A preparation method of Bacillus coagulans microcapsules

Abstract

The present invention relates to a method for preparing probiotic microcapsules, particularly a method for preparing Bacillus coagulans microcapsules. The method employs a combination of inulin and xanthan gum to encapsulate Bacillus coagulans microcapsules. The microcapsules are activated and microencapsulated, and the inulin and xanthan gum are used to prepare the wall material. Sterile physiological saline is used as a solvent, to which Bacillus coagulans is added. The solution is homogenized to obtain a uniform solution, and then freeze-dried to obtain the microcapsules. The method improves the survival rate and stability of Bacillus coagulans. The prepared Bacillus coagulans microcapsules have high resistance and strong barrier properties, overcoming the limitations of Bacillus coagulans in consumption and storage.

Description

Technical Field

[0001] The present invention relates to a method for preparing probiotic microcapsules, in particular to a method for preparing bacillus coagulans microcapsules, and belongs to the technical field of polymer material processing. Background Art

[0002] With the advancement of science and technology and the improvement of people's living standards, the demand for nutritious foods has become increasingly stringent. Traditional nutritious foods can only maintain nutritional content but cannot exert certain functional effects, thus failing to meet people's demand for health. In recent years, probiotics have become a new favorite in global research due to their unique functional properties. However, their low digestibility and storage stability have become a major problem during consumption, processing and storage.

[0003] Bacillus coagulans ( Bacillus coagulans Bacillus coagulans is a type of probiotic bacteria that possesses the advantages of endospore formation, aiding digestion, preventing constipation, and producing metabolites. These metabolites have anti-inflammatory and lipid-lowering properties. However, Bacillus coagulans only exerts its effects when it colonizes and ferments within the intestines. Studies have shown that direct consumption of Bacillus coagulans is only half effective. Although Bacillus coagulans can improve its survival in the digestive system, low gastric acid, various digestive enzymes, and bile salts can significantly damage it, preventing it from colonizing and fermenting in the intestines and rendering it inactive. While pure Bacillus coagulans possesses potent anti-inflammatory and antioxidant properties and can colonize and ferment within the body, gastric acid forces it into spore form, requiring germination to better compete and colonize the intestines.

[0004] Prior art has limitations in the consumption and processing of probiotics. Encapsulation technology for probiotics is a primary approach to addressing these challenges. By combining different types of proteins, polysaccharides, and fats in varying proportions, different physical and mechanical properties can be created to meet the activity requirements of different probiotics. Consequently, probiotic microcapsules have seen rapid development in recent years. A Chinese patent, with authorization publication number CN112544977B, discloses a method for preparing probiotic microcapsules, aiming to improve the activity and stability of probiotics. A Chinese patent, with authorization publication number CN106993813B, also discloses a method for preparing probiotic microcapsules, also aiming to improve the activity and stability of probiotics. This suggests that microcapsules can improve the stability of probiotics. Numerous references indicate that different methods and wall materials can be used to encapsulate probiotics, and different bacterial strains can also be used. Not all probiotics are suitable for the same wall material and preparation method; different preparation methods and wall materials can directly affect the encapsulation and stability of probiotics.

[0005] Therefore, it is urgent for those skilled in the art to solve the technical problems that need to be solved by selecting wall materials suitable for Bacillus coagulans and developing a preparation method suitable for Bacillus coagulans microcapsules. Summary of the Invention

[0006] The present invention is to solve the above technical problems and provides a method for preparing Bacillus coagulans microcapsules, which improves the survival rate and stability of Bacillus coagulans. The prepared Bacillus coagulans microcapsules have high resistance and strong barrier properties, overcoming the limitations of Bacillus coagulans in consumption and storage.

[0007] In order to solve the above technical problems, the present invention is achieved through the following technical solutions:

[0008] A preparation method of Bacillus coagulans microcapsules comprises the following steps: taking 1 part by mass of activated Bacillus coagulans, dissolving the dissolved solution in 97 parts by mass of sterile physiological saline to prepare a Bacillus coagulans suspension, adding 1 part by mass of sterilized inulin and 1 part by mass of xanthan gum to the Bacillus coagulans suspension for embedding, standing the solution to completely dissolve, and then homogenizing the solution to prepare primary liquid microcapsules, placing the primary liquid microcapsules in a low-temperature refrigerator for precooling to convert the liquid form into an ice crystal solid form, and then placing the microcapsules in the ice crystal form in a freeze dryer for freeze drying to prepare Bacillus coagulans microcapsules.

[0009] Preferably, the preparation method of the activated Bacillus coagulans is as follows: 1 part by mass of lyophilized Bacillus coagulans powder is dissolved in 99 parts by mass of sterile physiological saline, and the mixture is inoculated into a sterilized MRS plate culture medium, and cultured at 37°C for 24 hours to prepare a seed solution. A loopful of the seed solution is picked and inoculated into an MRS broth culture medium, and the culture is placed in a shaker at 37°C for 48 hours to complete activation.

[0010] Preferably, the embedding treatment is to centrifuge the Bacillus coagulans suspension at 4° C. and 2500 r / min for 10 min, collect the precipitate, and disperse the precipitate in the physiological saline.

[0011] Preferably, the standing time is 4 h.

[0012] Preferably, the freeze-drying time is 48 h.

[0013] Preferably, the homogenization process is performed at 12000 r / min for 2 min.

[0014] Preferably, the sterilization is to sterilize the inulin and xanthan gum at a high temperature of 121° C. for 15 minutes.

[0015] Preferably, the pre-cooling is to place the primary liquid microcapsules into a -80°C low-temperature refrigerator for pre-cooling for 8-12 hours.

[0016] Due to the adoption of the above technical solution, the present invention has the following characteristics and effects:

[0017] This patent discloses a method for preparing Bacillus coagulans microcapsules, which activates and microencapsulates Bacillus coagulans. The microcapsules are prepared using inulin and xanthan gum as the wall material. Sterile physiological saline is used as the solvent, to which Bacillus coagulans is added. A uniform solution is obtained by homogenization, and then freeze-drying is performed to obtain the microcapsules. The raw materials of the present invention are widely available and the cost is low. The prepared Bacillus coagulans microcapsules have high resistance and strong barrier properties, overcoming the limitations of Bacillus coagulans in consumption and storage, and compensating for the defects of Bacillus coagulans being easily killed and losing activity during processing and storage. By combining inulin and xanthan gum into a dense outer shell, the present invention effectively ensures that Bacillus coagulans can pass through the harsh digestive environment of the gastrointestinal tract with high activity. The main ingredient of the wall material used in the present invention is inulin, a macromolecular dietary fiber and prebiotic, which meets the current social demand for sugar-free and low-calorie diets and better maintains the activity of Bacillus coagulans. Xanthan gum serves as a stabilizer to ensure that the prepared microcapsules do not become loose powder after freeze-drying. Improved the survival rate and stability of Bacillus coagulans. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the effect of inulin concentration on the encapsulation efficiency.

[0019] Figure 2 shows the effect of xanthan gum concentration on the embedding efficiency.

[0020] Figure 3 shows the effect of the added bacteria ratio on the embedding efficiency.

[0021] Figure 4 shows the in vitro digestion results of microcapsules with different wall materials.

[0022] Figure 5 This is a scanning electron micrograph of the microcapsules.

[0023] Figure 6 is a Fourier transform infrared spectrum.

[0024] Figure 7 This is the result of microcapsule laser particle size analyzer.

[0025] Figure 8 shows the storage survival effect of microcapsules at 25°C.

[0026] Figure 9 shows the survival effect of microcapsules stored at 4°C. DETAILED DESCRIPTION

[0027] The following uses specific embodiments to further illustrate the above content of the present invention. However, this should not be interpreted as limiting the design concept of the present invention to the following examples. Any non-substantial changes to the above content of the present invention using this concept shall be deemed to infringe the scope of protection of the present invention. Example 1

[0028] A method for preparing Bacillus coagulans microcapsules comprises the following steps:

[0029] 1 part by mass of Bacillus coagulans freeze-dried powder was dissolved in 99 parts by mass of sterile physiological saline, and inoculated into a sterilized MRS plate medium, cultured at 37°C for 24 hours to prepare a seed liquid, a ring of seed liquid was picked, inoculated into an MRS broth medium, and cultured on a shaker at 37°C for 48 hours to complete activation, thereby preparing an activated Bacillus coagulans; 1 part by mass of the activated Bacillus coagulans was taken and dissolved in 97 parts by mass of sterile physiological saline to prepare a Bacillus coagulans suspension, and 1 part by mass of inulin and 1 part by mass of xanthan gum that had been sterilized were added to the Bacillus coagulans suspension for embedding treatment, and the embedding treatment was taking the Bacillus coagulans suspension and incubating it at 4°C and 2500 r / min The mixture was centrifuged for 10 minutes, and the precipitate was collected. The precipitate was dispersed in the physiological saline, allowed to stand and completely dissolved, and then homogenized to prepare primary liquid microcapsules. The primary liquid microcapsules were placed in a low-temperature refrigerator for pre-cooling to change the liquid form into an ice crystal solid form. The ice crystal microcapsules were then placed in a freeze dryer and freeze-dried to prepare Bacillus coagulans microcapsules.

[0030] The above-mentioned standing time is 4 hours, the above-mentioned freeze-drying time is 48 hours, the above-mentioned homogenization treatment is 12000 r / min homogenization treatment for 2 minutes, the above-mentioned sterilization is to sterilize the inulin and xanthan gum at a high temperature of 121°C for 15 minutes, and the precooling is to put the primary liquid microcapsules into a -80°C low-temperature refrigerator for precooling for 8-12 hours.

[0031] The test parameters of the Bacillus coagulans microcapsules prepared in this example were as follows: the embedding efficiency was 86.6% and the biological activity after storage at 25°C for 120 days was (2.24±0.11)×10 9 CFU g -1 The biological activity after storage at 4℃ for 120 days was (2.31±0.4)×10 11 CFU g -1 . Comparative Example 1

[0032] The preparation method of a Bacillus coagulans microcapsule is the same as that in Example 1, except that the xanthan gum concentration is set to 0.8%, the inulin concentration is set to 1.2%, and the activated Bacillus coagulans concentration is set to 1.5%.

[0033] The detection parameters of the Bacillus coagulans microcapsules prepared in this example: the embedding rate was 74.1%. Comparative Example 2

[0034] The preparation method of a Bacillus coagulans microcapsule is the same as that in Example 1, except that the xanthan gum concentration is set to 0.8%, the inulin concentration is set to 1%, and the activated Bacillus coagulans concentration is set to 1%.

[0035] The detection parameters of the Bacillus coagulans microcapsules prepared in this example: the embedding efficiency was 76.4%. Comparative Example 3

[0036] The preparation method of a Bacillus coagulans microcapsule is the same as that in Example 1, except that the xanthan gum concentration is set to 1%, the inulin concentration is set to 0.8%, and the activated Bacillus coagulans concentration is set to 1.5%.

[0037] The detection parameters of the Bacillus coagulans microcapsules prepared in this example: the embedding efficiency was 67.9% after detection.

[0038] The characteristics and effects of the present invention are further illustrated below by test data:

[0039] Test method:

[0040] Microcapsule embedding efficiency: 0.5 g of microcapsules prepared by the method of Example 1 of the present invention were rinsed three times with deionized water, then ground and dissolved. The viable bacterial count in the concentrate was measured by plate count (A (CFU / mL)). The total viable bacterial count in the concentrated bacterial solution used for microcapsule preparation was then measured (B (CFU / mL)). The microcapsule embedding efficiency was calculated using this method. The formula is as follows:

[0041]

[0042] Single-factor experiment: By controlling the variable method, we identified the single factor with the greatest impact on encapsulation efficiency. We studied the effect of the ratio of core material concentration to wall material on microencapsulation efficiency. Core material concentrations were set at 1%, 1.5%, 2%, 2.5%, and 3%, inulin concentrations at 0.8%, 1%, 1.2%, 1.4%, and 1.6%, and xanthan gum concentrations at 0.8%, 1%, 1.2%, 1.4%, and 1.6%.

[0043] Orthogonal experiment: Based on the single factor experiment, the concentration of core material Bacillus coagulans, inulin concentration, and xanthan gum concentration were used as independent variables, and the embedding rate was used as the evaluation index. The orthogonal experiment was used to optimize the preparation of microcapsules. The factor levels are shown in Table 1.

[0044] Table 1. Orthogonal experiment optimization factor level table for microcapsule preparation

[0045] level Inulin concentration Xanthan gum concentration Bacteria concentration -1 1.4% 1.2% 2% 0 1.2% 1% 1.5% 1 1% 0.8% 1%

[0046] Scanning electron microscopy (SEM): Based on the above results, the wall material of the Bacillus coagulans microcapsules of the present invention is a gel composed of 1% inulin and 1% xanthan gum; the wall material of the high-ester pectin group probiotic microcapsules is a gel composed of 1% high-ester pectin and 1% xanthan gum. After freeze-drying, the high-ester pectin group microcapsules and the inulin and xanthan gum group microcapsules were fixed to the sample stage using conductive glue. After vertical gold coating under vacuum conditions, the microcapsule morphology was characterized using SEM at an accelerating voltage of 30 kV.

[0047] Fourier transform infrared spectroscopy (FT-IR): The prepared microcapsules were passed through a 60-mesh sieve. 5 mg of the sieved high-ester pectin microcapsules and the microcapsule gel powder prepared by the method of Example 1 of the present invention were weighed, 200 mg of potassium bromide powder was added, and the two were thoroughly ground to uniformly mix. The mixed powder was then pressed into a transparent sheet using a tablet press. The sheet was scanned using an infrared spectrometer in the scanning spectral range of 4000-400 cm -1 , with a resolution of 4 cm -1 .

[0048] Laser Particle Size Analyzer: The average particle size and particle size distribution of the prepared microcapsules were determined using a Malvern nanometer laser particle size analyzer. The refraction angle was set at 90°, the measurement temperature was set at 25 ± 1°C, and the polydisperse emulsion mode was used. To minimize the influence of multiple scattering, each emulsion was diluted 100-fold with distilled water before testing.

[0049] Gastric and intestinal phase simulation experiments (in vitro): To simulate digestion in the stomach, the pH of the sample was adjusted to 1 mol L -1 Adjust the pH of the HCl solution to 1.6. Add porcine mucosal protease and candida nut lipase to concentrations of 0.03 g per 10 ml and 0.0009 mg per 10 ml, respectively. Incubate the sample at 37°C with agitation (150 rpm) for 2 hours in an environmental incubator shaker to simulate the gastric phase. After simulated digestion, adjust the pH of the sample to approximately 4.7 using a sterile alkaline solution (1 L contains 150 ml of 1 mol L -1NaOH and 14g NaH2PO4). Subsequently, bovine bile and porcine pancreatic enzymes were added to concentrations of 0.1g per 10ml and 0.01g per 10ml. The samples were simulated digested under the above conditions for 2 hours (enteric phase I). After 2 hours of simulated digestion, the sample pH was further raised to 7.1 using an alkaline solution. Furthermore, the concentrations of bile and pancreatic enzymes were adjusted to 0.1g per 10ml and 0.1g per 10ml. The samples were simulated digested under the above conditions for 24 hours to obtain a 24-hour assay evaluation. Bacillus coagulans colony activity was collected using a 3M plate count at 2, 4, 6, 9, 12, and 24-hour intervals, as shown below. All enzymatic hydrolysates used in the experiments were suspended in water and sterilized.

[0050] Stability test of Bacillus coagulans storage microcapsules: The microcapsules prepared by the method of Example 1 of the present invention were added to sterile physiological saline and stored at 4 and 25°C, respectively. Samples were taken for 3M plate count at 0, 15, 30, 45, 60, 75, 90, and 120 days.

[0051] Results and Analysis

[0052] Effect of different raw materials on embedding efficiency:

[0053] Inulin concentration: The effect of inulin concentration on the embedding efficiency of Bacillus coagulans is as follows: Figure 1 As shown, inulin alone has a moderate encapsulation effect on Bacillus coagulans. When inulin is used exclusively, the encapsulation efficiency reaches 71.4%. When the inulin concentration reaches 1%, the encapsulation efficiency increases with increasing inulin concentration. At inulin concentrations of 1%, 1.2%, and 1.4%, the encapsulation efficiency is relatively similar, at 86.6%, 85.9%, and 86.1%, respectively. However, it can be seen that at concentrations above 1%, the encapsulation efficiency decreases with increasing inulin concentration. The peak encapsulation efficiency occurs at 1%, with an encapsulation efficiency of 86.6%.

[0054] Xanthan gum concentration: The effect of xanthan gum concentration on the encapsulation efficiency of Bacillus coagulans is shown in Figure 2. Adding xanthan gum gradually decreased the encapsulation efficiency, likely due to xanthan gum acting as a stabilizer and gel thickener. Furthermore, the initial xanthan gum concentration was too low, which prevented the formation of a stable powder during the freeze-drying process.

[0055] The ratio of added bacteria: The effect of the ratio of added bacteria on the embedding rate is as follows: Figure 3As shown in the figure, the maximum encapsulation efficiency, 86.1%, was achieved at a 1% concentration. The trend then began to decline, before increasing slightly at 3%, a result of excessive bacterial substrate. Although freeze-drying is the process that causes the greatest loss of Bacillus coagulans during microencapsulation, a significant portion of the bacteria still survives, so the maximum concentration was set at 3%.

[0056] Orthogonal experiment: An orthogonal experiment was conducted based on the optimal microcapsule preparation conditions obtained from the single-factor experiment. The four-factor three-level orthogonal analysis method was used to explore the effects of inulin concentration, xanthan gum concentration, and Bacillus coagulans cell concentration on the microcapsule encapsulation effect of Bacillus coagulans to determine the final microcapsule preparation conditions. The results are shown in Table 2.

[0057] Table 2 Orthogonal experiment results

[0058] Serial number Inulin concentration Xanthan gum concentration The proportion of added bacteria Embedding rate 1 1 1 1 51.4 ± 2.4% 2 2 2 2 76.4 ± 1.7% 3 3 3 3 67.9 ± 3.1% 4 3 1 2 84.7 ± 2.4% 5 2 1 3 79.7 ± 4.1% 6 3 2 3 86.6 ± 3.2% 7 1 3 1 61.3 ± 4.1% 8 2 2 1 71.5 ± 2.3% 9 1 3 2 49.3 ± 3.7% <![CDATA[K1]]> 214.49 221.52 219.25 <![CDATA[K2]]> 232.19 241.30 239.12 <![CDATA[K3]]> 197.23 216.25 207.11 <![CDATA[K1]]> 74.50 77.80 76.80 <![CDATA[K2]]> 79.49 77.96 78.03 <![CDATA[K3]]> 79.03 77.26 78.00 R 3.89 0.91 1.69 Optimal level <![CDATA[A3]]> <![CDATA[B2]]> <![CDATA[C3]]>

[0059] Table 2 shows that the order of range (R) is A > C > B, indicating that inulin concentration has the greatest impact on microcapsule encapsulation efficiency. The highest value among K1, K2, and K3 represents the optimal solution for that level. Therefore, the A3B2C3 combination is the optimal preparation condition. Verification indicates that this condition achieved an encapsulation efficiency of (86.6 ± 3.20)%, significantly higher than the other combinations. Therefore, the A3B2C3 combination was selected for subsequent experiments, with encapsulation efficiency as the evaluation metric. The optimal process parameters were an inulin concentration of 1%, a xanthan gum concentration of 1%, and a bacterial addition ratio of 1%.

[0060] In vitro digestion simulation of microcapsules with different wall materials:

[0061] To exert their biological activity in vivo, probiotic cells must pass through the digestive system and withstand hydrolysis by digestive enzymes, reaching the small intestine and colon for effective absorption. Furthermore, to provide beneficial effects to consumers, probiotic cells must remain viable within the food matrix and reach the large intestine in sufficient quantities to promote colonization and proliferation. Therefore, in this study, the digestive stability of microcapsules A (high-ester pectin-based microcapsules) and B (microcapsules prepared by the method of Example 1 of the present invention) was investigated in a simulated gastrointestinal digestion model system and compared with free Bacillus coagulans. To examine their gastric acid resistance, the pH was lowered to 1.6 during the initial 2-hour gastric phase simulation experiment, and porcine mucosal protease and Candida alata lipase were added to simulate human protein- and fat-digesting enzymes.

[0062] Figure 4 shows the results of gastric and intestinal digestion simulations for Bacillus coagulans and two microcapsules. Comparison clearly shows that the encapsulated Bacillus coagulans exhibited significantly higher activity during the digestion simulations, with Group A showing over 100-fold higher activity and Group B even reaching 1,000-fold. This suggests that encapsulation of Bacillus coagulans with inulin or high-ester pectin and xanthan gum plays a crucial role in transporting Bacillus coagulans to the duodenum or colon, where they are most active, protected by the microcapsule's barrier.

[0063] The activity of the three groups of Bacillus coagulans dropped sharply in the first 2 hours, indicating that Bacillus coagulans is extremely sensitive to strong acidic conditions. In the subsequent in vitro digestion simulation experiments, the trend of its activity decline gradually slowed down. This is also the result of simulating the acidic conditions of the small intestine and colon and the gradual increase in pH. Many bacteria have proton pumps in their plasma membranes as a defense mechanism to protect the cytoplasm close to its physiological pH. If these regulatory systems cannot operate normally, intracellular acidification will occur and loss of activity will occur. The activity of the KB group in strong acidic conditions for 2 hours decreased from 14 lgCFU g -1 Directly down to 9 lgCFU g -1 In contrast, the microcapsules in groups A and B can still maintain 12 lgCFU g -1 However, in the intestinal digestion simulation experiment, the biological activities of Groups A and B gradually diverged. This is because the bovine bile added to the simulated intestinal phase has a certain harmful effect on high-ester pectin. Another reason why the microcapsules prepared by the method of Example 1 of the present invention are more active than the microcapsules in the high-ester pectin group in the presence of harmful substances such as gastric acid, digestive enzymes, and bile acids may be the result of their larger particle size and thicker shell.

[0064] Scanning electron microscopy (SEM): In Figure 5, KB: Unencapsulated Bacillus coagulans; A: High-ester pectin microcapsules; B: Microcapsules prepared by the method of Example 1 of the present invention. Figure 5 As can be seen in the figure, the free Bacillus coagulans freeze-dried powder is largely aggregated, with a small amount of dispersion. Figure 1 shows a 10µm scale, magnified 1000x; Figure 2 shows a 1µm scale, magnified 10,000x. The scale ratios of the scanning electron microscope are 10µm and 1µm, respectively. It is clearly visible that the majority of microcapsules in both groups exhibit a regular spherical shape, although irregular spherical and ellipsoidal shapes also exist. Figures A1, A2 and B1, B2 show the surface conditions of the microcapsules prepared using the method of Example 1 of the present invention, respectively, and the smoothness of the microcapsules in the high-ester pectin group is clearly smoother. In contrast, the surface smoothness of the microcapsules in Group B is significantly less, and the particles attached to their surface are more densely aggregated than those in Group A.

[0065] From the images obtained by scanning electron microscopy, one can notice that the microcapsules are interconnected by solid bonds, probably caused by the freezing process and freeze-binding. However, this may promote the penetration of water during the dissolution dispersion process due to the presence of capillaries in the agglomerates. In addition, there are no bacterial cells on the surface of the freeze-dried microcapsules, indicating that the Bacillus coagulans cells are well fixed within the core of the supramolecular biopolymer structure to form microcapsules. Compared with matrix capsules, storage capsules are considered to provide greater protection for probiotic cells. The former fixes the probiotics in the core area and is protected by the biopolymer wall; while matrix-type capsules disperse the cells in the granules, making the probiotics more vulnerable to environmental stress. However, when the polymer concentration is 10 g L -1 When the particle size of the aggregates was >50 μm, some particles agglomerated and condensed to form particles with different morphologies. The results of this study showed that the morphology of the aggregates was closely related to the type and concentration of the biopolymer used as the wall material.

[0066] Fourier transform infrared spectroscopy (FT-IR): Figure 6 In the figure, H is xanthan gum raw material; A is high ester pectin group microcapsules; B is microcapsules prepared by the method of Example 1 of the present invention. Figure 6 It is obvious that the permeability peaks of microcapsules in groups A and B are different from those in the xanthan gum raw material. -1 The peak surface changes in the range of 3000 - 2500 cm -1 The peak surface in the range also shrinks slightly; 2000 - 1000 cm -1 The permeability peaks of the three peaks of group A and group B shifted to the left or right, and the permeabilities of the three peaks also decreased; 1000 - 500 cm -1 The changes in the characteristic peaks of the microcapsule polymers in groups A and B indicate that the high-ester pectin, inulin and xanthan gum of the present invention undergo certain chemical changes or physical polymerization during the microcapsule production process.

[0067] The FT-IR spectrum of xanthan gum polysaccharide shows characteristic peaks of polymers at 3500 and 3000 cm -1 The peaks in the range are due to OH stretching vibrations indicating inter- and intramolecular hydrogen bonding. Studies on xanthan gum showed a peak at 3440 cm -1 There is a strong peak at 2854 cm, indicating OH stretching vibration, indicating the formation of hydrogen bonds between molecules. -1 and 2926 cm -1The presence of 1635 cm-1 is caused by the vibrational stretching of CH2 and CH functional groups. -1 and 1408 cm -1 The peaks at 1260 and 1100 cm-1 were assigned to C-OO asymmetric and symmetric stretching, respectively, which are typical features of galacturonic acid. -1 The microcapsules prepared from the high ester pectin group and the microcapsules prepared by the method of Example 1 of the present invention have a wavelength of 3500-3000 cm -1 It is not difficult to see that the key forces in the formation of microcapsules may be hydrogen bonds or van der Waals forces. It is clearly evident that the peak surface of group B is narrower than that of groups H and A, indicating a better bonding effect.

[0068] Particle Size: Figure 7 shows the particle size and distribution of the two groups of microcapsules, as measured by a laser particle size analyzer. Both groups exhibited a normal distribution with a narrow distribution range, indicating a relatively uniform particle size distribution. During the homogenization and freeze-drying process, the particle size distribution of both the high-ester pectin microcapsules and the microcapsules prepared by the method of Example 1 of the present invention was not uniform, indicating that microcapsules varied in size during their formation. The particle size of the high-ester pectin microcapsules was relatively small, primarily ranging from 150 to 350 μm. The average surface area diameter was 210.919 μm, while the average volume diameter was 321.16 μm. The particle size of the microcapsules prepared by the method of Example 1 of the present invention was significantly larger, with an average surface area diameter of 246.327 μm and an average volume diameter of 357.387 μm. Smaller capsules will have higher permeability, while larger capsules will have higher protection.

[0069] Survival effect during storage: In FIG8 , KB: unencapsulated Bacillus coagulans; A: high-ester pectin microcapsules; B: microcapsules prepared by the method of Example 1 of the present invention. Figure 8 The results of the storage experiment of microcapsules placed naturally at 25℃ for 120 days show that with the increase of time, the activity of KB, A and B groups all show a downward trend, among which the activity of KB group is the most obvious. The activity of Bacillus coagulans decreases rapidly from 0 to 60 days. At 60 days, it is as low as (3.27±0.65)×10 3 CFU / g, and at 75 days, its activity was almost completely lost. In contrast, the activity of the microcapsules in groups A and B only decreased by 2 lgCFUg in 0-60 days. -1 , respectively (5.25±0.15)×10 11 CFU -1 、(5.10±0.15)×1012 CFU -1 During the 120-day storage period, the microcapsules of groups A and B could still maintain 8-9 lgCFUg -1 The activity was (5.20±0.10)×10 8 CFU -1 and (2.24±0.11)×10 9 CFU -1 It can be seen that the activity of Group A microcapsules stored at 25°C was significantly higher than that of Group B microcapsules (p<0.05). This is because the inulin and xanthan gum are more tightly bound during the capsule preparation process.

[0070] Figure 9 The results of the storage experiment of microcapsules placed naturally at 4℃ for 120 days show that with the increase of time, the activity of KB, A and B groups all show a downward trend, among which the activity of KB group is the most obvious. During the period of 0-60 days, the activity of Bacillus coagulans decreases rapidly. At 120 days, it is as low as (2.33±0.12)×10 6 CFU / g, and the activity was almost completely lost after 135 days. In contrast, the activity of microcapsules in groups A and B only decreased by 3 lgCFU g from 0 to 120 days. -1 , respectively (2.64±0.58)×10 10 CFU -1 、(2.31±0.4)×10 11 CFU g -1 It can be seen that the activity of Group A microcapsules stored at 4°C was significantly higher than that of Group B microcapsules (p<0.05). This is also due to the tighter combination of inulin and xanthan gum during the capsule preparation process.

[0071] Test conclusion:

[0072] The optimal process for preparing microcapsules combining inulin and xanthan gum was determined through single-factor and orthogonal experiments. Freeze-drying was used to prepare the microcapsule powder, and the same process parameters were used to prepare high-ester pectin microcapsules in Group A, a control group. The physicochemical properties and characterization of the microcapsules in Groups A and B were investigated, along with their protective effects against Bacillus coagulans under simulated in vitro digestion. The main conclusions are as follows:

[0073] (1) The combination of inulin and xanthan gum to form a wall material has an excellent encapsulation effect on Bacillus coagulans. When the inulin concentration is 1%, the xanthan gum concentration is 1%, and the bacterial concentration is 1%, the encapsulation efficiency is as high as 86.6 ± 3.20%. Compared with group A, the encapsulation effect is 17.3% higher, which significantly improves the survival rate of Bacillus coagulans, indicating that inulin and xanthan gum have great potential as probiotic wall materials.

[0074] (2) The microcapsules of groups A and B significantly improved the survival efficiency of Bacillus coagulans in in vitro digestion simulation. The study found that the in vitro survival efficiency of group B was significantly higher than that of group A, and the activity difference reached 1 lg CFU g -1 FT-IR results showed that the raw materials in group B had more obvious electrostatic interactions, so the composite wall material formed had a better protective effect against Bacillus coagulans. The hydrogen bonds and van der Waals forces between high-ester pectin and xanthan gum were weaker, so its protective effect was relatively poor.

[0075] (3) Through SEM observation, the surface of group A is relatively smoother, while the surface of group B is relatively rougher. This is because the polysaccharide has better water solubility, and the residual polysaccharide makes the xanthan gum adhere to each other, resulting in a tight formation. The results of the laser particle size analyzer show that the particle size of the two groups is relatively small, and their distribution is basically uniform. The particle size distribution and particle size of group B are slightly larger than those of group A, indicating that the residual inulin has a certain adhesion effect on the xanthan gum. The results of the in vitro digestion simulation experiment also proved that group B has a more beneficial protective effect on Bacillus coagulans and can reach the duodenum more effectively.

[0076] (4) The study of its stable storage performance shows that Groups A and B have a barrier protection effect on Bacillus coagulans, isolating the adverse effects of external adverse factors on its activity. By exploring the storage stability at 25℃ and 4℃, both Groups A and B have excellent protection effects, and it is obvious that Group B is better than Group A. -1 After 120 days, both groups A and B could maintain 8-9 lg CFU g -1 biological activity.

Claims

1. A method for preparing Bacillus coagulans microcapsules, characterized in that The method comprises the following steps: taking 1 part by mass of activated Bacillus coagulans, dissolving the dissolved dissolved dissolved dissolved dissolved dissolved dissolved dissolved dissolved dissolved dissolved dissolved bacillus coagulans in 97 parts by mass of sterile physiological saline to prepare a Bacillus coagulans suspension, adding 1 part by mass of sterilized inulin and 1 part by mass of xanthan gum to the Bacillus coagulans suspension for embedding, standing the sterilized inulin and xanthan gum to completely dissolve the sterilized inulin, and then homogenizing the sterilized inulin to prepare primary liquid microcapsules, placing the primary liquid microcapsules in a low-temperature refrigerator for pre-cooling to convert the liquid form into an ice crystal solid form, and then placing the microcapsules in the ice crystal form into a freeze dryer for freeze-drying to prepare Bacillus coagulans microcapsules.

2. The method for preparing Bacillus coagulans microcapsules according to claim 1, characterized in that The activated Bacillus coagulans is prepared as follows: 1 part by mass of freeze-dried Bacillus coagulans powder is dissolved in 99 parts by mass of sterile physiological saline, and the mixture is inoculated into a sterilized MRS plate culture medium, and cultured at 37°C for 24 hours to prepare a seed solution. A loopful of the seed solution is picked and inoculated into an MRS broth culture medium, and the culture is placed on a shaker at 37°C for 48 hours to complete activation.

3. The method for preparing Bacillus coagulans microcapsules according to claim 1, characterized in that The embedding treatment is to centrifuge the Bacillus coagulans suspension at 4° C. and 2500 r / min for 10 minutes, collect the precipitate, and disperse the precipitate in the physiological saline.

4. The method for preparing Bacillus coagulans microcapsules according to claim 1, characterized in that The standing time is 4 h.

5. The method for preparing Bacillus coagulans microcapsules according to claim 1, characterized in that The freeze-drying time is 48 h.

6. The method for preparing Bacillus coagulans microcapsules according to claim 1, characterized in that The homogenization process is performed at 12000 r / min for 2 min.

7. The method for preparing Bacillus coagulans microcapsules according to claim 1, characterized in that The sterilization is to sterilize the inulin and xanthan gum at a high temperature of 121° C. for 15 minutes.

8. The method for preparing Bacillus coagulans microcapsules according to claim 1, characterized in that The pre-cooling is to put the primary liquid microcapsules into a -80°C low-temperature refrigerator for pre-cooling for 8-12 hours.

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