A probiotic feather coat based on surface coating technology and its preparation method and application

Abstract

The present invention discloses a probiotic feather coat based on surface coating technology and its preparation method and application. A layer of polysaccharide or protein feather coat is prepared on the surface of probiotics by surface coating technology. Specifically, probiotics at a certain pH are respectively combined with chitosan oligosaccharides or whey protein isolates in a certain proportion through interaction forces, and are resuspended in sterile water after stirring, oscillating, centrifuging, washing, vortexing and other treatments. The present invention induces polysaccharides or proteins to self-assemble on the surface of probiotics to form stable feather coats by lowering the pH value. The operation is simple, fast and low in cost. The feather coat can be coated on the surface of a single live probiotic. The polysaccharide feather coat or protein feather coat prepared by the present invention can improve the thermal stability, freeze-thaw stability and storage stability of probiotics, can reduce the damage of probiotics under adverse external environmental conditions, and improve the survival rate of probiotics.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, and in particular to the preparation and application of polysaccharide or protein probiotic feather coat. Background Art

[0002] As a living microorganism, probiotics can relieve nausea and bloating caused by lactose intolerance, regulate blood sugar metabolism, improve gastrointestinal function, enhance the body's immunity and anti-cancer activity, etc. However, probiotics will face various harsh environments during processing and storage, resulting in a low survival rate, which in turn affects the efficacy of probiotics. In order to improve the survival ability of probiotics, probiotic encapsulation technology is often used to prevent direct contact between probiotics and the external environment and between probiotics. Common encapsulation methods are divided into multi-cell encapsulation and single-cell encapsulation. Usually, multi-cell encapsulation methods such as emulsions and microspheres encapsulate a large number of probiotic individuals inside the carrier. The larger volume of micron-sized carriers is difficult to add to food, which will have a certain impact on the appearance, taste and stability of the product. In addition, the multi-cell encapsulation method may also have defects such as high cost and low encapsulation rate; while single-cell encapsulation can form a nano-coating on the surface of a single live probiotic, directly exerting its effect at a specific location without the need to be released from the encapsulation carrier. Among them, the layer-by-layer assembly method is one of the most popular methods in single-cell encapsulation, which mainly uses electrostatic interactions to adsorb substances with opposite charges onto the surface of probiotics. At present, natural food-grade biopolymers are often used as coating wall materials. In addition to the common polysaccharides and proteins from terrestrial plants and animals, polysaccharides from marine sources are also gaining more and more attention.

[0003] As shown in the following patent: Application number: 202211337194.5, Publication number: CN 115558659 A, Invention name: "A kind of plant lactobacillus emulsion and its preparation method and application", discloses a preparation method and application of plant lactobacillus encapsulated in a mixed protein-gellan gum emulsion. The emulsion prepared by this method can improve the storage stability of plant lactobacillus at 4°C and the survival rate under adverse external environmental conditions, but the application of the emulsion in food is limited.

[0004] As shown in the following patent: Application number: 202310286037.4, Publication number: CN 116349886 A, Invention name: "A probiotic encapsulation material, an encapsulated probiotic and its preparation method", discloses a probiotic encapsulation material composed of a protective layer and a functional layer. The prepared coating bacteria will not affect the vitality of the encapsulated probiotics, but the probiotics need a certain amount of time to break through the coating barrier.

[0005] Article "Use of gelatin and gum arabic for microencapsulation of probiotic cells from Lactobacillus plantarum The article "A method for encapsulating Lactobacillus plantarum by microcapsules prepared by combining double emulsification with complex coacervation" discloses a method for encapsulating Lactobacillus plantarum by microcapsules prepared by combining double emulsification with complex coacervation. The prepared microcapsules have good storage stability and improve the activity of Lactobacillus plantarum, but the preparation method is relatively complicated and not suitable for large-scale industrial production. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the primary purpose of the present invention is to provide a probiotic feather coat, wherein the feather coat is chitosan oligosaccharide or whey protein isolate.

[0007] The second object of the present invention is to provide a method for preparing the above-mentioned probiotic feather coat.

[0008] The third object of the present invention is to provide the application of the above-mentioned probiotic feather coat.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is:

[0010] 1) The frozen probiotics were revived in the culture medium. After a certain period of culture, the culture medium was centrifuged at 7000 rpm for 10 min, washed several times to ensure that there was no culture medium residue, the supernatant was removed, the bacterial sludge was collected, and the bacterial sludge was resuspended in 5 mL of sterile ultrapure water (the probiotic cell concentration was 1×10 10 ~1×10 11 CFU / mL), vortex for 10 s to obtain a uniformly dispersed bacterial suspension, and store it in a 4°C refrigerator for later use.

[0011] 2) Weigh a certain amount of polysaccharide and protein and dissolve them in sterile water. Stir at 500 rpm for 2 h to completely dissolve them. Place them in a 4°C refrigerator overnight to allow them to hydrate.

[0012] 3) The pH of the polysaccharide solution and the protein solution in step 2) was maintained at a fixed value by adding 0.1 M NaOH and HCl.

[0013] 4) Disperse 0.5 mL of the probiotic suspension prepared in step 1) in 4.5 mL of sterile ultrapure water, and maintain the pH at a fixed value by adding NaOH and HCl.

[0014] 5) The probiotic suspension in step 4) was added drop by drop into the polysaccharide solution and protein solution in the vortex in step 3), followed by mixing and stirring for 10 min and shaking on a shaker for 20 min.

[0015] 6) The mixed solution of probiotics and polysaccharides and the mixed solution of probiotics and protein obtained in step 5) are centrifuged at 7000 rpm for 10 min, washed twice with sterile water of the same pH, and the supernatant is removed to obtain probiotic mud covered with polysaccharide feathers or protein feathers.

[0016] 7) Re-disperse the centrifuged bacterial sludge in step 6) in sterile water of the same pH and store in a refrigerator at 4°C.

[0017] Preferably, the temperature of the frozen storage in step 1) is -80°C; the culture medium is MRS liquid culture medium; the probiotics are Lactobacillus plantarum (PL), the culture time is the logarithmic growth phase of the probiotics, and the culture temperature is 37°C.

[0018] Preferably, in step 2), the polysaccharide is chitosan oligosaccharide (COS), and the protein is whey protein isolate (WPI).

[0019] Preferably, in step 2), the concentration of polysaccharide is 2 mg / mL, and the concentration of protein is 2 mg / mL.

[0020] Preferably, the pH of the polysaccharide solution or protein solution in step 3) is 4.

[0021] Preferably, the pH of the probiotic suspension in step 4) is 4.

[0022] Preferably, in step 5), the volume ratio of the probiotic suspension to the polysaccharide solution or the protein solution is 1:5.

[0023] Compared with the prior art, the beneficial effects and advantages of the present invention are:

[0024] (1) The present invention uses chitosan oligosaccharide and whey protein isolate as feather materials. As natural polysaccharides and proteins, chitosan oligosaccharide and whey protein isolate have high biocompatibility and biosafety. Among them, chitosan oligosaccharide, as a marine polysaccharide, can also regulate the composition of intestinal flora, protect the gastrointestinal environment of the human body, and improve the body's immunity.

[0025] (2) The probiotic feathers prepared by the present invention are polysaccharides and proteins. By coating the surface of the probiotics with chitosan oligosaccharides or whey protein isolate, the probiotics can be isolated from adverse external environmental conditions to a certain extent. The prepared chitosan oligosaccharide feathers and whey protein isolate feathers can increase the survival rate of the probiotics and improve their thermal stability, freeze-thaw stability and storage stability.

[0026] (3) The chitosan oligosaccharide feathers and whey protein isolate feathers prepared by the present invention can improve the survival rate of probiotics without affecting the normal growth and reproduction of the probiotics themselves.

[0027] (4) The preparation method provided by the present invention is simple to operate, efficient and low-cost, suitable for industrial-scale production, and lays a foundation for the industrial application of probiotic feather coat. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Comparison of the Zeta potential of PL, PL@COS and PL@WPI as well as COS and WPI solutions.

[0029] Figure 2 Electric fluorescence microscopy images of PL@COS and PL@WPI under bright field and fluorescence field.

[0030] Figure 3 Fourier transform infrared spectra of PL, PL@COS, PL@WPI, COS and WPI powders.

[0031] Figure 4 The growth curves of PL, PL@COS and PL@WPI.

[0032] Figure 5 Pasteurization stability of PL, PL@COS and PL@WPI.

[0033] Figure 6 Freeze-thaw stability of PL, PL@COS and PL@WPI.

[0034] Figure 7 The storage stability of PL, PL@COS and PL@WPI. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with embodiments. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0036] The experimental methods used in the present invention are all conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0037] Example 1

[0038] The frozen Lactobacillus plantarum glycerol bacteria were revived in MRS broth, cultured to the logarithmic phase, and then centrifuged (7000 rpm, 10 min), washed several times, the supernatant was removed, the Lactobacillus plantarum sludge was collected, and the sludge was resuspended in 5 mL of sterile ultrapure water, and vortexed for 10 s to obtain a uniformly dispersed bacterial suspension. 0.5 mL of the bacterial suspension was diluted 10 times for use, and the pH was adjusted to 3. Chitooligosaccharides were accurately weighed to prepare 5 mL of chitooligosaccharide solution with a concentration of 2 mg / mL, and placed in a refrigerator at 4°C overnight to adjust the pH to 3. Subsequently, 1 mL of the diluted pH 3 bacterial suspension was added dropwise to the above 5 mL pH 3 chitooligosaccharide solution under vortex conditions, mixed and stirred for 10 min, shaken on a shaker for 20 min, and then centrifuged twice (7000 rpm, 10 min), washed with sterile water to remove excess chitooligosaccharides, and finally the sludge was resuspended in sterile water to obtain a Lactobacillus plantarum suspension covered with chitooligosaccharide feathers.

[0039] Example 2

[0040] The frozen Lactobacillus plantarum glycerol bacteria were revived in MRS broth, cultured to the logarithmic phase, and then centrifuged (7000 rpm, 10 min), washed several times, the supernatant was removed, the Lactobacillus plantarum sludge was collected, and the sludge was resuspended in 5 mL of sterile ultrapure water, and vortexed for 10 s to obtain a uniformly dispersed bacterial suspension. 0.5 mL of the bacterial suspension was diluted 10 times for use, and the pH was adjusted to 4. Chitooligosaccharides were accurately weighed to prepare 5 mL of chitooligosaccharide solution with a concentration of 2 mg / mL, and placed in a refrigerator at 4°C overnight to adjust the pH to 4. Subsequently, 1 mL of the diluted pH 4 bacterial suspension was added dropwise to the above 5 mL pH 4 chitooligosaccharide solution under vortex conditions, mixed and stirred for 10 min, shaken on a shaker for 20 min, and then centrifuged (7000 rpm, 10 min) twice, washed with sterile water to remove excess chitooligosaccharides, and finally the sludge was resuspended in sterile water to obtain a Lactobacillus plantarum suspension covered with chitooligosaccharide feathers.

[0041] Example 3

[0042] The frozen Lactobacillus plantarum glycerol bacteria were revived in MRS broth, cultured to the logarithmic phase, and then centrifuged (7000 rpm, 10 min), washed several times, the supernatant was removed, the Lactobacillus plantarum sludge was collected, and the sludge was resuspended in 5 mL of sterile ultrapure water, and vortexed for 10 s to obtain a uniformly dispersed bacterial suspension. 0.5 mL of the bacterial suspension was diluted 10 times for use, and the pH was adjusted to 5. Chitooligosaccharides were accurately weighed to prepare 5 mL of chitooligosaccharide solution with a concentration of 2 mg / mL, stored in a refrigerator at 4°C overnight, and the pH was adjusted to 5. Subsequently, 1 mL of the diluted pH 5 bacterial suspension was added dropwise to the above 5 mL pH 5 chitooligosaccharide solution under vortex conditions, mixed and stirred for 10 min, shaken on a shaker for 20 min, and then centrifuged twice (7000 rpm, 10 min), washed with sterile water to remove excess chitooligosaccharides, and finally the sludge was resuspended in sterile water to obtain a Lactobacillus plantarum suspension covered with chitooligosaccharide feathers.

[0043] Example 4

[0044] The frozen Lactobacillus plantarum glycerol bacteria were revived in MRS broth, cultured to the logarithmic phase, and then centrifuged (7000 rpm, 10 min), washed several times, the supernatant was removed, the Lactobacillus plantarum sludge was collected, and the sludge was resuspended in 5 mL of sterile ultrapure water, and vortexed for 10 s to obtain a uniformly dispersed bacterial suspension. 0.5 mL of the bacterial suspension was diluted 10 times for use, and the pH was adjusted to 6. Chitooligosaccharides were accurately weighed to prepare 5 mL of chitooligosaccharide solution with a concentration of 2 mg / mL, stored in a refrigerator at 4°C overnight, and the pH was adjusted to 6. Subsequently, 1 mL of the diluted pH 6 bacterial suspension was added dropwise to the above 5 mL pH 6 chitooligosaccharide solution under vortex conditions, mixed and stirred for 10 min, shaken on a shaker for 20 min, and then centrifuged (7000 rpm, 10 min) twice, washed with sterile water to remove excess chitooligosaccharides, and finally the sludge was resuspended in sterile water to obtain a Lactobacillus plantarum suspension covered with chitooligosaccharide feathers.

[0045] Example 5

[0046] The frozen Lactobacillus plantarum glycerol bacteria were revived in MRS broth, cultured to the logarithmic phase, and then centrifuged (7000 rpm, 10 min), washed several times, the supernatant was removed, the Lactobacillus plantarum sludge was collected, and the sludge was resuspended in 5 mL of sterile ultrapure water, and vortexed for 10 s to obtain a uniformly dispersed bacterial suspension. Take 0.5 mL of the bacterial suspension and dilute it 10 times for use, and adjust the pH to 3. Accurately weigh whey protein isolate, prepare 5 mL of whey protein isolate solution with a concentration of 2 mg / mL, store it in a refrigerator at 4°C overnight, and adjust the pH to 3. Then, under vortex conditions, take 1 mL of the diluted pH 3 bacterial suspension and add it dropwise to the above 5 mL pH 3 whey protein isolate solution, mix and stir for 10 min, shake on a shaker for 20 min, then centrifuge (7000 rpm, 10 min) twice, wash with sterile water to remove excess whey protein isolate, and finally resuspend the sludge in sterile water to obtain a Lactobacillus plantarum suspension coated with whey protein isolate feathers.

[0047] Example 6

[0048] The frozen Lactobacillus plantarum glycerol bacteria were revived in MRS broth, cultured to the logarithmic phase, and then centrifuged (7000 rpm, 10 min), washed several times, the supernatant was removed, the Lactobacillus plantarum sludge was collected, and the sludge was resuspended in 5 mL of sterile ultrapure water, and vortexed for 10 s to obtain a uniformly dispersed bacterial suspension. Take 0.5 mL of the bacterial suspension and dilute it 10 times for use, and adjust the pH to 4. Accurately weigh whey protein isolate, prepare 5 mL of whey protein isolate solution with a concentration of 2 mg / mL, store it in a refrigerator at 4°C overnight, and adjust the pH to 4. Then, under vortex conditions, take 1 mL of the diluted pH 4 bacterial suspension and add it dropwise to the above 5 mL pH 4 whey protein isolate solution, mix and stir for 10 min, shake on a shaker for 20 min, then centrifuge (7000 rpm, 10 min) twice, wash with sterile water to remove excess whey protein isolate, and finally resuspend the sludge in sterile water to obtain a Lactobacillus plantarum suspension coated with whey protein isolate feathers.

[0049] Example 7

[0050] The frozen Lactobacillus plantarum glycerol bacteria were revived in MRS broth, cultured to the logarithmic phase, and then centrifuged (7000 rpm, 10 min), washed several times, the supernatant was removed, the Lactobacillus plantarum sludge was collected, and the sludge was resuspended in 5 mL of sterile ultrapure water, and vortexed for 10 s to obtain a uniformly dispersed bacterial suspension. Take 0.5 mL of the bacterial suspension and dilute it 10 times for use, and adjust the pH to 5. Accurately weigh whey protein isolate, prepare 5 mL of whey protein isolate solution with a concentration of 2 mg / mL, store it in a refrigerator at 4°C overnight, and adjust the pH to 5. Then, under vortex conditions, take 1 mL of the diluted pH 5 bacterial suspension and add it dropwise to the above 5 mL pH 5 whey protein isolate solution, mix and stir for 10 min, shake on a shaker for 20 min, then centrifuge (7000 rpm, 10 min) twice, wash with sterile water to remove excess whey protein isolate, and finally resuspend the sludge in sterile water to obtain a Lactobacillus plantarum suspension coated with whey protein isolate feathers.

[0051] Example 8

[0052] The frozen Lactobacillus plantarum glycerol bacteria were revived in MRS broth, cultured to the logarithmic phase, and then centrifuged (7000 rpm, 10 min), washed several times, the supernatant was removed, the Lactobacillus plantarum sludge was collected, and the sludge was resuspended in 5 mL of sterile ultrapure water, and vortexed for 10 s to obtain a uniformly dispersed bacterial suspension. Take 0.5 mL of the bacterial suspension and dilute it 10 times for use, and adjust the pH to 6. Accurately weigh whey protein isolate, prepare 5 mL of whey protein isolate solution with a concentration of 2 mg / mL, store it in a refrigerator at 4°C overnight, and adjust the pH to 6. Then, under vortex conditions, take 1 mL of the diluted pH 6 bacterial suspension and add it dropwise to the above 5 mL pH 6 whey protein isolate solution, mix and stir for 10 min, shake on a shaker for 20 min, then centrifuge (7000 rpm, 10 min) twice, wash with sterile water to remove excess whey protein isolate, and finally resuspend the sludge in sterile water to obtain a Lactobacillus plantarum suspension coated with whey protein isolate feathers.

[0053] Experimental Example 1 Zeta potential test

[0054] Free Lactobacillus plantarum and the Lactobacillus plantarum covered with feathers prepared in Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, and Example 8 were used as experimental samples, and the original polysaccharide and protein solutions used in the preparation were used as controls. The potential of the samples was tested by a Malvern nanoparticle size analyzer.

[0055] pass Figure 1The experimental results show that free plant lactobacillus is negatively charged, which may be due to the rich lipoteichoic acid contained in the cell wall of plant lactobacillus. The chitosan oligosaccharide solution in embodiments 1 to 3 is positively charged, and the surface potential of the plant lactobacillus covered with chitosan oligosaccharide feathers prepared is reversed compared with free plant lactobacillus, indicating that electrostatic interaction may have occurred between the chitosan oligosaccharides in embodiments 1 to 3 and plant lactobacillus and combined together, successfully forming a coating on the plant lactobacillus surface. In embodiment 4, there is no reversal of charge after coating, which may be due to the fact that the chitosan oligosaccharide is less positively charged when pH 6, and the electrostatic interaction between plant lactobacillus and chitosan oligosaccharides is too weak to form a coating. Compared with embodiment 7 and embodiment 8, the whey protein isolate solution at pH 3 and pH 4 in embodiment 5 and embodiment 6 is positively charged, and the surface potential of the plant lactobacillus covered with whey protein isolate feathers prepared is similar to the protein solution potential corresponding to it, and also reverses the charge compared with free plant lactobacillus, indicating that the whey protein isolate in embodiment 5 and embodiment 6 is also successfully coated on the plant lactobacillus surface by electrostatic interaction.

[0056] Experimental Example 2: Motorized Fluorescence Microscope

[0057] Chitosan oligosaccharide and whey protein isolate were covalently cross-linked with FITC to obtain FITC-labeled chitosan oligosaccharide powder and whey protein isolate powder. Samples were then prepared according to the methods of Example 2 and Example 6, and observed using the bright field and fluorescent field of an electric fluorescence microscope.

[0058] according to Figure 2 The results show that the green fluorescence of FITC co-localizes with Lactobacillus plantarum under bright field, proving that chitosan oligosaccharides and whey protein isolate were successfully modified on the surface of Lactobacillus plantarum to form a layer of feather coat.

[0059] Experimental Example 3 Fourier Transform Infrared Spectroscopy

[0060] Free Lactobacillus plantarum and PL@COS and PL@WPI prepared in Example 2 and Example 6 were used as experimental samples. The samples were freeze-dried into powder (chitosan oligosaccharide powder and whey protein isolate powder were taken for comparison). The powder was mixed with KBr, ground and pressed into tablets. Fourier transform infrared spectrometer was used to obtain the wavelengths from 500 to 4000 cm -1 The spectral signal recorded was averaged over 64 scans with a resolution of 4 cm -1 .

[0061] according to Figure 3 The results show that the O-H and N-H stretching vibration peaks of chitosan oligosaccharide are located at 3386 cm -1 , C-H stretching vibration is located at 2892 cm -1 , which is at 1621 cm-1 , 1515 cm -1 、1319 cm -1 The characteristic peaks at 1158-890 cm-1 belong to the C═O stretching vibration of amide I, the N-H bending vibration of amide II, and the C-H bending vibration of amide Ш. -1 The absorption peak at 1646 cm corresponds to the typical 1,4-glycosidic bond of polysaccharides. After chitosan oligosaccharide binds to Lactobacillus plantarum, its absorption peak at amide I and amide II shifts to 1646 cm -1 and 1542 cm -1 and at 1230 cm -1 The new peak at 3386 cm -1 and 2892 cm -1 The O-H, N-H stretching vibration and C-H stretching vibration at 3390 cm -1 and 2927 cm -1 The hydrogen bond is also involved in the formation of chitosan oligosaccharide feather coat. -1 (O-H and N-H stretching vibration), 2962 cm -1 (C-H stretching vibration), 1647 cm -1 (C═O stretching vibration and amide I) and 1533 cm -1 The characteristic peaks of amide I and amide II of PL@WPI shifted to 1648 cm -1 and 1542 cm -1 , indicating that electrostatic interaction promotes the formation of whey protein isolate feathers. In addition, the hydroxyl group at 3291 cm -1 The stretching vibration at 3419 cm -1 The results show that hydrogen bonds are enhanced, that is, the binding of whey isolate protein to the surface of Lactobacillus plantarum enhances the hydrogen bonds in the structure.

[0062] Experimental Example 4 Growth Curve

[0063] Free Lactobacillus plantarum and PL@COS and PL@WPI prepared in Example 2 and Example 6 were used as experimental samples. The samples were diluted in MRS liquid culture medium to an optical density value (OD600) in the range of 0.1 to 0.2. The growth of the bacteria at 37°C was monitored every 0.5 h using a fully automatic high-throughput microbial droplet culture instrument, and a growth curve was drawn based on the results.

[0064] according to Figure 4The results show that the growth of PL@COS in 37℃ MRS liquid medium is better than that of PL, and the growth of PL@WPI is similar to that of PL. This may be because chitosan oligosaccharide, as a marine polysaccharide and prebiotic, can promote the growth and reproduction of Lactobacillus plantarum. Overall, the presence of the polysaccharide or protein coat does not affect the normal growth and proliferation of Lactobacillus plantarum.

[0065] Experimental Example 5 Pasteurization Stability

[0066] Free Lactobacillus plantarum and PL@COS and PL@WPI prepared in Example 2 and Example 6 were used as experimental samples. The bacterial suspension was added to a sterile tube, stored at 63°C for 30 min, taken out and cooled at room temperature, appropriately diluted and inoculated on MRS agar, and counted after being cultured at 37°C for 48 h.

[0067] according to Figure 5 The results show that the pasteurization stability of PL@COS and PL@WPI prepared in Example 2 and Example 6 is higher than that of PL. After heating at 63°C for 30 min, the viable counts of PL, PL@COS and PL@WPI decreased by 4.30, 3.57 and 3.74 Log CFU / mL, respectively. Compared with PL, chitosan oligosaccharide feathers and whey protein isolate feathers can reduce the heat damage of Lactobacillus plantarum to a certain extent, and the effect of chitosan oligosaccharide feathers is better than that of whey protein isolate feathers.

[0068] Experimental Example 6 Freeze-thaw stability

[0069] Free Lactobacillus plantarum and PL@COS and PL@WPI prepared in Example 2 and Example 6 were used as experimental samples, stored at -20°C for 22 h, and then thawed in a 30°C water bath for 2 h, which was a freeze-thaw cycle. Then, the thawed samples were frozen and thawed again, for a total of two cycles. At the end of the freeze-thaw cycle, the samples were collected, and 20 μL was inoculated on MRS agar after appropriate dilution, and counted after culturing at 37°C for 48 h.

[0070] according to Figure 6 The results show that after two freeze-thaw cycles, the viable counts of PL, PL@COS and PL@WPI decreased from 10.70 Log CFU / mL, 10.53 Log CFU / mL and 10.59 Log CFU / mL to 9.17 Log CFU / mL, 9.68 Log CFU / mL and 9.22 Log CFU / mL, respectively, that is, the freeze-thaw stability is PL@COS>PL@WPI>PL, indicating that both chitosan oligosaccharide feathers and whey protein isolate feathers can improve the freeze-thaw stability of Lactobacillus plantarum to a certain extent, and the effect of chitosan oligosaccharide feathers is better.

[0071] Experimental Example 7 Storage Stability

[0072] Under 4°C storage conditions, free Lactobacillus plantarum and PL@COS and PL@WPI prepared in Example 2 and Example 6 were used as experimental samples, and their storage activity was studied for 15 days, and the number of live bacteria was detected by colony counting.

[0073] according to Figure 7 The results show that after storage at 4°C for 15 days, the number of viable bacteria of PL was 7.23 Log CFU / mL, while the number of viable bacteria of PL@COS and PL@WPI were 9.00 and 7.97 Log CFU / mL, respectively. This indicates that the chitosan oligosaccharide coat and whey protein isolate coat can act as physical barriers to isolate adverse external environmental conditions to a certain extent and improve the storage stability of Lactobacillus plantarum.

[0074] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Application of chitosan oligosaccharide or whey protein isolate in the field of preparing probiotic feather coat, characterized in that: The probiotics are Lactobacillus plantarum; The application is to interact the probiotic suspension with the chitosan oligosaccharide or whey protein isolate solution, and after stirring, mixing, oscillating incubation, centrifugal washing and other treatments, resuspend it in sterile ultrapure water and vortex it to prepare the probiotic feather coat; specifically, it includes the following steps: 1) The frozen probiotics were revived in the culture medium. After culture, they were centrifuged at 7000 rpm for 10 min, washed several times to ensure that there was no culture medium residue, the supernatant was removed, the bacterial sludge was collected, and the bacterial sludge was resuspended in 5 mL of sterile ultrapure water. The probiotic cell concentration was 1×10 10 ~1×10 11 CFU / mL, vortex for 10 s to obtain a uniformly dispersed bacterial suspension, and store it in a 4°C refrigerator for later use; wherein the culture medium is MRS broth culture medium; the probiotics are Lactobacillus plantarum, the culture time is the logarithmic growth period of the probiotics, and the culture temperature is 37°C; 2) Weigh chitosan oligosaccharides and whey protein isolate and dissolve them in ultrapure water to prepare polysaccharide solution and protein solution respectively, stir at 500 rpm for 2 h to completely dissolve them, and place them in a refrigerator at 4°C overnight to hydrate them; the concentration of polysaccharide is 2 mg / mL, and the concentration of protein is 2 mg / mL; 3) maintaining the pH of the polysaccharide solution and the protein solution in step 2) at a fixed value by adding 0.1 M NaOH and HCl; wherein the pH of the polysaccharide solution or the protein solution is 4; 4) 0.5 mL of the probiotic suspension of step 1) was dispersed in 4.5 mL of sterile ultrapure water for dilution, and the pH was maintained at a fixed value by adding 0.1 M NaOH and HCl; wherein the pH of the probiotic suspension was 4; 5) Add the probiotic suspension in step 4) drop by drop into the polysaccharide solution and the protein solution in the vortex in step 3), then mix and stir for 10 minutes and shake on a shaker for 20 minutes; wherein the volume ratio of the probiotic suspension to the polysaccharide solution or the protein solution is 1:5; 6) centrifuging the mixed solution of probiotics and polysaccharides and the mixed solution of probiotics and protein obtained in step 5) at 7000 rpm for 10 min, washing twice with sterile water of the same pH, removing the supernatant, and obtaining probiotic mud covered with feathers; 7) Re-disperse the centrifuged bacterial sludge in step 6) in sterile water of the same pH and store in a refrigerator at 4°C.

Images