect the spores to obtain calcium-loaded spores; (3) Crush the calcium-loaded spores obtained in step (2) to obtain calcium-loaded spore nanoparticles, which are the probiotic desiccant.

5. The preparation method of the probiotic desiccant as described in claim 4, characterized in that, In step (3), the crushing process is as follows: the calcium-loaded spores are diluted with water to 1×10⁻⁶. 10 -1×10 11 Transfer CFU / mL to a grinding tube, then add grinding beads with a diameter of 0.1-0.5 mm, and grind at 5-7 m / s for 5-10 min.

6. The probiotic desiccant prepared by the method of any one of claims 1-5.

7. The use of the probiotic desiccant prepared by the method of any one of claims 1-5 in the preparation of probiotic formulations.

8. A probiotic preparation, characterized in that, The probiotic agent includes the probiotic desiccant and probiotics as described in claim 6.

9. The probiotic preparation according to claim 8, characterized in that, The mass ratio of the probiotics to the probiotic desiccant is 10:1 to 10.

10. The probiotic preparation according to claim 8, characterized in that, The probiotics are selected from Lactobacillus, Bifidobacterium, Streptococcus, Lactobacillus, Propionibacterium, Pediococcus, Staphylococcus, Kluyveromyces, or combinations thereof.

Images