Seaweed-source low-molecular-weight active polysaccharide probiotic embedded particles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of food processing, in particular to seaweed-source low-molecular-weight active polysaccharide probiotic embedded particles as well as a preparation method and application thereof. The preparation raw materials of the seaweed source low-molecular-weight active polysaccharide probiotic embedded particles comprise seaweed source low-molecular-weight active polysaccharide and probiotics, and the seaweed source low-molecular-weight active polysaccharide is obtained by carrying out enterococcus fermentative degradation treatment, alcohol precipitation, deproteinization, dialysis and freeze-drying treatment on carrageenan to obtain the seaweed source low-molecular-weight active polysaccharide. The preparation method has the beneficial effects that carrageenan is fermented and modified to obtain a combination of seaweed-source low-molecular-weight active polysaccharide and probiotic enterococcus, so that on one hand, the molecular weight can be reduced, active groups such as hydroxyl can be exposed, and the blood fat reducing activity and the like of the polysaccharide can be enhanced; on the other hand, the synergistic effect of the seaweed-source low-molecular-weight active polysaccharide and the beneficial bacteria is exerted, and the embedding rate and the viable count of the system are increased.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and in particular to seaweed-derived low-molecular-weight active polysaccharide probiotic embedded particles and a preparation method and application thereof. Background Art

[0002] Prebiotics (dietary factors such as active polysaccharides) and probiotic products show good prospects in regulating intestinal homeostasis imbalance, lowering blood lipids and blood sugar, and preventing various metabolic disorders. Enterococci and other bacteria are recognized as potential probiotics after Lactobacillus and Bifidobacterium. They have the functions of regulating the intestinal flora of the host, lowering blood lipids, lowering blood sugar, and enhancing immunity. However, probiotics are often affected by the fermentation environment and have poor biological activity stability. Seaweed is one of the important marine biological resources along the coast of my country. It contains active ingredients such as carrageenan and red hair algae polysaccharides. Among them, seaweed polysaccharides such as carrageenan, agar, and sodium alginate not only have good gelling effects, but are often used as microcapsule wall materials for embedding delivery systems. They can also be used as important carbon sources for intestinal probiotics. Fermentation produces active polysaccharides and their metabolites. Like non-gel polysaccharides such as red hair algae polysaccharides, they have good blood sugar and blood lipid lowering, regulating intestinal flora and other biological activities. They are potential sources of prebiotic raw materials, but they are often too high in molecular weight and are slowly degraded in the intestine, limiting their activity. Therefore, there is an urgent need to develop prebiotic-probiotic embedded particles with stable activity.

[0003] Yogurt is a well-known dairy product, which not only contains rich protein and minerals, but also contains a large number of probiotics such as live lactic acid bacteria. In recent years, many studies have used probiotics and prebiotics to produce functional yogurt, which can not only exert the physiological activity and health care functions of the functional ingredients themselves, but also improve the quality and characteristics of yogurt. The Chinese invention patent application with publication number CN113598231A discloses a sucrose-free yogurt with added composite dietary fiber and human probiotics and a preparation method thereof. The product is composed of skimmed fresh milk, water-soluble dietary fiber, xylitol, whey protein, stabilizer, aspartame, acesulfame potassium, trivalent chromium, thermophilic streptococcus ST81, and Lactobacillus delbrueckii subspecies bulgaricus. Trivalent chromium, which has the effect of lowering blood sugar, is added to yogurt to play a role in lowering blood sugar. The Chinese invention patent application with publication number CN107232305A discloses a composite probiotic fermented sugar-free laver yogurt and a preparation method thereof, which uses xylitol instead of sucrose, and is particularly suitable for consumers such as the middle-aged and elderly and diabetic patients, and has a simple process and low production cost. The technical solution of the above patent application does not focus on whether the addition of probiotic factors can increase the probiotics and texture quality stability in yogurt. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a seaweed-derived low molecular weight active polysaccharide probiotic embedded particles and a preparation method and application thereof, which can improve the survival rate of probiotics and the stability of texture quality of the obtained product.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: to provide a seaweed-derived low molecular weight active polysaccharide probiotic embedded granules, comprising the following raw materials: 20-35 parts by weight of sodium alginate, 30-40 parts by weight of calcium lactate, 0.5-1.5 parts by weight of low molecular weight active polysaccharide from seaweed, 1-2 parts by weight of xanthan gum, 10 parts by weight of probiotics, 35-45 parts by weight of calcium chloride, 120-140 parts by weight of xylitol, 20-40 parts by weight of cocoa powder, and 195-198 parts by weight of edible oil; The method for preparing the seaweed-derived low molecular weight active polysaccharide comprises: subjecting carrageenan to enterococcal fermentation degradation, alcohol precipitation, deproteinization, dialysis and freeze-drying treatment to obtain the seaweed-derived low molecular weight active polysaccharide.

[0006] Furthermore, in the above-mentioned seaweed-derived low molecular weight active polysaccharide probiotic embedded particles, the probiotics are enterococci.

[0007] Furthermore, in the above-mentioned seaweed-derived low molecular weight active polysaccharide probiotic embedded particles, the enterococcus fermentation degradation treatment is specifically as follows: preparing a carrageenan aqueous solution with a solute mass percentage of 1%-1.5%, pasteurizing at 65°C for 40 min, inoculating 1% volume ratio of enterococci, and culturing in a constant temperature shaker at 180 rpm and 37°C for 12-48h.

[0008] Furthermore, in the above-mentioned seaweed-derived low molecular weight active polysaccharide probiotic embedded particles, the seaweed-derived low molecular weight active polysaccharide is prepared by the following method: after the carrageenan is subjected to enterococcal fermentation and degradation treatment, the bacteria and insoluble impurities are removed by centrifugation; the supernatant is concentrated under reduced pressure to 1 / 5 of the original volume at 55°C, anhydrous ethanol is slowly added dropwise to a final ethanol concentration of 75% by mass, precipitated, centrifuged, the precipitate is collected, the precipitate is redissolved, the obtained precipitate aqueous solution is deproteinized by the Sevag method, and the supernatant is collected, the obtained supernatant is dialyzed with 100Da dialysis bag distilled water, the obtained sample is concentrated and freeze-dried to obtain the seaweed-derived low molecular weight active polysaccharide.

[0009] Another technical solution provided by the present invention is to provide a method for preparing the above-mentioned seaweed-derived low molecular weight active polysaccharide probiotic embedded particles, comprising the following steps: Step 1, mixing edible oil and distilled water, homogenizing and emulsifying, then adding xanthan gum and sodium alginate respectively, fully swelling and further homogenizing and mixing; adding low molecular weight active polysaccharide from seaweed, sterilizing to obtain a mixed system; Add probiotics to the mixed system and mix evenly, drip the obtained mixed system into a 3.5%-4.5% by mass calcium chloride solution to soak to obtain probiotic embedded particles, remove the probiotic embedded particles and wash them 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 and mix well. Use a dropper to suck it and drop it vertically into a sterile aqueous solution of calcium lactate. After soaking, take it out to obtain seaweed-derived low molecular weight active polysaccharide probiotic embedded particles.

[0010] Furthermore, the preparation method of the above-mentioned seaweed-derived low molecular weight active polysaccharide probiotic embedded particles 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 the mixture to obtain a mixed emulsion, and 1-2 parts by weight of xanthan gum and 5-10 parts by weight of sodium alginate are added respectively to fully swell and further homogenize and mix; Add 0.5-1.5 parts by weight of low molecular weight active polysaccharide from seaweed, and pasteurize at 65°C for 1 hour; Add enterococci to the mixed system and mix evenly, so that the final viable bacterial count of the system reaches 100 CFU / mL; transfer the mixed system to a 2 mL syringe, vertically drop it into a calcium chloride solution with a solute mass percentage of 3.5%-4.5%, and soak it for 20 seconds to obtain probiotic embedded particles, remove it, wash it with sterile water, and store it at 4°C for use; Step 2, prepare a cocoa solution with a solute mass percentage of 2%-4% by weight of 20-40 parts by weight of cocoa powder and sterile water, add 15-25 parts by weight of sodium alginate, stir and dissolve in a water bath at 80°C, add 12%-14% by weight of xylitol to fully dissolve after it swells completely, and then place it in a refrigerator at 4°C for 3-4 hours to defoam, to obtain a defoaming solution; Step 3, preparing a calcium lactate solution with a solute mass percentage of 2.5% to 3.5% by weight of calcium lactate and sterile water; The probiotic embedded particles obtained in step 1 are added to the defoaming solution obtained in step 2 at a mass ratio of 2%-3%, mixed evenly, sucked with a dropper, vertically dripped into the calcium lactate solution, soaked for 10 minutes and then fished out to obtain seaweed-derived low molecular weight active polysaccharide probiotic embedded particles.

[0011] Another technical solution of the present invention is to provide an application of the embedded particles prepared by the preparation method of the above-mentioned seaweed-derived low molecular weight active polysaccharide probiotic embedded particles in food.

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

[0013] Furthermore, in the above application, the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles are added to fresh milk, and Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus are added as fermentation agents, stirred evenly, and fermented to obtain yogurt.

[0014] The beneficial effect of the present invention is that the multi-level embedded particles formed by adding a combination of low molecular weight active polysaccharides from seaweed and probiotic enterococci can better maintain the biological activity of probiotics. The lipid-lowering, anti-colitis and other active effects of the obtained product and its stability against adverse environments are significantly improved. The prepared yogurt gel has high stability and good lipid-lowering activity, realizing the high-quality and high-value development of yogurt products. Specifically, it has the following beneficial effects: 1. The present invention combines fermented modified carrageenan (seaweed-derived low molecular weight active polysaccharide) with probiotic enterococci. Compared with traditional hot water-extracted seaweed polysaccharides, the seaweed-derived low molecular weight active polysaccharides can, on the one hand, reduce the molecular weight and expose active groups such as hydroxyl groups, thereby enhancing the lipid-lowering activity of the polysaccharide; on the other hand, the seaweed-derived low molecular weight active polysaccharide can promote the growth and reproduction of enterococci and their beneficial metabolites such as short-chain fatty acids, thereby exerting the synergistic effect of modified seaweed active polysaccharides and beneficial bacteria, and further improving the lipid-lowering activity of the product.

[0015] 2. The present invention can enhance the mechanical strength and mechanical properties of the wall material of sodium alginate microcapsule by adding seaweed-derived low molecular weight active polysaccharides, improve the system embedding rate and the number of viable bacteria, and has good acid resistance, UV resistance, enteric resistance and other properties. It is used to prepare functional yogurt, can well enhance and maintain the activity of probiotics and prebiotics in the product, and has better stability during transportation and storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is a comparison chart of the effects of low molecular weight active polysaccharides from seaweed and original carrageenan on the proliferation of probiotic enterococci according to a specific embodiment of the present invention.

[0017] Figure 2 Shown is a comparison chart of the lipid-lowering activities of the seaweed-derived low molecular weight active polysaccharide and the enterococcal fermentation product of original carrageenan according to a specific embodiment of the present invention.

[0018] Figure 3 It is a comparison chart showing the live count of enterococci in the gastrointestinal adverse environment of the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles of Examples 1-2 and Comparative Examples 1-3 according to the specific embodiments of the present invention.

[0019] Figure 4It is a comparison chart of the live count of enterococci in the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles in ultraviolet environment according to Examples 1-2 and Comparative Examples 1-3 of the specific embodiments of the present invention.

[0020] Figure 5 Shown is a comparison chart of the effects of seaweed-derived low molecular weight active polysaccharide probiotic embedded particles on the triglyceride content of adipocytes according to Examples 1-2 and Comparative Examples 1-3 of the specific embodiments of the present invention.

[0021] Figure 6 It is a comparison chart of the live probiotic counts of the functional yogurt prepared by the probiotic embedded particles of Example 1 and Comparative Example 2 according to the specific embodiments of the present invention and the yogurt without the probiotic embedded particles (blank control group). 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 the seaweed-derived low molecular weight active polysaccharide used in the specific embodiment of the present invention is: the seaweed-derived low molecular weight active polysaccharide is obtained by subjecting carrageenan to enterococcal fermentation degradation, alcohol precipitation, deproteinization, dialysis, and freeze-drying. The specific preparation method is as follows: 1% of a tube (specification 1 mL) of enterococcal bacterial liquid (purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd., No. BNCC341917) was inoculated into 10 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 1-2 days, and then preserved with glycerol (glycerol: bacterial liquid = 3:7) and stored at -80°C for subsequent experiments; 0.1 mL of glycerol-preserved enterococcal bacterial liquid was taken and activated by adding 9.9 mL of sterilized FT thioglycollate fluid culture medium, and cultured at 37°C under anaerobic conditions for 6 h, and then 0.1 mL of bacterial liquid was added to 10 mL of sterilized FT thioglycollate fluid culture medium for expansion culture, and cultured at 37°C under anaerobic conditions for 4 h, and the bacterial liquid was centrifuged, and the bacterial precipitate was resuspended with physiological saline (the number of viable bacteria in the bacterial suspension was about 1×10 8 CFU / mL), and then gradually diluted in ten-fold gradient until the number of viable bacteria was about 1×10 4 CFU / mL of inoculum solution.

[0024] Prepare a 1%-1.5% mass fraction carrageenan and other seaweed polysaccharide aqueous solution, pasteurize at 65℃ for 40 min, inoculate 1% enterococci (100 CFU / mL), and culture in a constant temperature shaker at 180 rpm and 37℃ for 12-48h. After fermentation and degradation, centrifuge to remove bacteria and insoluble impurities; the supernatant is concentrated to 1 / 5 of the original volume under reduced pressure at 55℃. Slowly add anhydrous ethanol to a final ethanol concentration of 75%, precipitate overnight, centrifuge, and collect the precipitate. The precipitate is redissolved, and the Sevag reagent is prepared by mixing chloroform and n-butanol in a volume ratio of 4:1 according to the Sevag method. The precipitated aqueous solution and the Sevag reagent are mixed in a ratio of 3:1, shaken, and centrifuged to remove the protein precipitate layer. The supernatant is recovered by the same method, repeated 3-5 times, and the supernatant obtained in the last time is separated and collected. The obtained supernatant is dialyzed with a 100Da dialysis bag for 2 days. After the sample is concentrated and freeze-dried, a low molecular weight active polysaccharide from seaweed is obtained.

[0025] In one embodiment of the present invention, a method for preparing seaweed-derived low molecular weight active polysaccharide probiotic embedded particles is provided, comprising the following steps: The seaweed-derived low molecular weight active polysaccharide probiotic embedded particles are made of the following raw materials: 35 parts by weight of sodium alginate, 40 parts by weight of calcium lactate, 1.5 parts by weight of seaweed-derived low molecular weight active polysaccharide, 1 part by weight of xanthan gum, 10 parts by weight of probiotics, 45 parts by weight of calcium chloride, 140 parts by weight of xylitol, 40 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 are mixed in a volume ratio of 1:4, and homogenized for 10-15 minutes to emulsify the mixture to obtain a mixed emulsion, and 1 part by weight of xanthan gum and 10 parts by weight of sodium alginate are added respectively, and the mixture is fully swollen and further homogenized and mixed; Add 1.5 parts by weight of low molecular weight active polysaccharide from seaweed, and pasteurize at 65°C for 1 hour; Enterococci were added into the mixed system and mixed evenly, so that the final viable bacterial count of the system reached 100 CFU / mL; the mixed system was transferred into a 2 mL syringe, and vertically dropped into a calcium chloride solution with a solute mass percentage of 4.5% to obtain probiotic embedded particles after soaking for 20 seconds, which were fished out, washed with sterile water, and stored at 4°C for use; Step 2, 40 parts by weight of cocoa powder and sterile water are prepared into a cocoa solution with a solute mass percentage of 4%, 25 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, 14% 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 to defoam, thereby obtaining a defoaming solution; Step 3, preparing a calcium lactate solution with a solute mass percentage of 3.5% by weight by adding 40 parts by weight of calcium lactate and sterile water; The probiotic embedded particles obtained in step 1 are added to the defoaming solution obtained in step 2 at a mass ratio of 3%, mixed evenly, sucked with a dropper, vertically dripped into the calcium lactate solution, soaked for 10 minutes and then fished out to obtain seaweed-derived low molecular weight active polysaccharide probiotic embedded particles.

[0026] In another embodiment of the present invention, another method for preparing seaweed-derived low molecular weight active polysaccharide probiotic embedded particles is provided, comprising the following steps: The seaweed-derived low molecular weight active polysaccharide probiotic embedded particles are made of the following raw materials: 25 parts by weight of sodium alginate, 35 parts by weight of calcium lactate, 1 part by weight of seaweed-derived low molecular weight active polysaccharide, 1 part by weight of xanthan gum, 10 parts by weight of probiotics, 40 parts by weight of calcium chloride, 125 parts by weight of xylitol, 30 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 10-15 minutes to emulsify the mixture to obtain a mixed emulsion, and 1 part by weight of xanthan gum and 10 parts by weight of sodium alginate were added respectively to fully swell and further homogenized and mixed; Add 1 part by weight of low molecular weight active polysaccharide from seaweed, and pasteurize at 65°C for 1 hour; Enterococci were added into the mixed system and mixed evenly, so that the final viable bacterial count of the system reached 100 CFU / mL; the mixed system was transferred into a 2 mL syringe, and vertically dropped into a 4% calcium chloride solution to soak for 20 seconds to obtain probiotic embedded particles, which were fished out, washed with sterile water, and stored at 4°C for later use; Step 2, 30 parts by weight of cocoa powder and sterile water are prepared into a cocoa solution with a solute mass percentage of 3%, 15 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, 13% 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 to defoam, thereby obtaining a defoaming solution; Step 3, preparing a calcium lactate solution with a solute mass percentage of 3% by weight by adding 40 parts by weight of calcium lactate and sterile water; The probiotic embedded particles obtained in step 1 are added to the defoaming solution obtained in step 2 at a mass ratio of 2.5%, mixed evenly, sucked with a dropper, vertically dripped into the calcium lactate solution, soaked for 10 minutes and then fished out to obtain seaweed-derived low molecular weight active polysaccharide probiotic embedded particles.

[0027] The seaweed-derived low molecular weight active polysaccharide probiotic embedded particles are added to fresh milk, and Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus are added as fermentation agents, stirred evenly, and fermented to obtain yogurt.

[0028] The beneficial effects of the present invention are: The seaweed-derived low molecular weight active polysaccharide probiotic embedded particles provided by the present invention and its preparation method and application, utilize enterococcus fermentation to extract seaweed polysaccharide carrageenan, compared with original carrageenan, the seaweed-derived low molecular weight active polysaccharide obtained by enterococcus degradation can promote the proliferation metabolism of beneficial bacteria enterococcus, and has higher blood lipid lowering activity, and the seaweed-derived low molecular weight active polysaccharide obtained by enterococcus degradation interacts stably with other raw materials and auxiliary materials such as sodium alginate, which can promote gelation, embedding layer by layer, and the obtained gel and embedding system with better mechanical properties make its probiotic survival rate higher, and the resistance in gastrointestinal and ultraviolet adverse environment is higher, and the blood lipid lowering activity of the product is significantly improved at the same time, which can be applied to the production of products such as yogurt, and the number of beneficial bacteria and blood lipid lowering activity are increased. Further, the inner layer of the probiotic embedded particles obtained by the present invention adopts calcium chloride to cross-link sodium alginate to form a dense core material, and the outer layer is reinforced by calcium lactate secondary curing to enhance the mechanical strength, and improve the survival rate of live bacteria in the gastric acid environment.

[0029] Example 1 A method for preparing seaweed-derived low molecular weight active polysaccharide probiotic embedded particles comprises the following steps: Raw materials preparation: 20 parts by weight of sodium alginate, 30 parts by weight of calcium lactate, 1 part by weight of xanthan gum, 0.5 parts by weight of seaweed-derived low molecular weight active polysaccharide, 10 parts by weight of enterococci, 40 parts by weight of calcium chloride, 120 parts by weight of xylitol, and 30 parts by weight of cocoa powder.

[0030] The seaweed-derived low molecular weight active polysaccharide is obtained by subjecting carrageenan to enterococcal fermentation degradation, alcohol precipitation, deproteinization, dialysis, and freeze-drying. Specifically, the seaweed-derived low molecular weight active polysaccharide is prepared by the following method: 1% of a tube (specification 1 mL) of enterococcal bacterial liquid (purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd., No. BNCC341917) was inoculated into 10 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 1-2 days, and then preserved with glycerol (glycerol: bacterial liquid = 3:7) and stored at -80°C for subsequent experiments; 0.1 mL of glycerol-preserved enterococcal bacterial liquid was taken and activated by adding 9.9 mL of sterilized FT thioglycollate fluid culture medium, and cultured at 37°C under anaerobic conditions for 6 h, and then 0.1 mL of bacterial liquid was added to 10 mL of sterilized FT thioglycollate fluid culture medium for expansion culture, and cultured at 37°C under anaerobic conditions for 4 h, and the bacterial liquid was centrifuged, and the bacterial precipitate was resuspended with physiological saline (the number of viable bacteria in the bacterial suspension was about 1×10 8 CFU / mL), and then gradually diluted in ten-fold gradient until the number of viable bacteria was about 1×10 4 CFU / mL of inoculum solution.

[0031] Prepare a 1%-1.5% mass fraction carrageenan and other seaweed polysaccharide aqueous solution, pasteurize at 65℃ for 40 min, inoculate 1% enterococci (100 CFU / mL), and culture in a constant temperature shaker at 180 rpm and 37℃ for 12-48h. After fermentation and degradation, centrifuge to remove bacteria and insoluble impurities; the supernatant is concentrated to 1 / 5 of the original volume under reduced pressure at 55℃. Slowly add anhydrous ethanol to a final ethanol concentration of 75%, precipitate overnight, centrifuge, and collect the precipitate. The precipitate is redissolved, and the Sevag reagent is prepared by mixing chloroform and n-butanol in a volume ratio of 4:1 according to the Sevag method. The precipitated aqueous solution and the Sevag reagent are mixed in a ratio of 3:1, shaken, and centrifuged to remove the protein precipitate layer. The supernatant is recovered by the same method, repeated 3-5 times, and the supernatant obtained in the last time is separated and collected. The obtained supernatant is dialyzed with a 100Da dialysis bag for 2 days. After the sample is concentrated and freeze-dried, a low molecular weight active polysaccharide from seaweed is obtained.

[0032] Preparation of seaweed-derived low molecular weight active polysaccharide probiotic embedded particles: 1. Preparation of calcium chloride solution and calcium lactate solution A 4% calcium chloride solution was prepared using 40 parts by weight of calcium chloride and sterile water, and a 3% calcium lactate solution was prepared using 30 parts by weight of calcium lactate and sterile water.

[0033] 2. Preparation of seaweed polysaccharide probiotic embedded particles 195 parts by weight of edible oil and distilled water were mixed in a volume ratio of 1:4, and then homogenized for 10-15 min to emulsify the mixed emulsion, and 1 part by weight of xanthan gum and 10 parts by weight of sodium alginate were added respectively, and the mixture was homogenized after being fully swollen. 0.5 parts by weight of seaweed polysaccharide was added, mixed evenly, and pasteurized at 65°C for 1h. After 10 parts by weight of enterococcal cells were washed with sterile water 2-3 times, they were added to the mixed system at a 1% inoculation amount and mixed evenly, so that the number of viable bacteria in the final system reached 100 CFU / mL. The mixed system prepared above was transferred to a 2 mL syringe, vertically dripped into a 4% calcium chloride solution and soaked for about 20 s to obtain probiotic embedded particles, which were removed and washed with sterile water and stored at 4°C for use.

[0034] 3. Preparation of seaweed-derived low molecular weight active polysaccharide probiotic embedded particles 30 parts by weight of cocoa powder and sterile water are prepared into a 3% cocoa solution, which is then dissolved in 15 parts by weight of sodium alginate in a water bath at 80°C with stirring. After the solution is completely swollen, 120 parts by weight of xylitol is added. After the solution is fully dissolved, the solution is placed in a refrigerator at 4°C for 3-4 hours for defoaming.

[0035] The seaweed polysaccharide probiotic embedded particles in step 2 are added in an amount of 2% by mass to the defoaming outer layer sodium alginate embedding solution and mixed evenly, and the calcium lactate solution is vertically dripped into the solution. After soaking for 10 minutes, the particles are taken out and washed with sterile water for 2-3 times to obtain seaweed-derived low molecular weight active polysaccharide probiotic embedded particles.

[0036] Preferably, the application in yogurt is characterized in that the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles are added to fresh milk, and fermentation agents such as Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are added, stirred evenly, and fermented to obtain high-stability seaweed polysaccharide functional yogurt.

[0037] Example 2 The difference from Example 1 is that the seaweed-derived low molecular weight active polysaccharide in the raw material is 1 part by weight, and the rest is the same as Example 1.

[0038] Comparative Example 1 The difference from Example 1 is that the low molecular weight active polysaccharide from seaweed in the raw material is replaced with original carrageenan (carrageenan that has not been treated by enterococcus fermentation degradation, alcohol precipitation, etc.). Accordingly, the steps of enterococcus fermentation treatment of carrageenan and preparation of low molecular weight carrageenan are not performed, and the rest are the same as Example 1.

[0039] Comparative Example 2 The difference from Example 1 is that the raw materials do not contain seaweed-derived low molecular weight active polysaccharides. Accordingly, there is no step of preparing seaweed-derived low molecular weight active polysaccharides and no step of adding seaweed-derived low molecular weight active polysaccharides to the probiotic stable embedding particles. The rest is the same as Example 1.

[0040] Comparative Example 3 The difference from Example 1 is that the sodium alginate in step 2 of preparing seaweed-derived low molecular weight active polysaccharide probiotic embedded particles is changed to 2.5 parts by weight, and the rest is the same as Example 1.

[0041] 1. Determination of the effects of low molecular weight active polysaccharides from seaweed and original carrageenan on the proliferation of probiotic enterococci See also Figure 1 , is a comparison chart of the effects of low molecular weight active polysaccharides from seaweed and original carrageenan on the proliferation of probiotic enterococci.

[0042] Enterococci were inoculated at 1% inoculum, activated and expanded (anaerobic culture to 1×10 4 CFU / mL), and subcultured 2-3 times. 1 mg / mL of seaweed-derived low molecular weight active polysaccharide and original carrageenan culture medium were prepared respectively, and the blank culture medium (no carbon source group) without seaweed polysaccharide carrageenan was used as the blank control group, and sterilized at 121℃ for 15 min.

[0043] The cultured enterococci were centrifuged at 4000 rpm / min for 8 min, the supernatant was discarded, and an equal volume of 0.9% (w / v) sterile saline was added for resuspending to obtain a bacterial suspension (viable count 1×10 4 CFU / mL), inoculate 1% enterococci into the culture medium to make the final colony count in the system 100 CFU / mL, adjust the pH of the culture medium (including 1 mg / mL of seaweed-derived low molecular weight active polysaccharides and original carrageenan culture medium, and a blank culture medium without seaweed polysaccharides and carrageenan) to 7.0±0.02 and culture at 37℃ for 48h in an anaerobic environment, and determine the number of viable enterococci by plate counting.

[0044] 2. Determination of the hypolipidemic activity of low molecular weight active polysaccharides from seaweed and the fermentation products of enterococci from carrageenan See also Figure 2 , which is a comparison chart of the lipid-lowering activity of low molecular weight active polysaccharides from seaweed and the enterococcal fermentation products of original carrageenan.

[0045] The lipid-lowering activity was evaluated by triglycerides in 3T3-L1 cells. After the 3T3-L1 cells grew to about 80%-90% adherence, the cells were cultured in D / A medium containing 10 µg / mL insulin, 0.5 mM IBMX (1-methyl-3-isobutylxanthine) and 1 μM dexamethasone (defined as day 0); after 2 days of culture, the D / A medium was replaced with D / M medium containing 10 µg / mL insulin and cultured for another 2 days (starting from day 3-4); the medium was switched to growth medium and renewed every two days until the cells were fully differentiated into adipocytes (day 8).

[0046] 50 μL / mL of low molecular weight active polysaccharides from seaweed and the fermentation product of enterococci from carrageenan (the product of enterococci cultured at 37°C in an anaerobic environment for 48 hours in method 1) were used for intervention on differentiation days 0-8. After the induction, the triglyceride level in the cells was measured using a triglyceride kit. The cells without the addition of seaweed polysaccharides were designated as the high lipid cell model group.

[0047] 3. Sensory score determination of seaweed-derived low molecular weight active polysaccharide probiotic embedded particles A sensory evaluation panel consisting of 10 graduate students majoring in food science conducted a sensory evaluation of the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles in terms of mouthfeel, flavor, chewing sensation, appearance, and degree of preference. The evaluation criteria are listed in Table 1.

[0048] Table 1 Sensory evaluation standards

[0049] 4. Determination of the anti-digestive properties of seaweed-derived low molecular weight active polysaccharide probiotic embedded particles See also Figure 3 , is the anti-digestion performance test of the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles obtained in Examples 1, 2 and Comparative Examples 1-3; The seaweed-derived low molecular weight active polysaccharide probiotic embedded particles were squeezed and crushed to simulate oral chewing, and 0.5 g of the crushed seaweed-derived low molecular weight active polysaccharide probiotic embedded particles 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 sample was shaken at high speed by a vortex oscillator to destroy the outer membrane of the embedded particles and release the probiotics therein, and the viable bacteria were calculated by the plate count method after diluting it with sterile water.

[0050] In addition, a group of seaweed-derived low molecular weight active polysaccharide probiotic embedded particles 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 completed after 4 hours. The seaweed-derived low molecular weight active polysaccharide probiotic embedded particles were decapsulated and their live bacterial counts were determined.

[0051] 5. Determination of UV resistance of seaweed-derived low molecular weight active polysaccharide probiotic embedded particles See also Figure 4 , is the anti-ultraviolet performance test of the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles obtained in Examples 1, 2 and Comparative Examples 1-3; The seaweed-derived low molecular weight active polysaccharide probiotic embedded particles were irradiated under a 254 nm ultraviolet lamp for 0.5 h, and then placed in the dark for 1 h. Then, appropriate amounts of phosphate buffer were added, and the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles were decapsulated, diluted, and coated to determine the number of viable bacteria.

[0052] 6. Determination of lipid-lowering activity of seaweed-derived low molecular weight active polysaccharide probiotic embedded particles See also Figure 5 , is the lipid-lowering activity determination of the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles obtained in Examples 1, 2 and Comparative Examples 1-3; The lipid-lowering activity was evaluated by triglyceride in 3T3-L1 cells. After 1.0 g of seaweed-derived low molecular weight active polysaccharide probiotic embedded particles were decapsulated, adipocytes were treated at a concentration of 50 μL / mL, and the triglyceride level in the cells was measured using a triglyceride kit according to the steps in method 2. The high-lipid cell model group was taken without the addition of seaweed-derived low molecular weight active polysaccharide probiotic embedded particles.

[0053] 7. Preparation of seaweed-derived low molecular weight active polysaccharide probiotic embedded yogurt particles See also Figure 6 and Table 3, which are the application tests of the probiotic embedded particles obtained in Example 1 and Comparative Example 2 in yogurt; In fresh milk, 1% seaweed-derived low molecular weight active polysaccharide probiotic embedded particles were added, and 5%-8% of Lactobacillus delbrueckii subspecies bulgaricus and thermophilic Streptococcus and other fermentation agents were added, stirred evenly, and fermented at 50℃ for 8-10h, and then fermented at 4℃ for more than 4h to obtain high-stability seaweed polysaccharide functional yogurt. Functional yogurt without probiotic embedded particles was used as the blank control group. The texture quality of each group of yogurt was determined by texture analyzer, and the number of viable bacteria was determined after dilution and coating.

[0054] 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.

[0055] Chart Description: Depend on Figure 1 and Figure 2 As shown, the enterococcal fermentation-degraded seaweed-derived low molecular weight active polysaccharides in Example 1 have significantly higher enterococcal viable bacteria count and lipid-lowering activity than the original carrageenan in Comparative Example 1 (p < 0.05), which may be related to the lower molecular weight of seaweed polysaccharides degraded by enterococci fermentation, and the glycolysis products contain higher levels of active monosaccharides such as mannose, glucose, and rhamnose, which are beneficial to the growth and reproduction of enterococci and increase the number of viable bacteria; at the same time, the growth and metabolism of enterococci can produce short-chain fatty acids such as acetic acid, which synergize with the low molecular weight active polysaccharides from seaweed to significantly enhance the lipid-lowering activity of the product.

[0056] As can be seen from Table 2, the sensory score of Example 1-2 is significantly higher than that of Comparative Example 1-3 (p < 0.05), mainly because the taste and appearance are significantly improved, accompanied by an increase in the degree of liking. Compared with Comparative Example 2, Example 1-2 found that the low molecular weight active polysaccharide from seaweed can promote the cross-linking of the wall material sodium alginate, and compared with the original carrageenan without enterococcus fermentation degradation in Comparative Example 1, the low molecular weight active polysaccharide from seaweed fermented and degraded by enterococcus in Example 1-2 can better promote the cross-linking of sodium alginate, enhance the gelation and chewy taste, and the prepared seaweed low molecular weight active polysaccharide probiotic embedded particles have a better sensory score. Compared with Comparative Example 3, Example 1-2 shows that a good interaction between different components requires a suitable amount of ingredients to be added.

[0057] Table 2 Sensory evaluation of seaweed-derived low molecular weight active polysaccharide probiotic embedded particles

[0058] from Figure 3and 4 It can be seen that Examples 1 and 2 can significantly improve the survival rate of enterococci in the stomach, intestines and ultraviolet adverse environment (p < 0.05), and increase with the increase of the concentration of seaweed-derived low molecular weight active polysaccharides. Examples 1 and 2 show a good survival rate of probiotics. On the one hand, it may be related to the high prebiotic effect of the seaweed-derived low molecular weight active polysaccharides produced by the degradation of enterococci, which contain higher active monosaccharides such as mannose, glucose, and rhamnose, which are conducive to the growth and reproduction of enterococci and increase the number of viable bacteria; on the other hand, it may be related to the fact that the seaweed-derived low molecular weight active polysaccharides promote the interaction of multiple components of sodium alginate, promote the gelation of the multi-level encapsulation system, form a just encapsulation system and mechanical properties and stability, thereby increasing the number of viable enterococci in the stomach, intestines and ultraviolet adverse environment.

[0059] In addition, from Figure 5 It can be seen that compared with comparative examples 1-3 and the model group, examples 1 and 2 can significantly reduce the triglyceride content in adipocytes (p < 0.05), indicating that the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles have better lipid-lowering activity. Compared with comparative example 1, examples 1 and 2 showed just the lipid-lowering activity, which may be related to the lipid-lowering activity of the seaweed-derived low molecular weight active polysaccharide molecules themselves degraded by enterococcus fermentation ( Figure 2 ), on the other hand, it may be related to the fact that the enterococcal fermentation and degradation of low molecular weight active polysaccharide molecules from seaweed has a higher prebiotic effect, which can promote the proliferation and metabolism of beneficial bacteria enterococci, increase the number of probiotics and beneficial metabolites, and improve the lipid-lowering activity.

[0060] The probiotic embedded particles prepared in Example 1 and Comparative Example 2 were used to study their application in yogurt. The mass fraction of the probiotic embedded particles added was 1% based on the mass of fresh milk, and the probiotic embedded particles without addition were used as the blank group. The number of viable probiotic bacteria in each group was as follows: Figure 6 As shown in the results, it was found that the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles can improve the survival rate of probiotics in yogurt products and have potential lipid-lowering activity; at the same time, as shown in Table 3, after adding seaweed-derived low molecular weight active polysaccharide probiotic embedded particles, the quality of yogurt such as gel strength, adhesion and cohesion is better.

[0061] Table 3 Texture quality of yogurt products

[0062] In summary, the seaweed-derived low-molecular-weight active polysaccharide probiotic-embedded particles provided by the present invention, as well as their preparation method and application, utilize enterococcus fermentation to extract seaweed polysaccharide carrageenan. Compared with the original carrageenan, the seaweed-derived low-molecular-weight active polysaccharide obtained by enterococcus degradation can better promote the proliferation and metabolism of beneficial bacteria enterococci, and has higher lipid-lowering activity. The seaweed-derived low-molecular-weight active polysaccharide obtained by enterococcus degradation can stably interact with other raw and auxiliary materials such as sodium alginate, an embedding wall material, to promote gelation, and embedding layer by layer to obtain a gel and an embedding system with better mechanical properties, so that the survival rate of the probiotics is higher, and the resistance to the gastrointestinal and ultraviolet adverse environments is higher. At the same time, the lipid-lowering activity of the product is significantly improved, and it can be applied to the production of products such as yogurt to increase the number of beneficial bacteria and the lipid-lowering activity.

[0063] 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 seaweed-derived low molecular weight active polysaccharide probiotic embedded granule, characterized in that: Made from the following ingredients: 20-35 parts by weight of sodium alginate, 30-40 parts by weight of calcium lactate, 0.5-1.5 parts by weight of low molecular weight active polysaccharide from seaweed, 1-2 parts by weight of xanthan gum, 10 parts by weight of probiotics, 35-45 parts by weight of calcium chloride, 120-140 parts by weight of xylitol, 20-40 parts by weight of cocoa powder, and 195-198 parts by weight of edible oil; The method for preparing the seaweed-derived low molecular weight active polysaccharide comprises: subjecting carrageenan to enterococcal fermentation degradation, alcohol precipitation, deproteinization, dialysis and freeze-drying treatment to obtain the seaweed-derived low molecular weight active polysaccharide.

2. The seaweed-derived low molecular weight active polysaccharide probiotic embedded particles according to claim 1, characterized in that: The probiotics are enterococci.

3. The seaweed-derived low molecular weight active polysaccharide probiotic embedded particles according to claim 1, characterized in that: The enterococcus fermentation degradation treatment is specifically as follows: preparing a carrageenan aqueous solution with a solute mass percentage of 1%-1.5%, pasteurizing at 65°C for 40 min, inoculating 1% enterococci by volume, and culturing in a constant temperature shaker at 180 rpm and 37°C for 12-48h.

4. The seaweed-derived low molecular weight active polysaccharide probiotic embedded particles according to claim 1, characterized in that: The seaweed-derived low molecular weight active polysaccharide is prepared by the following method: After carrageenan is fermented and degraded by enterococci, the bacteria and insoluble impurities are removed by centrifugation; the supernatant is concentrated under reduced pressure at 55°C to 1 / 5 of the original volume of the probiotics, and anhydrous ethanol is slowly added dropwise to a final ethanol concentration of 75% by mass of the probiotics, precipitated, centrifuged, and the precipitate is collected. The precipitate is re-dissolved, and the obtained precipitate aqueous solution is deproteinized by the Sevag method and the supernatant is collected. The obtained supernatant is dialyzed with distilled water in a 100Da dialysis bag, and the obtained sample is concentrated and freeze-dried to obtain a seaweed-derived low molecular weight active polysaccharide.

5. A method for preparing the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles 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, then adding xanthan gum and sodium alginate respectively, fully swelling and further homogenizing and mixing; adding low molecular weight active polysaccharide from seaweed, sterilizing to obtain a mixed system; Add probiotics to the mixed system and mix evenly, drip the obtained mixed system into a 3.5%-4.5% by mass calcium chloride solution to soak to obtain probiotic embedded particles, remove the probiotic embedded particles and wash them 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 and mix well. Use a dropper to suck it and drop it vertically into a sterile aqueous solution of calcium lactate. After soaking, take it out to obtain seaweed-derived low molecular weight active polysaccharide probiotic embedded particles.

6. The method for preparing the seaweed-derived low molecular weight active polysaccharide probiotic embedded particles according to claim 5, specifically comprising 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 the mixture to obtain a mixed emulsion, and 1-2 parts by weight of xanthan gum and 5-10 parts by weight of sodium alginate are added respectively to fully swell and further homogenize and mix; Add 0.5-1.5 parts by weight of low molecular weight active polysaccharide from seaweed, and pasteurize at 65°C for 1 hour; Enterococci were added into the mixed system and mixed evenly, so that the final viable bacterial count of the system reached 100 CFU / mL; the mixed system was transferred into a 2 mL syringe, and vertically dropped into a calcium chloride solution with a solute mass percentage of 3.5%-4.5% and soaked for 20 seconds to obtain probiotic embedded particles, which were fished out, washed with sterile water, and stored at 4°C for use; Step 2, prepare a cocoa solution with a solute mass percentage of 2%-4% by weight of 20-40 parts by weight of cocoa powder and sterile water, add 15-25 parts by weight of sodium alginate, stir and dissolve in a water bath at 80°C, add 12%-14% by weight of xylitol to fully dissolve after it swells completely, and then place it in a refrigerator at 4°C for 3-4 hours to defoam, to obtain a defoaming solution; Step 3, preparing a calcium lactate solution with a solute mass percentage of 2.5% to 3.5% by weight of calcium lactate and sterile water; The probiotic embedded particles obtained in step 1 are added to the defoaming solution obtained in step 2 at a mass ratio of 2%-3%, mixed evenly, sucked with a dropper, vertically dripped into the calcium lactate solution, soaked for 10 minutes and then fished out to obtain seaweed-derived low molecular weight active polysaccharide probiotic embedded particles.

7. Use of the embedded particles prepared by the method for preparing seaweed-derived low molecular weight active polysaccharide probiotic embedded particles according to any one of claims 5 to 6 in food.

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

9. The use according to claim 8, characterized in that: The seaweed-derived low molecular weight active polysaccharide probiotic embedded particles are added into fresh milk, and Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus are added as fermentation agents, stirred evenly, and fermented to obtain yogurt.

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