Probiotic microcapsule and preparation method thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a probiotic microcapsule and a preparation method thereof. The preparation method comprises the following steps: preparing a bacterial suspension of probiotics; mixing a polyvinyl alcohol solution, nanocellulose and nanocellulose crystals, and heating in a water bath until the mixture is completely dissolved to prepare a PCC suspension; the obtained PCC suspension liquid is subjected to high-pressure sterilization, and the nanocellulose-based hydrogel microcapsule is obtained; and mixing the nanocellulose-based hydrogel microcapsule with a bacterial suspension to obtain the probiotic microcapsule. The preparation method has the beneficial effects that the nano-crystalline cellulose fibers, the nano-crystalline cellulose crystals and the polyvinyl alcohol are combined to form a compact network structure, so that the stability and the activity of the probiotics in a severe environment can be effectively enhanced. Furthermore, the prepared nanocellulose microcapsule encapsulates the probiotics, so that the survival rate of the probiotics under freeze drying and heating conditions can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a probiotic microcapsule and a preparation method thereof. Background Art

[0002] As a key component of the human intestinal microbiome, probiotics help maintain and enhance the balance of intestinal microorganisms. The appropriate intake of active probiotics can promote intestinal health, protect the intestinal mucosa from pathogens and other harmful microorganisms, and enhance the immune system. Specific probiotic strains can also regulate the metabolism of nutrients, bringing additional benefits such as regulating blood sugar and controlling blood pressure, which is essential for maintaining overall health. Studies have found that the activity of probiotics may be significantly reduced during digestion, especially in the acidic environment of the stomach, which may result in the inability of active probiotics to reach sufficient numbers in the small intestine to exert the expected benefits. To overcome this problem, researchers have proposed using microencapsulation technology to create a protective coating to protect probiotics from harsh external conditions and increase their survival rate in the intestine.

[0003] As renewable green nanomaterials, nanocellulose fibers (CNF) and nanocellulose crystals (CNC) have great potential in probiotic delivery. Studies have found that insoluble fibers can be treated with acid to remove lignin and hemicellulose to obtain CNF, and further acid hydrolysis to destroy the non-crystalline region can obtain CNC. Since the acid ions replace the surface hydroxyl groups of nanocellulose after acid treatment, it has rich negative charges and unique biocompatibility and physicochemical properties. CNC can prevent cell damage caused by rapid freezing and ice crystal formation during freeze-drying. Using whey protein isolate (WPI), CNC and inulin (INU) as wall materials, microcapsules prepared by freeze-drying process can further improve the survival rate of probiotics in the gastrointestinal tract. In addition, CNC-doped microgels significantly enhance the resistance of probiotics by filling pores to reduce the entry of gastric acid. When nanocellulose is combined with other macromolecules such as polyvinyl alcohol (PVA), new microcapsules can be prepared by electrospinning, which show excellent performance in protecting probiotics and promoting intestinal colonization. However, the stability of probiotics encapsulated in the existing composite fiber materials formed by nanocellulose fibers (CNF) and PVA is still not high enough, and cannot guarantee the stability and activity of probiotics in harsh environments. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a probiotic microcapsule which can improve the stability and activity of probiotics under freeze-drying and heating conditions and a preparation method thereof.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a probiotic microcapsule and a preparation method thereof, comprising the following steps: 1) Preparing a probiotic bacterial suspension; 2) heating the polyvinyl alcohol solution, nanocellulose fibers and nanocellulose crystals in a water bath until they are completely dissolved to prepare a PCC suspension; the PCC suspension is evenly dispersed under the action of a magnetic stirrer, and then ultrasonically dispersed; 3) sterilizing the PCC suspension obtained in step 2) by high pressure sterilization at 110-130° C. to obtain nanocellulose-based hydrogel microcapsules; 4) mixing the nanocellulose-based hydrogel microcapsules with a bacterial suspension, using a vortex oscillator to fully and evenly disperse the bacteria in the bacterial suspension into the nanocellulose-based hydrogel microcapsules, incubating in a shaker and then freezing, and freeze-drying to obtain probiotic microcapsules.

[0006] Furthermore, in the preparation method of the above-mentioned probiotic microcapsules, the step 1) is specifically as follows: after the probiotic strains stored at -80°C are thawed, they are cultured at 37°C for 24-48 hours to activate to the third generation, the third generation bacterial solution is aspirated and centrifuged to remove the culture medium, the sterile saline is centrifuged and washed twice, and the bacterial suspension is resuspended with sterile saline to prepare a bacterial suspension for use.

[0007] Furthermore, in the above-mentioned method for preparing probiotic microcapsules, the centrifugation setting conditions of step 1) are: 5500 rpm, 15 min, and the probiotics are one or more of Bifidobacterium, Lactobacillus fermentum or Lactobacillus plantarum.

[0008] Furthermore, in the above-mentioned method for preparing probiotic microcapsules, the concentrations of the solutes in the PCC suspension in step 2) are as follows: the nanocellulose fiber content is 0.5wt%, the nanocellulose crystal content is 5wt%, and the polyvinyl alcohol content is 4wt%.

[0009] Furthermore, in the above-mentioned method for preparing probiotic microcapsules, the conditions for ultrasonic dispersion in step 2) are temperature 40-50° C., power 200 W, and time 2 h.

[0010] Furthermore, in the above-mentioned method for preparing probiotic microcapsules, the step 3) is specifically: sterilizing the PCC suspension obtained in step 2) under high pressure at 121° C. for 15 minutes to obtain nanocellulose-based hydrogel microcapsules.

[0011] Furthermore, in the preparation method of the above-mentioned probiotic microcapsules, the step 4) is specifically: mixing the nanocellulose-based hydrogel microcapsules and the bacterial suspension in a volume ratio of 1:1, using a vortex oscillator to fully and evenly disperse the bacteria in the bacterial suspension in the nanocellulose-based hydrogel microcapsules, incubating in a shaker at 37°C for 1 hour, freezing at -80°C for 12 hours, and freeze-drying for 24 hours to obtain the probiotic microcapsules.

[0012] Furthermore, in the above-mentioned method for preparing probiotic microcapsules, the method for preparing nanocellulose fibers comprises the following steps: Grinding: Grinding the bamboo shoots into powder to obtain bamboo shoot powder; Degreasing: Soak bamboo shoot powder in 4 volumes of ethyl acetate for 1 h to remove lipids, and filter with distilled water three times to obtain a defatted sample; Washing: The defatted sample was soaked in 80°C water at a mass ratio of 1:30 for 2 h, filtered after cooling, and the residue was washed three times with 78% solute mass percentage ethanol to obtain a washed sample; Drying and crushing: Place the washed sample in an oven at 55-60°C for drying, then crush it in a grinder and pass it through a 100-mesh sieve to obtain a sieved sample; Delignification: The sieved sample was immersed in 5% solute mass percentage NaOH at 50°C at a mass ratio of 1:20 and stirred for 4 h. The precipitate was collected and washed with distilled water for 3 times to obtain a delignified sample. Decolorization: The delignified sample was immersed in 8% solute mass percentage H2O2 at a mass ratio of 1:20, and allowed to stand for 24 h to obtain a decolorized sample; Hydrolysis: The decolorized sample was added to 5% hydrochloric acid at a mass ratio of 1:25, and ultrasonicated at 160 W and 50°C for 90 min. The precipitate was collected by centrifugation. The precipitate was added to 10% hydrochloric acid at a mass ratio of 1:25, and ultrasonicated at 160 W and 40°C for 60 min. The precipitate was collected and washed to obtain nanocellulose fibers.

[0013] Furthermore, in the above-mentioned method for preparing probiotic microcapsules, the method for preparing nanocellulose crystals comprises the following steps: The nanocellulose obtained in claim 8 and 62 wt% sulfuric acid were stirred at 37°C in a mass ratio of 1:20, and after 1 hour, 5 times the volume of distilled water was added to stop the acid hydrolysis to obtain a hydrolyzate; Washing and freeze-drying: The hydrolyzate was centrifuged at 8000 r / min for 10 min, then the precipitate was washed with distilled water until the supernatant was turbid, homogenized for 5 min, dialyzed to neutrality, and freeze-dried to obtain nanocellulose crystals.

[0014] Another technical solution provided by the present invention is to provide a probiotic microcapsule prepared by the preparation method of the probiotic microcapsule.

[0015] The beneficial effects of the present invention are: 1) The present invention uses waste bamboo shoot shell plant fibers as raw materials, and the nanocellulose fibers and nanocellulose crystals prepared by enzyme acid hydrolysis combined with physical means are combined with polyvinyl alcohol to form a dense network structure, which can effectively enhance the stability and activity of probiotics in harsh environments. 2) The present invention uses the prepared nanocellulose microcapsules to encapsulate probiotics, which can improve the survival rate of probiotics under freeze-drying and heating conditions. 3) Nanocellulose fibers, nanocellulose crystals, and polyvinyl alcohol are widely available and safe and reliable; the process conditions for preparing probiotic microcapsules by combining the three are simple and suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a flow chart of a method for preparing probiotic microcapsules according to Example 1 of a specific embodiment of the present invention; Figure 2 Scanning electron microscope images of probiotic microcapsules prepared in Example 1 of the specific embodiment of the present invention; A: PVA-CNF cross-sectional structure diagram; B: PVA-CNC cross-sectional structure diagram; C: PVA-CNF-CNC cross-sectional structure diagram; D: probiotic microcapsule surface structure diagram; E: probiotic microcapsule internal structure diagram; Figure 3 This is a comparative diagram of Example 2 of a specific embodiment of the present invention analyzing the survival rates of different strains under freeze-drying; Figure 4 This is a comparison chart of analyzing the heat resistance of probiotic microcapsules in Example 3 of a specific embodiment of the present invention; Figure 5 This is a comparison diagram of the simulated gastrointestinal digestion analysis of the probiotic microcapsules in Example 4; Figure 6 This is a comparison chart of the analysis of the storage properties of probiotic microcapsules in Example 5. DETAILED DESCRIPTION

[0017] In order to explain the technical content, achieved objectives and effects of the present invention in detail, the following is an explanation in combination with the implementation modes and the accompanying drawings.

[0018] The key concept of the present invention is to use nanocellulose crystals (CNC), nanocellulose fibers (CNF) and polyvinyl alcohol (PVA) derived from bamboo shoot waste to construct a new type of nanocellulose hydrogel probiotic microcapsule system to protect the activity of probiotics in food processing and intestinal colonization. Specifically, the above-mentioned bamboo shoot shell nanocellulose fibers and nanocellulose crystals are prepared by acid hydrolysis combined with physical means. The surfaces of nanocellulose fibers and nanocellulose crystals are rich in negative charges and hydroxyl groups, which are conducive to combining with macromolecular substances through hydrogen bonding and electrostatic effects. A dense network structure is formed through the direct hydrogen bonding of CNC, CNF and PVA, and the probiotics are encapsulated in the prepared nanocellulose microcapsule material through electrostatic and hydrogen bonding to enhance the stability and activity of the probiotics.

[0019] The nanocellulose fibers (CNF) and nanocellulose crystals (CNC) used in the embodiments of the present application are prepared by the following method: Grinding: Grinding the bamboo shoots into powder to obtain bamboo shoot powder; Degreasing: Soak bamboo shoot powder in 4 volumes of ethyl acetate for 1 h to remove lipids, and filter with distilled water three times to obtain a defatted sample; Washing: The defatted sample was soaked in 80℃ water with a mass ratio of 1:30 for 2 h, filtered after cooling, and the residue was washed three times with 78% mass percent ethanol to obtain a washed sample; Drying and crushing: Place the washed sample in an oven at 55-60℃ for drying, then crush it in a grinder and pass it through a 100-mesh sieve to obtain the sieved sample; Delignification: The sieved sample was immersed in 5% NaOH at 50°C at a mass ratio of 1:20 and stirred for 4 h. The precipitate was collected and washed with distilled water for 3 times to obtain a delignified sample. Decolorization: The delignified sample was immersed in 8% by mass percentage H2O2 at a mass ratio of 1:20, and allowed to stand for 24 h to obtain a decolorized sample; Hydrolysis: the decolorized sample was added to 5% hydrochloric acid at a mass ratio of 1:25, ultrasonicated at 160 W and 50 ° C for 90 min, and the precipitate was collected by centrifugation. The precipitate was added to 10% hydrochloric acid at a mass ratio of 1:25, ultrasonicated at 160 W and 40 ° C for 60 min, and the precipitate was collected and washed to obtain nanocellulose fibers. Nanocellulose fibers and 62 wt% sulfuric acid were stirred at 37°C in a mass ratio of 1:20, and 5 times the volume of distilled water was added after 1 h to stop the acid hydrolysis to obtain a hydrolyzate; Washing and freeze-drying: The hydrolyzate was centrifuged at 8000 r / min for 10 min, then the precipitate was washed with distilled water until the supernatant was turbid, homogenized for 5 min, dialyzed to neutrality, and freeze-dried to obtain nanocellulose crystals.

[0020] Example 1 A method for preparing probiotic microcapsules comprises the following steps (see flow chart Figure 1 ): 1) Preparation of PVA-CNF / PVA-CNC / PVA-CNF-CNC suspension: Weigh 4 g of polyvinyl alcohol in 100 mL of distilled water, heat in a water bath at 90 °C and stir until dissolved to prepare a polyvinyl alcohol solution. Weigh 0.5 wt% of CNF and add it to the polyvinyl alcohol solution and stir until CNF is evenly dispersed in the solution. Weigh 5 wt% of CNC and add it to the PVA-CNF liquid and stir until CNF and CNC are evenly dispersed in the solution. Take it out and ultrasonicate it at 50 °C for 2 h to prepare a PVA-CNF-CNC suspension.

[0021] The same method can be used to prepare PVA-CNC suspension and PVA-CNC suspension separately; dense network microcapsules are formed by freeze drying (temperature is -50℃, vacuum degree is 0.1 kPa), and the cross-sectional structure of the prepared microcapsules is observed by liquid nitrogen treatment. The results are shown in Figure 2 .

[0022] 2) Formation of probiotic microcapsules: The prepared PVA-CNF-CNC suspension was sterilized by high pressure at 121°C for 15 minutes, and the sterile nanocellulose-based hydrogel microcapsules were taken out and cooled at room temperature for later use. The second or third generation bacterial suspension was centrifuged and resuspended to the original concentration (about 10 9 ~10 10 CFU / mL), the resuspended bacterial solution was mixed with nanocellulose hydrogel microcapsules at a ratio of 1:1 (v / v), and the probiotics were fully encapsulated by vortexing, and then incubated in a shaker at 37°C for 1 hour. The microcapsules were taken out and pre-frozen at -80°C for 12 hours, and freeze-dried for 24 hours, so that the probiotics were encapsulated in the microcapsules for subsequent use.

[0023] 3) The bacterial solution in step (2) was centrifuged (1000 rpm, 5 min), and the probiotic microcapsules prepared by vacuum freeze drying were stored in a refrigerator at 0-4°C.

[0024] The morphological structure of the formed microcapsules was analyzed by setting up five groups for the following treatments: A: PVA-CNC group, B: PVA-CNC group, C: PVA-CNF-CNC, D: PVA-CNF-CNC+probiotics (PCC) surface structure group, and E: PVA-CNF-CNC+probiotics (PCC) internal cross-sectional structure group. The prepared nanocellulose hydrogel microcapsules were treated with liquid nitrogen to analyze their morphological structure. The results are shown in Figure 2 .

[0025] Figure 2 This is the SEM image of nanocellulose hydrogel microcapsules, where Figure 2 A is a scanning electron microscopy image of the cross-section pores of polyvinyl alcohol composite nanocellulose fibers (PVA-CNF, 4% PVA-0.5%CNF). Figure 2 B is a scanning electron microscopy image of the cross-section pores of polyvinyl alcohol composite nanocellulose crystals (PVA-CNC, 4% PVA-5% CNC). Figure 2 C is a scanning electron micrograph of the cross-sectional pores of polyvinyl alcohol composite nanocellulose fibers and nanocellulose crystals (PVA-CNF-CNC, 4% PVA - 0.5% CNF-5% CNC). By comparison, it can be found that the hydrogel microcapsules with only CNF added present a layered network structure with larger pore size on the cross section. When CNC is added, the pore size of the nanocellulose hydrogel microcapsules becomes smaller and the network structure becomes more compact. However, the addition of CNC alone will cause the microcapsules to form a larger cross-sectional pore size due to its rigid structure. The combination of PVA, CNF, and CNC through hydrogen bonding will form a denser layered network structure, which is beneficial to protect the probiotics from the digestion of gastric juice.

[0026] The concentrated bacterial solution was mixed with nanocellulose hydrogel microcapsules and freeze-dried to prepare nanocellulose hydrogel probiotic microcapsules (PCC). Figure 2 D shows the SEM image of the PCC surface. Through observation and analysis, it can be found that there is no void on the surface of the probiotic microcapsule, but there is slight shrinkage due to freeze drying, and there are fewer free probiotics, and the probiotic encapsulation effect is good. Figure 2 E shows the SEM image of the PCC cross-section. It can be observed from the figure that there are a large number of probiotics in the internal cross-section, and their structure is smooth and rod-shaped. In addition, it can be found that the bacteria are tightly bound to the nanocellulose hydrogel, and the nanocellulose hydrogel forms a dense film on the surface of the probiotics, firmly wrapping the bacteria inside the microcapsule.

[0027] Example 2 Analysis of probiotic activity in probiotic microcapsules Experimental methods: The preparation method of probiotic microcapsules is as described in Example 1. Survival rate calculation method: Add 0.1 g of freeze-dried microcapsules to 10 ml of 2% sterile sodium citrate solution, adjust the pH to about 7, and stir magnetically at room temperature for 10 minutes. During this process, the microcapsules will open and release bacterial cells. Subsequently, the sample is diluted with sterile saline, cultured on MRS agar medium, and incubated at 37°C for 48 hours. Finally, the number of bacteria (N) is counted by the plate count method. Embedding survival rate: EY=N / N0; N is the number of live bacteria encapsulated in the microcapsules, log 10 CFU / g, N0 is the number of viable bacteria initially added, log 10 CFU / g.

[0028] The nanocellulose hydrogel microcapsules were divided into three parts, and Bifidobacterium, Lactobacillus fermentum and Lactobacillus plantarum were respectively embedded to prepare probiotic microcapsules, and the live bacterial counts of naked bacteria were compared and analyzed, with the initial live bacterial counts as the control. Figure 3 .

[0029] Experimental results: Depend on Figure 3 It can be seen that the survival of probiotics of different strains encapsulated in the prepared probiotic microcapsules was improved, and the average viable count of Bifidobacterium could be increased to 7.84 log 10 CFU / mL, the average viable count of Lactobacillus fermentum can be increased to 7.66 log 10 CFU / mL, the average viable count of Lactobacillus plantarum can be increased to 8.59 log 10 CFU / mL, and the embedding survival rate can reach more than 80%. It can be seen that the prepared nanocellulose hydrogel microcapsules can effectively protect probiotics and reduce the damage to probiotics caused by ice crystals under rapid freezing.

[0030] Example 3 Analysis of heat resistance of probiotic microcapsules Experimental methods: Probiotic microcapsules were prepared using Lactobacillus plantarum as the experimental strain. The preparation method of probiotic microcapsules was referred to the above Example 1. 1 g of microcapsules were weighed and added to 9 mL of phosphate buffer, placed in a water bath at the corresponding temperature (55°C, 65°C and 75°C) for 10 min, and fully shaken and disintegrated with a vortex oscillator. The colony count was performed, and 1 mL of the same concentration of bacterial suspension was used as a control experiment.

[0031] Experimental results: Depend on Figure 4It can be seen that probiotic microcapsules also showed significant protective effects under high temperature conditions. The study found that the survival rate of probiotic microcapsules under 55℃, 65℃ and 75℃ water bath treatment was significantly higher than that of naked probiotics. In particular, the survival rate of probiotic microcapsules was significantly higher than that of naked probiotics in the 75℃ water bath treatment, indicating that nanocellulose hydrogel microcapsules can form a denser protective layer, effectively protecting probiotics from heat damage, thereby improving their stability and survival rate during processing and storage.

[0032] Example 4 Analysis of simulated gastrointestinal digestion of probiotic microcapsules Experimental methods: Lactobacillus plantarum was used as the experimental strain to prepare probiotic microcapsules. The preparation method of the probiotic microcapsules was referred to the above Example 1.

[0033] 0.1 g of probiotic microcapsules (1 mL of original bacterial solution) were added to SGF (10 mL, pH 2.5) for 2 hours, and then added to SIF (10 mL, pH 7.0) for 2 hours. The samples collected before digestion (0 h), after simulated gastric digestion (2 h), and after simulated gastric digestion and small intestinal digestion (4 h) were serially diluted (1-10 5 Then, the MRS plates were incubated anaerobically at 37°C for 48 h for viable cell counting.

[0034] Among them, simulated gastric fluid preparation: pepsin (enzyme activity: 2000U / mL), CaCl2 (0.15mM), HCl (adjust pH to 2-3) Add 5mg CaCl2 and hydrochloric acid to 300mL distilled water to adjust pH and filter and sterilize to prepare simulated gastric fluid (SGF), add pepsin (20mg) and filter with a sterile filter head for later use. Simulated small intestinal fluid preparation: trypsin (enzyme activity: 100U / mL), CaCl2 (0.6mM), bile salt (10mM) (adjust pH to 7) Dissolve CaCl2 (20mg) in 300mL phosphate buffer (5 mM PBS, pH 7) and filter and sterilize to prepare simulated intestinal fluid (SIF), add bile salt (1.35g) and trypsin (12mg) and filter with a sterile filter head for later use. Results are shown in Figure 5 .

[0035] Experimental results: In vitro simulated digestion is an effective method to evaluate the efficiency of probiotic delivery systems. It allows for a preliminary assessment of whether the microcapsules can maintain stable probiotic activity. Probiotic microcapsules were subjected to simulated digestion, initial viable counts were assessed, and 2h gastric digestion and 2h intestinal digestion were compared with unencapsulated probiotics. The results are shown in Figure 2. Figure 5 The survival rate of non-encapsulated probiotics after gastric digestion was 9.34 log 10CFU / mL dropped sharply to 6.91 log 10 CFU / mL, the survival rate was 74.08%, and then 3.54 log 10 The CFU / mL survival rate was 34.95%. In contrast, the probiotic microcapsules showed 91.01% (8.49 log 10 CFU / mL, maintained at 86.21% (8.04 log 10 CFU / mL). Compared with probiotic microcapsules prepared with other macromolecular polysaccharides, such as highland barley non-starch polysaccharide-Lactobacillus plantarum co-microcapsules, after the simulated gastrointestinal digestion, the viable bacterial counts were measured to be 5.70 lg (CFU / mL) and 6.3 lg (CFU / mL), respectively; with sodium alginate (Alg) and N,O-carboxymethyl chitosan (CMCS) as capsule materials, the survival rate of probiotics after passing through the gastrointestinal simulated fluid was (7.6±0.65)log 10 CFU / mL. Compared with traditional freeze-drying protective agents, such as the optimal composite freeze-drying protective agent: glycerol 2%, skim milk 6%, ascorbic acid 0.6%, trehalose 9% embedded probiotics, the survival rate after treatment with simulated gastric juice was only 61%. These results show that probiotic microcapsules can effectively protect probiotics from the impact of the digestive process and stably maintain the activity of probiotics.

[0036] Example 5 Analysis of the storage properties of probiotic microcapsules Experimental methods: Probiotic microcapsules were prepared using Lactobacillus plantarum as the experimental strain. The preparation method of the probiotic microcapsules was referred to Example 1 above. The prepared probiotic microcapsules were stored at 37°C and 0°C, respectively. Samples were taken at 0d, 7d, 14d, 21d, 28d, 35d, 42d, 52d, and 62d, respectively. The plate count method was used to determine the viable bacterial count. The control group was composed of non-encapsulated probiotics, and the same method as Example 2 was used to evaluate its viability under the same storage time. The two experimental groups were compared, and the conclusions were drawn. Figure 6 .

[0037] Experimental results: The storage of probiotics is crucial to the entire industry. According to China’s “Probiotic Health Food Evaluation Regulations”, during storage, the live bacterial count of probiotic health foods should not be less than 10 6 CFU / mL (g). In this study, probiotic microcapsule samples were stored at 37°C and 0°C, and their viable bacterial counts were tested regularly and compared with the control group (unencapsulated probiotics). Figure 6At 37°C, the viable count of the unembedded group was undetectable after 21 days, whereas at 0°C, the viable count of the unembedded group remained active until the 28th day, gradually decreasing to 5.25 log on the 62nd day. 10 CFU / mL, and the survival rate was 55.54%. Compared with the unencapsulated group, the probiotic microcapsule samples significantly improved the probiotic activity at 37°C. Specifically, the survival rate of the microcapsule sample group on day 62 was 38.80%, and the viable bacterial count was 3.75 log 10 CFU / mL. Under 0°C, the microcapsule sample group performed best in maintaining probiotic activity, with a survival rate of 87.44% and a viable bacterial count of 8.35 log after 62 days. 10 CFU / mL. It can be concluded that probiotic microcapsules significantly maintain the probiotic activity compared with unencapsulated probiotics.

[0038] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing probiotic microcapsules, characterized in that: The steps include: 1) Preparing a probiotic bacterial suspension; 2) heating the polyvinyl alcohol solution, nanocellulose fibers and nanocellulose crystals in a mixed water bath until they are completely dissolved to prepare a PCC suspension; the PCC suspension is evenly dispersed under the action of a magnetic stirrer, and then ultrasonically dispersed; 3) sterilizing the PCC suspension obtained in step 2) by high pressure sterilization at 110-130° C. to obtain nanocellulose-based hydrogel microcapsules; 4) mixing the nanocellulose-based hydrogel microcapsules with a bacterial suspension, using a vortex oscillator to fully and evenly disperse the bacteria in the bacterial suspension into the nanocellulose-based hydrogel microcapsules, incubating in a shaker and then freezing, and freeze-drying to obtain probiotic microcapsules.

2. The method for preparing probiotic microcapsules according to claim 1, characterized in that: The step 1) specifically comprises: thawing the probiotic strain stored at -80°C, culturing at 37°C for 24-48h to activate to the third generation, aspirating the third generation bacterial solution by centrifugation to remove the culture medium, washing twice by centrifugation with sterile saline, and resuspending with sterile saline to prepare a bacterial suspension for standby use.

3. The method for preparing probiotic microcapsules according to claim 2, characterized in that: The centrifugation setting conditions of step 1) are: 5500 rpm, 15 min, and the probiotics are one or more of Bifidobacterium, Lactobacillus fermentum or Lactobacillus plantarum.

4. The method for preparing probiotic microcapsules according to claim 1, characterized in that: The concentrations of the solutes in the PCC suspension in step 2) are as follows: the nanocellulose fiber content is 0.5 wt %, the nanocellulose crystal content is 5 wt %, and the polyvinyl alcohol content is 4 wt %.

5. The method for preparing probiotic microcapsules according to claim 1, characterized in that: The conditions for ultrasonic dispersion in step 2) are temperature 40-50° C., power 200 W, and time 2 h.

6. The method for preparing probiotic microcapsules according to claim 1, characterized in that: The step 3) is specifically as follows: sterilizing the PCC suspension obtained in step 2) under high pressure at 121° C. for 15 minutes to obtain nanocellulose-based hydrogel microcapsules.

7. The method for preparing probiotic microcapsules according to claim 1, characterized in that: The step 4) is specifically as follows: the nanocellulose-based hydrogel microcapsules and the bacterial suspension are mixed in a volume ratio of 1:1, the bacteria in the bacterial suspension are fully and evenly dispersed in the nanocellulose-based hydrogel microcapsules by a vortex oscillator, incubated in a shaker at 37° C. for 1 hour, frozen at -80° C. for 12 hours, and freeze-dried for 24 hours to obtain probiotic microcapsules.

8. The method for preparing probiotic microcapsules according to claim 1, characterized in that: The preparation method of the nanocellulose fiber comprises the following steps: Grinding: Grinding the bamboo shoots into powder to obtain bamboo shoot powder; Degreasing: Soak bamboo shoot powder in 4 volumes of ethyl acetate for 1 h to remove lipids, and filter with distilled water three times to obtain a defatted sample; Washing: The defatted sample was soaked in 80°C water at a mass ratio of 1:30 for 2 h, filtered after cooling, and the residue was washed three times with 78% solute mass percentage ethanol to obtain a washed sample; Drying and crushing: Place the washed sample in an oven at 55-60°C for drying, then crush it in a grinder and pass it through a 100-mesh sieve to obtain a sieved sample; Delignification: The sieved sample was immersed in 5% solute mass percentage NaOH at 50°C at a mass ratio of 1:20 and stirred for 4 h. The precipitate was collected and washed with distilled water for 3 times to obtain a delignified sample. Decolorization: The delignified sample was immersed in 8% solute mass percentage H2O2 at a mass ratio of 1:20, and allowed to stand for 24 h to obtain a decolorized sample; Hydrolysis: The decolorized sample was added to 5% hydrochloric acid at a mass ratio of 1:25, and ultrasonicated at 160 W and 50°C for 90 min. The precipitate was collected by centrifugation. The precipitate was added to 10% hydrochloric acid at a mass ratio of 1:25, and ultrasonicated at 160 W and 40°C for 60 min. The precipitate was collected and washed to obtain nanocellulose fibers.

9. The method for preparing probiotic microcapsules according to claim 1, characterized in that: The preparation method of the cellulose nanocrystals comprises the following steps: The nanocellulose obtained in claim 8 and 62 wt% sulfuric acid were stirred at 37°C in a mass ratio of 1:20, and after 1 hour, 5 times the volume of distilled water was added to stop the acid hydrolysis to obtain a hydrolyzate; Washing and freeze-drying: The hydrolyzate was centrifuged at 8000 r / min for 10 min, and then the precipitate was washed with distilled water until the supernatant was turbid, and then homogenized for 5 min, dialyzed to neutrality, and freeze-dried to obtain nanocellulose crystals.

10. The probiotic microcapsules prepared according to the method for preparing probiotic microcapsules according to any one of claims 1 to 9.

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