A method of preparing a marine composite prebiotic product

Abstract

The application belongs to the technical field of preparation of composite prebiotic products, and particularly relates to a preparation method of a marine composite prebiotic product, which mainly comprises the following steps: (1) preparing a mixed solution of chitosan oligosaccharide and fucoidan; (2) continuously stirring the mixed solution in step (1); (3) after the mixed solution in step (2) is stirred, freeze-drying is performed to obtain a fucoidan-chitosan oligosaccharide compound; (4) preparing a fucoidan solution; (5) immersing the fucoidan-chitosan oligosaccharide compound in step (3) in the fucoidan solution in step (4); (6) continuously stirring the solution in step (5); (7) after uniform stirring, centrifugal collection is performed on the precipitate; (8) drying the precipitate; (9) repeating steps 3-8 in steps (4)-(8) for 3-8 times, and finally, the marine composite prebiotic product is obtained by drying the precipitate in the last time. The marine composite prebiotic product can effectively alleviate the inhibition of chitosan oligosaccharide on protein digestion, and has the advantages of low input cost, simple operation and short time consumption.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of composite prebiotic products, and particularly relates to a preparation method of a marine composite prebiotic product. Background Art

[0002] Compound prebiotics are organic compounds composed of several prebiotics. These compounds are not digested or absorbed by the host, but instead reach the colon, selectively stimulating the growth and reproduction of one or more beneficial bacteria (such as bifidobacteria and lactic acid bacteria) within the intestine. Compound prebiotics are crucial for improving the intestinal microbiome and promoting lipid, protein, and mineral metabolism. Consequently, they are widely used in food and feed applications, such as compound prebiotic solid beverages containing oligofructose and inulin, and compound prebiotic fruit and vegetable jellies.

[0003] Chitosan oligosaccharides are oligosaccharides with a degree of polymerization (DP) of 2-10, made from chitosan through enzymatic hydrolysis with papain (or papain and cellulase), filtration, and spray drying. These oligosaccharides are composed of N-acetyl-D-glucosamine bound by β-1,4-glycosidic bonds. Since 2014, chitosan oligosaccharides have been approved as a new food ingredient by the Chinese government and can be used in the food industry. Chitosan oligosaccharides exhibit numerous biological activities, including antioxidant, antibacterial, anti-tumor, immune-enhancing, and gut microbial balance. They are a common marine prebiotic and are widely used in the food and nutrition sectors.

[0004] Fucoidan is a water-soluble sulfated polysaccharide extracted from brown algae that exhibits multiple biological activities. It is primarily composed of L-fucose and small amounts of galactose, glucose, and uronic acid. Numerous studies have shown that fucoidan possesses diverse biological activities, including protecting the intestinal barrier, lowering blood sugar, enhancing immunity, anti-coagulation, regulating intestinal flora, and anti-tumor, anti-inflammatory, anti-diabetic, antioxidant, and antiviral effects.

[0005] Chitosan oligosaccharides can interact with proteins and digestive enzymes, affecting protein digestion and absorption. Protein is an essential nutrient for the human body and plays an important role in maintaining human health. Therefore, there is an urgent need to develop a marine composite prebiotic product to eliminate the adverse effects of chitosan oligosaccharides on protein absorption. Summary of the Invention

[0006] [Technical Issues]

[0007] The technical problem to be solved by the present invention is that oligosaccharides in the existing marine composite prebiotic products interact with proteins and digestive enzymes, thereby affecting the digestion and absorption of proteins.

[0008] [Technical solution]

[0009] In order to solve the above problems, the present invention uses chitosan oligosaccharide and fucoidan as raw materials, provides a preparation method of a marine composite prebiotic product, and develops a marine composite prebiotic product that can effectively alleviate the inhibition of chitosan on protein digestion.

[0010] The first object of the present invention is to provide a method for preparing a marine composite prebiotic product, comprising the following steps:

[0011] (1) Chitosan oligosaccharide (COS) and fucoidan (FUC) were weighed and dissolved in a solvent to prepare a chitosan oligosaccharide and fucoidan mixed solution;

[0012] (2) maintaining the mixed solution of step (1) at a temperature of 30-70° C. and a stirring speed of 200-800 rpm, and continuing stirring for 1-4 hours;

[0013] (3) After the mixed solution of step (2) is stirred, freeze-dried to obtain a fucoidan-chitosan oligosaccharide complex;

[0014] (4) Weighing fucoidan and dissolving it in water to prepare a fucoidan solution;

[0015] (5) soaking the fucoidan-chitosan oligosaccharide complex of step (3) in the fucoidan solution of step (4);

[0016] (6) Maintain the temperature at 20-70°C and the stirring speed at 300-700 rpm and continue stirring for 1-6 hours;

[0017] (7) After stirring evenly, centrifuge for 10-20 min at 8000-12000 r / min and collect the precipitate;

[0018] (8) drying the precipitate from step (7) in an oven at 50-80°C for 4-8 hours;

[0019] (9) Repeat steps 3-8 times from step (4) to step (8), and the final precipitation and drying are performed to obtain the marine composite prebiotic product.

[0020] In one embodiment of the present invention, the concentration of the mixed solution of chitosan oligosaccharide and fucoidan in step (1) is 10-50 mg / mL, and the mass ratio of chitosan oligosaccharide to fucoidan is 0.5-3.

[0021] In one embodiment of the present invention, the freezing conditions of step (3) are: vacuum degree 1-10 Pa, cold trap temperature -40 to -60°C.

[0022] In one embodiment of the present invention, the concentration of the fucoidan solution in step (4) is 5 to 80 mg / mL.

[0023] In one embodiment of the present invention, the concentration of the fucoidan-chitosan oligosaccharide complex in step (5) is 80-150 mg / mL.

[0024] In one embodiment of the present invention, the specific process of step (9) is as follows: the precipitate dried in step (8) is immersed in the fucoidan solution in step (4) again, stirred evenly, centrifuged, and the precipitate is dried to obtain the marine composite prebiotic product.

[0025] The second object of the present invention is to claim protection for a marine composite prebiotic product prepared according to the above-mentioned preparation method of the present invention.

[0026] The present invention also seeks to protect the use of the marine composite prebiotic product in inhibiting protein digestion in the gastrointestinal tract.

[0027] [Beneficial Effects]

[0028] 1. The present invention prepares a fucoidan-chitosan oligosaccharide complex and coats the complex with a fucoidan solution to prepare a marine composite prebiotic product. Compared with chitosan oligosaccharide and a mixed powder of chitosan oligosaccharide and fucoidan, the present invention can effectively alleviate the inhibition of chitosan oligosaccharide on protein digestion and has low investment cost, simple operation and short time consumption.

[0029] 2. The present invention prepares a fucoidan-chitooligosaccharide complex and coats the complex with a fucoidan solution to produce a marine composite prebiotic product that can alleviate the reduction in protein digestibility caused by chitosan oligosaccharide. Compared to not adding chitosan, the addition of chitosan oligosaccharide reduced the degree of hydrolysis of whey protein from 54% to 18%. Furthermore, compared to the addition of chitosan oligosaccharide, the addition of the marine composite prebiotic product and the addition of a mixed powder of chitosan oligosaccharide and fucoidan significantly increased the degree of protein hydrolysis to 41% and 31%, respectively, significantly alleviating the inhibition of chitosan oligosaccharide on protein digestion. The marine composite prebiotic product exhibited a superior alleviating effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the appearance of the marine composite prebiotic product obtained in Example 1;

[0031] Figure 2 This is a scanning electron micrograph (1000×) of the marine composite prebiotic product obtained in Example 1;

[0032] Figure 3 This is a transmission electron micrograph (15000×) of the marine composite prebiotic product obtained in Example 1;

[0033] Figure 4 This is the distribution diagram of sulfur elements on the surface of the marine composite prebiotic product obtained in Example 1;

[0034] Figure 5The figure shows the effects of the same concentration of chitosan oligosaccharide, chitosan oligosaccharide and fucoidan powder, and the marine composite prebiotic product obtained in Example 1 on the hydrolysis degree of whey protein. As shown in the figure, compared with the chitosan oligosaccharide group and the group added with chitosan oligosaccharide and fucoidan powder, the protein hydrolysis degree in the group added with the marine composite prebiotic product was the highest. DETAILED DESCRIPTION

[0035] The embodiments of the present invention are described in detail below with reference to the examples. In the examples, if the specific conditions are not specified, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be obtained commercially.

[0036] Elemental Distribution Testing Method: The surface morphology and elemental distribution of the prebiotic composite were evaluated using a scanning electron microscope (JSM-7800F) coupled with EDS. 2 mg of dry prebiotic composite powder was adhered to a sample stage and placed in an ion sputtering instrument, where it was coated with a conductive gold film. The composite prebiotic product was observed and measured at 1000x magnification using an electron beam accelerating voltage of 3 kV.

[0037] Potential test method: Using distilled water as a dispersant, COS and compound prebiotics were prepared into a solution with a concentration of 1.0 mg / mL. The prepared solution was injected into a laser particle size analyzer (Zetasizer 3000HSA) using a 20mL syringe to measure the ζ-potential of the sample surface. Each sample was measured three times.

[0038] Example 1

[0039] A method for preparing a marine composite prebiotic product comprises the following steps:

[0040] (1) Accurately weigh 5 g of dry chitosan oligosaccharide (COS) and 5 g of dry fucoidan (FUC) and add them to a 250 mL beaker. Add 100 mL of deionized water and turn on the magnetic stirrer (rotor speed 600 rpm) to stabilize the temperature at 40 °C. Stir evenly to obtain a mixed solution of chitosan oligosaccharide and fucoidan, which contains insoluble COS and FUC complexes.

[0041] (2) The mixed solution of step (1) was kept at a temperature of 40°C and a stirring speed of 600 rpm and stirred for 2 hours;

[0042] (3) After the mixed solution of step (2) is stirred, it is freeze-dried under the conditions of a vacuum degree of 5 Pa and a cold trap temperature of -50°C to obtain a fucoidan-chitosan oligosaccharide complex;

[0043] (4) Accurately weigh 3 g of dried fucoidan and add it to a 250 mL beaker. Pour in 100 mL of deionized water and turn on the magnetic stirrer (rotor speed 600 rpm) to stabilize the temperature at 40°C. Dissolve the fucoidan under stirring with the magnetic stirrer to obtain a clear and transparent fucoidan solution.

[0044] (5) soaking the fucoidan-chitosan oligosaccharide complex obtained in step (3) in the fucoidan solution in step (4);

[0045] (6) Maintain the temperature at 40°C and the stirring speed at 600 rpm and continue stirring for 4 hours;

[0046] (7) After stirring evenly, centrifuge for 15 min at 10,000 rpm and collect the precipitate;

[0047] (8) Dry the precipitate from step (7) in an oven at 60°C for 6 h;

[0048] (9) The precipitate obtained in step (8) is used as a fucoidan-chitosan oligosaccharide complex and the steps (4) to (8) are repeated 5 times. The final precipitate is dried to obtain a marine composite prebiotic product.

[0049] The prepared marine composite prebiotic product, such as Figure 1 Shown as reddish brown powder.

[0050] Figure 2 This is a scanning electron micrograph (1000×) of the marine composite prebiotic product obtained in Example 1. Figure 2 As shown, the marine composite prebiotic product presents a smooth flake-like morphology.

[0051] Figure 3 This is a transmission electron micrograph (15000×) of the marine composite prebiotic product obtained in Example 1. Figure 3 As shown, the marine composite prebiotic product presents a film structure.

[0052] Figure 4 This is the sulfur element distribution diagram on the surface of the marine composite prebiotic product obtained in Example 1. Figure 4 As shown, sulfur element is evenly distributed on the surface of the marine composite prebiotic product.

[0053] Table 1 is a table of element distribution on the surface of the marine composite prebiotic product obtained in Example 1. As shown in Table 1, compared with chitosan oligosaccharides, the nitrogen content on the surface of the marine composite prebiotic product is significantly reduced and the sulfur content is significantly increased. The S content on the surface of the marine composite prebiotic product prepared by the present invention is 8-18%, indicating that the surface of the prebiotic product is successfully coated with fucoidan.

[0054] Table 1 surface element distribution table of marine composite prebiotics product obtained in Example 1

[0055]

[0056] Table 2 is the potential table of marine composite prebiotics product obtained in Example 1, as shown in Table 2, the surface of marine composite prebiotics product carries negative charge, which is significantly changed compared with chitooligosaccharide, the potential of marine composite prebiotics product prepared by the present application is-50~ -10mV, indicating that the outside of chitooligosaccharide has been successfully coated by fucoidan.

[0057] Table 2 potential table of marine composite prebiotics product obtained in Example 1

[0058]

[0059] A method for verifying that marine composite prebiotics product relieves chitooligosaccharide from inhibiting the digestion of whey protein in gastrointestinal tract, comprising the following steps:

[0060] S1, preparing simulated gastric juice, simulated intestinal juice and ninhydrin color developing solution, the method is as follows:

[0061] (1) simulated gastric juice: accurately weigh 1.0g NaCl, 0.4g KCl, 0.2g NaHCO3 and 0.05g CaCl2·2H2O, dissolve in 300mL deionized water, and use 1mol / L HCl to adjust the pH of the solution to 1.5, add 600mg pepsin (≥400U / mg) to the above solution to obtain simulated gastric juice, and the simulated gastric juice is prepared and used immediately;

[0062] (2) simulated intestinal juice: accurately weigh 1.8g NaCl, 0.22g KCl and 0.11g CaCl2·2H2O, dissolve in 300mL deionized water, and use 1mol / L NaHCO3 to adjust the pH to 6.5, add 600mg trypsin (4U) and 3600mg cholate to the above solution to obtain simulated intestinal juice, and the simulated intestinal juice is prepared and used immediately;

[0063] (3) ninhydrin color developing solution: accurately weigh 0.25g ninhydrin, 0.15g fructose, 5g Na2HPO4·10H2O and 3g KH2PO4, use deionized water to constant volume to 50mL, and store at 4℃ in the dark;

[0064] S2, take 12 10mL centrifuge tubes, weigh 8mg of dried whey protein isolate (WPI), 8mg of dried chitosan oligosaccharide (COS) and 8mg of dried whey protein isolate (WPI), 8mg of dried chitosan oligosaccharide (COS) and 8mg of dried fucoidan (FUC) and 8mg of dried whey protein isolate (WPI), 8mg of dried marine composite prebiotic product and 8mg of dried whey protein isolate (WPI), then draw 8mL of the above-mentioned simulated gastric fluid and transfer it to each centrifuge tube, mix well and adjust the pH to 1.5 with 1mol / L hydrochloric acid solution, and prepare a mixed solution in which the concentration of whey protein isolate, chitosan oligosaccharide and fucoidan and marine composite prebiotic product is 1mg / mL, respectively named WPI, COS+WPI, COS+FUC+WPI, COS-FUC+WPI, 4 parallels for each sample;

[0065] S3. Place the centrifuge tube containing the above samples in a constant temperature oscillator and oscillate for 60 minutes (37°C, 140 rpm). Pipette 1 mL of each sample and transfer it to a 2 mL centrifuge tube and place it in a boiling water bath to inactivate the enzyme for 10 minutes. Then quickly cool and centrifuge (8000 rpm, 5 minutes, 4°C) to obtain the supernatant. Then, use 1 mol / L NaOH solution to inactivate pepsin in the sample after simulated gastric digestion, adjust the pH to 6.5, and mix it with simulated intestinal fluid at a volume ratio of 1:1. Then place the mixture in a constant temperature oscillator and stir for 120 minutes (37°C, 140 rpm). Pipette 1 mL of each sample and transfer it to a 2 mL centrifuge tube and place it in a boiling water bath to inactivate the enzyme for 10 minutes. Then quickly cool and centrifuge (8000 rpm, 5 minutes, 4°C) to obtain the supernatant. Then determine the degree of protein hydrolysis.

[0066] S4. The free amino acid content and whey protein hydrolysis degree of the solution after simulated gastrointestinal digestion were determined by ninhydrin colorimetry. The results were as follows: Figure 5 shown.

[0067] The results showed that after gastrointestinal digestion, chitosan oligosaccharides had a significant inhibitory effect on the hydrolysis of whey protein, and the degree of hydrolysis decreased from 54% to 18%; the addition of compound prebiotics and fucoidan alleviated the inhibition of chitosan oligosaccharides on whey protein hydrolysis, and the addition of fucoidan powder alleviated the inhibition of chitosan oligosaccharides on protein hydrolysis, and the degree of hydrolysis increased to 31%. In addition, the addition of marine compound prebiotics had the most significant effect on alleviating the inhibition of chitosan oligosaccharides on protein hydrolysis, and the degree of hydrolysis increased to 41%.

[0068] This embodiment may also include pre-processing steps such as solution preparation and ninhydrin color development method operation.

[0069] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a marine composite prebiotic product, characterized in that: The steps include: (1) Weigh chitosan oligosaccharide and fucoidan to prepare a mixed solution; the concentrations of chitosan oligosaccharide and fucoidan in the mixed solution are both 50 mg / mL, and the mass ratio of chitosan oligosaccharide to fucoidan is 1:1; (2) The mixed solution of step (1) was stirred continuously at a temperature of 40°C and a stirring speed of 600 rpm for 2 hours; (3) After the mixed solution in step (2) is stirred, it is freeze-dried to obtain a fucoidan-chitosan oligosaccharide complex; (4) Prepare fucoidan solution; the concentration of fucoidan solution is 30 mg / mL; (5) Soaking the fucoidan-oligochitosan complex prepared in step (3) in the fucoidan solution prepared in step (4); the concentration of the fucoidan-oligochitosan complex is 80-150 mg / mL; (6) The solution obtained in step (5) was stirred continuously at a temperature of 40°C and a stirring speed of 600 rpm for 4 hours; (7) After stirring evenly, centrifuge and collect the precipitate; (8) Dry the precipitate from step (7) in an oven at 50-80°C for 4-8 hours; (9) The precipitate obtained in step (8) is used as a fucoidan-chitosan oligosaccharide complex and the steps from step (4) to step (8) are repeated 3 to 8 times. The final precipitate is dried to obtain a marine composite prebiotic product.

2. The method for preparing a marine composite prebiotic product according to claim 1, characterized in that: The centrifugal conditions in step (7) are a rotation speed of 8000-12000 r / min and a centrifugal time of 10-20 min.

3. The method for preparing a marine composite prebiotic product according to claim 1, characterized in that: The specific process of step (9) is as follows: the precipitate dried in step (8) is immersed in the fucoidan solution in step (4) again, stirred evenly, centrifuged, and the precipitate is dried to obtain the marine composite prebiotic product.

4. A marine composite prebiotic product prepared according to the method according to any one of claims 1 to 3.

5. Use of the marine composite prebiotic product according to claim 4 in alleviating the inhibition of whey protein digestion in the gastrointestinal tract by chitosan oligosaccharides.

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