CN118165976A

CN118165976A - Probiotic collard based on surface coating technology and preparation method and application thereof

Info

Publication number: CN118165976A
Application number: CN202410591848.XA
Authority: CN
Inventors: 魏子淏; 张子墨
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2024-05-14
Filing date: 2024-05-14
Publication date: 2024-06-11

Abstract

The invention discloses a probiotic feather garment based on a surface coating technology, a preparation method and application thereof, wherein a layer of polysaccharide or protein feather garment is prepared on the surface of the probiotic through the surface coating technology, specifically, the probiotic under a certain pH value is respectively combined with chitosan oligosaccharide or whey protein isolate according to a certain proportion through interaction force, and the probiotic is resuspended in sterile water after being subjected to treatment such as stirring, oscillating, centrifuging, washing, swirling and the like. According to the invention, the pH value is reduced to induce polysaccharide or protein to self-assemble on the surface of the probiotics to form the stable collard, so that the operation is simple, convenient and quick, the cost is low, and the collard can be coated on the surface of a single living probiotics. The polysaccharide coat or protein coat prepared by the method can improve the thermal stability, freeze thawing stability and storage stability of the probiotics, can reduce the damage of the probiotics under the external adverse environmental conditions, and can improve the survival rate of the probiotics.

Description

Probiotic collard based on surface coating technology and preparation method and application thereof

Technical Field

The invention relates to the technical field of probiotics, in particular to preparation and application of polysaccharide or protein probiotic plumule.

Background

The probiotics as a living microorganism can relieve nausea and abdominal distention caused by lactose intolerance, regulate blood sugar metabolism, improve gastrointestinal function, enhance organism immunity and anticancer activity, etc. However, the probiotics can face various severe environments in the processing and storage processes, so that the survival rate of the probiotics is low, and the efficacy of the probiotics is further affected. In order to increase the viability of probiotics, probiotic encapsulation techniques are often used to prevent direct contact between the probiotics and the external environment and probiotics. Common encapsulation methods are divided into multicellular encapsulation and single-cell encapsulation. In general, a multicellular encapsulation method such as emulsion, microsphere, etc., a large number of probiotic individuals are encapsulated inside a carrier, and a large volume of micron-sized carrier is difficult to be added into food, so that the appearance, taste and stability of the probiotic can be affected to some extent. In addition, the multicellular encapsulation method may have the defects of high cost, low encapsulation efficiency and the like; whereas single cell encapsulation can form a nanocoating on the surface of a single living probiotic, acting directly at a specific site and without release from the encapsulation carrier. Among them, the layer-by-layer assembly method is one of the most popular methods in single cell encapsulation, mainly using electrostatic interactions to adsorb oppositely charged substances to the probiotic surface. Currently, natural food grade biopolymers are often used as coating wall materials, and apart from the usual polysaccharides and proteins of terrestrial plant and animal origin, polysaccharides of marine origin are also of increasing interest.

As shown in the following patents: application number: 202211337194.5 with publication number CN 115558659A, the invention discloses an embedded lactobacillus plantarum emulsion, a preparation method and application thereof, and discloses a preparation method and application of mixed protein-gellan gum emulsion embedded lactobacillus plantarum. The emulsion prepared by the method can improve the storage stability of lactobacillus plantarum at 4 ℃ and the survival rate of lactobacillus plantarum under the condition of external bad environment, but the application of the emulsion in food is limited.

As shown in the following patents: application number: 202310286037.4 with publication number CN 116349886A, the invention is named as a probiotic packaging material, an encapsulated probiotic and a preparation method thereof, and discloses a probiotic packaging material composed of a protective layer and a functional layer. The prepared coating bacteria can not influence the activity of the encapsulated probiotics, but the probiotics can take a certain time to break through the coating barrier.

Article "Use of gelatin and gum arabic for microencapsulation of probiotic cells from Lactobacillus plantarum by a dual process combining double emulsification followed by complex coacervation" discloses a method for encapsulating lactobacillus plantarum by double emulsification in combination with complex coacervation. The microcapsule prepared by the method has good storage stability and improves the activity of lactobacillus plantarum, but the preparation method is complex and is not suitable for large-scale industrial production.

Disclosure of Invention

In order to overcome the defects of the prior art, the primary purpose of the invention is to provide a probiotic plume, wherein the plume is chitosan oligosaccharide or whey protein isolate.

The second object of the invention is to provide a preparation method of the probiotic collard.

A third object of the present invention is to provide the use of the probiotic plume as described above.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

1) Resuscitates the frozen and stored probiotics in a culture medium, cultures the probiotics for a certain time, centrifugates the probiotics at 7000 rpm for 10 min, washes the probiotics for a plurality of times to ensure no culture medium residue, removes supernatant, collects bacterial sludge, resuspends the bacterial sludge in 5 mL sterile ultrapure water (the concentration of probiotic cells is 1X 10 ¹⁰~1×10¹¹ CFU/mL), and vortex 10 s obtains evenly dispersed bacterial suspension which is stored in a refrigerator at 4 ℃ for standby.

2) A certain amount of polysaccharide and protein are weighed and dissolved in sterile water, 500 rpm is stirred for 2h to be completely dissolved, and the mixture is placed in a refrigerator at 4 ℃ for overnight to be hydrated.

3) The pH of the polysaccharide and protein solutions described in step 2) was maintained at a fixed value by the addition of 0.1M NaOH and HCl.

4) The probiotic bacterial suspension of step 1) was dispersed in 4.5 mL sterile ultra pure water at a pH maintained at a fixed value by the addition of NaOH and HCl, taking 0.5 mL.

5) The probiotic bacterial suspension in the step 4) is respectively dropwise added into the polysaccharide solution and the protein solution in the vortex in the step 3), and then is mixed and stirred for 10 min, and the shaking table oscillates for 20 min.

6) Centrifuging the mixed solution of the probiotics and the polysaccharide and the mixed solution of the probiotics and the protein obtained in the step 5) for 10 min at 7000 rpm, washing twice with sterile water with the same pH value, and removing the supernatant to obtain the probiotics bacterial mud covered with the polysaccharide feathers or the protein feathers.

7) And (3) re-dispersing the bacterial sludge subjected to the centrifugation in the step (6) in sterile water with the same pH value, and storing in a refrigerator at the temperature of 4 ℃.

Preferably, the temperature of the frozen storage in step 1) is-80 ℃; 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 ℃.

Preferably, in step 2) the polysaccharide is Chitosan Oligosaccharide (COS) and the protein is Whey Protein Isolate (WPI).

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

Preferably, the polysaccharide solution or protein solution in step 3) has a pH of 4.

Preferably, the pH of the probiotic bacterial suspension in step 4) is 4.

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

Compared with the prior art, the invention has the following beneficial effects:

(1) The chitosan oligosaccharide and the whey protein isolate are used as the feather materials, and the chitosan oligosaccharide and the whey protein isolate are used as natural polysaccharide and protein, so that the invention has higher biocompatibility and biosafety. Wherein, the chitosan oligosaccharide is used as marine polysaccharide, and can also regulate the composition of intestinal flora, protect the gastrointestinal tract environment of human body and improve the immunity of the organism.

(2) The probiotics feather coat prepared by the invention is polysaccharide and protein, and the chitosan oligosaccharide or whey protein isolate can isolate the external bad environmental conditions to a certain extent by coating the chitosan oligosaccharide or whey protein isolate on the surface of the probiotics, so that the survival rate of the probiotics can be improved, and the thermal stability, the freeze thawing stability and the storage stability of the probiotics can be improved.

(3) The chitosan oligosaccharide coat and whey isolated protein coat prepared by the method can improve the survival rate of probiotics and can not influence the normal growth and propagation of the probiotics.

(4) The preparation method provided by the invention is simple to operate, high-efficiency and low in cost, is suitable for industrial large-scale production, and lays a foundation for industrial application of the probiotics collard.

Drawings

FIG. 1 is a Zeta potential comparison plot of PL, PL@COS and PL@WPI and COS and WPI solutions.

FIG. 2 is an electrokinetic fluorescence microscopy image of PL@COS and PL@WPI in bright field and fluorescent field.

FIG. 3 is a Fourier transform infrared spectrum of PL, PL@COS and PL@WPI and COS and WPI powders.

FIG. 4 is a graph of growth curves for PL, PL@COS and PL@WPI.

Figure 5 is the pasteurization stability of PL, pl@cos and pl@wpi.

FIG. 6 is the freeze-thaw stability of PL, PL@COS and PL@WPI.

FIG. 7 shows the storage stability of PL, PL@COS and PL@WPI.

Detailed Description

The invention is further illustrated below with reference to examples. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

The test method used in the invention is a conventional method unless specified otherwise; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Example 1

Resuscitates the frozen stored lactobacillus plantarum glycerol bacteria in MRS broth, cultures to centrifugation (7000 rpm,10 min) after log phase, washes for multiple times, removes supernatant, collects lactobacillus plantarum bacterial puree, resuspensions the puree in 5 mL sterile ultrapure water, and vortexes 10 s to obtain a uniformly dispersed bacterial suspension. Diluting the bacterial suspension of 0.5 mL by 10 times for later use, and adjusting the pH to 3. The chitosan oligosaccharide is accurately weighed, a chitosan oligosaccharide solution with the concentration of 2 mg/mL of 5 mL is prepared, the temperature is 4 ℃ and the refrigerator is kept overnight, and the pH is adjusted to 3. Then under vortex condition, 1 mL of diluted bacterial suspension with pH 3 is dropwise added into the 5 mL of chitosan oligosaccharide solution with pH 3, 10 min are mixed and stirred, 20min are oscillated by a shaking table, then centrifugation (7000 rpm,10 min) is carried out twice, the solution is washed by sterile water to remove superfluous chitosan oligosaccharide, and finally bacterial mud is resuspended in sterile water to obtain the lactobacillus plantarum bacterial suspension coated with chitosan oligosaccharide plume.

Example 2

Resuscitates the frozen stored lactobacillus plantarum glycerol bacteria in MRS broth, cultures to centrifugation (7000 rpm,10 min) after log phase, washes for multiple times, removes supernatant, collects lactobacillus plantarum bacterial puree, resuspensions the puree in 5 mL sterile ultrapure water, and vortexes 10 s to obtain a uniformly dispersed bacterial suspension. Diluting the bacterial suspension of 0.5 mL by 10 times for later use, and adjusting the pH to 4. The chitosan oligosaccharide is accurately weighed, a chitosan oligosaccharide solution with the concentration of 2 mg/mL of 5 mL is prepared, the temperature is 4 ℃ and the refrigerator is kept overnight, and the pH is adjusted to 4. Then under vortex condition, 1 mL of diluted bacterial suspension with pH 4 is dropwise added into the 5 mL of chitosan oligosaccharide solution with pH 4, 10 min is mixed and stirred, 20min is oscillated by a shaking table, then centrifugation (7000 rpm,10 min) is carried out twice, the solution is washed by sterile water to remove superfluous chitosan oligosaccharide, and finally bacterial mud is resuspended in sterile water to obtain the lactobacillus plantarum bacterial suspension coated with chitosan oligosaccharide plume.

Example 3

Resuscitates the frozen stored lactobacillus plantarum glycerol bacteria in MRS broth, cultures to centrifugation (7000 rpm,10 min) after log phase, washes for multiple times, removes supernatant, collects lactobacillus plantarum bacterial puree, resuspensions the puree in 5mL sterile ultrapure water, and vortexes 10 s to obtain a uniformly dispersed bacterial suspension. Diluting the bacterial suspension of 0.5 mL by 10 times for later use, and adjusting the pH to 5. The chitosan oligosaccharide is accurately weighed, a chitosan oligosaccharide solution with the concentration of 2 mg/mL of 5mL is prepared, the temperature is 4 ℃ and the refrigerator is kept overnight, and the pH is adjusted to 5. Then under vortex condition, 1 mL diluted bacterial suspension with pH 5 is dropwise added into the 5mL chitosan oligosaccharide solution with pH 5, 10min is mixed and stirred, 20 min is oscillated by a shaking table, then centrifugation (7000 rpm,10 min) is carried out twice, the solution is washed by sterile water to remove superfluous chitosan oligosaccharide, and finally bacterial mud is resuspended in sterile water to obtain the lactobacillus plantarum bacterial suspension coated with chitosan oligosaccharide plume.

Example 4

Resuscitates the frozen stored lactobacillus plantarum glycerol bacteria in MRS broth, cultures to centrifugation (7000 rpm,10 min) after log phase, washes for multiple times, removes supernatant, collects lactobacillus plantarum bacterial puree, resuspensions the puree in 5 mL sterile ultrapure water, and vortexes 10 s to obtain a uniformly dispersed bacterial suspension. Diluting the bacterial suspension of 0.5 mL by 10 times for later use, and adjusting the pH to 6. The chitosan oligosaccharide is accurately weighed, a chitosan oligosaccharide solution with the concentration of 2 mg/mL of 5 mL is prepared, and the pH is adjusted to 6 by a refrigerator overnight at 4 ℃. Then under vortex condition, 1 mL of diluted bacterial suspension with pH 6 is dropwise added into the 5 mL of chitosan oligosaccharide solution with pH 6, 10 min are mixed and stirred, 20min are oscillated by a shaking table, then centrifugation (7000 rpm,10 min) is carried out twice, the solution is washed by sterile water to remove superfluous chitosan oligosaccharide, and finally bacterial mud is resuspended in sterile water to obtain the lactobacillus plantarum bacterial suspension coated with chitosan oligosaccharide plume.

Example 5

Resuscitates the frozen stored lactobacillus plantarum glycerol bacteria in MRS broth, cultures to centrifugation (7000 rpm,10 min) after log phase, washes for multiple times, removes supernatant, collects lactobacillus plantarum bacterial puree, resuspensions the puree in 5mL sterile ultrapure water, and vortexes 10 s to obtain a uniformly dispersed bacterial suspension. Diluting the bacterial suspension of 0.5 mL by 10 times for later use, and adjusting the pH to 3. Whey protein isolate was precisely weighed, a whey protein isolate solution was prepared at a concentration of 5mL to 2 mg/mL, and the pH was adjusted to 3 at 4℃overnight in a refrigerator. Then under vortex condition, 1mL of diluted bacterial suspension with pH 3 is dropwise added into the above-mentioned whey protein isolate solution with pH 3, 10min is mixed and stirred, 20min is oscillated by a shaking table, then centrifuged (7000 rpm,10 min) twice, the solution is washed by sterile water to remove superfluous whey protein isolate, and finally bacterial mud is resuspended in sterile water to obtain the lactobacillus plantarum bacterial suspension coated with whey protein isolate coat.

Example 6

Resuscitates the frozen stored lactobacillus plantarum glycerol bacteria in MRS broth, cultures to centrifugation (7000 rpm,10 min) after log phase, washes for multiple times, removes supernatant, collects lactobacillus plantarum bacterial puree, resuspensions the puree in 5mL sterile ultrapure water, and vortexes 10 s to obtain a uniformly dispersed bacterial suspension. Diluting the bacterial suspension of 0.5 mL by 10 times for later use, and adjusting the pH to 4. Whey protein isolate was precisely weighed, a whey protein isolate solution was prepared at a concentration of 5mL to 2 mg/mL, and the pH was adjusted to 4 overnight in a refrigerator at 4 ℃. Then under vortex condition, 1 mL of diluted bacterial suspension with pH 4 is dropwise added into the above-mentioned whey protein isolate solution with pH 4, 10 min is mixed and stirred, 20 min is oscillated by a shaking table, then centrifuged (7000 rpm,10 min) twice, the solution is washed by sterile water to remove superfluous whey protein isolate, and finally bacterial mud is resuspended in sterile water to obtain the lactobacillus plantarum bacterial suspension coated with whey protein isolate coat.

Example 7

Resuscitates the frozen stored lactobacillus plantarum glycerol bacteria in MRS broth, cultures to centrifugation (7000 rpm,10 min) after log phase, washes for multiple times, removes supernatant, collects lactobacillus plantarum bacterial puree, resuspensions the puree in 5mL sterile ultrapure water, and vortexes 10 s to obtain a uniformly dispersed bacterial suspension. Diluting the bacterial suspension of 0.5mL by 10 times for later use, and adjusting the pH to 5. Whey protein isolate was precisely weighed, a whey protein isolate solution was prepared at a concentration of 5mL to 2 mg/mL, and the pH was adjusted to 5at 4℃overnight in a refrigerator. Then under vortex condition, 1mL of diluted bacterial suspension with pH 5 is dropwise added into the 5mL of whey protein isolate solution with pH 5, 10min is mixed and stirred, 20 min is oscillated by a shaking table, then centrifugation (7000 rpm,10 min) is carried out twice, the solution is washed by sterile water to remove superfluous whey protein isolate, and finally bacterial mud is resuspended in the sterile water to obtain the lactobacillus plantarum bacterial suspension coated with whey protein isolate feather.

Example 8

Resuscitates the frozen stored lactobacillus plantarum glycerol bacteria in MRS broth, cultures to centrifugation (7000 rpm,10 min) after log phase, washes for multiple times, removes supernatant, collects lactobacillus plantarum bacterial puree, resuspensions the puree in 5mL sterile ultrapure water, and vortexes 10 s to obtain a uniformly dispersed bacterial suspension. Diluting the bacterial suspension of 0.5mL by 10 times for later use, and adjusting the pH to 6. Whey protein isolate was precisely weighed, a whey protein isolate solution was prepared at a concentration of 5mL to 2 mg/mL, and the pH was adjusted to 6 at 4℃overnight in a refrigerator. Then under vortex condition, 1mL of diluted bacterial suspension with pH 6 is dropwise added into the above-mentioned whey protein isolate solution with pH 6, 10min is mixed and stirred, 20 min is oscillated by a shaking table, then centrifuged (7000 rpm,10 min) twice, the solution is washed by sterile water to remove superfluous whey protein isolate, and finally bacterial mud is resuspended in sterile water to obtain the lactobacillus plantarum bacterial suspension coated with whey protein isolate coat.

Experimental example 1 Zeta potential test

The sample potential was measured by a malvern nanoparticle analyzer using free lactobacillus plantarum and the lactobacillus plantarum coated with the feathers prepared in examples 1, 2, 3, 4, 5, 6, 7, and 8 as experimental samples and the raw polysaccharide and protein solution used for the preparation as a control.

From the experimental results in FIG. 1, it can be seen that free Lactobacillus plantarum is negatively charged, probably due to the rich lipoteichoic acid contained in the cell wall of Lactobacillus plantarum. The chitosan oligosaccharide solution in examples 1-3 is positively charged, and the surface potential of the prepared lactobacillus plantarum coated with chitosan oligosaccharide feathers is reversed in charge compared with that of free lactobacillus plantarum, which indicates that electrostatic interaction between the chitosan oligosaccharide in examples 1-3 and lactobacillus plantarum may occur and are combined together, so that a coating is successfully formed on the surface of the lactobacillus plantarum. In example 4, no charge reversal occurred after coating, probably because the chitosan oligosaccharide was less positively charged at pH 6, the electrostatic interaction between lactobacillus plantarum and chitosan oligosaccharide was too weak to combine to form a coating. The whey protein isolate solutions at pH 3 and pH 4 in examples 5 and 6 were positively charged compared to examples 7 and 8, the surface potential of the prepared Lactobacillus plantarum coated with whey protein isolate coat was similar to the corresponding protein solution potential, and charge reversal occurred also compared to free Lactobacillus plantarum, indicating that the whey protein isolate in examples 5 and 6 was also successfully coated on Lactobacillus plantarum surface by electrostatic interactions.

Experimental example 2 electric fluorescence microscope

FITC-labeled chitosan oligosaccharide powder and whey protein isolate powder were obtained by covalently cross-linking chitosan oligosaccharide and whey protein isolate, respectively, with FITC. Samples were prepared by the method described in example 2 and example 6, and observed using the bright field and fluorescent field of an electrokinetic fluorescent microscope.

From the results of fig. 2, it can be seen that the green fluorescence of FITC co-localizes with lactobacillus plantarum in the bright field, which proves that both chitosan oligosaccharide and whey protein isolate are successfully modified on the surface of lactobacillus plantarum to form a layer of feather coat.

Experimental example 3 Fourier transform Infrared Spectrometry

The free lactobacillus plantarum and the PL@COS and PL@WPI prepared in example 2 and example 6 are taken as experimental samples, the samples are freeze-dried into powder (the chitosan oligosaccharide powder is taken to be compared with the whey protein isolate powder), and the powder is mixed with KBr, ground and pressed into tablets. And (3) obtaining a spectrum signal recorded from the wave number of 500-4000 cm ^-1 by using a Fourier transform infrared spectrometer, and carrying out average scanning for 64 times, wherein the resolution is 4 cm ^-1.

As can be seen from the results of FIG. 3, the O-H and N-H stretching vibration peaks of the chitosan oligosaccharide are located at 3386 cm ^-1, the C-H stretching vibration is located at 2892 cm ^-1, the characteristic peaks at 1621 and cm ^-1、1515 cm^-1、1319 cm^-1 respectively belong to C ═ O stretching vibration of amide I, N-H bending vibration of amide II and C-H bending vibration of amide III, and the absorption peak at 1158-890 cm ^-1 corresponds to typical 1, 4-glycosidic bond of polysaccharide. After binding of the chitosan oligosaccharide to lactobacillus plantarum, its absorption peaks at amide i and amide ii shift to 1646 cm ^-1 and 1542 cm ^-1 and a new peak appears at 1230 cm ^-1, indicating that the positively charged amino groups of the chitosan oligosaccharide are involved in electrostatic interactions. It moves to 3390 cm ^-1 and 2927 cm ^-1 at the O-H, N-H stretching vibration and C-H stretching vibration of 3386 cm ^-1 and 2892 cm ^-1, which indicate that hydrogen bonds are also involved in the formation of chitosan oligosaccharide plumule. Whereas whey protein isolate showed its characteristic peaks at 3291 cm ^-1 (O-H and N-H stretching vibrations), 2962 cm ^-1 (C-H stretching vibrations), 1647 cm ^-1 (C ═ O stretching vibrations and amide I) and 1533 cm ^-1 (amide II). The amide i and amide ii of pl@wpi move to 1648 cm ^-1 and 1542 cm ^-1, respectively, indicating that the electrostatic interaction is involved in promoting the formation of whey protein isolate coat. In addition, the stretching vibration of the hydroxyl group at 3291 cm ^-1 is transferred to 3419 at cm ^-1, showing an enhancement of hydrogen bonding, i.e. the binding of whey protein isolate to the lactobacillus plantarum surface enhances hydrogen bonding in the structure.

Experimental example 4 growth curve

The free lactobacillus plantarum, PL@COS and PL@WPI prepared in example 2 and example 6 are taken as experimental samples, the samples are diluted in an MRS liquid culture medium until the optical density value (OD 600) is within a range of 0.1-0.2, a full-automatic high-flux microorganism liquid drop incubator is adopted to monitor the growth condition of bacteria at 37 ℃ every 0.5 h, and a growth curve is drawn according to the result.

From the results of FIG. 4, it can be seen that the growth conditions of PL@COS in MRS liquid medium at 37℃are better than PL, and the growth conditions of PL@WPI are similar to PL, which is probably that chitosan oligosaccharide is used as a marine polysaccharide and prebiotic, and can promote the growth and reproduction of Lactobacillus plantarum. In general, the presence of the polysaccharide or protein coat does not affect the normal growth and proliferation of lactobacillus plantarum.

Experimental example 5 pasteurization stability

The free lactobacillus plantarum, PL@COS and PL@WPI prepared in example 2 and example 6 are taken as experimental samples, the bacterial suspension is added into a sterile tube, stored at 63 ℃ for 30min, taken out, cooled at room temperature, properly diluted, inoculated on MRS agar, cultured at 37 ℃ for 48h and counted.

As can be seen from the results of FIG. 5, the PL@COS and PL@WPI prepared in example 2 and example 6 are higher in pasteurization stability than PL, and after heating at 63 ℃ for 30min, the viable count of PL, PL@COS and PL@WPI is reduced by 4.30, 3.57 and 3.74 Log CFU/mL, respectively. Compared with PL, the chitosan oligosaccharide coat and the whey isolated protein coat can reduce the heat damage to lactobacillus plantarum to a certain extent, and the effect of the chitosan oligosaccharide coat is better than that of the whey isolated protein coat.

Experimental example 6 Freeze thawing stability

The free lactobacillus plantarum, PL@COS and PL@WPI prepared in example 2 and example 6 are taken as experimental samples, and are preserved at-20 ℃ to 22 h, and then are thawed in a water bath at 30 ℃ to 2h, so that a freeze thawing cycle is adopted. The thawed samples were then again subjected to freeze thawing for a total of two cycles. At the end of the freeze-thaw cycle, samples were collected, diluted appropriately and 20 μl was inoculated onto MRS agar and counted after incubation at 37 ℃ for 48 h.

As can be seen from the results of FIG. 6, after two freeze-thawing cycles, the viable count of PL, PL@COS and PL@WPI is respectively reduced 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, namely, the freeze-thawing stability is PL@COS > PL@WPI > PL, which indicates that the freeze-thawing stability of lactobacillus plantarum can be improved to a certain extent by the chitosan oligosaccharide plume and whey isolated protein plume, and the effect of the chitosan oligosaccharide plume is better.

Experimental example 7 storage stability

Under the storage condition of 4 ℃, free lactobacillus plantarum, PL@COS and PL@WPI prepared in example 2 and example 6 are taken as experimental samples, the storage activity of the experimental samples is studied for 15 days, and the number of viable bacteria is detected by colony counting.

As can be seen from the results of FIG. 7, after 15 days of storage at 4 ℃, the number of viable PL bacteria remained 7.23 Log CFU/mL, while the numbers of viable PL@COS and PL@WPI bacteria were 9.00 and 7.97 Log CFU/mL, respectively, indicating that the chitosan oligosaccharide coat and whey protein isolate coat as physical barriers can isolate the external adverse environmental conditions to a certain extent, and improve the storage stability of Lactobacillus plantarum.

It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or equally substituted without departing from the spirit and scope of the technical solution of the present invention, and it should be covered 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 collard is provided.

2. The probiotic collard based on the surface coating technology is characterized in that the component of the collard is chitosan oligosaccharide or whey protein isolate.

3. The preparation method of the probiotic plume is characterized by comprising the steps of enabling probiotic suspension and polysaccharide or protein solution to interact, and re-suspending the probiotic suspension in sterile ultrapure water and vortex after treatment such as stirring and mixing uniformly, shaking and incubating, centrifugal washing and the like, so as to prepare the probiotic plume.

4. The method for preparing the probiotic collard according to claim 3, which is characterized by comprising the following steps:

1) Resuscitating the frozen and stored probiotics in a culture medium, centrifuging for 10 min at 7000 rpm after culturing, washing for many times to ensure no culture medium residue, removing supernatant, collecting bacterial mud, re-suspending the bacterial mud in 5 mL sterile ultrapure water, wherein the cell concentration of the probiotics is 1X 10 ¹⁰~1×10¹¹ CFU/mL, swirling for 10 s to obtain uniformly dispersed bacterial suspension, and storing in a refrigerator at 4 ℃ for later use;

2) Weighing polysaccharide and protein, dissolving in ultrapure water to prepare polysaccharide solution and protein solution respectively, stirring 500 rpm for 2h to dissolve completely, and standing in a refrigerator at 4deg.C overnight to hydrate;

3) Maintaining the pH of the polysaccharide and protein solutions of step 2) at fixed values by adding NaOH and HCl at 0.1M;

4) Dispersing the probiotic bacterial suspension in step 1) in 4.5 mL sterile ultrapure water to dilute the probiotic bacterial suspension, and maintaining the pH at a fixed value by adding NaOH and HCl of 0.1M;

5) Dropwise adding the probiotic bacterial suspension in the step 4) into the polysaccharide solution and the protein solution in the vortex in the step 3), mixing and stirring 10 min, and oscillating 20 min by a shaking table;

6) Centrifuging the mixed solution of the probiotics and the polysaccharide and the mixed solution of the probiotics and the protein obtained in the step 5) for 10 min at 7000 rpm, washing twice with sterile water with the same pH value, and removing the supernatant to obtain the probiotic bacterial sludge coated with the feathers;

5. The method of claim 4, wherein the culture medium in step 1) is MRS broth; the probiotics are lactobacillus plantarum, the culture time is the logarithmic growth phase of the probiotics, and the culture temperature is 37 ℃.

6. The method of claim 4, wherein the polysaccharide in step 2) is chitosan oligosaccharide and the protein is whey protein isolate.

7. The method of claim 4, wherein the polysaccharide concentration in step 2) is 2 mg/mL and the protein concentration is 2 mg/mL.

8. The method of claim 4, wherein the polysaccharide or protein solution in step 3) has a pH of 4.

9. The method of claim 4, wherein the pH of the probiotic suspension in step 4) is 4.

10. The method of claim 4, wherein the volume ratio of the probiotic suspension to the polysaccharide solution or the protein solution in step 5) is 1:5.