Soybean peptide embedded probiotic particles targeting intestinal tracts and preparation method of soybean peptide embedded probiotic particles

Abstract

The invention discloses soybean peptide embedded probiotic particles targeting intestinal tracts and a preparation method of the soybean peptide embedded probiotic particles, belongs to the technical field of probiotics, and provides the soybean peptide embedded probiotic particles targeting intestinal tracts, which comprise probiotics, soluble soybean peptides and insoluble soybean peptide nano dispersed particles. The preparation method comprises the following steps: performing enzymolysis on a soybean protein isolate solution to obtain soluble soybean peptide and insoluble soybean peptide; the preparation method comprises the following steps: carrying out hydration and ice-bath ultrasonic treatment on insoluble soybean peptide to obtain an insoluble soybean peptide nano dispersed particle solution, mixing the insoluble soybean peptide nano dispersed particle solution with freeze-dried soluble soybean peptide, then mixing with lactobacillus rhamnosus, and carrying out spray drying to obtain the soybean peptide embedded probiotic particles. According to the invention, the soluble soybean peptide and the insoluble soybean peptide nano dispersed particles are compounded, and the lactobacillus rhamnosus is embedded, so that the activity, the gastrointestinal fluid digestion resistance, the storage stability and the intestinal cell adhesion capacity of the probiotics are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and in particular relates to intestinal targeted soybean peptide-embedded probiotic particles and a preparation method thereof. Background Art

[0002] Probiotics are an important health-promoting factor, which is defined as "a class of live microorganisms that can benefit the health of the host after oral administration in sufficient amounts." In recent years, people's health awareness has continued to improve, and the probiotic market has developed rapidly. Spray drying has the advantages of high efficiency, low energy consumption, low cost, and controllable particle morphology and size. It has broad prospects for being used to produce probiotic powder. However, heat stress, dehydration stress, and oxidative stress during the drying process can easily cause damage to bacterial cells and inactivation of a large number of them. The damaged bacteria have a weaker tolerance to digestive juices, making it impossible to achieve intestinal accessibility of live bacteria, resulting in loss of physiological functions, which greatly limits the development of the high-activity spray-dried probiotic powder manufacturing industry. Therefore, it is necessary to improve the activity level of spray-dried probiotics and the ability of intestinal targeted delivery.

[0003] Usually, probiotics are encapsulated with proteins or polysaccharides to alleviate the stress of probiotics during the spray drying process. However, polysaccharides have poor resistance to gastrointestinal stress, and animal proteins are not friendly to people with allergies. Soy protein is a plant-based protein with a wide range of sources and good functionality, and has good application potential. At present, researchers generally use soy protein isolate (SPI) in combination with other substances or improve its encapsulation ability for bioactive substances through enzymatic modification. The enzymatic hydrolysis products of SPI, soluble soy peptides (SSP) and insoluble soy peptides (ISP), are rich in nutrients, but ISP is insoluble in water and is often treated as waste, resulting in a waste of resources. SSP has extremely high water solubility. If it is used alone as a probiotic carrier to prepare granular powder, it may lead to a lack of effective protection of the bacteria in the digestive juice, and the hygroscopicity of the particles will also be high, and the practicality cannot be guaranteed. Therefore, how to effectively improve the activity of probiotics after drying and spraying remains to be solved. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a soybean peptide-embedded probiotic granule targeting the intestine and a preparation method thereof, wherein two enzymatic hydrolysates of soybean protein isolate (SPI), soluble soybean peptide (SSP) and insoluble soybean peptide nanodispersed particles (NISP), are used in combination as protective agents to embed Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), which improves its activity, resistance to gastrointestinal digestion, storage stability, and intestinal cell adhesion after spray drying.

[0005] To achieve the above object, the present invention provides a soybean peptide-embedded probiotic particle targeting the intestine, wherein the soybean peptide-embedded probiotic particle comprises probiotics and a soybean peptide composite carrier, and the soybean peptide composite carrier comprises soluble soybean peptide and insoluble soybean peptide nano-dispersed particles.

[0006] Preferably, the total effective viable count of the probiotics is 10 9 CFU / mL, the probiotic is Lactobacillus rhamnosus, and the Lactobacillus rhamnosus is Lactobacillus casei rhamnosus ( Lactobacillus rhamnosus ) GG strain.

[0007] Preferably, the soluble soybean peptide is obtained by enzymatic hydrolysis of isolated soybean protein, and the insoluble soybean peptide nano-dispersed particles are obtained by enzymatic hydrolysis of isolated soybean protein, hydration, and ice bath ultrasonic treatment.

[0008] The present invention also provides a method for preparing the soybean peptide-embedded probiotic particles, comprising the following steps: (1) The soy protein isolate solution is mixed with the composite protease, enzymatically hydrolyzed to obtain the soy protein isolate protease hydrolyzate, centrifuged three times, and the supernatant is combined as the soluble soy peptide, and the obtained precipitate is the insoluble soy peptide; (2) The poorly soluble soybean peptide obtained in step (1) is first freeze-dried to obtain a poorly soluble soybean peptide freeze-dried powder, the poorly soluble soybean peptide freeze-dried powder is mixed with ultrapure water, stirred, and hydrated at 4° C. for 10 to 14 hours to obtain a poorly soluble soybean peptide dispersion, and subjected to ice bath ultrasonic treatment to obtain a poorly soluble soybean peptide nano-dispersed particle solution; (3) The soluble soybean peptide obtained in step (1) is subjected to a second freeze-drying treatment to obtain a soluble soybean peptide freeze-dried powder, and the soluble soybean peptide freeze-dried powder is mixed with the insoluble soybean peptide nano-dispersed particle solution obtained in step (2) to obtain a soybean peptide composite carrier solution; (4) Lactobacillus rhamnosus is mixed with the soybean peptide composite carrier solution obtained in step (3) to obtain a bacterial suspension, and the mixture is spray-dried to obtain soybean peptide-encapsulated probiotic particles.

[0009] Preferably, the mass concentration of the soy protein isolate solution in step (1) is 3% to 5%; the mass ratio of the soy protein isolate solution to the composite protease in step (1) is 400:1.5; the enzymolysis in step (1) is carried out in a constant temperature water bath at 45 to 55° C., the pH is maintained constant during the enzymolysis process, and the enzymolysis time is 5 to 7 hours.

[0010] Preferably, the centrifugal force of the centrifugation in step (1) is 7500-8500 g, the centrifugal temperature is 20-30° C., and the centrifugal time is 10-20 min.

[0011] Preferably, the temperature of the first freeze-drying treatment in step (2) is -18~-25°C, and the time of the first freeze-drying treatment is 40~50h; the mixing ratio of the poorly soluble soybean peptide freeze-dried powder and ultrapure water in step (2) is 0.5~2g:100mL; the stirring time in step (2) is 1~3h, and the stirring speed is 800~1200rpm; the ultrasonic power of the ice bath ultrasonic treatment in step (2) is 180~220W, the output frequency of the ice bath ultrasonic treatment is 18~22kHz, the ice bath ultrasonic treatment is ultrasonicated for 1s and stopped for 2s, and the time of the ice bath ultrasonic treatment is 15~25min.

[0012] Preferably, the temperature of the second freeze-drying treatment in step (3) is -18~-25°C, and the time of the second freeze-drying treatment is 40~50h; the mixing ratio of the soluble soybean peptide freeze-dried powder and the insoluble soybean peptide nano-dispersed particle solution in step (3) is 1g:10mL.

[0013] Preferably, the Lactobacillus rhamnosus in step (4) is Lactobacillus rhamnosus GG strain, and the total effective viable count of Lactobacillus rhamnosus is 10 9 CFU / mL; the mixing ratio of the Lactobacillus rhamnosus and the soybean peptide composite carrier solution in step (4) is 1~3g:40~60mL.

[0014] Preferably, the feed rate of the spray drying in step (4) is 10-20 mL / min, the inlet air temperature of the spray drying is 120-160° C., and the spray drying time is 3-8 min.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention proposes a soybean peptide-encapsulated probiotic particle targeting the intestine and a preparation method thereof. Two enzymatic hydrolysis products of soybean protein isolate (SPI), soluble soybean peptide (SSP) and insoluble soybean peptide nanodispersed particles (NISP), are used as protective agents. After ultrasonic treatment, insoluble soybean peptide (ISP) can self-assemble to form nanodispersed particles (NISP), which have the potential for intestinal targeted delivery. Therefore, the "disadvantage" of ISP's water insolubility can be converted into an advantage, acting as a protective barrier for the bacteria in the later storage and digestion process. Encapsulation of Lactobacillus rhamnosus improves its activity after spray drying, resistance to gastrointestinal fluid digestion, storage stability, and intestinal cell adhesion.

[0016] 1) The present invention is the first to use two soybean peptides after SPI enzymatic hydrolysis to synergistically encapsulate probiotics, which helps to protect the activity of probiotics during spray drying, enhances the intestinal targeted delivery function of the powder when it is orally ingested, realizes resource recycling, and is green and environmentally friendly; 2) The probiotic microcapsule powder prepared by this method has a certain ability to resist gastric digestion stress and good intestinal cell adhesion ability, which helps the probiotics to colonize in the intestine and exert their probiotic effects. At the same time, the good storage stability helps to reduce production costs and improve the quality of probiotic powder products. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The figure is a graph showing the effect of different spray drying temperatures on the number of viable probiotic bacteria in the soybean peptide-embedded probiotic bacteria particles of Example 1, Example 2 and Comparative Example 1. In the figure, SSP-LGG represents Comparative Example 1, 0.5% NISP + SSP-LGG represents Example 2, and 2% NISP + SSP-LGG represents Example 1. In the figure, abcd represents the significance of the difference; Figure 2 Graph showing the effect of in vitro simulated digestion on the number of viable probiotic bacteria in soybean peptide-encapsulated probiotic particles, in which SSP-LGG-120°C represents Comparative Example 1, SSP-LGG-140°C represents Comparative Example 1 with the spray drying temperature changed to 140°C, SSP-LGG-160°C represents Comparative Example 1 with the spray drying temperature changed to 160°C, 0.5% NISP-SSP-LGG-120°C represents Example 2, 0.5% NISP-SSP-LGG-140°C represents Example 2 with the spray drying temperature changed to 140°C, 0.5% NISP-SSP-LGG-160°C represents Example 2 with the spray drying temperature changed to 160°C, 2% NISP-SSP-LGG-120°C represents Example 1, 2% NISP-SSP-LGG-140°C represents Example 1 with the spray drying temperature changed to 140°C, and 2% NISP-SSP-LGG-160°C represents Example 1 with the spray drying temperature changed to 160°C; Figure 3It is the storage stability of the soybean peptide-embedded probiotic particles treated with different spray drying temperatures. In the figure, 2% NPL-120℃-4℃ represents that the soybean peptide-embedded probiotic particles in Example 1 are stored at 4℃, and 2% NPL-140℃-4℃ in the figure represents that the soybean peptide-embedded probiotic particles prepared by Example 1 with the spray drying temperature changed to 140℃ are stored at 4℃, and 2% NPL-160℃-4℃ in the figure represents that the soybean peptide-embedded probiotic particles prepared by Example 1 with the spray drying temperature changed to 160℃ are stored at 4℃, and 2% NPL-120℃-25℃ in the figure represents that the soybean peptide-embedded probiotic particles in Example 1 are stored at 25℃, and 2% NPL-140℃-25℃ in the figure represents that the soybean peptide-embedded probiotic particles prepared by Example 1 with the spray drying temperature changed to 140℃ are stored at 25℃, and 2% NPL-160℃-25℃ in the figure represents that the soybean peptide-embedded probiotic particles prepared by Example 1 with the spray drying temperature changed to 160℃ are stored at 25℃; Figure 4 This figure shows the effect of different spray drying temperature treatments on the adhesion ability of Caco-2 cells of soy peptide-embedded probiotic particles. In the figure, LGG represents the untreated Lactobacillus rhamnosus GG strain, 2% NPL-120°C represents the soy peptide-embedded probiotic particles prepared in Example 1, 2% NPL-140°C represents the soy peptide-embedded probiotic particles prepared in Example 1 with the spray drying temperature changed to 140°C, 2% NPL-160°C represents the soy peptide-embedded probiotic particles prepared in Example 1 with the spray drying temperature changed to 160°C, and ab in the figure represents the significance of the difference. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] The source of the materials used in the present invention: Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) GG strain was purchased from American Culture Company; soy protein isolate was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; composite protease Protamex was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; MRS broth medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; pepsin and trypsin were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; potassium chloride, potassium dihydrogen phosphate, sodium bicarbonate, sodium chloride, magnesium chloride hexahydrate, anhydrous calcium chloride and ammonium carbonate were purchased from Sinopharm Chemical Reagent Co., Ltd.; constant temperature oscillator was purchased from Qun'an Experimental Instrument Co., Ltd.; spray dryer was purchased from Shanghai Yacheng Instrument Equipment Co., Ltd.

[0025] The preparation of the simulated gastric fluid (SGF) stock solution and the simulated intestinal fluid (SIF) stock solution used in the present invention is shown in Table 1 below.

[0026] Table 1 Formulas of simulated gastric fluid stock solution and simulated intestinal fluid stock solution ;

[0027] The Lactobacillus rhamnosus GG strain used in the experiment of the present invention: the Lactobacillus rhamnosus GG strain stored in glycerol was taken out of the -80°C refrigerator, thawed, inoculated into MRS broth medium, and statically cultured at 37°C for 12 hours. Subsequently, the bacterial suspension was transferred to an MRS agar plate (containing 52g / L MRS broth powder and 20g / L agar). The inoculated plate was statically cultured at 37°C for 48 hours, and then stored at 4°C, and the plate was streaked and subcultured every week. The single colony on the plate was transferred to 10mL of sterile MRS broth medium, and then statically cultured at 37°C for 12 hours. The resulting bacterial suspension was transferred to fresh MRS broth medium with an inoculation volume of 2% (v / v), and incubated at 37°C for 24 hours to ensure that the cell concentration reached 10 9 CFU / mL or more. Subsequently, the mixture was centrifuged at 4°C and 8000 g for 10 min to collect the precipitated Lactobacillus rhamnosus GG strain (LGG), and then the precipitate was washed twice with 0.85% (w / v) NaCl solution under the same centrifugal conditions to remove impurities, and the Lactobacillus rhamnosus GG strain was obtained for use.

[0028] Example 1 (1) Accurately weigh a certain amount of soy protein isolate and dissolve it in deionized water. Stir it at room temperature for 2 hours to obtain a soy protein isolate solution with a mass concentration of 4%. The soy protein isolate solution and the composite protease are mixed at a mass ratio of 400:1.5, and enzymolyzed in a water bath in a constant temperature oscillator at 50°C for 6 hours. During the enzymolysis process, 1M NaOH or 1M HCl is used to maintain a constant pH. After the enzymolysis is completed, the pH of the enzymolysis solution is adjusted to 7 with 1M NaOH or 1M HCl, and the solution is placed in a 90°C water bath and heated for 10 minutes to inactivate the enzyme. The solution is cooled to room temperature in an ice bath to obtain a soy protein isolate hydrolyzate. The solution is centrifuged at 8000g at 25°C for 10 minutes for three times. The supernatants are combined as soluble soy peptides, and the precipitates are insoluble soy peptides. (2) The poorly soluble soybean peptide was first freeze-dried at -20°C for 48 hours to obtain a poorly soluble soybean peptide freeze-dried powder, the poorly soluble soybean peptide freeze-dried powder was mixed with ultrapure water at a ratio of 2 g:100 mL, stirred at 1000 rpm for 2 hours, and hydrated at 4°C for 12 hours to obtain a poorly soluble soybean peptide dispersion, which was subjected to a probe-type ice bath ultrasonic treatment with an ultrasonic power of 200 W and an output frequency of 20 kHz. The ultrasonic treatment was performed for 1 second and then 2 seconds. The temperature was controlled below 25°C during the entire ultrasonic process. The ultrasonic treatment was performed for 20 minutes to obtain a poorly soluble soybean peptide nano-dispersed particle solution with a mass concentration of 2%; (3) Soluble soybean peptide, second freeze-drying treatment at -20°C for 48 hours to obtain soluble soybean peptide freeze-dried powder, the soluble soybean peptide freeze-dried powder and the insoluble soybean peptide nano-dispersed particle solution are mixed at a ratio of 1g:10mL to obtain a soybean peptide composite carrier solution; (4) Lactobacillus rhamnosus GG strain was mixed with soybean peptide composite carrier solution in a ratio of 2 g: 50 mL to obtain bacterial suspension. The spray drying instrument was cleaned, and then the heater was turned on until the inlet air temperature reached 120 °C and then operated for 15 min. The feed rate was set to 15 mL / min and the fan speed was 30 m / min. 3 / h, the inlet air temperature was set to 120℃ and spray-dried for 5 minutes, and the bacterial powder was collected immediately to obtain soybean peptide-encapsulated probiotic granules 2% NISP-10% SSP-LGG (2% NPL), which were stored in a refrigerator at 4℃.

[0029] Example 2 (1) Accurately weigh a certain amount of soy protein isolate and dissolve it in deionized water. Stir it at room temperature for 2 hours to obtain a soy protein isolate solution with a mass concentration of 4%. The soy protein isolate solution and the composite protease are mixed at a mass ratio of 400:1.5, and enzymolyzed in a water bath in a constant temperature oscillator at 50°C for 6 hours. During the enzymolysis process, 1M NaOH or 1M HCl is used to maintain a constant pH. After the enzymolysis is completed, the pH of the enzymolysis solution is adjusted to 7 with 1M NaOH or 1M HCl, and the solution is placed in a 90°C water bath and heated for 10 minutes to inactivate the enzyme. The solution is cooled to room temperature in an ice bath to obtain a soy protein isolate hydrolyzate. The solution is centrifuged at 8000g at 25°C for 10 minutes for three times. The supernatants are combined as soluble soy peptides, and the precipitates are insoluble soy peptides. (2) The poorly soluble soybean peptide was first freeze-dried at -20°C for 48 hours to obtain a poorly soluble soybean peptide freeze-dried powder, the poorly soluble soybean peptide freeze-dried powder was mixed with ultrapure water at a ratio of 0.5 g:100 mL, stirred at 1000 rpm for 2 hours, and hydrated at 4°C for 12 hours to obtain a poorly soluble soybean peptide dispersion, which was subjected to probe-type ice bath ultrasonic treatment with an ultrasonic power of 200 W and an output frequency of 20 kHz. The ultrasonic treatment was performed for 1 second and then 2 seconds. The temperature was controlled below 25°C during the entire ultrasonic process. The ultrasonic treatment lasted for 20 minutes to obtain a poorly soluble soybean peptide nano-dispersed particle solution with a mass concentration of 0.5%; (3) Soluble soybean peptide, second freeze-drying treatment at -20°C for 48 hours to obtain soluble soybean peptide freeze-dried powder, the soluble soybean peptide freeze-dried powder and the insoluble soybean peptide nano-dispersed particle solution are mixed at a ratio of 1g:10mL to obtain a soybean peptide composite carrier solution; (4) Lactobacillus rhamnosus GG strain was mixed with soybean peptide composite carrier solution in a ratio of 2 g: 50 mL to obtain bacterial suspension. The spray drying instrument was cleaned, and then the heater was turned on until the inlet air temperature reached 120 °C and then operated for 15 min. The feed rate was set to 15 mL / min and the fan speed was 30 m / min. 3 / h, the inlet air temperature was set to 120℃ and spray-dried for 5 minutes, and the bacterial powder was collected immediately to obtain soybean peptide-encapsulated probiotic granules 0.5% NISP-10% SSP-LGG (0.5% NPL), which were stored in a refrigerator at 4℃.

[0030] Example 3 (1) Accurately weigh a certain amount of soy protein isolate and dissolve it in deionized water. Stir it at room temperature for 2 hours to obtain a soy protein isolate solution with a mass concentration of 3%. The soy protein isolate solution and the composite protease are mixed at a mass ratio of 400:1.5, and enzymolyzed in a water bath in a constant temperature oscillator at 45°C for 7 hours. During the enzymolysis process, 1M NaOH or 1M HCl is used to maintain a constant pH. After the enzymolysis is completed, the pH of the enzymolysis solution is adjusted to 7 with 1M NaOH or 1M HCl, and the solution is placed in a 90°C water bath and heated for 10 minutes to inactivate the enzyme. The solution is cooled to room temperature in an ice bath to obtain a soy protein isolate hydrolyzate. The solution is centrifuged at 7500g at 20°C for 15 minutes for three times. The supernatants are combined as soluble soy peptides, and the precipitates are insoluble soy peptides. (2) The poorly soluble soybean peptide was first freeze-dried at -18°C for 48 hours to obtain a poorly soluble soybean peptide freeze-dried powder, the poorly soluble soybean peptide freeze-dried powder was mixed with ultrapure water at a ratio of 1 g: 100 mL, stirred at 1000 rpm for 1 hour, and hydrated at 4°C for 10 hours to obtain a poorly soluble soybean peptide dispersion, which was subjected to probe-type ice bath ultrasonic treatment with an ultrasonic power of 180 W and an output frequency of 18 kHz, with an ultrasonic cycle of 1 second and a rest of 2 seconds. During the entire ultrasonic process, the temperature was controlled below 25°C and the ultrasonic process was performed for 25 minutes to obtain a poorly soluble soybean peptide nano-dispersed particle solution with a mass concentration of 1%; (3) Soluble soybean peptide, second freeze-drying treatment at -18°C for 48 hours to obtain soluble soybean peptide freeze-dried powder, the soluble soybean peptide freeze-dried powder and the insoluble soybean peptide nano-dispersed particle solution are mixed at a ratio of 1g:10mL to obtain a soybean peptide composite carrier solution; (4) Lactobacillus rhamnosus GG strain and soybean peptide composite carrier solution were mixed in a ratio of 2g:50mL to obtain a bacterial suspension. The spray drying instrument was cleaned, and then the heater was turned on until the inlet air temperature reached 120°C and then operated for 15 minutes. The feed rate was set to 10mL / min and the fan speed was 30m 3 / h, the inlet air temperature was set to 140℃ and spray-dried for 5 min, the bacterial powder was collected immediately to obtain soybean peptide-encapsulated probiotic particles, which were stored in a refrigerator at 4℃.

[0031] Example 4 (1) Accurately weigh a certain amount of soy protein isolate and dissolve it in deionized water. Stir it at room temperature for 2 hours to obtain a soy protein isolate solution with a mass concentration of 5%. The soy protein isolate solution and the composite protease are mixed in a mass ratio of 98.5:1.5, and enzymolyzed in a water bath in a constant temperature oscillator at 55°C for 5 hours. During the enzymolysis process, 1M NaOH or 1M HCl is used to maintain a constant pH. After the enzymolysis is completed, the pH of the enzymolysis solution is adjusted to 7 with 1M NaOH or 1M HCl, and the solution is placed in a 90°C water bath and heated for 10 minutes to inactivate the enzyme. The solution is cooled to room temperature in an ice bath to obtain a soy protein isolate hydrolyzate. The solution is centrifuged at 8500g at 25°C for 5 minutes for three times. The supernatants are combined as soluble soy peptides, and the precipitates are insoluble soy peptides. (2) The poorly soluble soybean peptide was first freeze-dried at -25°C for 48 hours to obtain a poorly soluble soybean peptide freeze-dried powder, the poorly soluble soybean peptide freeze-dried powder was mixed with ultrapure water at a ratio of 1 g: 100 mL, stirred at 1000 rpm for 3 hours, and hydrated at 4°C for 14 hours to obtain a poorly soluble soybean peptide dispersion, which was subjected to probe-type ice bath ultrasonic treatment with an ultrasonic power of 220 W and an output frequency of 22 kHz. The ultrasonic treatment was performed for 1 second and then for 2 seconds. The temperature was controlled below 25°C during the entire ultrasonic process and the ultrasonic treatment was performed for 25 minutes to obtain a poorly soluble soybean peptide nano-dispersed particle solution with a mass concentration of 1%; (3) Soluble soybean peptide, second freeze-drying treatment at -25°C for 48 hours to obtain soluble soybean peptide freeze-dried powder, the soluble soybean peptide freeze-dried powder and the insoluble soybean peptide nano-dispersed particle solution are mixed at a ratio of 1g:10mL to obtain a soybean peptide composite carrier solution; (4) Lactobacillus rhamnosus GG strain was mixed with soybean peptide composite carrier solution in a ratio of 2 g: 50 mL to obtain bacterial suspension. The spray drying instrument was cleaned, and then the heater was turned on until the inlet air temperature reached 120 °C and then operated for 15 min. The feed rate was set to 20 mL / min and the fan speed was 30 m / min. 3 / h, the inlet air temperature was set to 160℃ and spray-dried for 5 min, the bacterial powder was collected immediately to obtain soybean peptide-encapsulated probiotic particles, which were stored in a refrigerator at 4℃.

[0032] Comparative Example 1 (1) Accurately weigh a certain amount of soy protein isolate and dissolve it in deionized water. Stir it at room temperature for 2 hours to obtain a soy protein isolate solution with a mass concentration of 4%. The soy protein isolate solution and the composite protease are mixed at a mass ratio of 98.5:1.5, and enzymolyzed in a water bath in a constant temperature oscillator at 50°C for 6 hours. During the enzymolysis process, 1M NaOH or 1M HCl is used to maintain a constant pH. After the enzymolysis is completed, the pH of the enzymolysis solution is adjusted to 7 with 1M NaOH or 1M HCl, and the solution is placed in a 90°C water bath and heated for 10 minutes to inactivate the enzyme. The solution is cooled to room temperature in an ice bath to obtain a soy protein isolate hydrolyzate, and the solution is centrifuged at 8000g at 25°C for 10 minutes for 3 times. The supernatants are combined to obtain soluble soybean peptides. (2) Soluble soybean peptide, second freeze-drying treatment at -20°C for 48 hours to obtain soluble soybean peptide freeze-dried powder, and the soluble soybean peptide freeze-dried powder is mixed with ultrapure water at a ratio of 1g:10mL to obtain a soluble soybean peptide solution; (4) Lactobacillus rhamnosus GG strain was mixed with soluble soybean peptide solution at a ratio of 2 g:50 mL to obtain a bacterial suspension. The spray drying apparatus was cleaned and the heater was turned on until the inlet air temperature reached 120 °C and then operated for 15 min. The feed rate was set to 15 mL / min and the fan speed was 30 m / min. 3 / h, the inlet air temperature was set to 120℃ and spray-dried for 5 minutes, and the bacterial powder was collected immediately to obtain soybean peptide-encapsulated probiotic particles 10% SSP-LGG (SPL), which were stored in a refrigerator at 4℃.

[0033] Experimental example 1. Bacterial activity test Accurately weigh 0.01 g of sample (2% NPL, 0.5% NPL and SPL of soybean peptide-embedded probiotic granules prepared in Example 1, Example 2 and Comparative Example 1) and add it to 1 mL of sterile saline, dilute and spread it, and culture it in a 37°C incubator for 48 hours before reading the number of single colonies to determine bacterial activity.

[0034] The preparation methods described in Example 1, Example 2 and Comparative Example 1 were adjusted to spray drying temperatures of 140° C. and 160° C. to prepare soybean peptide-embedded probiotic particles, respectively, and the bacterial activity was determined in the same manner as above.

[0035] The results are as follows Figure 1 As shown in the figure, after spray drying, the number of live LGG bacteria in the three particles is higher than 9 log CFU / g, where SSP-LGG represents the SPL of Comparative Example 1, 0.5% NISP + SSP-LGG represents the 0.5% NPL of Example 2, and 2% NISP + SSP-LGG represents the 2% NPL of Example 1. Among them, the retention of live bacteria in the 2% NPL sample of Example 1 is more prominent at an inlet air temperature of 120°C. The encapsulated body has a high resistance to spray drying stress, which verifies the effectiveness of the encapsulation system in protecting the activity of LGG.

[0036] 2. Test of resistance to digestive stress To prepare simulated gastric fluid (SGF), take 8 mL of SGF stock solution, add 5 μL of 0.3 M CaCl2 solution (final concentration of 0.15 mmol / L), 1.95 mL of ultrapure water, adjust the solution pH to 3.0, and then add 50 mg of pepsin (enzyme activity 2000 U / mL).

[0037] To prepare simulated intestinal fluid (SIF), take 8 mL of SIF stock solution, add 20 μL of 0.3 M CaCl2 solution (final concentration 0.6 mmol / L), 0.08 g of bile salt, and 1.945 mL of ultrapure water to make the solution 10 mL, adjust the solution pH to 7.0, and then add 125 mg of pancreatic enzyme (enzyme activity 100 U / mL).

[0038] The preparation methods described in Example 1, Example 2 and Comparative Example 1 were used to prepare soybean peptide-embedded probiotic particles respectively after adjusting the spray drying temperature to 140° C. and 160° C.

[0039] Accurately weigh 0.1 g of the spray-dried soybean peptide-embedded probiotic granule sample and add it to 10 mL of SGF working solution with a pH of 3.0, and shake it in a water bath at 37°C and 50 rpm for 2 h.

[0040] After the simulated gastric digestion phase, 10 mL of SIF working solution was added to the gastric digestion product, and the pH of the final mixture was adjusted to 7.0, and then the mixture was shaken in a water bath for 2 h under the same conditions. 200 μL of samples were taken at 0, 1, and 2 h after simulated gastric digestion, and 1 and 2 h after simulated intestinal digestion. The samples were counted for viable cells. Each simulated digestion experiment was performed in triplicate.

[0041] The results are as follows Figure 2 As shown in the figure, after 2 hours of simulated gastric digestion, the survival rate of LGG in all groups was maintained above 80%, and the number of viable bacteria was above 8 log CFU / g. This shows that under the spray drying conditions of 120~160℃, the prepared soybean peptide-encapsulated probiotic granules powders have good resistance to gastric digestion. After 4 hours of simulated digestion, the survival rate of microencapsulated LGG was above 40.9%, and the number of viable LGG in the soybean peptide-encapsulated probiotic granules (2% NPL-120℃) prepared in Example 1 was still 6 logCFU / mL, which shows that the SSP-NISP encapsulation system showed a certain protective effect during the in vitro digestion of LGG.

[0042] 3. Storage stability The preparation method described in Example 1 was used, and the spray drying temperature was adjusted to 140° C. and 160° C. to prepare soybean peptide-embedded probiotic particles respectively.

[0043] The soybean peptide-encapsulated probiotic granules were stored in a dry environment at 4°C and 25°C. Every 2 weeks, 0.01 g of dry bacterial powder was taken and dissolved in 1 mL of sterile saline to reconstitute the powder sample. The plate count method was used to determine the change in the number of viable bacteria during the storage period.

[0044] The results are as follows Figure 3As shown, the soybean peptide-embedded probiotic particles prepared in Example 1 and the spray drying temperature were adjusted to 140°C and 160°C to prepare the soybean peptide-embedded probiotic particles, which were stored at 4°C for 8 weeks. Figure 3 The survival rate of LGG was still as high as 97% at 2% NPL-120℃-4℃, 2% NPL-140℃-4℃ and 2% NPL-160℃-4℃, indicating that the soybean peptide-embedded probiotic particles prepared by the preparation method of the present invention have good storage stability.

[0045] 4. Intestinal cell adhesion ability The preparation method described in Example 1 was used, and the spray drying temperature was adjusted to 140° C. and 160° C. to prepare soybean peptide-embedded probiotic particles respectively.

[0046] Caco-2 cell monolayer was used to simulate the human intestinal environment. Soy peptide-encapsulated probiotic particles were resuspended in sterile water, and LGG cells were collected by centrifugation and resuspended in PBS. Cells were cultured to a monolayer in a 12-well plate, 1 mL of LGG cell suspension was added, and incubated at 37°C for 2 h. The monolayer was washed with PBS to remove free LGG cells. Subsequently, 0.25% trypsin-0.02% EDTA solution was added and incubated at 37°C for 5 min to break the monolayer. The mixture was collected in a centrifuge tube, and the number of viable adherent bacteria was calculated by the plate count method.

[0047] The results are as follows Figure 4 As shown, compared with LGG that has not been spray-dried, the soybean peptide-embedded probiotic particles prepared in Example 1 and the soybean peptide-embedded probiotic particles prepared by adjusting the spray drying temperature to 140°C and 160°C, that is, Figure 4 At 2% NPL-120℃, 2% NPL-140℃ and 2% NPL-160℃, LGG still has good adhesion ability to Caco-2 cells.

[0048] The present invention utilizes two kinds of soybean peptides obtained after proteolysis of soybean isolated proteins for the first time to synergistically encapsulate probiotics, which helps to protect the activity of the probiotics during spray drying; the soybean peptide-encapsulated probiotics prepared by the present invention have a certain ability to resist gastric digestion stress and good intestinal cell adhesion ability, which helps the probiotics to colonize in the intestine and exert their probiotic effects, and at the same time, good storage stability helps to reduce production costs and improve the quality of probiotic powder products.

[0049] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A soybean peptide-embedded probiotic granule targeting the intestinal tract, characterized in that: The soybean peptide-embedded probiotic particles comprise probiotics and a soybean peptide composite carrier, and the soybean peptide composite carrier comprises soluble soybean peptide and insoluble soybean peptide nano-dispersed particles.

2. The soybean peptide-embedded probiotic granules according to claim 1, characterized in that: The total effective viable count of the probiotics is 10 9 CFU / mL, the probiotic is Lactobacillus rhamnosus, and the Lactobacillus rhamnosus is Lactobacillus casei rhamnosus ( Lactobacillus rhamnosus ) GG strain.

3. The soybean peptide-embedded probiotic granules according to claim 1, characterized in that: The soluble soybean peptide is obtained by enzymatic hydrolysis of isolated soybean protein, and the insoluble soybean peptide nano-dispersed particles are obtained by enzymatic hydrolysis of isolated soybean protein, hydration, and ice bath ultrasonic treatment.

4. The method for preparing soybean peptide-embedded probiotic particles according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) The soy protein isolate solution is mixed with the composite protease, enzymatically hydrolyzed to obtain the soy protein isolate protease hydrolyzate, centrifuged three times, and the supernatant is combined as the soluble soy peptide, and the obtained precipitate is the insoluble soy peptide; (2) The poorly soluble soybean peptide obtained in step (1) is first freeze-dried to obtain a poorly soluble soybean peptide freeze-dried powder, the poorly soluble soybean peptide freeze-dried powder is mixed with ultrapure water, stirred, and hydrated at 4° C. for 10 to 14 hours to obtain a poorly soluble soybean peptide dispersion, and subjected to ice bath ultrasonic treatment to obtain a poorly soluble soybean peptide nano-dispersed particle solution; (3) The soluble soybean peptide obtained in step (1) is subjected to a second freeze-drying treatment to obtain a soluble soybean peptide freeze-dried powder, and the soluble soybean peptide freeze-dried powder is mixed with the insoluble soybean peptide nano-dispersed particle solution obtained in step (2) to obtain a soybean peptide composite carrier solution; (4) Lactobacillus rhamnosus is mixed with the soybean peptide composite carrier solution obtained in step (3) to obtain a bacterial suspension, and the mixture is spray-dried to obtain soybean peptide-encapsulated probiotic particles.

5. The preparation method according to claim 4, characterized in that: The mass concentration of the soy protein isolate solution in step (1) is 3% to 5%; the mass ratio of the soy protein isolate solution to the composite protease in step (1) is 400:1.5; the enzymolysis in step (1) is carried out in a constant temperature water bath at 45 to 55° C., the pH is maintained constant during the enzymolysis process, and the enzymolysis time is 5 to 7 hours.

6. The preparation method according to claim 4, characterized in that: The centrifugal force of the centrifugation in step (1) is 7500-8500 g, the centrifugal temperature is 20-30° C., and the centrifugal time is 10-20 min.

7. The preparation method according to claim 4, characterized in that: The temperature of the first freeze-drying treatment in step (2) is -18~-25°C, and the time of the first freeze-drying treatment is 40~50h; the mixing ratio of the poorly soluble soybean peptide freeze-dried powder and ultrapure water in step (2) is 0.5~2g:100mL; the stirring time in step (2) is 1~3h, and the stirring speed is 800~1200rpm; the ultrasonic power of the ice bath ultrasonic treatment in step (2) is 180~220W, the output frequency of the ice bath ultrasonic treatment is 18~22kHz, the ice bath ultrasonic treatment is ultrasonicated for 1s and stopped for 2s, and the time of the ice bath ultrasonic treatment is 15~25min.

8. The preparation method according to claim 4, characterized in that: The temperature of the second freeze-drying treatment in step (3) is -18~-25°C, and the time of the second freeze-drying treatment is 40~50h; the mixing ratio of the soluble soybean peptide freeze-dried powder and the insoluble soybean peptide nano-dispersed particle solution in step (3) is 1g:10mL.

9. The preparation method according to claim 4, characterized in that: The Lactobacillus rhamnosus in step (4) is Lactobacillus rhamnosus GG strain, and the total effective viable count of Lactobacillus rhamnosus is 10 9 CFU / mL; the mixing ratio of the Lactobacillus rhamnosus and the soybean peptide composite carrier solution in step (4) is 1~3g:40~60mL.

10. The preparation method according to claim 4, characterized in that: The feed rate of the spray drying in step (4) is 10-20 mL / min, the inlet air temperature of the spray drying is 120-160° C., and the spray drying time is 3-8 min.

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