Explosive blasting bead containing fucosan and Ackerman's bacteria as well as preparation method and application of explosive blasting bead containing fucosan and Ackerman's bacteria

Abstract

The invention relates to the technical field of food processing, in particular to popping capsules containing fucosan and ackermania as well as a preparation method and application of the popping capsules. The popping bead is prepared from the following raw materials: fucosan and ackermania. The fucosan is obtained by drying kelp, autoclaving, fermenting with lactobacillus plantarum, extracting with water, precipitating with alcohol, deproteinizing, dialyzing and freeze-drying. The preparation method disclosed by the invention has the beneficial effects that the popping beads with high immune induction activity are formed by adding the fucosan modified by autoclaving-fermentation and the probiotics Ackermania, and the popping beads with high immune induction activity are prepared by utilizing good cross-linking and gelling effects of the modified fucosan and the sodium alginate; the probiotic activity, the acid resistance, the ultraviolet resistance, the enteric coating resistance and the like of the popping bead product are further enhanced, and the popping bead can be applied to preparation of bread foods.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and in particular to explosive beads containing fucoidan and Akkermansia, and a preparation method and application thereof. Background Art

[0002] Akkermansia muciniphila ( Akkermansia muciniphila ) is a potential probiotic recognized after Lactobacillus and Bifidobacterium, and has the functions of regulating the host's intestinal flora, intestinal inflammation, and immune response. Kelp is an important large economic seaweed along the coast of my country, containing active ingredients such as sodium alginate and fucoidan. Sodium alginate has a good gelling effect and is often used as a microcapsule wall material for encapsulation and delivery systems, while fucoidan has good immunomodulatory activity, regulates intestinal flora, and has multiple biological activities such as anti-tumor and antioxidant. In addition, a large number of studies have shown that the use of microbial fermentation such as Lactobacillus plantarum to assist in the extraction of polysaccharides has better biological activity.

[0003] Probiotics can be used as immune activators to stimulate and improve the immune system by increasing the activity of intestinal flora and the production of effector molecules. Polysaccharides are common prebiotic factors that can increase the relative abundance of intestinal probiotics, change the structure of intestinal flora, and thus improve intestinal health. Studies have found that encapsulation and delivery of probiotics by microencapsulation of polysaccharides and external ion gels can effectively protect probiotics from environmental damage in the upper digestive tract and achieve release in the lower digestive tract. For example, the invention patent "A composite sodium alginate probiotic microcapsule and its preparation method" (CN118556869A) discloses the use of sodium alginate, modified attapulgite, modified inulin, carrageenan, nano-silica, arabinoxylan, and fucoidan to prepare probiotic microcapsules encapsulating bifidobacteria, lactobacilli, Clostridium butyricum, and enterococci. Modified attapulgite, modified inulin, and carrageenan can be used to improve the viscosity, mechanical strength, and mechanical properties of the sodium alginate microcapsule wall. However, there are still the following limitations: the system embedding rate and viable bacteria count are low, and probiotics are selected from traditional strains. Existing fucoidan extraction processes mostly use single autoclave or fermentation treatments, resulting in high molecular weight of polysaccharides and low bioavailability, which cannot effectively cooperate with probiotics and promote probiotic proliferation. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a fucoidan and Akkermansia-containing popping bead which can improve the survival rate and immune induction activity of probiotics, and a preparation method and application thereof.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: to provide a popping bead containing fucoidan and Akkermansia, which is made of the following raw materials: 20-26 parts by weight of sodium alginate, 30-40 parts by weight of calcium lactate, 1-2 parts by weight of fucoidan, 1 part by weight of xanthan gum, 10 parts by weight of Akkermansia, 140-180 parts by weight of xylitol, 50-80 parts by weight of cocoa powder, and 195-198 parts by weight of edible oil; The preparation method of fucoidan comprises the following steps: drying, heat-pressing, microbial fermentation, water extraction, alcohol precipitation, protein removal, dialysis and freeze-drying the kelp to obtain the fucoidan.

[0006] Furthermore, in the above-mentioned popping beads containing fucoidan and Akkermansia, the pressure heat treatment is specifically as follows: after the kelp powder is dried to constant weight, an aqueous solution is prepared according to a volume ratio of 1:40, and the solution is placed in a high pressure sterilizer and treated at 121°C and 0.1 MPa for 15 min.

[0007] Furthermore, in the above-mentioned popping beads containing fucoidan and Akkermansia, the microbial fermentation treatment is specifically as follows: after the solution obtained by autoclave treatment is cooled, 5% volume ratio of Lactobacillus plantarum is inoculated and cultured in a constant temperature shaker at 180 rpm and 37°C for 24-48h.

[0008] Furthermore, in the above-mentioned explosive beads containing fucoidan and Akkermansia, the preparation method of the fucoidan is specifically as follows: After the kelp powder is dried to constant weight, an aqueous solution is prepared according to a volume ratio of 1:40, placed in a high pressure sterilizer, treated at 121°C and 0.1 MPa for 15 min, inoculated with 5% by volume of Lactobacillus plantarum after cooling, and cultured in a constant temperature shaker at 180 rpm and 37°C for 24-48 hours to obtain fermented kelp; The fermented kelp is extracted with hot water at 90° C. for 2 h, filtered with a 200-mesh filter cloth, the filtrate is centrifuged at 4000 rpm for 20 min, the supernatant is concentrated to 1 / 3 of the volume by rotary evaporation, the pH is adjusted to 0.8±0.02 with HCl, and then precipitated at 4° C. overnight, the supernatant is taken, adjusted to 7.0±0.02 with NaOH, and then precipitated with 75% ethanol, the precipitate is collected after centrifugation, and freeze-dried to obtain crude polysaccharide, the crude polysaccharide aqueous solution is deproteinized according to the Sevag method, and the supernatant is collected, the obtained supernatant is dialyzed with a 3.5 KDa dialysis bag, and then rotary evaporation and freeze-dried to obtain fucoidan.

[0009] Another technical solution provided by the present invention is to provide a method for preparing the above-mentioned popping beads containing fucoidan and Akkermansia, comprising the following steps: Step 1, mixing edible oil and distilled water, homogenizing and emulsifying them to obtain a mixed emulsion, adding xanthan gum and sodium alginate respectively, and homogenizing and mixing them after they are fully swollen, adding fucoidan, and mixing them evenly to obtain a mixed system; adding Akkermansia cells to the mixed system; The obtained mixed system is vertically dropped into a calcium chloride solution to obtain probiotic embedded particles, and the probiotic embedded particles are fished out and washed with sterile water for later use; Step 2, cocoa powder is prepared into cocoa solution with sterile water, sodium alginate is added, and the solution is stirred and dissolved in a water bath, and xylitol is added to fully dissolve the solution after the solution is completely swollen, followed by defoaming to obtain a defoaming solution; Step 3: Add the probiotic embedded particles obtained in step 1 to the defoaming solution obtained in step 2 at a mass ratio of 2%-3% and mix well. Suck them in with a dropper and drop them vertically into a sterile aqueous solution of calcium lactate. After soaking, take them out and wash them with sterile water to obtain the popping beads containing fucoidan and Akkermansia.

[0010] Furthermore, the preparation method of the above-mentioned popping beads specifically comprises the following steps: Step 1, 195-198 parts by weight of edible oil and distilled water are mixed in a volume ratio of 1:4, and homogenized for 10-15 minutes to emulsify them to obtain a mixed emulsion, 1 part by weight of xanthan gum and 10-12 parts by weight of sodium alginate are added respectively, and after they are fully swollen, they are homogenized and mixed, and 1-2 parts by weight of fucoidan are added to mix them evenly to obtain a mixed system; After washing Akkermansia cells with sterile water for 2-3 times, add them into the mixed system at a volume ratio of 1% and mix them evenly to make the final live bacterial concentration of Akkermansia system 1×10 2 CFU / mL; The obtained mixed system was transferred to a 2 mL syringe, and vertically dripped into a 4% solute mass percentage calcium chloride solution to soak for 20 s to obtain probiotic embedded particles. The probiotic embedded particles were fished out, washed with sterile water, and stored at 4°C for use; Step 2, 50-80 parts by weight of cocoa powder are prepared into a 5%-8% cocoa solution with sterile water, 10-14 parts by weight of sodium alginate are added, and the solution is stirred and dissolved in a water bath at 80°C, and after the solution is completely swollen, 14-18 parts by weight of xylitol are added to fully dissolve the solution, and then the solution is placed in a refrigerator at 4°C for 3-4 hours for defoaming treatment to obtain a defoaming solution; Step 3, prepare a calcium lactate solution with a solute mass percentage of 3%-4% with 30-40 parts by weight of calcium lactate and sterile water, add the probiotic embedded particles obtained in step 1 to the defoaming solution obtained in step 2 at a mass ratio of 2%-3% and mix well, suck it with a dropper, vertically drip it into the calcium lactate solution, soak it for 10 minutes, then take it out, wash it with sterile water 2-3 times to obtain the explosive beads containing fucoidan and Akkermansia.

[0011] Another technical solution of the present invention is to provide an application of the popping beads prepared by the above-mentioned preparation method of the popping beads in food.

[0012] Furthermore, in the above application, the food is bread.

[0013] Furthermore, in the above application, the amount of the popping beads added to the bread is 0.5%-2.0% of the mass of flour.

[0014] The beneficial effects of the present invention are as follows: the present invention provides a high immune-inducing activity explosive bead containing fucoidan and Akkermansia and a preparation method and application thereof, which has the following beneficial effects: 1. The present invention combines autoclave-fermented fucoidan with the probiotic Akkermansia, and autoclaves and Lactobacillus plantarum ferment the modified fucoidan. Compared with the polysaccharides extracted by traditional hot water, the purity of the polysaccharides can be improved, the total sugar and galactose contents can be increased, the molecular weight can be reduced, and the immune-inducing activity of the polysaccharides can be enhanced. On the other hand, the autoclave-fermented fucoidan can promote the growth and reproduction of Akkermansia and the production of beneficial metabolites, exert the synergistic effect of the modified fucoidan and Akkermansia, and further improve the immune-inducing activity of the product.

[0015] 2. The present invention can enhance the mechanical strength and mechanical properties of the sodium alginate microcapsule wall material by adding autoclave-fermented fucoidan, improve the system embedding rate and the number of viable bacteria, have good acid resistance, UV resistance, enteric resistance and other properties, and have better stability during transportation and storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure shows the effects of the autoclave-fermented fucoidan of Example 1, the autoclave-treated fucoidan of Comparative Example 1, and the fermentation-treated fucoidan of Comparative Example 2 on the growth of Akkermansia according to a specific embodiment of the present invention.

[0017] Figure 2 Shown are the live bacterial counts of Akkermansia of the explosive beads of Examples 1-2 and Comparative Examples 1-4 according to the specific embodiments of the present invention in the gastrointestinal adverse environment.

[0018] Figure 3 Shown are the live bacterial counts of Akkermansia in the explosive beads of Examples 1-2 and Comparative Examples 1-4 according to the specific embodiments of the present invention in an ultraviolet environment.

[0019] Figure 4 Shown is the amount of ROS produced by immune cells by the supernatant of the Explosive Beads digestion of Examples 1-2 and Comparative Examples 1-4 according to the specific embodiments of the present invention.

[0020] Figure 5 Shown is the amount of NO produced by immune cells from the supernatant of the Explosive Beads digestion of Examples 1-2 and Comparative Examples 1-4 according to the specific embodiments of the present invention.

[0021] Figure 6Shown is the number of live Akkermansia in bread prepared with the popping beads of Example 2 and Comparative Example 3 according to the specific embodiments of the present invention. DETAILED DESCRIPTION

[0022] In order to explain the technical content, achieved objectives and effects of the present invention in detail, the following is an explanation in combination with the implementation modes and the accompanying drawings.

[0023] The preparation method of fucoidan used in the specific embodiment of the present invention is: After the kelp powder was dried to constant weight, an aqueous solution was prepared according to a volume ratio of 1:40, placed in an autoclave, and treated at 121°C and 0.1 MPa for 15 min. After cooling, 5% volume ratio of Lactobacillus plantarum (1×10 8 CFU / mL), cultured in a constant temperature shaker at 180 rpm and 37°C for 24-48h to obtain fermented kelp; The above-mentioned Lactobacillus plantarum was purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd. (No. JYLP-326). The Lactobacillus plantarum powder was inoculated into sterilized MRS medium (Guangdong Huankai Microbiological Technology Co., Ltd.) at a 1% inoculation rate, activated and expanded to 1×10 8 CFU / mL, serially passaged 2-3 times.

[0024] The fermented kelp is extracted with hot water at 90°C for 2 h, filtered with a 200-mesh filter cloth, the filtrate is centrifuged at 4000 rpm for 20 min, the supernatant is concentrated to 1 / 3 of the volume, the pH is adjusted to 0.8±0.02 with HCl, and then precipitated at 4°C overnight, the supernatant is taken, adjusted to 7.0±0.02 with NaOH, and then precipitated with 75% ethanol, centrifuged, and the precipitate is collected and freeze-dried to obtain crude polysaccharide; According to the Sevag method, a 4:1 volume ratio of chloroform: n-butanol was used to prepare the Sevag reagent. The crude polysaccharide aqueous solution was mixed with the Sevag reagent in a 4:1 ratio, shaken, and centrifuged to remove the protein precipitate layer. The supernatant was recovered by the same method, repeated 3-4 times, and the supernatant obtained for the last time was separated and collected. The supernatant was dialyzed with a 3.5 KDa dialysis bag and then rotary evaporated and freeze-dried to obtain fucoidan.

[0025] In one embodiment of the present invention, a method for preparing explosive beads containing fucoidan and Akkermansia is provided, comprising the following steps: The popping beads are made of the following raw materials: 26 parts by weight of sodium alginate, 40 parts by weight of calcium lactate, 2 parts by weight of fucoidan, 1 part by weight of xanthan gum, 10 parts by weight of Akkermansia, 180 parts by weight of xylitol, 80 parts by weight of cocoa powder, and 198 parts by weight of edible oil; Step 1, 198 parts by weight of edible oil and distilled water were mixed in a volume ratio of 1:4, and homogenized for 15 minutes to emulsify them to obtain a mixed emulsion, 1 part by weight of xanthan gum and 12 parts by weight of sodium alginate were added respectively, and after they were fully swollen, they were homogenized and mixed, and 2 parts by weight of fucoidan were added to mix them evenly to obtain a mixed system; After washing Akkermansia cells with sterile water for 2-3 times, add them into the mixed system at a volume ratio of 1% and mix them evenly to make the final live bacterial concentration of Akkermansia system 1×10 2 CFU / mL; The obtained mixed system was transferred to a 2 mL syringe, and vertically dripped into a 4% solute mass percentage calcium chloride solution to soak for 20 s to obtain probiotic embedded particles. The probiotic embedded particles were fished out, washed with sterile water, and stored at 4°C for use; Step 2, 80 parts by weight of cocoa powder is prepared into 8% cocoa solution with sterile water, 14 parts by weight of sodium alginate is added, and the solution is stirred and dissolved in a water bath at 80°C, and after the solution is completely swollen, 180 parts by weight of xylitol is added to fully dissolve, and then the solution is placed in a refrigerator at 4°C for 3-4 hours for defoaming treatment to obtain a defoaming solution; Step 3, prepare a calcium lactate solution with a solute mass percentage of 3%-4% with 40 parts by weight of calcium lactate and sterile water, add the probiotic embedded particles obtained in step 1 to the defoaming solution obtained in step 2 at a mass ratio of 2%-3% and mix well, suck it with a dropper, vertically drip it into the calcium lactate solution, soak it for 10 minutes, then take it out, wash it with sterile water 2-3 times to obtain the explosive beads containing fucoidan and Akkermansia.

[0026] The popping beads obtained above can be added to bread, and the amount added to the bread is 0.5% of the mass of flour.

[0027] In another embodiment of the present invention, another method for preparing explosive beads containing fucoidan and Akkermansia is provided, comprising the following steps: The popping beads are made of the following raw materials: 23 parts by weight of sodium alginate, 35 parts by weight of calcium lactate, 2 parts by weight of fucoidan, 1 part by weight of xanthan gum, 10 parts by weight of Akkermansia, 160 parts by weight of xylitol, 60 parts by weight of cocoa powder, and 197 parts by weight of edible oil; Step 1, 197 parts by weight of edible oil and distilled water were mixed in a volume ratio of 1:4, and homogenized for 15 minutes to emulsify them to obtain a mixed emulsion, 1 part by weight of xanthan gum and 12 parts by weight of sodium alginate were added respectively, and after they were fully swollen, they were homogenized and mixed, and 2 parts by weight of fucoidan were added to mix them evenly to obtain a mixed system; After washing Akkermansia cells with sterile water for 2-3 times, add them into the mixed system at a volume ratio of 1% and mix them evenly to make the final live bacterial concentration of Akkermansia system 1×102 CFU / mL; The obtained mixed system was transferred to a 2 mL syringe, and vertically dripped into a 4% solute mass percentage calcium chloride solution to soak for 20 s to obtain probiotic embedded particles. The probiotic embedded particles were fished out, washed with sterile water, and stored at 4°C for use; Step 2, 60 parts by weight of cocoa powder are prepared into 8% cocoa solution with sterile water, 11 parts by weight of sodium alginate is added, and the solution is stirred and dissolved in a water bath at 80°C, and after the solution is completely swollen, 160 parts by weight of xylitol is added to fully dissolve the solution, and then the solution is placed in a refrigerator at 4°C for 3-4 hours for defoaming treatment to obtain a defoaming solution; Step 3, prepare a calcium lactate solution with a solute mass percentage of 3%-4% with 35 parts by weight of calcium lactate and sterile water, add the probiotic embedded particles obtained in step 1 to the defoaming solution obtained in step 2 at a mass ratio of 2%-3% and mix well, suck it with a dropper, vertically drip it into the calcium lactate solution, soak it for 10 minutes, then take it out, wash it with sterile water 2-3 times to obtain the explosive beads containing fucoidan and Akkermansia.

[0028] The popping beads obtained above can be added to bread, and the amount added to the bread is 1.5% of the mass of flour.

[0029] The beneficial effect of the present invention is that: the present invention utilizes pressure-heat combined with fermentation to extract fucoidan, and compared with fucoidan extracted by single pressure-heat and single fermentation, the pressure-heat combined with fermentation fucoidan can better promote the growth and proliferation of the beneficial bacteria Akkermansia, and the pressure-heat combined with fermentation fucoidan interacts with other raw materials and auxiliary materials such as sodium alginate, the wall material of the popping beads, to obtain the popping beads with better gel and mechanical properties, so that the survival rate of probiotics is higher, and the resistance to gastrointestinal and ultraviolet adverse environments is higher, and the cellular immune activity is better. The inner layer of the popping beads obtained by the present invention uses calcium chloride to cross-link sodium alginate to form a dense core material, and the outer layer is secondary solidified with calcium lactate to enhance the mechanical strength, thereby improving the survival rate of live bacteria in the gastric acid environment.

[0030] Example 1 A method for preparing explosive beads containing fucoidan and Akkermansia comprises the following steps: Raw materials: 20 parts by weight of sodium alginate, 30 parts by weight of calcium lactate, 1 part by weight of fucoidan (autoclaved-fermented fucoidan), 1 part by weight of xanthan gum, 10 parts by weight of Akkermansia, 140 parts by weight of xylitol, 50 parts by weight of cocoa powder, and 195 parts by weight of edible oil; The preparation method of fucoidan is as follows: kelp powder is dried to constant weight and then prepared into an aqueous solution at a ratio of 1:40 (v / v), placed in an autoclave (121°C, 0.1 MPa) for 15 min, cooled and then inoculated with 5% plant lactobacillus (viable count 1×10 8CFU / mL) and cultured in a constant temperature shaker at 180 rpm and 37°C for 24-48 h.

[0031] The fermented kelp was extracted with hot water at 90°C for 2 h, filtered with a 200-mesh filter cloth, and the filtrate was centrifuged at 4000 rpm for 20 min. The supernatant was concentrated to 1 / 3 volume by rotary evaporation, and the pH was adjusted to 0.8±0.02 with HCl and then precipitated at 4°C overnight. The supernatant was adjusted to 7.0±0.02 with NaOH, and then precipitated with 75% ethanol. After centrifugation, the precipitate was collected and freeze-dried to obtain crude polysaccharide. According to the Sevag method, chloroform and n-butanol were mixed in a volume ratio of 4:1 to prepare Sevag reagent. The crude polysaccharide aqueous solution was mixed with the Sevag reagent in a ratio of 4:1, shaken, and centrifuged to remove the protein precipitate layer. The supernatant was recovered by the same method, repeated 3-4 times, and the supernatant obtained in the last time was separated and collected. The obtained supernatant was dialyzed with a 3.5 KDa dialysis bag and then rotary evaporated and freeze-dried to obtain autoclaved-fermented fucoidan.

[0032] 1. Preparation of calcium chloride solution, calcium lactate solution and activation and expansion of Akkermansia strains A 4% calcium chloride solution with a solute mass percentage was prepared using 40 parts by weight of calcium chloride, and a 3% calcium lactate solution was prepared using 30 parts by weight of calcium lactate and sterile water.

[0033] A tube (specification 0.02 g) of Akkermansia freeze-dried powder (purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd., No. BNCC341917) was inoculated into 15 mL of sterilized FT thioglycollate fluid culture medium (purchased from Guangdong Huankai Microbiological Technology Co., Ltd.), and cultured at 37°C under anaerobic conditions for 4-5 days, and then preserved with glycerol (glycerol: bacterial solution = 3:7) and stored at -80°C for subsequent experiments; 1 mL of glycerol-preserved Akkermansia was taken and activated by adding 100 mL of sterilized FT thioglycollate fluid culture medium, and cultured at 37°C under anaerobic conditions for 9 h, and then 1 mL of bacterial solution was added to 100 mL of sterilized FI thioglycollate fluid culture medium for expansion culture, and cultured at 37°C under anaerobic conditions for 4 h, and the bacterial solution was centrifuged, and the bacterial precipitate was resuspended in sterile water (the number of viable bacteria in the bacterial suspension was about 1×10 7 CFU / mL) and then gradually diluted in ten-fold gradients until the number of viable bacteria was about 1×10 4 CFU / mL of inoculum solution.

[0034] 2. Preparation of fucoidan and Akkermansia embedded particles 195 parts by weight of edible oil and distilled water were mixed in a volume ratio of 1:4 and homogenized for 10-15 min to obtain a mixed emulsion. 1 part by weight of xanthan gum and 10 parts by weight of sodium alginate were added respectively and homogenized after they were fully swollen. 1 part by weight of autoclaved-fermented fucoidan was added and mixed evenly. 10 parts by weight of Akkermansia cells (viable count 1×10 4 CFU / mL) was washed 2-3 times with sterile water, and then added into the mixed system at a 1% inoculum amount and mixed evenly, so that the final live bacterial concentration of Akkermansia system was 1×10 2 CFU / mL. The mixed system prepared above was transferred to a 2 mL syringe and vertically dropped into a 4% solute mass percentage calcium chloride solution and soaked for about 20 s to obtain probiotic embedded particles, which were fished out and washed with sterile water and stored at 4°C for use.

[0035] 3. Preparation of outer layer sodium alginate embedding solution 50 parts by weight of cocoa powder were prepared into 5% cocoa solution with sterile water, 10 parts by weight of sodium alginate was added, and the solution was stirred and dissolved in a water bath at 80°C. After the solution was completely swollen, 140 parts by weight of xylitol was added and fully dissolved. The solution was then placed in a refrigerator at 4°C for 3-4 hours for defoaming.

[0036] 4. Add 2% of the fucoidan and Akkermansia embedding particles in step 1 to the defoaming outer layer sodium alginate embedding solution and mix well. Suck it in with a dropper and vertically drip into the calcium lactate solution. After soaking for 10 minutes, take it out and wash it with sterile water for 2-3 times to obtain the explosive beads containing fucoidan and Akkermansia.

[0037] Example 2 The difference from Example 1 is that the amount of fucoidan in the raw material is 2 parts by weight, and the rest is the same as Example 1.

[0038] Comparative Example 1 The difference from Example 1 is that the autoclave-fermented fucoidan in the raw material is replaced with a single autoclave-treated fucoidan. Accordingly, the fucoidan solution is not subjected to 5% Lactobacillus plantarum fermentation treatment during preparation, and the rest is the same as Example 1.

[0039] Comparative Example 2 The difference from Example 1 is that the autoclave-fermented fucoidan in the raw material is replaced with a single fermentation-treated fucoidan. Accordingly, the fucoidan solution is not placed in an autoclave (121°C, 0.1MPa) for 15 min during preparation, and the rest is the same as Example 1.

[0040] Comparative Example 3 The difference from Example 1 is that the raw material does not contain fucoidan. Accordingly, there is no step of preparing fucoidan and no step of adding fucoidan to the Akkermansia embedding particles. The rest is the same as Example 1.

[0041] Comparative Example 4 The difference from Example 1 is that the sodium alginate in the raw material is changed to 10 parts by weight, and correspondingly, the sodium alginate in the fucoidan and Akkermansia embedding particles and the outer layer sodium alginate embedding solution is changed to 5 parts by weight, and the rest is the same as Example 1.

[0042] Various indicators of the fucoidan prepared in the above Example 1 and Comparative Examples 1-2 and the explosive beads prepared in the above Example 1-2 and Comparative Examples 1-4 were measured.

[0043] 1. Determination of the effect of fucoidan on the proliferation of probiotic Akkermansia The Akkermansia frozen at -80℃ was inoculated, activated and expanded (anaerobic culture) at a 1% inoculum, and subcultured 2-3 times. A 1.0 mg / mL fucoidan culture medium was prepared and sterilized at 121℃ for 15 min. The expanded Akkermansia culture was centrifuged at 5000 rpm / min for 5 min, the supernatant was discarded and diluted to 1×10 4 CFU / mL, inoculated into the fucoidan medium at a 1% inoculation volume, so that the final colony count in the system was 1×10 2 CFU / mL, adjust the medium pH to 7.0±0.02 and culture at 37℃ in an anaerobic environment for 60h, and measure the corresponding OD 600 .

[0044] 2. Sensory score determination of popping pearls A sensory evaluation panel consisting of 10 graduate students majoring in food science conducted a sensory evaluation of the popping pearls in terms of mouthfeel, flavor, chewing sensation, appearance, and degree of preference. The evaluation criteria are shown in Table 1.

[0045] Table 1 Sensory evaluation standards

[0046] 3. Determination of the resistance of Explosive Beads to adverse digestive environment The popping beads were crushed to simulate oral chewing, and 0.5 g of the crushed popping beads were weighed, 9 mL of artificial gastric juice was added, and the mixture was shaken at 180 rpm in a 37°C constant temperature shaker to simulate gastric digestion. After 2 h of gastric digestion, the sample was taken out, the artificial gastric juice was discarded by centrifugation, and 5 mL of PBS solution was added. The outer membrane of the popping beads was destroyed by high-speed vortex oscillator to release the Akkermansia in it, and the live bacteria were calculated by plate counting method after diluting it with sterile water.

[0047] In addition, a group of Exploding Beads products were prepared. After the simulated gastric digestion was completed, 10 mL of artificial intestinal fluid was added to simulate the intestinal fluid digestion process under the same conditions. The intestinal digestion process was ended after 4 hours. The number of live bacteria was determined after the Exploding Beads were dissected.

[0048] 4. Determination of UV resistance of Explosive Beads The Explosive Beads were irradiated under a 254 nm UV lamp for 0.5 h, and then placed in the dark for 1 h. Then, appropriate amounts of phosphate buffer were added, and the Explosive Beads were decapsulated, diluted, and smeared to determine the number of viable bacteria.

[0049] 5. Cellular immune activity assay Cellular immune activity was evaluated by the generation of reactive oxygen species (ROS) and NO in RAW264.7 cells. 1.0 g of the Explosive Beads were dissected and treated with 50 μL / mL of RAW 264.7 cells to measure immune activity. LPS (2 μg / mL) was used as a positive control group. The generation of reactive oxygen species (ROS) and NO in cells were measured using the DCFH-DA probe and Griess reagent, respectively.

[0050] Data Analysis: Each test was repeated three times, and the data were analyzed and compared using Tukey's multiple comparison method. p < 0.05 was considered statistically significant.

[0051] Chart Description: Depend on Figure 1 As shown, the growth rate of Akkermansia by the autoclaved-fermented fucoidan in Example 1 was significantly higher than that of the fucoidan treated with single autoclave and single fermentation in Comparative Examples 1-2 (p < 0.05). In contrast, the autoclaved-fermented fucoidan showed the best prebiotic potential, which may be related to its higher galactose content and lower molecular weight.

[0052] As can be seen from Table 2, the sensory score of Example 1-2 is significantly higher than that of Comparative Example 1-4 (p < 0.05), mainly because the taste and chewiness are significantly improved, accompanied by the improvement of appearance and preference. Compared with Comparative Example 3, Example 1-2 shows that fucoidan can promote the cross-linking of sodium alginate. Compared with the fucoidan treated with single autoclave and single fermentation in Comparative Example 1-2, the autoclave-fermented fucoidan used in Example 1-2 promotes the cross-linking of sodium alginate more significantly, and the prepared popping beads have better taste and chewiness.

[0053] Table 2 Sensory evaluation of popping pearls

[0054] from Figure 2 and 3It can be seen that Examples 1 and 2 can significantly improve the survival rate of Akkermansia in the stomach, intestine and ultraviolet adverse environment (p < 0.05), and increase with the increase of the concentration of autoclaved-fermented fucoidan. Examples 1 and 2 show a good probiotic survival rate, which may be related to the higher prebiotic effect of autoclaved-fermented fucoidan itself on the one hand, and on the other hand, it may be related to the stronger cross-linking of autoclaved-fermented fucoidan with seaweed yogurt, and better gel and mechanical properties, thereby improving the survival rate of Akkermansia in adverse environment.

[0055] In addition, from Figure 4 and 5 It can be seen that compared with Comparative Example 3, similar to the positive control LPS induction, Examples 1 and 2 can significantly increase the production of ROS and NO in immune cells (p < 0.05), indicating that autoclave-fermented fucoidan can improve the cellular immune induction activity. Compared with Comparative Examples 1-2, Examples 1 and 2 show better cellular immune activity, which may be related to the higher prebiotic effect of autoclave-fermented fucoidan itself. Compared with fucoidan treated with single autoclave and single fermentation, autoclave-fermented fucoidan has a lower molecular weight and higher uronic acid, and the polysaccharide itself has better cellular immune regulation activity; on the other hand, it may be related to the autoclave-fermented fucoidan promoting the proliferation and survival rate of Akkermansia, promoting the proliferation and metabolism of the probiotic Akkermansia, and enhancing cellular immunity.

[0056] The popping beads prepared in Example 1 and Comparative Example 3 were used to study their application in bread. The mass fraction of the popping beads added was 1% based on the mass of flour. The number of live Akkermansia in each group was as follows: Figure 6 As shown, it was found that autoclave-fermented fucoidan could improve the probiotic survival rate of Akkermansia popping beads in bread products.

[0057] In summary, the present invention provides a method for preparing a fucoidan with high immune-inducing activity and Akkermansia blasting beads and their application, wherein the fucoidan is extracted by combining pressure-heat with fermentation. Compared with fucoidan extracted by single pressure-heat or single fermentation, the obtained pressure-heat combined fermentation fucoidan can better promote the growth and proliferation of the beneficial bacteria Akkermansia. The pressure-heat combined fermentation fucoidan interacts with other raw materials and auxiliary materials such as sodium alginate, which is the embedding wall material of the blasting beads, to obtain blasting beads with better gel and mechanical properties, which have a higher survival rate of probiotics, higher resistance to gastrointestinal and ultraviolet adverse environments, and better cellular immune activity.

[0058] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A popping bead containing fucoidan and Akkermansia, characterized in that: Made from the following ingredients: 20-26 parts by weight of sodium alginate, 30-40 parts by weight of calcium lactate, 1-2 parts by weight of fucoidan, 1 part by weight of xanthan gum, 10 parts by weight of Akkermansia, 140-180 parts by weight of xylitol, 50-80 parts by weight of cocoa powder, and 195-198 parts by weight of edible oil; The preparation method of fucoidan comprises the following steps: drying, heat-pressing, microbial fermentation, water extraction, alcohol precipitation, protein removal, dialysis and freeze-drying the kelp to obtain the fucoidan.

2. The explosive beads containing fucoidan and Akkermansia according to claim 1, characterized in that: The autoclave treatment is specifically as follows: the kelp powder is dried to constant weight, an aqueous solution is prepared according to a volume ratio of 1:40, and the solution is placed in a high pressure sterilizer and treated at 121° C. and 0.1 MPa for 15 min.

3. The explosive beads containing fucoidan and Akkermansia according to claim 1, characterized in that: The microbial fermentation treatment is specifically as follows: after the solution obtained by the autoclave treatment is cooled, 5% by volume of Lactobacillus plantarum is inoculated and cultured in a constant temperature shaker at 180 rpm and 37° C. for 24-48 hours.

4. The explosive beads containing fucoidan and Akkermansia according to claim 1, characterized in that: The preparation method of the fucoidan is specifically as follows: After the kelp powder is dried to constant weight, an aqueous solution is prepared according to a volume ratio of 1:40, placed in a high pressure sterilizer, treated at 121°C and 0.1MPa for 15 min, inoculated with 5% by volume of Lactobacillus plantarum after cooling, and cultured in a constant temperature shaker at 180 rpm and 37°C for 24-48 hours to obtain fermented kelp; The fermented kelp is extracted with hot water at 90° C. for 2 h, filtered with a 200-mesh filter cloth, the filtrate is centrifuged at 4000 rpm for 20 min, the supernatant is concentrated to 1 / 3 of the volume by rotary evaporation, the pH is adjusted to 0.8±0.02 with HCl, and then precipitated at 4° C. overnight, the supernatant is taken, adjusted to 7.0±0.02 with NaOH, and then precipitated with 75% ethanol, centrifuged, collected, and freeze-dried to obtain crude polysaccharides, the crude polysaccharide aqueous solution is deproteinized according to the Sevag method, and the supernatant is collected, the obtained supernatant is dialyzed with a 3.5 KDa dialysis bag, and then rotary evaporated and freeze-dried to obtain fucoidan.

5. A method for preparing the explosive beads containing fucoidan and Akkermansia according to any one of claims 1 to 4, characterized in that: The steps include: Step 1, mixing edible oil and distilled water, homogenizing and emulsifying them to obtain a mixed emulsion, adding xanthan gum and sodium alginate respectively, and homogenizing and mixing them after they are fully swollen, adding fucoidan, and mixing them evenly to obtain a mixed system; adding Akkermansia cells to the mixed system; The obtained mixed system is vertically dropped into a calcium chloride solution to obtain probiotic embedded particles, and the probiotic embedded particles are fished out and washed with sterile water for later use; Step 2, cocoa powder is prepared into cocoa solution with sterile water, sodium alginate is added, and the solution is stirred and dissolved in a water bath, and xylitol is added to fully dissolve the solution after the solution is completely swollen, followed by defoaming to obtain a defoaming solution; Step 3: Add the probiotic embedded particles obtained in step 1 to the defoaming solution obtained in step 2 at a mass ratio of 2%-3% and mix well. Suck them in with a dropper and drop them vertically into a sterile aqueous solution of calcium lactate. After soaking, take them out and wash them with sterile water to obtain the popping beads containing fucoidan and Akkermansia.

6. The method for preparing popping beads according to claim 5, characterized in that: The specific steps include: Step 1, 195-198 parts by weight of edible oil and distilled water are mixed in a volume ratio of 1:4, and homogenized for 10-15 minutes to emulsify them to obtain a mixed emulsion, 1 part by weight of xanthan gum and 10-12 parts by weight of sodium alginate are added respectively, and after they are fully swollen, they are homogenized and mixed, and 1-2 parts by weight of fucoidan are added to mix them evenly to obtain a mixed system; After washing Akkermansia cells with sterile water for 2-3 times, add them into the mixed system at a volume ratio of 1% and mix them evenly, so that the final live bacterial concentration of Akkermansia system is 1×10 2 CFU / mL; The obtained mixed system was transferred to a 2 mL syringe, and vertically dropped into a 4% solute mass percentage calcium chloride solution and soaked for 20 seconds to obtain probiotic embedded particles. The probiotic embedded particles were fished out, washed with sterile water, and stored at 4°C for use; Step 2, 50-80 parts by weight of cocoa powder are prepared into a 5%-8% cocoa solution with sterile water, 10-14 parts by weight of sodium alginate are added, and the solution is stirred and dissolved in a water bath at 80°C, and after the solution is completely swollen, 14-18 parts by weight of xylitol are added to fully dissolve the solution, and then the solution is placed in a refrigerator at 4°C for 3-4 hours for defoaming treatment to obtain a defoaming solution; Step 3, prepare a calcium lactate solution with a solute mass percentage of 3%-4% with 30-40 parts by weight of calcium lactate and sterile water, add the probiotic embedded particles obtained in step 1 to the defoaming solution obtained in step 2 at a mass ratio of 2%-3% and mix well, suck it with a dropper, vertically drip it into the calcium lactate solution, soak it for 10 minutes, then take it out, wash it with sterile water 2-3 times to obtain the explosive beads containing fucoidan and Akkermansia.

7. Use of the popping beads prepared by the method for preparing popping beads according to any one of claims 5 to 6 in food.

8. The use according to claim 7, characterized in that: The food is bread.

9. The use according to claim 8, characterized in that: The amount of the popping beads added to the bread is 0.5%-2.0% of the mass of flour.

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