A probiotic microcapsule and a preparation method and application thereof

Abstract

The present application provides a kind of probiotic microcapsule and its preparation method and application, the probiotic microcapsule includes composite probiotic core material and the composite wall material of the composite probiotic core material, the composite probiotic core material includes lactobacillus rhamnosus and paracasei, the composite wall material includes sodium alginate and stachyose.The probiotic microcapsule of the present application is lactobacillus rhamnosus and paracasei as composite core material, stachyose and sodium alginate as composite wall material, embedding forms microcapsule, stachyose and sodium alginate synergistic effect can not only improve the embedding rate of composite probiotic, but also can improve the metabolic activity and acid resistance, digestion resistance of probiotic, ensure more live bacteria reach intestinal tract.

Description

Technical Field

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

[0002] Probiotics, as active microorganisms that regulate gut microbiota, inhibit pathogenic bacteria, enhance immunity, and prevent disease, are widely used in fermented foods, dairy products, and health supplements. However, their survival conditions are extremely demanding; they are highly sensitive to oxygen, gastric acid, enzymes, and bile salts, and are easily inactivated in large quantities during processing, storage, transportation, and digestion. This leads to the destruction of the bacteria in the stomach, making it difficult for them to reach the intestines and exert their effects. Traditional powders, tablets, or ordinary capsules have low survival rates in acidic and bile environments. While single-layer coating technology can slightly increase acid resistance, its protective effect is limited. Furthermore, environmental factors such as temperature and humidity significantly reduce the number of live bacteria during long-term storage. In addition, existing formulations lack precise controlled-release technology, resulting in disordered release and difficulty in targeted colonization of probiotics in the digestive tract, thus limiting their health benefits and bioavailability.

[0003] Prebiotics are carbohydrates that cannot be digested and utilized by the human body but can selectively promote the proliferation and metabolism of beneficial intestinal bacteria. Prebiotics play an important role in the host's gut health; adding a certain amount of prebiotics can enhance the function of lactic acid bacteria. Different lactic acid bacteria exhibit significant differences in their affinity for specific prebiotics. Biological preparations or microecological preparations that combine probiotics with suitable prebiotics are also known as synbiotics. Synbiotics work synergistically, providing nutrients to probiotics, selectively stimulating their growth and activating their metabolism, giving probiotics a competitive advantage in the gut, and thus beneficially influencing the host.

[0004] Microencapsulation technology, as an effective way to protect probiotics, can significantly improve their survival rate in the gastrointestinal tract, ensuring that probiotics reach the intestines and exert their effects. To improve the survival rate of probiotics and better regulate the balance of intestinal flora, existing technologies typically encapsulate probiotics and prebiotics together to form microcapsules. For example, Chinese invention patent CN120323652A discloses a composite prebiotic-encapsulated probiotic microcapsule, its preparation method, and its application. It uses Lactobacillus acidophilus to ferment green tea extract, and then uses inulin, sodium alginate, and modified dietary fiber as the main wall materials of the microcapsules to encapsulate the green tea fermentation liquid and probiotics. This improves the stability of active substances and probiotics in the green tea fermentation liquid, enabling them to play a protective and sustained-release role during simulated digestion. However, it still suffers from low encapsulation rate and low metabolic activity. Summary of the Invention

[0005] In view of this, the present invention aims to propose a probiotic microcapsule, its preparation method and application, using Lactobacillus rhamnosus and Lactobacillus paracasei as composite core materials and stachyose and sodium alginate as composite wall materials to encapsulate and form microcapsules, thereby improving the metabolic activity and encapsulation rate of probiotics.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a probiotic microcapsule, the probiotic microcapsule comprising a composite probiotic core material and a composite wall material coating the composite probiotic core material, the composite probiotic core material comprising Lactobacillus rhamnosus and Lactobacillus paracasei, and the composite wall material comprising sodium alginate and stachyose.

[0007] Furthermore, the *Lactobacillus rhamnosus* (LGG) strain is numbered ATCC7469 and classified as *Lactobacillus rhamnosus*. The *Lactobacillus paracasei* (LP33) strain is numbered ATCC25302 and classified as *Lactobacillus paracasei* subsp. *paracasei*.

[0008] Furthermore, the Lactobacillus rhamnosus and Lactobacillus paracasei are mixed in any proportion.

[0009] Furthermore, the probiotic microcapsules are prepared by mixing a composite probiotic core material solution and a composite wall material solution, followed by emulsification and drying. Preferably, the number of bacteria in the composite probiotic core material solution is not less than 1×10⁻⁶. 11 CFU / mL; Preferably, the mass concentration of the composite wall material solution is 1% to 5%; more preferably, it is 2%. Preferably, the volume of the composite probiotic core material solution is 5% to 20% of the volume of the composite wall material solution; more preferably, it is 10%.

[0010] Furthermore, the mass ratio of Lactobacillus rhamnosus to Lactobacillus paracasei in the composite probiotic core material is 2-4:1; preferably 3:1.

[0011] Secondly, the present invention provides a method for preparing probiotic microcapsules, the method comprising the following steps: S1. Mix the compound probiotic core material with physiological saline to prepare a compound probiotic core material solution, wherein the compound probiotic core material includes Lactobacillus rhamnosus and Lactobacillus paracasei. S2. Mix the composite wall material with deionized water to prepare a composite wall material solution, wherein the composite wall material includes sodium alginate and stachyose. S3. Mix the composite probiotic core material solution obtained in step S1 with the composite wall material solution obtained in step S2, and homogenize and emulsify to obtain an emulsion. S4. Dry the emulsion obtained in step S3 to obtain the probiotic microcapsules.

[0012] Furthermore, the concentration of the saline solution is 0.85%.

[0013] Furthermore, the number of bacteria in the compound probiotic core material solution is not less than 1×10⁻⁶. 11 The composite wall material solution has a mass concentration of 1% to 5% (CFU / mL); preferably, the mass concentration of the composite wall material solution is 10%.

[0014] Furthermore, the mass ratio of sodium alginate to stachyose in the composite wall material is 2-4:1; preferably 3:1.

[0015] Furthermore, the volume of the composite probiotic core material solution is 5% to 20% of the volume of the composite wall material solution; preferably 10%.

[0016] Furthermore, the drying method in step S3 is spray drying; Preferably, the inlet air temperature of the spray dryer is 120~150℃ and the feed rate is 20%~50%; more preferably, the inlet air temperature of the spray dryer is 140℃ and the feed rate is 40%.

[0017] Furthermore, the preparation method of the composite probiotic core material includes the following steps: S11. Inoculate Lactobacillus rhamnosus and Lactobacillus paracasei into the culture medium and subculture for 2-3 consecutive times to obtain activated strains; S12. After inoculating the activated bacterial strain into sterilized MRS broth and shaking culture, the composite probiotic core material is collected by centrifugation. Preferably, in step S11, the inoculum amount is 1%~2%, the culture temperature is 37℃, and the culture time is 24~48h; Preferably, in step S12, the inoculum amount is 1%~2%, the culture temperature is 37℃, the shaking rotation speed is 100~200 rpm, and the culture time is 8~15h; Preferably, in step S12, the centrifugation temperature is 1~5℃, the centrifugation speed is 3000~5000rpm, and the centrifugation time is 10~20min.

[0018] Thirdly, the present invention provides the use of probiotic microcapsules as described in the first aspect or the preparation method as described in the second aspect in food, health products or pharmaceuticals.

[0019] Compared with existing technologies, the probiotic microcapsules, their preparation method, and applications described in this invention have the following advantages: The probiotic microcapsules of this invention use Lactobacillus rhamnosus and Lactobacillus paracasei as composite core materials and stachyose and sodium alginate as composite wall materials to encapsulate and form microcapsules. The synergistic effect of stachyose and sodium alginate can not only improve the encapsulation rate of the composite probiotics, but also improve the metabolic activity and acid resistance and digestive resistance of the probiotics, ensuring that more live bacteria reach the intestines. Detailed Implementation

[0020] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0021] The present invention will now be described in detail with reference to embodiments.

[0022] Example 1

[0023] The preparation method of probiotic microcapsules in this embodiment includes the following steps: (1) Probiotic activation After thawing Lactobacillus rhamnosus (LGG) and Lactobacillus paracasei (LP33) stored at -80℃, they were inoculated into MRS liquid medium at an inoculum of 1% and cultured at 37℃ for 36 hours. This was repeated three times to obtain fully activated strains (activated bacteria). The liquid medium used in this example consisted of 10 parts peptone, 10 parts beef extract, 5 parts yeast extract, 20 parts glucose, 5 parts sodium acetate, 2 parts diammonium citrate, 1 part Tween 80, 0.5 parts magnesium sulfate, 0.25 parts manganese sulfate, 2 parts dipotassium hydrogen phosphate, and 15 parts agar powder. After thoroughly mixing all components, the pH was adjusted to 6.2–6.4, and the mixture was autoclaved at 121℃ and 101 kPa for 15 minutes.

[0024] (2) Preparation of bacterial suspension Take the activated bacteria obtained in step 1 and inoculate 1% of the bacteria into 10 mL of sterile MRS broth. Incubate at 37°C and 150 rpm for 12 h. Collect the bacterial cells by centrifugation at 4°C and 4000 rpm for 15 min, and wash twice with sterile 0.85% physiological saline. Resuspend the bacterial cells in 10 mL of 0.85% physiological saline and adjust the bacterial count to approximately 1 × 10⁻⁶. 11 CFU / mL was used to obtain a mixed bacterial suspension of LGG and LP33, which was then stored at 4℃ for later use.

[0025] (3) Preparation of probiotic microcapsules In this embodiment, sodium alginate and stachyose were selected as wall materials. They were mixed in a mass ratio of 3:1 and deionized water was added to prepare a wall material solution with a mass fraction of 2.00%. The pH was adjusted to 6.5-7.0. The solution was then cooled after being sterilized by high-pressure steam at 121℃.

[0026] Take the LGG and LP33 mixed bacterial suspension obtained in step (2) as the core material, add it at 10% of the wall material solution volume and mix well; place it in a high-speed homogenizer for emulsification to make the bacterial solution uniformly dispersed in the wall material phase. Send the emulsion into a preheated spray drying equipment for drying, with the inlet air temperature set at 140℃ and the feed rate at 40%; collect the obtained microcapsule powder as the target product.

[0027] Example 2

[0028] The difference from Example 1 is that in step (3) of this example, sodium alginate and stachyose are mixed in a mass ratio of 3.2:0.8, while the other steps are the same as in Example 1.

[0029] Example 3

[0030] The difference from Example 1 is that in step (3) of this example, sodium alginate and stachyose are mixed in a mass ratio of 2.67:1.33, and the other steps are the same as in Example 1.

[0031] Comparative Example 1 The difference from Example 1 is that in step (3) of this comparative example, sodium alginate is used as the wall material, and deionized water is added to prepare a wall material solution with a mass fraction of 2.00%. Stachyose is not added. Other steps are the same as in Example 1.

[0032] Comparative Example 2 The difference from Example 1 is that in step (3) of this comparative example, stachyose is used as the wall material, and deionized water is added to prepare a wall material solution with a mass fraction of 2.00%. Sodium alginate is not added. Other steps are the same as in Example 1.

[0033] Comparative Example 3 The difference from Example 1 is that in this comparative example, stachyose is added to the bacterial suspension in step (2), sodium alginate is used as the wall material in step (3), and deionized water is added to prepare a wall material solution with a mass fraction of 2.00%. The mass ratio of sodium alginate to stachyose is 4:1. Other steps are the same as in Example 1.

[0034] Comparative Example 4 The difference from Example 1 is that in this comparative example, stachyose is added to the bacterial suspension in step (2), sodium alginate is used as the wall material in step (3), and deionized water is added to prepare a wall material solution with a mass fraction of 2.00%. The mass ratio of sodium alginate to stachyose is 3:1. Other steps are the same as in Example 1.

[0035] Comparative Example 5 The difference from Example 1 is that sodium alginate and inulin are used as wall materials in step (3) of this comparative example, wherein sodium alginate and inulin are mixed in a mass ratio of 3:1, and the other steps are the same as in Example 1.

[0036] Performance test examples (1) Encapsulation rate The method for testing the total bacterial count after spray drying is as follows: Dissolve 0.5g of microcapsules in 20mL of PBS buffer, place in a constant temperature shaker at 37℃ and 150r / min for 1h to uncapsule, then use a pipette to take 0.1mL of the uncapsulated solution for serial dilution, spread it on MRS solid medium, and incubate in a constant temperature incubator at 37℃ for 48h. Calculate the number of bacteria contained in each gram of microcapsule, and then multiply it by the corresponding mass to obtain the total bacterial count after spray drying.

[0037] The encapsulation rates of Examples 1-3 and Comparative Examples 1-5 were calculated using the following formulas, and the results are shown in Table 1: Encapsulation efficiency = (Number of viable bacteria before spray drying / Number of viable bacteria after spray drying) × 100% Table 1. Encapsulation Rate Test Results

[0038] As shown in Table 1, the microcapsules prepared using stachyose and sodium alginate as wall materials in this application exhibit a more stable encapsulation rate compared to those prepared in Comparative Example 1 without stachyose or in Comparative Example 5 using inulin and sodium alginate as wall materials. The microcapsules show higher initial encapsulation rates, smaller batch-to-batch fluctuations, and better retention after low-temperature storage. This is because stachyose has high solubility and a fixed degree of polymerization, allowing for more consistent rheology with the spray drying and gelation processes. In contrast, inulin has a wide degree of polymerization distribution, easily introducing fluctuations and leading to significant differences in encapsulation rates between different batches.

[0039] (2) Simulating in vitro gastrointestinal digestion The anti-digestion properties of the microcapsules prepared in Example 1 and Comparative Examples 1-5 were analyzed using the following methods: Simulated gastric digestive fluid (SGF): 0.1 g NaCl, 350 μL 0.1 mol / L HCl dissolved in 50 mL of water, pH adjusted to 2.0, containing 2% (w:v) pepsin; Simulated intestinal digestive fluid (SIF): 0.34 g KH2PO4, 9.5 mL 0.1 mol / L NaOH dissolved in 50 mL of water, pH adjusted to 7.4, containing 0.2% (w:v) trypsin and 1.2% (w:v) ox bile salts. Sterilization was achieved by filtration through a 0.22 μm microporous membrane. 0.25 g of microcapsules were placed in 10 mL of SGF and slowly shaken at 120 rpm in a constant temperature shaker at 37°C. At 0, 0.5, 1.0, and 2.0 h, 1 mL of the sample was taken and dissolved in 9 mL of sodium citrate solution for 1 h to uncapsule the microcapsules. The uncapsulated solution was then diluted appropriately for plate counts. Subsequently, the microcapsule suspension, after 2 h of simulated gastric fluid treatment, was adjusted to pH 7.4 with 1 mol / L NaOH, and 5 mL was added to 5 mL of SGF. The solution was placed in a constant temperature shaker at 37°C and slowly shaken at 120 rpm. At 2.5, 4.5, and 6.0 h, 0.1 mL of the sample was serially diluted and plate counts were performed to calculate the survival rate. The side-view results of Example 1 are shown in Table 2.

[0040] Survival rate is calculated using the formula: Survival rate / % = A / B × 100% In the formula: A is the number of viable bacteria in the sample after treatment with the simulated digestion solution, CFU / mL; B is the number of viable bacteria in the sample before treatment with the simulated digestion solution, CFU / mL.

[0041] Table 2. Results of simulated in vitro gastrointestinal digestion

[0042] As shown in Table 2, after treatment with simulated gastric juice, the microcapsules prepared using stachyose and sodium alginate as wall materials in this application exhibited a significantly higher retention rate of culturable bacteria than those in comparative examples 1-5. This is because stachyose possesses both permeability and drying protection, as well as a rapid carbon source for recovery, and can synergistically work with sodium alginate to form a denser colloidal network, thereby mitigating acid inhibition and dehydration damage.

[0043] (3) Growth curve determination Equal amounts of microcapsules from each group were inoculated into MRS broth medium. The culture flasks were placed in a shaker at 37°C and 100 rpm. When the bacteria reached the logarithmic growth phase (logarithmic growth in LGG is 4-5.5 h (OD)... 600 ≈0.6-0.9). LP33 (logarithm 5-6.5h (OD) 600(≈0.6-0.9) Re-inoculated into MRS medium, incubated at 37°C on a shaker at 100 rpm, and monitoring of indicators began; samples were taken at 0, 4, 8, and 12 hours of incubation, and the pH value was measured using a pH meter. The optical density (OD) of the culture was measured at the same time points. 600 To observe the cell density of each group, the results of Example 1 are shown in Table 3, and the results of Comparative Examples 1-5 are shown in Table 4.

[0044] Table 3. Growth curve measurement results of Example 1

[0045] Table 4. Results of growth curve measurements for Comparative Examples 1-5

[0046] According to the results in Tables 3 and 4, the microcapsules prepared using sodium alginate and stachyose as wall materials in this application showed higher metabolic activity (acid production) and growth levels of probiotics compared to those prepared using sodium alginate alone in Comparative Example 1, stachyose alone in Comparative Example 2, and inulin and sodium alginate as wall materials in Comparative Example 5. Quantitative studies have shown that this combination (sodium alginate + stachyose mixed as wall materials) is superior to using probiotics alone and significantly superior to inulin. The synergistic effect of stachyose and sodium alginate can not only improve the encapsulation rate of compound probiotics, but also improve the metabolic activity, acid resistance, and digestive resistance of probiotics, ensuring that more live bacteria reach the intestines. Compared to Comparative Examples 3 and 4, where stachyose was added to the core material, the addition of stachyose to the wall material in this application also showed higher metabolic activity (acid production) and growth levels. This indicates that stachyose added to the wall material can serve as an readily available energy source for probiotics. Stachyose can be rapidly utilized by lactobacilli directly or through induced enzyme systems, upregulating glycolysis / acid production-related enzymes, resulting in a faster pH decrease and a steeper growth curve. However, adding stachyose directly to the core material cannot achieve the above effects. Compared to the same time point, the stachyose group had a higher OD600 and a lower pH than the group without stachyose and the group with inulin as a prebiotic.

[0047] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A probiotic microcapsule, characterized in that: The probiotic microcapsule includes a composite probiotic core material and a composite wall material covering the composite probiotic core material. The composite probiotic core material includes Lactobacillus rhamnosus and Lactobacillus paracasei, and the composite wall material includes sodium alginate and stachyose.

2. The probiotic microcapsule according to claim 1, characterized in that: The probiotic microcapsules are prepared by mixing a composite probiotic core material solution and a composite wall material solution, followed by emulsification and drying. Preferably, the number of bacteria in the composite probiotic core material solution is not less than 1×10⁻⁶. 11 CFU / mL; Preferably, the mass concentration of the composite wall material solution is 1% to 5%; more preferably, it is 2%. Preferably, the volume of the composite probiotic core material solution is 5% to 20% of the volume of the composite wall material solution; more preferably, it is 10%.

3. The probiotic microcapsule according to claim 1 or 2, characterized in that: The mass ratio of sodium alginate to stachyose in the composite wall material is 2-4:1; preferably 3:

1.

4. A method for preparing probiotic microcapsules, characterized in that, The preparation method includes the following steps: S1. Mix the compound probiotic core material with physiological saline to prepare a compound probiotic core material solution, wherein the compound probiotic core material includes Lactobacillus rhamnosus and Lactobacillus paracasei. S2. Mix the composite wall material with deionized water to prepare a composite wall material solution, wherein the composite wall material includes sodium alginate and stachyose. S3. Mix the composite probiotic core material solution obtained in step S1 with the composite wall material solution obtained in step S2, and homogenize and emulsify to obtain an emulsion. S4. Dry the emulsion obtained in step S3 to obtain the probiotic microcapsules.

5. The preparation method according to claim 4, characterized in that: The number of bacteria in the compound probiotic core material solution is not less than 1×10⁻⁶. 11 The composite wall material solution has a mass concentration of 1% to 5% (CFU / mL); preferably, the mass concentration of the composite wall material solution is 10%.

6. The preparation method according to claim 4, characterized in that: The mass ratio of sodium alginate to stachyose in the composite wall material is 2-4:1; preferably 3:

1.

7. The preparation method according to claim 4, characterized in that: The volume of the composite probiotic core material solution is 5% to 20% of the volume of the composite wall material solution.

8. The preparation method according to claim 4, characterized in that: The drying method in step S3 is spray drying; Preferably, the inlet air temperature of the spray dryer is 120~150℃ and the feed rate is 20%~50%; more preferably, the inlet air temperature of the spray dryer is 140℃ and the feed rate is 40%.

9. The preparation method according to claim 4, characterized in that, The preparation method of the composite probiotic core material includes the following steps: S11. Inoculate Lactobacillus rhamnosus and Lactobacillus paracasei into the culture medium and subculture for 2-3 consecutive times to obtain activated strains; S12. After inoculating the activated bacterial strain into sterilized MRS broth and shaking culture, the composite probiotic core material is collected by centrifugation. Preferably, in step S11, the inoculum amount is 1%~2%, the culture temperature is 37℃, and the culture time is 24~48h; Preferably, in step S12, the inoculum amount is 1%~2%, the culture temperature is 37℃, the shaking rotation speed is 100~200rpm, and the culture time is 8~15h; Preferably, in step S12, the centrifugation temperature is 1~5℃, the centrifugation speed is 3000~5000rpm, and the centrifugation time is 10~20min.

10. The use of the probiotic microcapsules as described in any one of claims 1 to 3 or the preparation method as described in any one of claims 4 to 9 in food, health products or pharmaceuticals.

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