Fucoidin-gamma-polyglutamic acid hydrogel entrapped with probiotics as well as preparation method and application of fucoidin-gamma-polyglutamic acid hydrogel

Abstract

The invention discloses a preparation method of fucoidin-gamma-polyglutamic acid hydrogel entrapped with probiotics, which comprises the following steps: (1) preparing gamma-polyglutamic acid modified by 3-aminophenylboronic acid through amidation reaction, namely gamma-PGAB; (2) respectively dissolving gamma-PGAB (gamma-polyglutamic acid) and fucoidin in resuspended lactobacillus plantarum LP90 bacterial liquid, and mixing, so as to obtain the fucoidin-gamma-polyglutamic acid hydrogel entrapped with the probiotics, wherein the gamma-PGAB and the fucoidin are respectively dissolved in the resuspended lactobacillus plantarum LP90 bacterial liquid; the fucoidin-gamma-polyglutamic acid hydrogel entrapped with the probiotics, provided by the invention, has good mechanical properties and biocompatibility; the lactobacillus plantarum can be well coated by a specific porous structure inside the hydrogel, and the lactobacillus plantarum can be protected by a structure which is contracted due to deprotonation of abundant carboxyl groups of the external gamma-polyglutamic acid in a gastric acid environment, so that the lactobacillus plantarum can be kept in a harsh gastric acid and bile salt environment, and the number of viable bacteria reaching intestinal tracts is increased; the method can be used for preparing probiotic food.

Description

Technical Field

[0001] The present invention relates to the field of food technology, and in particular to a probiotic-encapsulated fucoidan-gamma-polyglutamic acid hydrogel, and a preparation method and application thereof. Background Art

[0002] Probiotics, defined as living microorganisms, have many health benefits to the host after ingestion of a certain amount. For example, they can relieve lactose intolerance, boost the immune system, lower serum cholesterol, resist gastrointestinal (GI) infections, relieve constipation, etc. However, the low survival rate of probiotics under harsh gastric acid and bile salt conditions and the low retention time in the intestine greatly limit their health benefits. Therefore, there is a need for a carrier that can encapsulate probiotics so that they can survive the gastric acid environment and reach the intestine to exert their function of regulating intestinal flora.

[0003] Hydrogel loading and delivery of probiotics has been considered an effective and safe strategy. However, a large number of chemical modifications and non-food raw materials limit its application in food. Fuco, a water-soluble polysaccharide extracted from seaweed, has been established as an active prebiotic with multiple functions such as anti-inflammatory, regulating intestinal flora and immune response, and is often used as a functional food in the food industry and medicine. However, the gel properties of fucoidan alone are poor, and usually require the mediation of other conditions to form a gel. The current related treatment methods include promoting gelation at low temperature, adding other polysaccharides such as chitosan, carrageenan, etc. to form a gel blend, and adding Ca 2+ , Fe 3+ Gelation occurs through ion-mediated connection of polymer chains, etc. Summary of the invention

[0004] In view of the above problems existing in the prior art, the present invention provides a fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics, and a preparation method and application thereof. The hydrogel of the present invention has good cell compatibility and shows good microbial compatibility when co-incubated with probiotics. Compared with traditional probiotic protection hydrogels, the present invention uses natural fucoidan and natto extract γ-polyglutamic acid as raw materials, both of which are substances allowed to be added to food, which broadens its application in the food field, has actual production value, and opens up a new path for active edible probiotic foods.

[0005] The technical solution of the present invention is as follows:

[0006] The first object of the present invention is to provide a method for preparing a fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics, the preparation method comprising the following steps:

[0007] (1) Preparation of γ-polyglutamic acid modified with 3-aminophenylboronic acid, i.e., γ-PGA B ;

[0008] (2) γ-PGA B The resuspended bacterial solution is mixed with the fucoidan resuspended bacterial solution to prepare the fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics;

[0009] Before the γ-polyglutamic acid is modified, its carboxyl group is activated. The activation method is as follows: after the γ-polyglutamic acid is completely dissolved in deionized water at room temperature, N-hydroxysuccinimide and carbodiimide are added for activation for 10 to 60 minutes.

[0010] In one embodiment of the present invention, the molecular weight of γ-polyglutamic acid is 50-100 kDa.

[0011] In one embodiment of the present invention, the mass ratio of γ-polyglutamic acid, N-hydroxysuccinimide and carbodiimide is 10:(3-6):(3-6); the carbodiimide is one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, dicyclohexylcarbodiimide, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, and N,N'-diisopropylcarbodiimide.

[0012] In one embodiment of the present invention, in step (1), γ-PGA B The preparation method is as follows: 3-aminophenylboronic acid is dissolved in anhydrous ethanol, and then added dropwise to the activated γ-PGA solution, and reacted at room temperature for 10 to 24 hours at a speed of 600 rpm on a magnetic stirrer, and then packaged in a dialysis bag with a molecular weight cutoff of 8000 to 14000 kDa, and dialyzed in deionized water for 3 to 5 days, with the water being replaced 3 to 5 times a day, and freeze-dried at -80°C to -50°C for 3 to 5 days to obtain the γ-PGA. B In one embodiment of the present invention, the mass ratio of activated γ-polyglutamic acid to 3-aminophenylboronic acid is 10:(3-6).

[0013] In one embodiment of the present invention, in step (2), γ-PGA B Resuspended bacteria in γ-PGA B Dissolved in resuspended bacterial solution, γ-PGA B The concentration is 16-24wt%.

[0014] In one embodiment of the present invention, in step (2), the fucoidan re-suspension bacterial solution is prepared by dissolving fucoidan in the re-suspension bacterial solution, wherein the concentration of fucoidan is 8-12 wt %.

[0015] In one embodiment of the present invention, the resuspended bacterial solution is obtained by culturing Lactobacillus plantarum LP90 (deposit number: CGMCC No. 10453) overnight in MRS broth for 36 to 48 hours, centrifuging at 8000 rpm for 3 minutes, and resuspending in deionized water with a volume of 1 / 10 of the original bacterial solution; its concentration is 1.0×10 10 ~9.0×10 11 CFU / mL. The resuspended bacterial solution is used as a hydrogel medium.

[0016] In one embodiment of the present invention, in step (2), γ-PGA B The volume ratio of the resuspended bacterial solution to the fucoidan resuspended bacterial solution is 1:1.

[0017] Preferably, γ-PGA B The mass ratio of fucoidan to cellulose is 2:1.

[0018] The second object of the present invention is to provide a fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics.

[0019] The third object of the present invention is to provide an application of fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics for preparing probiotic food.

[0020] The beneficial technical effects of the present invention are:

[0021] The γ-polyglutamic acid (γ-PGA) used in the present invention is a safe natural edible polymer fermented by microorganisms. Its abundant surface carboxyl groups not only enable it to wrap probiotics to survive the harsh gastric acid environment, but also enable it to have the prerequisite for preparing hydrogels with natural high-molecular polysaccharides rich in hydroxyl groups.

[0022] The hydrogel of the present invention also shows good probiotic activity protection ability after co-incubation with artificial gastric acid and bile salts. After co-incubation with artificial intestinal fluid, the probiotics are gradually released to achieve the effect of probiotic stable delivery.

[0023] The nuclear magnetic resonance spectrometer and infrared spectroscopy indicators proved that it was not a simple physical mixture but a new substance was generated; the vial inversion experiment and rheological experiment proved the formation of its gel structure and excellent rheological properties; the cryo-scanning electron microscope observed that the porous structure in the gel provided conditions for the attachment of probiotics; the growth curve of Lactobacillus plantarum after co-incubation with the gel and the activity measurement of Caco-2 cells showed that γ-PGA B The probiotic protection hydrogel with fucoidan has good biocompatibility; after incubation with gastric acid and bile salts, it was found that the hydrogel has the ability to protect probiotics through the complex body fluid environment and maintain their activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the hydrogel prepared in Example 5.

[0025] Figure 2 This is the appearance of the hydrogel prepared in Example 1-4.

[0026] Figure 3 This is the Fourier transform infrared spectrum of the hydrogel prepared in Example 5.

[0027] Figure 4 The modulus of the hydrogels prepared in Example 1 and Example 5 varies with strain, frequency and time.

[0028] Figure 5 The thixotropic recovery diagram of the hydrogels prepared in Example 1 and Example 5.

[0029] Figure 6 This is a diagram showing the cell safety evaluation results of the hydrogel prepared in Example 5.

[0030] Figure 7 This is a diagram showing the biocompatibility evaluation results of the hydrogel prepared in Example 5.

[0031] Figure 8 These are cryo-scanning electron micrographs of the hydrogels prepared in Examples 1 and 5.

[0032] Fig. 9 This is a graph showing the acid and bile salt resistance results of the hydrogel prepared in Example 1. Fig.10 This is the acid and bile salt resistance result diagram of Example 1. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0034] Example 1

[0035] A fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics, the preparation method comprising the following steps:

[0036] Step 1: Dissolve 500 mg of food-grade γ-PGA in 15 mL of deionized water at room temperature, then add 262 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 159 mg of N-hydroxysuccinimide (NHS), and activate for 30 minutes to obtain activated γ-PGA;

[0037] 208 mg of 3-aminophenylboronic acid was dissolved in 2 mL of anhydrous ethanol and added dropwise to the activated γ-PGA solution. The mixture was reacted at 600 rpm on a magnetic stirrer at room temperature for 12 h. The mixture was packaged in a dialysis bag with a molecular weight cutoff of 8000-14000 kDa and dialyzed in deionized water for 3 days. The water was changed 3 times a day. The hydrogel precursor skeleton (γ-PGA) was obtained by freeze drying at -80 °C. B ).

[0038] Step 2: After culturing the revived Lactobacillus plantarum LP90 overnight in MRS broth, centrifuge at 8000 rpm for 3 min, and resuspend in deionized water with a volume of 1 / 10 of the original bacterial solution to obtain a concentration of 9.0×10 11 The resuspended bacterial solution with a concentration of CFU / mL is the hydrogel medium.

[0039] Step 3: 32 mg of γ-PGA B Dissolved in 200 μL of resuspended bacterial solution to obtain 16 wt% γ-PGA B Resuspend the bacterial solution; dissolve 16 mg of fucoidan in 200 μL of the resuspended bacterial solution to obtain 8 wt % fucoidan resuspended bacterial solution; mix the above two solutions in equal volumes to obtain the fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics.

[0040] Example 2

[0041] A fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics, the preparation method comprising the following steps:

[0042] Step 1: Dissolve 500 mg of food-grade γ-PGA in 15 mL of deionized water at room temperature, then add 262 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 159 mg of N-hydroxysuccinimide (NHS), and activate for 30 minutes to obtain activated γ-PGA;

[0043] 208 mg of 3-aminophenylboronic acid was dissolved in 2 mL of anhydrous ethanol and added dropwise to the activated γ-PGA solution. The mixture was reacted at 600 rpm on a magnetic stirrer at room temperature for 12 h. The mixture was packaged in a dialysis bag with a molecular weight cutoff of 8000-14000 kDa and dialyzed in deionized water for 3 days. The water was changed 3 times a day. The hydrogel precursor skeleton (γ-PGA) was obtained by freeze drying at -80 °C. B ).

[0044] Step 2: After culturing the revived Lactobacillus plantarum LP90 overnight in MRS broth, centrifuge at 8000 rpm for 3 min, and resuspend in deionized water with a volume of 1 / 10 of the original bacterial solution to obtain a concentration of 9.0×10 11The resuspended bacterial solution with a concentration of CFU / mL is the hydrogel medium.

[0045] Step 3: 48 mg of γ-PGA B Dissolved in 200 μL of resuspended bacterial solution to obtain 24 wt% γ-PGA B Resuspend the bacterial solution; dissolve 16 mg of fucoidan in 200 μL of the resuspended bacterial solution to obtain 8 wt % fucoidan resuspended bacterial solution; mix the above two solutions in equal volumes to obtain the fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics.

[0046] Example 3

[0047] A fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics, the preparation method comprising the following steps:

[0048] Step 1: Dissolve 500 mg of food-grade γ-PGA in 15 mL of deionized water at room temperature, then add 262 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 159 mg of N-hydroxysuccinimide (NHS), and activate for 30 minutes to obtain activated γ-PGA;

[0049] 208 mg of 3-aminophenylboronic acid was dissolved in 2 mL of anhydrous ethanol and added dropwise to the activated γ-PGA solution. The mixture was reacted at 600 rpm on a magnetic stirrer at room temperature for 12 h. The mixture was packaged in a dialysis bag with a molecular weight cutoff of 8000-14000 kDa and dialyzed in deionized water for 3 days. The water was changed 3 times a day. The hydrogel precursor skeleton (γ-PGA) was obtained by freeze drying at -80 °C. B ).

[0050] Step 2: After culturing the revived Lactobacillus plantarum LP90 overnight in MRS broth, centrifuge at 8000 rpm for 3 min, and resuspend in deionized water with a volume of 1 / 10 of the original bacterial solution to obtain a concentration of 9.0×10 11 The resuspended bacterial solution with a concentration of CFU / mL is the hydrogel medium.

[0051] Step 3: 32 mg of γ-PGA B Dissolved in 200 μL of resuspended bacterial solution to obtain 16 wt% γ-PGA B Resuspend the bacterial solution; dissolve 24 mg of fucoidan in 200 μL of the resuspended bacterial solution to obtain a 12 wt % fucoidan resuspended bacterial solution; mix the above two solutions in equal volumes to obtain the fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics.

[0052] Example 4

[0053] A fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics, the preparation method comprising the following steps:

[0054] Step 1: Dissolve 500 mg of food-grade γ-PGA in 15 mL of deionized water at room temperature, then add 262 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 159 mg of N-hydroxysuccinimide (NHS), and activate for 30 minutes to obtain activated γ-PGA;

[0055] 208 mg of 3-aminophenylboronic acid was dissolved in 2 mL of anhydrous ethanol and added dropwise to the activated γ-PGA solution. The mixture was reacted at 600 rpm on a magnetic stirrer at room temperature for 12 h. The mixture was packaged in a dialysis bag with a molecular weight cutoff of 8000-14000 kDa and dialyzed in deionized water for 3 days. The water was changed 3 times a day. The hydrogel precursor skeleton (γ-PGA) was obtained by freeze drying at -80 °C. B ).

[0056] Step 2: After culturing the revived Lactobacillus plantarum LP90 overnight in MRS broth, centrifuge at 8000 rpm for 3 min, and resuspend in deionized water with a volume of 1 / 10 of the original bacterial solution to obtain a concentration of 9.0×10 11 The resuspended bacterial solution with a concentration of CFU / mL is the hydrogel medium.

[0057] Step 3: 48 mg of γ-PGA B Dissolved in 200 μL of resuspended bacterial solution to obtain 24 wt% γ-PGA B Resuspend the bacterial solution; dissolve 24 mg of fucoidan in 200 μL of the resuspended bacterial solution to obtain a 12 wt % fucoidan resuspended bacterial solution; mix the above two solutions in equal volumes to obtain the fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics.

[0058] Example 5

[0059] A fucoidan-γ-polyglutamic acid hydrogel, the preparation method of which comprises the following steps:

[0060] Step 1: After dissolving 500 mg of food-grade γ-PGA in 15 mL of deionized water, 262 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 159 mg of N-hydroxysuccinimide (NHS) were added to the γ-PGA solution to activate its carboxyl group for 30 minutes to obtain activated γ-PGA.

[0061] 208 mg of 3-aminophenylboronic acid was dissolved in 2 mL of anhydrous ethanol and added dropwise to the activated γ-PGA solution. The mixture was reacted at 600 rpm on a magnetic stirrer at room temperature for 12 h. The mixture was packaged in a dialysis bag with a molecular weight cutoff of 8000-14000 kDa and dialyzed in deionized water for 3 days. The water was changed 3 times a day. The hydrogel precursor skeleton (γ-PGA) was obtained by freeze drying at -80 °C. B ).

[0062] Step 2: 32 mg of γ-PGA B Dissolve in 200 μL deionized water to obtain 16 wt% γ-PGA B Solution; dissolving 16 mg of fucoidan in 200 μL of deionized water to obtain an 8 wt % fucoidan solution; mixing the above two solutions in equal volumes to obtain the fucoidan-γ-polyglutamic acid hydrogel.

[0063] Test example:

[0064] 1. Nuclear magnetic resonance hydrogen spectrum: Use the nuclear magnetic resonance spectrometer produced by Bruker, Germany. The specific operation is as follows: select a 5mm sample tube, weigh 10-15mg of solid sample, and finally prepare a solution with a height of about 4cm for testing. 1 H-NMR nuclear magnetic resonance spectrum.

[0065] Figure 1 This is the nuclear magnetic resonance hydrogen spectrum of fucoidan-γ-polyglutamic acid hydrogel obtained in Example 5. It can be seen from the figure that compared with the nuclear magnetic resonance hydrogen spectrum of γ-PGA, γ-PGA B The characteristic peak of the benzene ring belonging to 3-aminophenylboronic acid appeared at 7.5ppm, indicating that 3-aminophenylboronic acid was successfully grafted onto γ-PGA, indicating the generation of new substances.

[0066] 2. Vial inversion experiment: Place 400 μL of fucoidan-γ-polyglutamic acid hydrogel encapsulating Lactobacillus plantarum LP90 in a 3 mL white-capped sample vial, wipe the edge of the hydrogel to make it in the same plane, invert the sample vial and take a photo.

[0067] Figure 2 This is the appearance of the hydrogel obtained in Example 1-4. It can be seen from the figure that the probiotic hydrogel is in a slightly yellow state and does not fall down when inverted, indicating the formation of a gel state.

[0068] 3. Infrared spectroscopy: A Fourier transform infrared spectrometer produced by Platinum Elmer Co., Ltd. was used, and the potassium bromide tablet method was adopted. The specific operation was as follows: spectrally pure potassium bromide powder and different samples were weighed in a ratio of 150:1, placed in a mortar and ground until the powder size was less than 2.5 μm. After grinding, the sample was taken out and placed in a tablet press to make a transparent or translucent disc of uniform thickness. The sample was taken out and tested.

[0069] Figure 3 This is the Fourier infrared spectrum of the hydrogel prepared in Example 5. The FT-IR spectrum shows that 1333 cm -1 The peak at 3349 cm is the -CH stretching vibration peak of the benzene ring of the boric acid group in 3-aminophenylboronic acid. This characteristic peak also appears in Example 5, further supporting the occurrence of the grafting reaction. -1 There is a characteristic peak of OH stretching vibration at B After mixing, this stretching shifts to lower wavenumbers, which is due to the formation of hydrogen bonds involving -OH groups. This proves that the formation of the gel in Example 5 is due to the generation of new structures.

[0070] 4. Determination of gel rheological properties: The samples were scanned using a Discovery HR-2 hybrid rheometer (TAInstruments) to measure the storage modulus and loss modulus, respectively. The hydrogel properties prepared in Examples 1 and 5 were tested using a rheometer to measure the storage modulus (G') and loss modulus (G") of the hydrogel material with strain (0.1-1000%), time (0-300s) and frequency (0.1-100Hz) to study the properties of the material.

[0071] Figure 4 The graphs of the viscosity of the hydrogels obtained in Example 1 and Example 5 as a function of shear rate and the modulus as a function of strain, frequency and time are shown below. Figure 4 It can be seen from A that both Example 1 (blue line) and Example 5 (gray line) show shear thinning when subjected to shear. Figure 4 As can be seen from C and D, for Example 5 (red line), when the strain is between 0.1% and 8%, G' and G" are in the linear viscoelastic range, and when the strain is equal to 11%, a gel-sol transition point appears; the linear viscoelastic range of its frequency is 0.1-10 Hz. Figure 4B It can be seen that when the strain and frequency in the linear viscoelastic range are selected for time scanning, the G' value of Example 1 is measured to be stable at about 980Pa. For Example 1 (black line), the linear viscoelastic range of its strain is between 0.1% and 1%. When the strain is equal to 9%, a gel-sol transition point appears. The linear viscoelastic range of its frequency is 0.1-10Hz. When the strain and frequency in the linear viscoelastic range are selected for time scanning, the G' value of Example 1 is measured to be stable at about 730Pa, indicating that the hydrogel has excellent mechanical properties.

[0072] 5. Thixotropic recovery: Five stages are set. The first, third and fifth stages use 1% strain and 300s time scanning. The second and fourth stages use 1000% strain and 300s time scanning. The frequency of the five stages is 1Hz and the temperature is 25℃. Study the self-healing ability of the material.

[0073] Figure 5 The thixotropic recovery diagram of the hydrogels prepared in Example 1 (black line) and Example 5 (red line) can be seen from the figure that when the oscillating stress is alternately at 1% and 1000% and scanned for 300s, the high strain of 1000% destroys the network structure of the hydrogel and makes its G" value greater than the G' value; while at the low strain of 1%, the G' value can quickly recover to its initial value, indicating that the structure has been repaired and it has good self-healing performance.

[0074] 6. Cell safety verification:

[0075] CCK-8 cell proliferation and cytotoxicity assay: Caco-2 cells (10,000 cells / well) were inoculated in a 96-well plate containing 100 μL complete medium. After culturing for 24-48 h until they adhered to the wall and then the medium was washed away. 8 wt% Fuco solution, 16 wt% γ-PGA B The solution was added to the well plate with a combined hydrogel (red) at a volume ratio of 1 / 1. The cells incubated with pure complete medium were used as the control group (blue). After the cells were incubated at 37°C in an incubator containing 5% CO2 for 24 hours, the incubation solution was removed, washed with DPBS solution, and then 10% CCK-8 solution was added. The blank group was a well without cells. After incubation for 4 hours in the dark, the absorbance at 450nm was observed.

[0076] Figure 6 The figure is the result of cell safety evaluation of the hydrogel prepared in Example 5. It can be seen that after being treated with Example 5 (Hydrogel), the activity of Caco-2 cells has no significant difference with that of the group treated with pure Caco-2 cell complete medium, indicating that Example 5 has good cell safety.

[0077] Calcein-AM and PI live / dead cell staining: Caco-2 cells (200,000 cells / well) were seeded in a 12-well plate containing 1 mL of complete medium. After culturing for 24-48 h until they attached to the wall, the medium was washed off and 4 wt% Fuco solution and 8 wt% γ-PGA were added. B The solution and Example 5 (Hydrogel) were added to a 12-well plate containing cells, and cells incubated with pure Caco-2 cell complete medium were used as the control group. The cells were incubated in an incubator at 37°C and 5% CO2 for 24 hours. The incubation solution was removed by suction, and washed with DPBS solution, and then 0.1% Calcein stain was added and incubated for 15-20 minutes, and then the stain was washed away with PBS, and then 0.1% PI stain was added and incubated for 15-20 minutes, and then the stain was washed away, and 1 mL of DPBS was added and observed under a fluorescent inverted microscope.

[0078] Figure 7 Example 5 and 4wt% Fuco, 8wt% γ-PGA B Live / dead cell staining fluorescence microscopy photos of cells after treatment. It can be seen that after Example 5 and 4wt% Fuco, 8wt% γ-PGA B After treatment, the active cell area (Calcein staining is green) and the number of dead cells (PI staining is red) are similar to those of the pure Caco-2 cell complete medium treatment group, indicating that Example 5 and 4wt% Fuco, 8wt% γ-PGA B Has good cell safety.

[0079] 7. Biocompatibility evaluation:

[0080] The revived Lactobacillus plantarum LP90 was cultured in MRS broth overnight, centrifuged at 8000 rpm for 3 min, and resuspended in deionized water with a volume of 1 / 10 of the original bacterial solution to obtain a concentration of 9.0×10 11 The resuspended bacterial solution with a concentration of CFU / mL is the hydrogel medium.

[0081] 32 mg of γ-PGA B Dissolve in 200 μL of resuspended bacterial solution to obtain 16 wt% PGA B Solution; 16 mg of fucoidan was dissolved in 200 μL of resuspended bacterial solution to obtain an 8 wt% fucoidan solution. ① Example 1 (grey line): The above two solutions were mixed in equal volumes to obtain 200 μL of fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics; ② Blue line: 16 mg of γ-PGA B Dissolve in 200 μL of resuspended bacterial solution to obtain 200 μL of 8 wt% γ-PGA BProbiotic solution; ③ Black line: 8 mg of fucoidan was dissolved in 200 μL of resuspended bacterial solution to obtain 200 μL of 4wt% fucoidan probiotic solution: ④ Red line: 200 μL of resuspended bacterial solution was used as the control group. After incubating the above groups with probiotics for 30 minutes, 200 μL of glucanase was used to release the probiotics in ①, and 200 μL of deionized water was added to the other groups to control the volume. Cultured at 37°C in a shaking incubator at a speed of 150 rpm / min, taking points every 1 hour, cultured to the stable period, and plotted to observe the effects of different treatments on the growth of probiotics.

[0082] Figure 8 Example 1 and 4wt% Fuco, 8wt% γ-PGA B The biocompatibility evaluation results of the prepared hydrogels are shown in the figure. As can be seen from the figure, after Example 1 (gray line) and 4wt% Fuco (black line), 8wt% γ-PGA B (Blue line) The growth curve of Lactobacillus plantarum LP90 after treatment and the growth trend of the control group (red line) and the OD when reaching the stable period 600 Similar, indicating that Example 1 and 4wt% Fuco, 8wt% γ-PGA B It has good biocompatibility and will not affect the normal growth of Lactobacillus plantarum.

[0083] 8. Cryo-scanning electron microscopy: A cryo-scanning electron microscope (SU8010 / PP3010T) was used to characterize the internal microstructure of the hydrogel. 5-10 μL of Example 1 and Example 5 were placed on the sample stage and then frozen in liquid nitrogen. The frozen samples were fractured to observe their internal structure. After sublimation at -90°C for 30 min, they were gold-plated for 60 s. Observation and imaging were performed under low temperature conditions.

[0084] Fig. 9 The cryo-SEM images of the hydrogels prepared in Example 1 and Example 5 are shown in FIG. B -The fucoidan hydrogel exhibits a typical three-dimensional porous structure. When Lactobacillus plantarum LP90 is encapsulated in the hydrogel, its porous structure can still be clearly observed, indicating that encapsulation of Lactobacillus plantarum has no effect on the internal structure of the hydrogel. The circular cross-section and rod-shaped longitudinal section of Lactobacillus plantarum embedded in the hydrogel are consistent with the normal morphology of Lactobacillus plantarum, indicating that the encapsulation of the hydrogel does not affect the morphological characteristics of Lactobacillus plantarum.

[0085] 9. Acid and bile salt resistance test: In order to simulate the low pH gastric environment, 0.1 mol·L -1HCL was used to prepare MRS medium with a pH of 2.0. In order to simulate the bile salt environment, 0.3wt% MRS medium was prepared using ox bile salt. The revived Lactobacillus plantarum was inoculated into MRS broth at an inoculum size of 3% and incubated at 37°C for 24h. The Lactobacillus plantarum was collected by centrifugation at 8000rpm for 3min, washed three times with 0.85% saline, and resuspended in MRS broth with 1 / 10 of the original medium.

[0086] 32 mg of γ-PGA B Dissolve in 200 μL of resuspended bacterial solution to obtain 16 wt% PGA B Solution; 16 mg of fucoidan was dissolved in 200 μL of resuspended bacterial solution to obtain 8 wt% fucoidan solution. ① Example 1 (red): The above two solutions were mixed in equal volumes to obtain 200 μL of γ-PGA-fucoidan probiotic protection hydrogel; ② Blue: 200 μL of resuspended bacterial solution was used as the control group. After incubating the above groups with probiotics for 30 minutes, 200 μL of glucanase was used to release the probiotics in Example 1, and 200 μL of deionized water was added to the other groups to control the volume.

[0087] Gastric acid resistance test: The above different treatment groups were added to 800 μL MRS medium with a pH of 2.0, cultured in a shaking incubator at 37°C and 120 rpm / min, and samples were taken at 1 and 2 hours, diluted to a suitable concentration with 0.85% saline and then applied. After 36 hours of culture in a constant temperature biochemical incubator, the number of viable bacteria was calculated.

[0088] Bile salt resistance test: The above different treatment groups were added to 800 μL of MRS medium with a bile salt concentration of 0.3%, and cultured in a shaking incubator at 37°C and 120 rpm / min. Samples were taken at 2 and 4 hours, respectively, and diluted to the appropriate concentration with 0.85% saline before coating. After 36 hours of culture in a constant temperature biochemical incubator, the number of viable bacteria was calculated.

[0089] Fig.10 This is the acid and bile salt resistance result diagram of Example 1.

[0090] For gastric acid resistance test ( Fig.10 A): After 2 h of incubation in SGF, the number of viable bacteria in the LAB group increased from 1.9×10 10 CFU / mL decreased to 1.5×10 10 CFU / mL, while the number of viable bacteria in the γ-PGAB-fucoidan Example 1 group increased from 2.40×10 10 CFU / mL increased to 4×10 10 CFU / mL.

[0091] For the bile salt tolerance test ( Fig.10 B): The number of viable bacteria in the LAB group was 0.2×10 at 2 h after bile salt incubation. 9 CFU / mL, the probiotics were completely inactivated at 4h. However, the number of live bacteria in the Example 1 group continued to grow. The results of the Example 1 group showed a significant improvement compared with the single Lactobacillus plantarum group, indicating that γ-PGA B - Fucoidan hydrogel can largely protect probiotics from adverse conditions and maintain their normal physiological activity.

[0092] In summary, the method of the present invention can prepare a fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics, and the hydrogel has a porous continuous structure of a traditional hydrogel. The use of this set of technology can obtain a probiotic protection measure against gastric acid and bile salts, which has a good market prospect.

[0093] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics, characterized in that: The preparation method comprises the following steps: (1) Preparation of γ-polyglutamic acid modified with 3-aminophenylboronic acid, i.e., γ-PGA B ; (2) γ-PGA B The resuspended bacterial solution is mixed with the fucoidan resuspended bacterial solution to prepare the fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics; Before the γ-polyglutamic acid is modified, its carboxyl group is activated. The activation method is as follows: after the γ-polyglutamic acid is completely dissolved in deionized water at room temperature, N-hydroxysuccinimide and carbodiimide are added for activation for 10 to 60 minutes.

2. The preparation method according to claim 1, characterized in that: The molecular weight of γ-polyglutamic acid is 50-100 kDa.

3. The preparation method according to claim 1, characterized in that: The mass ratio of γ-polyglutamic acid, N-hydroxysuccinimide and carbodiimide is 10:(3-6):(3-6); the carbodiimide is one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, dicyclohexylcarbodiimide, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N,N'-diisopropylcarbodiimide.

4. The preparation method according to claim 1, characterized in that: In step (1), γ-PGA B The preparation method is as follows: dissolving 3-aminophenylboronic acid in anhydrous ethanol, adding dropwise to the activated γ-PGA solution, stirring and reacting at room temperature for 10 to 24 hours, placing in a dialysis bag for 3 to 5 days, and finally freeze-drying at -80°C to -50°C for 3 to 5 days to obtain the γ-PGA B .

5. The preparation method according to claim 4, characterized in that: The mass ratio of activated γ-polyglutamic acid to 3-aminophenylboronic acid is 10:(3-6); the specification of the dialysis bag is 8000-14000 kDa; During the dialysis process: dialyze in deionized water for 3 to 5 days, changing the water 2 to 3 times a day.

6. The preparation method according to claim 1, characterized in that: In step (2), γ-PGA B Resuspended bacteria in γ-PGA B Dissolved in resuspended bacterial solution, γ-PGA B The concentration of the fucoidan is 16-24wt%; the fucoidan re-suspension bacterial solution is prepared by dissolving fucoidan in the re-suspension bacterial solution, wherein the concentration of the fucoidan is 8-12wt%.

7. The preparation method according to claim 6, characterized in that: The resuspended bacterial solution was obtained by culturing Lactobacillus plantarum in MRS broth for 36 to 48 hours overnight, centrifuging at 8000 rpm for 3 minutes, and resuspending it in deionized water with a volume of 1 / 10 of the original bacterial solution; Its concentration is 1.0×10 10 ~9.0×10 11 CFU / mL.

8. The preparation method according to claim 1, characterized in that: In step (2), γ-PGA B The volume ratio of the resuspended bacterial solution to the fucoidan resuspended bacterial solution is 1:

1.

9. A fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics prepared by the preparation method according to any one of claims 1 to 8.

10. An application of the fucoidan-γ-polyglutamic acid hydrogel encapsulating probiotics according to claim 9, characterized in that: Used to prepare probiotic foods.

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