A stable probiotic capsule and preparation method thereof

Abstract

The present invention belongs to the field of biomedicine technology. Specifically, it relates to a stable probiotic capsule and a preparation method thereof. The inventors have found through research that the combination of zein or its hydrolyzate with feruloyl oligosaccharides from corn or wheat as microcapsule wall materials can not only increase the mechanical strength of the microcapsules, but also reduce the permeability of the microcapsules to water vapor and oxygen. Experiments show that when the two are combined in a certain weight ratio as a wall material for embedding probiotics, the survival rate of the probiotics in the in vitro simulated digestion process can be greatly improved, and the tolerance of the probiotics to adverse environments and storage stability are improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a stable probiotic capsule and a preparation method thereof, and more specifically relates to a microcapsule using zein or its hydrolyzate and feruloyl oligosaccharide as wall materials to embed probiotics and a preparation method thereof. Background Art

[0002] Probiotic microencapsulation can provide protection for originally sensitive probiotics, improving their tolerance to oxygen, freezing and acid environments during processing and storage, as well as during passage through the digestive tract. Currently, the wall materials used to encapsulate probiotics are mostly natural polymer materials, and have the following characteristics: good biocompatibility and film-forming properties, stable properties, compatibility with the core material, viscosity, permeability, hydrophilicity and solubility requirements, and biodegradability. These wall materials mainly include: carbohydrates, such as sodium alginate, chitosan, starch; plant gums, such as gum arabic and κ-carrageenan; animal proteins, such as milk and gelatin; bacterial polysaccharides, such as gellan, xanthan gum, etc. Zhang Yulei et al. [1] Using a mixed colloid of 1% sodium alginate and 3% gelatin as the wall material, spraying it into a CaCl2 solution to make microcapsules of Lactobacillus acidophilus, studies have shown that microencapsulation can improve the resistance of Lactobacillus acidophilus strains to adverse conditions such as acid, pepsin, bile salts, trypsin and high osmotic pressure, ensuring the performance of its physiological functions. Jiang Longfa et al. [2] Using chitosan as the wall material to embed viscous red yeast, the study found that when the chitosan mass concentration was 7% and the sodium tripolyphosphate mass concentration was 6% to 9%, the embedding rate reached more than 95%.

[0003] Zein molecules have fewer polar amino acids, more sulfur-rich amino acids and hydrophobic amino acids. The molecules are connected by strong hydrogen bonds, disulfide bonds and hydrophobic bonds to form a dense network structure. This special amino acid composition gives it good film-forming properties, and its film has certain moisture resistance, oxygen resistance and enteric solubility. However, the lack of charged acidic, alkaline and polar amino acids in zein molecules makes it have the disadvantages of fragile film formation, low tensile strength and poor mechanical properties. Some studies have prepared zein into drug coatings, such as wrapping aspirin with a zein outer coating, which can effectively protect the active ingredients in the drug from being digested and decomposed in the stomach, and can also control the release rate of the active ingredients in the drug in the small intestine, playing a role of sustained release. However, zein is brittle in film formation, and because it is insoluble in water and only soluble in 85-95% ethanol, it increases the difficulty of post-processing. At present, there is no report on the use of zein or its hydrolyzate as a wall material to embed probiotics.

[0004] Feruloylated oligosaccharides (FOs) are compounds formed by ester bonding between ferulic acid and oligosaccharides. Studies have shown that FOs have stronger synergistic antioxidant capacity, anti-glycation effect, immunomodulatory function and anti-tumor activity. At present, there are no reports on the use of feruloylated oligosaccharides and zein or its hydrolyzate as a wall material for encapsulating probiotics.

[0005] [1] Zhang Yulei, Wu Rongrong, Zhang Bijian, et al. Study on the physiological characteristics of microencapsulated Lactobacillus acidophilus under extreme environment [J]. China Dairy Industry, 2006, 34(12): 7-12.

[0006] [2] Jiang Longfa, Zhang Suoxin, Xue Wanli, et al. Research on preparation and application of chitosan biomicrocapsules [J]. Journal of Huaihai Institute of Technology, 2001, 10(2): 50-53. Summary of the invention

[0007] The inventors have found through research that combining zein or its hydrolyzate with feruloyl oligosaccharides derived from corn or wheat as microcapsule wall materials can not only increase the mechanical strength of the microcapsules, but also reduce the permeability of the microcapsules to water vapor and oxygen; experiments have shown that when the two are used as wall materials for encapsulating probiotics in a certain weight ratio, the survival rate of the probiotics in the simulated digestion process in vitro can be greatly improved, and the tolerance of the probiotics to adverse environments and the storage stability can be improved.

[0008] The above object of the present invention is achieved through the following technical scheme: a stable probiotic microcapsule, the microcapsule comprises a core material and a wall material, and the wall material is prepared from zein or its hydrolyzate and feruloyl oligosaccharide.

[0009] As mentioned above, zein alone is brittle and difficult to process. However, since the ferulic acid in feruloyl oligosaccharide can react with cysteine, lysine, etc. in protein to cross-link the protein, the structure of the film is more compact. Therefore, when feruloyl oligosaccharide is combined with zein to prepare microcapsules, the mechanical strength of the microcapsules can be increased, and the permeability of the microcapsules to oxygen and water vapor can be lowered.

[0010] The inventors further discovered that since feruloyl oligosaccharides remain stable under low pH and high temperature conditions, the inventors had a sudden idea: Can this composite wall material be used to encapsulate probiotics? Through experiments, it was found that adding feruloyl oligosaccharides to the microcapsule wall material can enhance the tolerance of probiotics to acid environments. After being treated in gastric juice for 4 hours, the survival rate was 57.52%, while the survival rate of microcapsules prepared using zein alone as the wall material was only 12.79%, but this mechanism is not yet fully understood.

[0011] The inventors found that compared with zein, the partially hydrolyzed zein product has higher water solubility and thermal stability, and the combination with feruloyl oligosaccharide as a wall material provides greater protection for probiotics during gastrointestinal digestion simulation. Further studies have shown that the tolerance of probiotics to high bile salt environments is enhanced, and the combination of zein and feruloyl oligosaccharide alone does not have the above effect. It is speculated that it may be because the small molecule protein produced after the hydrolysis of zein has a promoting effect on the proliferation of probiotics, or it enhances the resistance of probiotics to adverse environments. However, the inventors found that the strengthening effect of the above probiotics on acidic environments and high-salt environments only appears within a specific hydrolysis degree range (less than 20%). When the hydrolysis degree of zein exceeds 20%, the above effect is difficult to observe.

[0012] As mentioned above, preferably, the weight ratio of the zein hydrolysate to the feruloyl oligosaccharide is 6:1 to 3, preferably 6:2. When the zein hydrolysate and feruloyl oligosaccharide are used to prepare microcapsules, within a specific weight ratio range of 6:1 to 3, the embedding yield increases with the increase of the feruloyl oligosaccharide content in the wall material, and then decreases, and the peak appears when the ratio of the two is 6:2.

[0013] Preferably, the zein hydrolysate described herein is obtained by enzymolysis of zein with alkaline protease. In particular, due to the unique and complex internal structure of zein, it has a strong resistance to the "enzymatic attack" of protease. In addition, since zein is insoluble in water, the probability of contacting with protease when suspended in an aqueous solution is small. Therefore, in order to achieve the required degree of hydrolysis, zein can be pre-treated before hydrolysis, such as heating, radiation, mechanical vibration, crushing, ultrasound and the like.

[0014] As for the enzyme used for enzymatic hydrolysis, screening tests have shown that alkaline protease enzymatic hydrolysis is ideal. The specific steps of alkaline protease hydrolysis are as follows:

[0015] The zein is prepared into a suspension with distilled water, the pH of the suspension is adjusted to 9.0-10.0, alkaline protease is added to the suspension for enzymolysis, and during the enzymolysis process, the substrate concentration is controlled to be 1-3 g / (100 mL), the enzyme-substrate ratio is 50-80 mAU / g, and the temperature is 50-55° C. After the enzymolysis is completed, the enzyme is inactivated in a water bath at 80-95° C. for 10-20 minutes; after cooling, the hydrolyzate is collected, centrifuged, and the supernatant is taken to obtain the product.

[0016] Another object of the present invention is to provide a method for preparing stable probiotic microcapsules, comprising the following steps:

[0017] Step a: obtaining zein or its hydrolyzate;

[0018] Step b: preparing the zein or its hydrolyzate into a suspension, adding feruloyl oligosaccharide into the suspension, ultrasonically treating for 1 to 6 minutes, and placing in a water bath at 60 to 75° C. for 30 minutes to terminate the reaction and obtain a wall material;

[0019] Step c: mixing the probiotics and the material in step b, and homogenizing them to obtain a liquid;

[0020] Step d: spray drying the feed liquid to obtain probiotic microcapsules.

[0021] Preferably, the inlet air temperature of the spray drying is 150-180°C; the outlet air temperature is 70-100°C.

[0022] Preferably, in step b, the weight ratio of zein or its hydrolyzate to feruloyl oligosaccharide is 6:1-3; preferably 6:2.

[0023] In step c, the weight ratio of probiotics to wall material is 1:3-8, preferably 1:3, 1:5 or 1:8.

[0024] Preferably, the probiotics are one or more of Lactobacillus bulgaricus, Lactobacillus reuteri and Bifidobacterium. The probiotics of the present invention can be cultured and activated by conventional techniques in the art.

[0025] The feruloyl oligosaccharide described herein can be derived from corn bran or wheat bran, preferably from wheat bran. Specifically, the feruloyl oligosaccharide can be extracted from wheat bran by enzyme method, acid method or high temperature cooking method; the composite wall material of the present invention preferably uses feruloyl oligosaccharide obtained by high temperature cooking method. The specific preparation steps refer to the prior art (Ge Lihua, Preparation of feruloyl oligosaccharide and study on its antioxidant properties [D], Northeastern University, 2007-04-01). DETAILED DESCRIPTION

[0026] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0027] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0028] ① Preparation of Zein Hydrolysate F1:

[0029] Zein was prepared into a suspension with a concentration of 10 mg / mL using distilled water, and the suspension was sealed in a sealed bag and placed in an ultrasonic tank for 5 minutes, with the ultrasonic power controlled at 35 kHz;

[0030] The pH value of the suspension after ultrasonic treatment was adjusted to 9.0 with 0.2 mol / L sodium hydroxide solution, and alkaline protease was added to the suspension for enzymolysis for 43 min, and the substrate concentration, enzyme substrate ratio and temperature were controlled to be 1.0 g / (100 mL) and 80 mAU / g, respectively, during the enzymolysis process. The degree of hydrolysis was measured during the enzymolysis process, and after the enzymolysis was completed, the pH value was adjusted to 7.0 with 0.2 mol / L HCL solution. The enzyme was inactivated in a water bath at 90°C for 15 min. The hydrolyzate was collected after cooling to room temperature, centrifuged and the supernatant was taken to obtain a zein hydrolyzate F1. The degree of hydrolysis was measured to be 12.52% with the pH-stat method.

[0031] ② Preparation of Zein Hydrolysate F2:

[0032] The zein was prepared into a suspension with a concentration of 10 mg / mL using distilled water, and the suspension was sealed in a sealed bag and placed in an ultrasonic tank for 8 minutes, with the ultrasonic power controlled at 40 kHz;

[0033] The pH value of the suspension after ultrasonic treatment was adjusted to 9.0 with 0.2 mol / L sodium hydroxide solution, and alkaline protease was added to the suspension for enzymolysis for 65 min, and the substrate concentration, enzyme substrate ratio and temperature were controlled to be 1.0 g / (100 mL) and 80 mAU / g, respectively, during the enzymolysis process. The degree of hydrolysis was measured during the enzymolysis process, and after the enzymolysis was completed, the pH value was adjusted to 7.0 with 0.2 mol / L HCL solution. The enzyme was inactivated in a water bath at 90°C for 15 min. The hydrolyzate was collected after being cooled to room temperature, centrifuged and the supernatant was taken to obtain a zein hydrolyzate F2. The degree of hydrolysis was measured to be 17.16% with the pH-stat method.

[0034] ③ Preparation of Zein Hydrolysate F3:

[0035] The zein was prepared into a suspension with a concentration of 10 mg / mL using distilled water, and the suspension was sealed in a sealed bag and placed in an ultrasonic tank for 12 minutes, with the ultrasonic power controlled at 60 kHz;

[0036] The pH value of the suspension after ultrasonic treatment was adjusted to 9.0 with 0.2 mol / L sodium hydroxide solution, and alkaline protease was added to the suspension for enzymolysis for 122 min, and the substrate concentration, enzyme substrate ratio and temperature were controlled to be 1.0 g / (100 mL) and 80 mAU / g respectively during the enzymolysis process, respectively, and the temperature was 50°C. The degree of hydrolysis was measured during the enzymolysis process, and the pH value was adjusted to 7.0 with 0.2 mol / L HCL solution after the enzymolysis was completed. The enzyme was inactivated in a water bath at 90°C for 15 min. The hydrolyzate was collected after being cooled to room temperature, centrifuged and the supernatant was taken to obtain the zein hydrolyzate F3. The degree of hydrolysis was measured to be 21.47% by pH-stat method.

[0037] ④ Preparation of Zein Hydrolyzate F4:

[0038] The zein was prepared into a suspension with a concentration of 10 mg / mL using distilled water, and the suspension was sealed in a sealed bag and placed in an ultrasonic tank for 20 minutes, with the ultrasonic power controlled at 68 kHz;

[0039] The pH value of the suspension after ultrasonic treatment was adjusted to 9.0 by using 0.2 mol / L sodium hydroxide solution, and alkaline protease was added to the suspension for enzymolysis for 139 min, and the substrate concentration, enzyme substrate ratio and temperature were controlled to be 1.0 g / (100 mL) and 80 mAU / g respectively during the enzymolysis process, and the temperature was 50°C. The degree of hydrolysis was measured during the enzymolysis process, and the pH value was adjusted to 7.0 by using 0.2 mol / L HCL solution after the enzymolysis was completed. The enzyme was inactivated by keeping the suspension in a water bath at 90°C for 15 min. The hydrolyzate was collected after being cooled to room temperature, centrifuged and the supernatant was taken to obtain the zein hydrolyzate F4. The degree of hydrolysis was measured to be 23.50% by using the pH-stat method.

[0040] ⑤ Preparation of zein hydrolysate F5: Compared with F1, the difference is that neutral protease is used instead of alkaline protease. During the enzymatic hydrolysis of neutral protease, the pH is 7.0, the temperature is 50°C, the substrate concentration is 1.0g / (100mL), the enzyme-substrate ratio is 43mAU / g, the enzymatic hydrolysis time is 120min, and the hydrolysis degree is measured by pH-stat method to be 19.38%.

[0041] Example 1. Preparation of probiotic microcapsules

[0042] Step a: Cultivation and activation of bifidobacteria: inoculate bifidobacteria into MRS liquid culture medium after high temperature sterilization and cooling, culture at 37°C for 24 hours, centrifuge at 3000 r / min and wash with 0.85% (v / v) physiological saline, collect concentrated bacterial solution, detect the number of viable bacteria in the bacterial solution by plate counting, and store at 4°C;

[0043] Step b: The zein hydrolysate F1 is prepared into a solution with a concentration of 10 mg / mL using distilled water, and feruloyl oligosaccharide is added to the solution to control the weight ratio of the two to be 6:2, ultrasonic treatment is performed for 5 minutes, and the reaction is terminated in a water bath at 70° C. for 30 minutes to obtain a wall material;

[0044] Step c: mixing the bacterial solution and the wall material obtained in step b in a weight ratio of 1:3, and homogenizing to obtain a feed solution;

[0045] Step d: spray drying the feed liquid to obtain probiotic microcapsules; the inlet air temperature of the spray drying is 180°C; and the outlet air temperature is 100°C.

[0046] The difference between Example 2 and Example 1 is that the weight ratio of zein hydrolysate F1 to feruloyl oligosaccharide is 6:1, and the other parameters are the same as those of Example 1.

[0047] Example 3 is different from Example 1 in that the weight ratio of zein hydrolysate F1 to feruloyl oligosaccharide is 6:3, and the other parameters are the same as those of Example 1.

[0048] Example 4 is different from Example 1 in that zein is used to replace the zein hydrolysate F1 in Example 1, and the other parameters are the same as those in Example 1.

[0049] Example 5 is different from Example 1 in that the zein hydrolysate F2 is used to replace the zein hydrolysate F1 in Example 1, and the other parameters are the same as those in Example 1.

[0050] Comparative Example 1 is different from Example 1 in that zein hydrolysate F3 is used to replace zein hydrolysate F1 in Example 1, and the other parameters are the same as those in Example 1.

[0051] Comparative Example 2 is different from Example 1 in that zein hydrolysate F4 is used to replace zein hydrolysate F1 in Example 1, and the other parameters are the same as those in Example 1.

[0052] Comparative Example 3 is different from Example 1 in that the zein hydrolysate F5 is used to replace the zein hydrolysate F1 in Example 1, and the other parameters are the same as those in Example 1.

[0053] Comparative Example 4 is different from Example 1 in that no feruloyl oligosaccharide is added to the wall material, and the other parameters are the same as those of Example 1.

[0054] Study 1: Determination of microcapsule embedding yield

[0055] 1 g of microcapsules of Examples 1 to 5 and Comparative Examples 1 to 4 were respectively added to 9 mL of preheated 0.1 mol / L PBS buffer (37° C., pH 7.4), shaken at 37° C. for 10 min until the microcapsules were completely decapsulated, and then gradiently diluted with sterile saline and viable bacteria were counted by plate counting method. The results are shown in Table 1 below.

[0056] Embedding yield % = G1×V1×M / (N0×V0×M0)×100%

[0057] Wherein, G1: number of viable bifidobacteria in 1 mL of decapsulation fluid (CFU / mL); V1: total volume of decapsulation fluid (mL); N0: number of viable bacteria per unit volume of original bacterial solution before embedding (CFU / mL); V0: volume of original bacterial solution used to prepare microcapsules (mL); M: total weight of obtained microcapsules (g); M0: weight of weighed microcapsules (g).

[0058] Table 1: Microcapsule embedding yield

[0059]

[0060]

[0061] The experiment shows that the embedding yield of zein as wall material alone is 52.33%, and the embedding yield of zein combined with Fos can reach 76.57%; the embedding rate of bifidobacterium embedded in zein hydrolysate with a hydrolysis degree of less than 20% and feruloyl oligosaccharide can reach more than 80%; further analysis of the data found that within the range exceeding 20%, with the increase of the hydrolysis degree of zein hydrolysate, the embedding yield of the composite wall material decreased significantly (see comparative examples 1 and 2); in addition, within the range of zein hydrolysis degree less than 20%, the embedding yield increased with the increase of feruloyl oligosaccharide content in the wall material, and the peak appeared when the ratio of the two was 6:2, at which time the embedding yield was 89.35%. The reason for this may be that the addition of feruloyl oligosaccharide makes the cross-linking between zein closer, improves the stability of the capsule wall, and improves the embedding yield.

[0062] Study 2: Simulated gastric fluid stress tolerance test

[0063] Take 16.4 mL of dilute hydrochloric acid, add about 800 mL of water and 10 g of pepsin, stir well and add water to make up to 1000 mL to obtain simulated gastric fluid; add 1 g of microcapsules to 9 mL of simulated gastric fluid, mix well, shake at 200 r / min in a shaker at 37°C for 4 hours, take out, wash with 0.85% saline, and count the viable bacteria using the plate count method, repeat 3 times, and the results are shown in Table 2 below.

[0064]

[0065] Wherein, W1 represents the number of viable bacteria in the microcapsule sample after being treated in simulated gastric fluid for a specific period of time; W2 represents the number of viable bacteria in the microcapsule sample before the acid resistance test.

[0066] Table 2: Acid resistance comparison results (%)

[0067]

[0068]

[0069] From the results, it can be seen that the use of zein hydrolyzate with a hydrolysis degree of less than 20% and feruloyl oligosaccharide as wall materials to embed bifidobacteria can greatly improve their survival rate in gastric juice. After being treated in gastric juice for 4 hours, the survival rate can still reach 90.17%, which significantly improves the acid resistance of bifidobacterium microcapsules; and the effect is significantly better than that of microcapsules formed by zein hydrolyzate with a hydrolysis degree of more than 20% and feruloyl oligosaccharide as wall materials. Analysis of the results of comparative example 3 shows that the type of enzyme in the enzymatic hydrolysis process has a greater impact on the acid resistance of the composite wall material. The acid resistance of the composite wall material formed by zein hydrolyzate and feruloyl oligosaccharide obtained by enzymatic hydrolysis of neutral protease is significantly worse than that of the composite wall material using alkaline protease.

[0070] Study 3: Enteric Coating Test

[0071] Take 6.8g of potassium dihydrogen phosphate, add 500mL of water, and use 0.4% sodium hydroxide solution to adjust the pH to 6.8; take another 10g of trypsin and add appropriate amount of water to dissolve, mix the two liquids, and add water to make up to 1000mL to obtain simulated intestinal fluid. Take 1g of microcapsules and add them to 9mL of simulated intestinal fluid, mix them thoroughly, shake them in a shaker at 37℃ and 200r / min for 30, 60, 90, and 120min, absorb 3mL of the solution, and measure its transmittance at a wavelength of 600nm. Repeat 3 times for each group. The results are shown in Table 3.

[0072] Table 3: Dissolution of different microcapsules in artificial simulated intestinal fluid

[0073]

[0074] Since the microcapsules are lysed and bifidobacteria are released, the substances in the solution increase and the transmittance decreases. It can be seen from the above table that the transmittance of the bifidobacterium microcapsules prepared in Example 1 drops rapidly to about 15% in the first 30 minutes, and then the transmittance remains basically stable in 60-120 minutes, indicating that the microcapsules can be completely disintegrated at about 60 minutes, with good dissolution performance and good enteric solubility. The transmittance of Comparative Examples 1 and 2 is basically stable after 30 minutes, and the complete disintegration time is slightly shorter than that of Example 1, which may be due to the increase in the hydrolysis degree of zein hydrolysate, The enteric solubility of the microcapsules is improved; the microcapsules of Example 4 group remain basically stable after 90 minutes.

[0075] Study 4: Bile Salt Tolerance Test

[0076] Sodium cholate was added to the simulated intestinal fluid to prepare sodium cholate solutions with concentrations of 0.3% (w / v) and 0.5% (w / v), which were sterilized at 121°C. 1 g of microcapsule samples from each group were placed in 9 mL of the above solution and incubated in a water bath at 37°C for 3 h. The bile was discarded, the microcapsules were collected, and after being completely disintegrated with cystolysis fluid, a bacterial suspension was prepared. The live bacteria were counted by the plate count method, and the survival rate was calculated. The results are shown in Table 4 below.

[0077]

[0078] Wherein, W1 represents the number of viable bacteria in the microcapsule sample after being treated in bile for 3 hours; W2 represents the number of viable bacteria in the microcapsule sample before the bile salt resistance test.

[0079] Table 4: Changes in the number of viable bacteria in microcapsules under different concentrations of bile (survival rate, %)

[0080] Group 0.3%(w / v) 0.5%(w / v) Embodiment 1 86.38％ 80.67％ Embodiment 2 83.24％ 78.02％ Embodiment 3 82.52％ 75.31％ Embodiment 4 55.38％ 30.12％ Embodiment 5 80.05％ 76.37％ Comparative Example 1 65.49％ 55.24％ Comparative Example 2 59.78％ 46.62％ Comparative Example 3 72.29％ 60.17％ Comparative Example 4 32.08％ 17.35％

[0081] It can be found from the above table that the tolerance of the microcapsules formed by the hydrolyzate of zein with a specific hydrolysis degree and feruloyl oligosaccharides to artificial bile is much greater than that of the combination of zein and feruloyl oligosaccharides. As the bile salt concentration increases, the number of viable bacteria in each group of microcapsules shows a downward trend. However, among the groups, the number of viable bacteria in Examples 1 to 3 and Example 5 groups after being treated in bile at 0.3% and 0.5% concentrations for 3 hours still remains in a relatively high range.

[0082] Study 5: Storage stability

[0083] Each group of microcapsules was stored at 4°C for 90 days, and after being completely disintegrated with a capsule disintegration solution, a bacterial suspension was prepared, and live bacteria were counted using a plate count method. The results are shown in Table 5 below.

[0084] Table 5 Changes in viable bacterial count after storage at 4°C for 90 days (survival rate, %)

[0085] Group 30d 90d Embodiment 1 93.18％ 86.31％ Embodiment 4 82.36％ 71.29％ Comparative Example 1 86.41％ 76.90％ Comparative Example 2 82.67％ 70.04％ Comparative Example 3 86.35％ 77.12％ Comparative Example 4 72.27％ 63.54％

[0086] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A stable probiotic microcapsule, characterized in that: It is prepared by the following steps: Step a: Cultivation and activation of bifidobacteria: inoculate bifidobacteria into MRS liquid culture medium after high temperature sterilization and cooling, culture at 37°C for 24 hours, centrifuge at 3000 r / min and wash with 0.85% (v / v) physiological saline, collect concentrated bacterial liquid, detect the number of viable bacteria in the bacterial liquid by plate counting, and store at 4°C; Step b: The zein hydrolysate F1 was prepared into a solution with a concentration of 10 mg / mL using distilled water, and feruloyl oligosaccharide was added to the solution to control the weight ratio of the two to be 6:2, and ultrasonic treatment was performed for 5 minutes, and the reaction was terminated in a water bath at 70° C. for 30 minutes to obtain a wall material; Step c: mixing the bacterial solution and the wall material obtained in step b in a weight ratio of 1:3, and homogenizing to obtain a feed solution; Step d: spray drying the feed liquid to obtain probiotic microcapsules; the inlet air temperature of the spray drying is 180°C; the outlet air temperature is 100°C; The preparation method of the corn alcohol-soluble protein hydrolysate F1 is as follows: corn alcohol-soluble protein is prepared into a suspension with a concentration of 10 mg / mL by using distilled water, the suspension is sealed in a sealed bag and placed in an ultrasonic tank for ultrasonic treatment for 5 minutes, and the ultrasonic power is controlled to be 35kHz; the pH value of the suspension after ultrasonic treatment is adjusted to 9.0 by using a 0.2mol / L sodium hydroxide solution, alkaline protease is added to the suspension for enzymolysis for 43 minutes, and the substrate concentration during the enzymolysis process is controlled to be 1.0g / (100mL), the enzyme-substrate ratio is 80mAU / g, and the temperature is 50°C; the degree of hydrolysis is measured during the enzymolysis process, and after the enzymolysis is completed, the pH value is adjusted to 7.0 by using a 0.2mol / L HCL solution; the enzyme is inactivated in a water bath at 90°C for 15 minutes; the hydrolyzate is collected after cooling to room temperature, centrifuged, and the supernatant is taken to obtain the corn alcohol-soluble protein hydrolysate F1; and the degree of hydrolysis is measured by the pH-stat method to be 12.52%.

2. A stable probiotic microcapsule, characterized in that: It is prepared by the following steps: Step a: Cultivation and activation of bifidobacteria: inoculate bifidobacteria into MRS liquid culture medium after high temperature sterilization and cooling, culture at 37°C for 24 hours, centrifuge at 3000 r / min and wash with 0.85% (v / v) physiological saline, collect concentrated bacterial liquid, detect the number of viable bacteria in the bacterial liquid by plate counting, and store at 4°C; Step b: The zein hydrolysate F2 is prepared into a solution with a concentration of 10 mg / mL using distilled water, and feruloyl oligosaccharide is added to the solution to control the weight ratio of the two to be 6:2, ultrasonic treatment is performed for 5 minutes, and the reaction is terminated in a water bath at 70° C. for 30 minutes to obtain a wall material; Step c: mixing the bacterial solution and the wall material obtained in step b in a weight ratio of 1:3, and homogenizing to obtain a feed solution; Step d: spray drying the feed liquid to obtain probiotic microcapsules; the inlet air temperature of the spray drying is 180°C; the outlet air temperature is 100°C; The preparation method of the corn alcohol-soluble protein hydrolysate F2 is as follows: corn alcohol-soluble protein is prepared into a suspension with a concentration of 10 mg / mL by using distilled water, the suspension is sealed in a sealed bag and placed in an ultrasonic tank for ultrasonic treatment for 8 minutes, and the ultrasonic power is controlled to be 40kHz; the pH of the above-mentioned suspension after ultrasonic treatment is adjusted to 9.0 by using 0.2mol / L sodium hydroxide solution, alkaline protease is added to the suspension for enzymolysis for 65 minutes, and the substrate concentration during the enzymolysis process is controlled to be 1.0g / (100mL), the enzyme-substrate ratio is 80mAU / g, and the temperature is 50°C; the degree of hydrolysis is measured during the enzymolysis process, and after the enzymolysis is completed, the pH value is adjusted to 7.0 by using 0.2mol / L HCL solution; the enzyme is kept in a water bath at 90°C for 15 minutes to inactivate the enzyme; the hydrolyzate is collected after cooling to room temperature, centrifuged, and the supernatant is taken to obtain the corn alcohol-soluble protein hydrolysate F2; the degree of hydrolysis is measured by the pH-stat method to be 17.16%.

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