Preparation method of novel spirulina protein and application of novel spirulina protein in functional food

Abstract

The invention provides a novel spirulina protein preparation method and application thereof in functional food. The extracted spirulina protein can be used for preparing gel. The preparation method of the gel comprises the following steps: adding the spirulina protein into a sodium alginate solution, adjusting the pH value to 7.0 + / -0.1, heating the compound in a water bath at 85 DEG C for 30 minutes, quickly cooling with running water for 5 minutes, and preserving at 4 DEG C for 24 hours to form the gel. According to the method disclosed by the invention, the wall breaking rate of the spirulina protein can reach 40%, the content after extraction can reach 306.85 mg / g, the recovery rate is 74.11%, the composition and content of essential amino acids are rich, and the polysaccharide content is relatively low and is 2.27 + / -0.21 g / 100g. According to the invention, spirulina protein and sodium alginate are compounded to prepare the gel, and texture property research shows that the compounded gel prepared by the invention has relatively strong elasticity, stickiness, chewiness and hardness and a more compact and stable structure.

Description

Technical Field

[0001] The invention belongs to the technical field of marine food and biological product preparation, and specifically relates to a novel spirulina protein preparation method and application thereof in functional food. Background Art

[0002] Spirulina belongs to the genus Spirulina and the phylum Cyanophyta. It is a filamentous cyanobacteria. As an important variety of commercial microalgae in my country, it has the characteristics of large cultivation area and high yield. According to the research of the Food and Agriculture Organization of the United Nations, Spirulina is recognized as one of the best sources of protein. Spirulina is rich and balanced in nutrients, containing a variety of bioactive substances, such as phycobiliprotein, spirulina polysaccharide, unsaturated fatty acids, β-carotene, vitamin E, various trace elements, etc. It has multiple physiological functions such as anti-oxidation, lowering blood pressure, lowering blood lipids, improving body immunity, preventing cancer and inhibiting cancer, and delaying aging. The protein content in Spirulina is about 60-70%, and the amino acids it contains are complete, including 8 essential amino acids for the human body, and the proportions of various amino acids are very close to the recommended standards of the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO). In addition to general nutritional proteins, it also contains a considerable amount of special proteins with special physiological functions and medical and health functions, natural pigment proteins-phycobiliproteins.

[0003] Spirulina protein has multiple activities. It shows different gelling effects under different conditions and can be combined with polysaccharides and other substances to thicken and solidify. Therefore, Spirulina protein can be used as a gel component in food production.

[0004] However, during the extraction of spirulina protein, the cell wall of the microalgae will hinder the outflow of biomass. At present, mechanical or non-mechanical methods such as freeze-thaw, high-pressure homogenization, enzyme treatment, and chemical treatment are used to treat the cell wall of microalgae. Generally speaking, the higher the degree of cell disruption, the greater the protein yield, but the violent cell disruption method will also cause a large amount of other impurities such as polysaccharides in the cell to be released, which is not conducive to the subsequent extraction and separation. For example, the freeze-thaw method has a long extraction time and is generally only suitable for the treatment of a small amount of algae in the laboratory; the bead milling method is limited by the scale of operation and has high energy consumption; very fine cell fragments will be produced in the high-pressure homogenization method; the longer the ultrasonic disruption method is operated, the higher the feed temperature will be. Long-term operation will cause protein denaturation and this method will also release a large amount of impurities in the cell, which is not conducive to subsequent protein purification; the chemical treatment method is not conducive to food safety due to the increase in residual chemical reagents added. Spirulina protein can be prepared by coupling a variety of different treatment technologies to break the wall and combining efficient protein extraction and separation methods.

[0005] As age increases, the amount of saliva secreted by the human body decreases slightly, the softening (soaking) effect of food becomes worse, the muscles of the jaw and throat will be slightly weakened, the effect of chewing and swallowing will decrease, and the elderly are more likely to have chewing, swallowing difficulties and choking. At present, the elderly food on the market is mainly health food, but the standard definition of elderly food is to improve the physical properties of food and (or) adjust the types and contents of dietary (nutritional) ingredients to adapt to the physiological characteristics of elderly people with decreased chewing and (or) swallowing functions and malnutrition, and meet their dietary needs or nutritional needs. A kind of special dietary food. Therefore, it is necessary to develop a gel-type functional food containing a new type of spirulina protein, which can reduce the difficulty of chewing solid food, slow down the flow rate of fluid food, prevent the elderly from choking, and adjust the food texture to ensure the safety of the elderly diet on the basis of nutritional supplementation. Summary of the invention

[0006] The present invention provides a novel method for preparing spirulina protein and application thereof in functional foods. The extracted spirulina protein can be used to prepare functional foods with moderate viscosity and capable of preventing and improving choking caused by dysphagia.

[0007] The present invention first provides a novel spirulina protein, and the preparation method thereof comprises the following steps:

[0008] 1) adding spirulina to 8-10 volumes of PBS (0.001-0.1M, pH=6.8-7.2) solution, dispersing at 18-20° C. for 30 min, adding 1.0-3.0% (w / w) cellulase or pectinase, performing enzymolysis at 45-55° C. for 2-4 h, and then ultrasonicating at 350-550 W for 10-20 min to obtain a wall-broken spirulina solution;

[0009] 2) adding 0.5-1.5M NaOH solution to the broken spirulina solution to adjust the pH to 8-10, adjusting the mass / volume ratio of the feed liquid (i.e., g of spirulina: ml of PBS solution) to 1:30-1:45 at a treatment temperature of 20-40°C, and maintaining the ratio for 3-5 hours, and obtaining an extract after centrifugation;

[0010] 3) adjusting the pH of the extract, separating by isoelectric precipitation method with ammonium sulfate salting out, standing at 4-10° C. for 0.5-4 h, and taking 40%-60% of the salted protein precipitate to obtain a precipitate;

[0011] 4) adding 5 to 8 times the volume of deionized water at 18 to 25° C. to dissolve the precipitate obtained in step 3) to obtain a primary protein solution, removing the residual insoluble protein and impurities with a 0.22 μm filter membrane, desalting, concentrating, sterilizing with an ultrafiltration membrane with a molecular weight cutoff of 200, and freeze-drying to obtain spirulina protein powder.

[0012] The novel spirulina protein prepared by the present invention can be used to prepare food or functional biological products;

[0013] The food or functional biological product is a gel food or a functional biological product.

[0014] In another aspect, the present invention provides a functional gel product that prevents choking, which is prepared using the spirulina protein and sodium alginate prepared by the present invention as raw materials;

[0015] Furthermore, the anti-choking gel functional product is prepared by adding the prepared spirulina protein to a 0.5% to 2.5% (W / V) sodium alginate solution, adjusting the pH to 6.8 to 7.2, heating in a water bath at 75 to 90° C. for 25 to 35 minutes, rapidly cooling in running water for 5 to 10 minutes, and storing at 4° C. for 20 to 24 hours to form a gel, and obtaining the anti-choking gel functional food after sterilization and packaging.

[0016] Furthermore, the final concentration of the spirulina protein added is 3-6% (W / V), and the concentration of the sodium alginate solution is 2.5%.

[0017] The method provided by the present invention can achieve a wall-breaking release rate of spirulina protein of 53%, and the extraction rate of spirulina protein after further separation is 72.30%, of which the extraction rate of phycobiliprotein can reach 85.63%, the purity is 83.82%, and the polysaccharide content is low, which is 2.27±0.21g / 100g. The present invention uses a novel spirulina protein and sodium alginate to prepare a gel-type functional food containing microalgae protein, and determines that the optimal sodium alginate concentration is 2.5%. The texture property research shows that the composite gel prepared by the present invention has strong elasticity, adhesiveness, chewiness, hardness, moderate viscosity, and a more compact and stable structure, which avoids the choking cough of edible water-soluble products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The efficiency diagram of extracting and releasing Spirulina protein under different cell wall breaking methods is shown in Figure 2.

[0019] Figure 2 The efficiency diagram of the extraction and release of Spirulina protein by ultrasound-assisted shearing at different ultrasound times, treatment temperatures, and treatment times.

[0020] Figure 3 The efficiency diagram of extracting Spirulina protein under different pH, temperature, time and material-liquid ratio.

[0021] Figure 4 The efficiency diagram of separating Spirulina protein under different ammonium sulfate concentrations is shown in Figure 2.

[0022] Figure 5The amino acid composition and polysaccharide content of the extracted Spirulina protein are shown in Figure 2.

[0023] Figure 6 The surface hydrophobicity and free thiol content of sodium alginate gel food with different concentrations are shown in Figure 2.

[0024] Figure 7 The texture of sodium alginate gel food with different concentrations is shown in Figure 1.

[0025] Figure 8 These are scanning electron micrographs of sodium alginate gel foods with different concentrations. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with embodiments and drawings.

[0027] Example 1 Extraction and release of spirulina protein using different cell wall breaking methods

[0028] 1. Ultrasonic method (U): Weigh 5 g of spirulina powder, add PBS (0.1 M, pH = 6.8) at a solid-liquid ratio of 1:8, pre-soak for 30 minutes, then put it into a 700 W ultrasonic instrument for 30 minutes, work for 1 minute, rest for 1 minute, add ice during the process to prevent overheating, centrifuge at 10000 r / min and 4°C for 25 minutes, discard the precipitate, collect the supernatant for testing, and set up three parallels for each sample.

[0029] 2. Ultrasonic assisted shearing method (ZU): Weigh 5 g of Spirulina powder, add PBS (0.1 M, pH = 6.8) at a solid-liquid ratio of 1:8, pre-soak for 30 min, then ultrasonicate for 3 min, disperse and shear at 25 ° C for 2 h, centrifuge at 10000 r / min and 4 ° C for 25 min, discard the precipitate, collect the supernatant for testing, and set up three parallels for each sample.

[0030] 3. Tissue homogenization method (ZY): Weigh 5 g of Spirulina powder, add PBS (0.1 M, pH = 6.8) at a solid-liquid ratio of 1:8, pre-soak for 30 minutes, then treat with a tissue homogenizer for 30 minutes, work for 1 minute, rest for 1 minute, centrifuge at 10000 r / min, 4°C for 25 minutes, discard the precipitate, collect the supernatant for testing, and set up three parallels for each sample.

[0031] 4. Repeated freeze-thaw method (F): Weigh 5g of Spirulina powder, add PBS (0.1M, pH=6.8) at a solid-liquid ratio of 1:8, pre-soak for 30 minutes and freeze in a -20℃ refrigerator. Take out the algae solution after it is fully frozen for 6-8 hours, thaw at room temperature, freeze and thaw three times, centrifuge at 10000r / min and 4℃ for 25 minutes, discard the precipitate, collect the supernatant for testing, and set up three parallels for each sample.

[0032] 5. High-pressure homogenization method (HPM): Weigh 5 g of spirulina powder, add PBS (0.1 M, pH = 6.8) at a solid-liquid ratio of 1:8, pre-soak for 30 minutes and stir evenly, place in a high-pressure homogenizer (homogenization pressure: 1000 bar), centrifuge at 10000 r / min and 4°C for 25 minutes, discard the precipitate, collect the supernatant for testing, and set up three parallels for each sample.

[0033] 6. Tissue grinding method (M): Weigh 5 g of Spirulina powder, add PBS (0.1 M, pH = 6.8) at a solid-liquid ratio of 1:8, pre-soak for 30 minutes, then add an appropriate amount of grinding beads, put the suspension in a tissue ball mill, operate at 60 Hz for 50 seconds, rest for 10 seconds, and cycle 30 times. After grinding, centrifuge at 10000 r / min and 4°C for 25 minutes, discard the precipitate, collect the supernatant for testing, and set up three parallels for each sample.

[0034] 7. Pectinase method (PX): Weigh 5 g of spirulina powder, add PBS (0.1 M, pH = 6.8) according to a solid-liquid ratio of 1:8, pre-soak for 30 minutes and adjust the pH to 4.0 ± 0.1, add 2% (W / W) pectinase and hydrolyze in a 45°C water bath for 3.5 hours. After the hydrolysis, inactivate the enzyme at 100°C for 10 minutes, centrifuge at 10000 r / min and 4°C for 25 minutes, discard the precipitate, collect the supernatant for testing, and set up three parallels for each sample.

[0035] 8. Cellulase method (CX): Weigh 5 g of spirulina powder, add PBS (0.1 M, pH = 6.8) at a solid-liquid ratio of 1:8, pre-soak for 30 min and adjust the pH to 5.5 ± 0.1, add 2% (W / W) cellulase and hydrolyze in a 50°C water bath for 3.5 h. After the hydrolysis, inactivate the enzyme at 100°C for 10 min, centrifuge at 10000 r / min and 4°C for 25 min, discard the precipitate, collect the supernatant for testing, and set up three parallels for each sample.

[0036] like Figure 1 As shown in the figure, the protein content and wall breaking rate obtained by physical biological wall breaking extraction such as ultrasound (U), ultrasound-assisted shearing (ZU), tissue homogenization (ZY), repeated freeze-thaw method (F), high pressure homogenization (HPM), tissue grinding (M), pectinase (PX), and cellulase (CX) are different. Among them, when the enzymatic method is used alone to break the wall, the wall breaking effect is poor, and the protein content and wall breaking rate are 19.34-21.23 mg / mL and 24.92-27.36%, respectively. The protein content and wall breaking rate obtained by ultrasound-assisted shearing method are higher, which are 51.38±0.44 mg / mL and 50.03±0.45%, respectively. Therefore, the enzymatic method can be coupled with the ultrasound-assisted shearing method, and the wall is broken after enzyme treatment to release Spirulina protein.

[0037] Example 2

[0038] Based on the results of Example 1, this example provides a method for extracting and separating Spirulina protein, comprising the following steps:

[0039] S1. Spirulina is passed through an 80-mesh sieve and dispersed evenly. Under a microscope, the spirulina is in a dispersed state. 8 to 10 times the volume of PBS (0.001 to 0.1 M, pH = 6.8 to 7.2) solution is added. After dispersion at 18 to 20° C. for 30 min, 1.0 to 3.0% (w / w) cellulase or pectinase is added. The spirulina is enzymatically hydrolyzed at 45 to 55° C. for 2 to 4 h. Ultrasound is performed at 350 to 550 W for 10 to 20 min, with a working time of 1 min and an interval of 1 min. The pulverization and dispersion are achieved through the cavitation effect. The spirulina is extracted under shear conditions, which is beneficial to the destruction of the cell wall. The cell wall is synergistically destroyed by the enzymatic method and the assistance of sound waves, and the wall-breaking efficiency of the spirulina is improved to obtain a wall-broken spirulina solution.

[0040] S2, add 0.5-1.5M NaOH solution to the broken spirulina solution to adjust the pH to 8-10, adjust the feed liquid mass / volume ratio to 1:30-1:45 at a treatment temperature of 20-40°C, and maintain for 3-5 hours, centrifuge at 10000r / min and 4°C for 20 minutes to obtain an extract, continue to extract the precipitate 3 times, and combine the extracts;

[0041] S3, taking the obtained extract, adjusting the pH, separating by isoelectric precipitation method with ammonium sulfate salting out, standing at 4-10°C for 0.5-4h, taking 40%-60% of the salted out protein precipitate to obtain precipitate A;

[0042] S4. At 18-25°C, add 5-8 times the volume of deionized water, dissolve the precipitate A for 10-15 minutes to obtain the initial protein solution B, remove the residual insoluble protein and impurities with a 0.22 microporous filter membrane, use an ultrafiltration membrane with a molecular weight cutoff of 200 for desalination, concentration, sterilization, and freeze-drying to obtain Spirulina protein powder.

[0043] like Figure 2 , with the extension of ultrasonic time, the wall breaking rate and protein content of Spirulina first increased and then decreased, reaching the highest at 15min. Ultrasound not only destroys the cell structure, but also increases heat production, resulting in a decrease in wall breaking efficiency. With the increase of treatment temperature, the wall breaking rate and protein content of Spirulina also showed a trend of first increasing and then decreasing, reaching the highest at 35℃, and significantly decreasing above 35℃ (P<0.05). Higher temperature has an effect on the extraction of protein from Spirulina wall breaking. With the increase of treatment time, the wall breaking rate and protein content of Spirulina are higher at 4h, and continue to increase with time. The wall breaking rate and protein content show a trend of first decreasing and then slightly increasing, but it takes too long. Therefore, the ultrasonic time is selected as 15min, the treatment temperature is 35℃, and the treatment time is 4h.

[0044] Example 3 Extraction and separation of spirulina protein after cell wall breaking, determination of amino acid composition and polysaccharide content

[0045] The extraction solution after cell wall breaking extraction was adjusted to pH 7, 8, 9, 10, 11, 12 with NaOH solution, and protein extraction was performed at a solid-liquid ratio of 1:10 to 1:60, an extraction temperature of 15 to 65°C, and an extraction time of 1 to 6 hours. The solution was centrifuged at 10,000 r / min and 4°C for 20 minutes, the precipitate was discarded, the supernatant was collected, and after dilution by a certain multiple, the extraction content of Spirulina protein was determined and calculated using the Bradford method. Solid (NH4)2SO4 was added to the extract, and the isoelectric precipitation method and salting-out method were used to separate the saturated concentration of (NH4)2SO4 to 30%, 40%, 50%, 60%, 70%, and 80%. After standing, the solution was centrifuged at 10,000 r / min and 4°C for 15 minutes, and the protein content in the supernatant and precipitate was determined by the Bradford method.

[0046] Amino acid composition determination: The amino acid composition was determined according to the national standard method for amino acid determination (GB 5009.124-2016). The specific steps are as follows: (1) Sample weighing: According to the protein content in the sample (20-30 mg), weigh an appropriate amount of sample into an ampoule. (2) Digestion: Digest with HCl (6 mol / L, containing 5‰ mercaptoethanol), add 10 mL to each tube, fill with nitrogen, and seal the ampoule with an alcohol burner. Place in an oven at 110°C for 22 hours. (3) Volume adjustment and acid removal: Cool, and use 0.02 mol / L HCl to adjust the volume in a 50 mL volumetric flask. Transfer 1 mL of hydrolysis liquid nitrogen to blow dry, add a small amount of ultrapure water to blow dry to remove acid, and add three times in total. (4) Redissolve: Finally, use 2 mL, 0.02 mol / L HCl to redissolve. Pass through a 0.22 μm water filter membrane in a liquid phase bottle as the test solution. (5) On-machine determination: The amino acid composition of the samples was determined using Hitachi L-8900 automatic amino acid analyzer.

[0047] Determination of polysaccharide content: 0, 50, 100, 150, 200, 250, 300 μL of 0.1 mg / mL standard glucose solution was measured respectively, and water was added to 0.5 mL, and then 500 μL of 5% phenol and 2.5 mL of concentrated sulfuric acid were added. After vortexing, the solution was heated in a boiling water bath for 10 min, and the optical density was measured at 490 nm after cooling to room temperature (standing for 30 min). The concentration of the standard glucose solution was used as the horizontal axis, and the corresponding optical density value was used as the vertical axis to draw a standard curve to obtain the standard curve equation. The test samples were Spirulina raw materials and Spirulina protein. After the sample solution was prepared into a certain concentration (the raw materials must be digested first), 300 μL of the test solution was accurately drawn, and water was added to 0.5 mL. After that, the solution was operated in parallel with the standard monosaccharide, and the optical density value at 490 nm was measured, and the total sugar content of the crude polysaccharide was calculated according to the standard curve equation based on the average value of the three measurements.

[0048] like Figure 3 , with the increase of pH, the content of extracted spirulina protein increases, and the dissolution rate is better in alkaline environment, but the pigment group is inactivated after pH exceeds 9.5, and the pH should not be too high. With the increase of extraction time, the content of extracted spirulina protein reaches the highest at 3h, and it decreases significantly after exceeding 3h; with the increase of extraction temperature, the content of spirulina protein in the extract is the highest at 25℃, and there is no significant difference with 55℃, but the content decreases significantly when the temperature is increased; with the decrease of solid-liquid ratio, the content of extracted spirulina protein increases first and then decreases, reaching the highest at 1:40. Therefore, the extraction time is selected as 4h, the extraction temperature is 25℃, and the solid-liquid ratio is 1:40.

[0049] like Figure 4 With the increase of salt concentration, the protein content in the supernatant first decreased and then increased. When the concentration was 50%, the protein content in the supernatant was the lowest, which was 4.46%. The protein content in the precipitate first increased and then decreased. When the concentration was 50%, the protein content was the highest, which was 68.96%.

[0050] like Figure 5 , it was determined that the protein content of Spirulina protein obtained by ultrasonic assisted shearing extraction-isoelectric point synergistic 50% saturated (NH4)2SO4 salting out separation was 82.13±0.24g / 100g, and the essential amino acid composition was rich, with threonine content of 6.21g / 100g, isoleucine 4.42g / 100g, leucine 8.80g / 100g, and valine 4.51g / 100g. Compared with other high-quality proteins (egg white isolate protein, soy isolate protein, pea isolate protein, etc.), Spirulina protein has a higher content of essential amino acids. The standard curve equation of standard glucose solution concentration is y=30.725x+0.0795(R 2=0.9982), the polysaccharide content in the spirulina raw material was 9.68±0.29g / 100g, and the polysaccharide content in the spirulina protein obtained after ultrasound-assisted shearing extraction-isoelectric point synergistic 50% saturated (NH4)2SO4 salting-out separation was 2.27±0.21g / 100g. Analysis of the properties of components such as protein, amino acids and polysaccharides showed that ultrasound-assisted shearing extraction-isoelectric point synergistic 50% saturated (NH4)2SO4 salting-out separation of spirulina protein had a better effect.

[0051] Example 4 Extraction rate of Spirulina protein, extraction rate of phycobiliprotein and purity determination

[0052] The extraction rate of Spirulina protein obtained by isoelectric precipitation method and ammonium sulfate salting-out separation was calculated. The extract was desalted by ultrafiltration membrane, concentrated, sterilized and freeze-dried to obtain Spirulina protein powder. The extraction rate and purity of phycobiliprotein in the Spirulina protein powder could be calculated by further removing impurities and purifying the Spirulina protein powder.

[0053] Spirulina protein extraction rate determination: Spirulina protein extraction rate (%) = (Spirulina protein concentration (g / mL) × extract volume (mL)) / Spirulina dry matter protein content (g) × 100

[0054] Determination of the extraction rate of phycobiliprotein: extraction rate of phycobiliprotein (%) = phycobiliprotein content (g) / Spirulina protein content (g) × 100.

[0055] Purity determination of phycobiliprotein: Take an appropriate amount of phycobiliprotein, dissolve it to an appropriate concentration and then determine the purity of A 620 and A 652 The concentration of phycocyanin and allophycocyanin is calculated according to the empirical formula. The total concentration of phycobiliprotein is equal to the sum of the concentrations of the first two, and the purity of phycobiliprotein is calculated. The concentration calculation formula is as follows:

[0056]

[0057] A 620 —Absorbance at 620nm

[0058] A 652 —Absorbance at 650nm

[0059] The extraction rate of Spirulina protein was calculated to be 72.30±1.3%, which contained a large amount of phycobiliprotein and a small amount of impurity protein. The impurity protein could be further separated and adsorbed to obtain phycobiliprotein. After further impurity removal and purification, the extraction rate of phycobiliprotein could reach 85.63±1.5%. The purity of phycobiliprotein was determined to be 83.82±1.2%.

[0060] Example 5 Preparation of Spirulina Protein Functional Food / Product

[0061] To an equal volume of a fully hydrated 10% (W / V) spirulina protein solution, 1%, 2%, 3%, 4%, 5% (W / V) sodium alginate solution was added, and the pH was adjusted to 7.0±0.1. The uniformly treated composite was heated in a water bath at 85°C for 30 minutes, quickly cooled in running water for 5 minutes, stored at 4°C for 24 hours to form a gel, and packaged after sterilization, wherein the concentration of sodium alginate in the composite gel was 0.5%, 1.0%, 1.5%, 2.0%, 2.5% (W / V).

[0062] Surface hydrophobicity determination: Spirulina protein composite gel was diluted to a protein concentration of 1 mg / mL with phosphate buffer, and then 200 μL of 1 mg / mL bromophenol blue (BPB) solution was added to 1 mL of diluent. After mixing, the sample and control group were shaken at room temperature for 10 min, then centrifuged at 4000 r / min for 15 min, the supernatant was diluted 10 times, and the absorbance was measured at 595 nm using a UV-visible spectrophotometer. In the control group, 200 μL of 1 mg / mL BPB solution was supplemented to 1 mL of phosphate buffer. Surface hydrophobicity is expressed as the amount of BPB bound, and the calculation formula is as follows:

[0063]

[0064] Determination of free thiol content: About 0.1g of Spirulina protein composite gel was dissolved in 5mL Tris-glycine buffer (0.086M Tris, 0.09M glycine, 4mM Na2EDTA, pH 8.0). The mixed solution was incubated in a dark 25°C water bath for 24 hours, and then centrifuged at 5000r / min for 20 minutes. Then, 3mL of supernatant was mixed with 0.03mL Ellman reagent (4mgDTNB / mL Tris-glycine buffer) and incubated at 25°C for 15 minutes. The absorbance was measured at a wavelength of 412nm using an enzyme reader. Tris-glycine buffer was used as a blank control during the measurement. The protein content in the supernatant was determined using a BCA kit.

[0065] like Figure 6, it was determined that with the increase of sodium alginate polysaccharide concentration, the BPB binding amount of spirulina protein composite gel decreased, indicating that its surface hydrophobicity decreased. When the sodium alginate concentration was 0.5%, the BPB content was 165.73μg. When the sodium alginate polysaccharide concentration increased to 2.5%, the BPB content significantly decreased to 23.42μg (p<0.05). Surface hydrophobicity can reflect the distribution of hydrophobic amino acids on the protein surface. The addition of sodium alginate promoted the covalent cross-linking between spirulina protein and the sugar chains containing more hydrophilic hydroxyl groups in the polysaccharide. The formed complex hindered the binding of protein and BPB, and the hydrophilicity of the protein microenvironment was enhanced. When the concentration of sodium alginate polysaccharide was 2.5%, the free thiol content was 355.36 μmol / g, which was 12.36% less than that of 0.5%, because the polysaccharide absorbed the water around the protein, concentrated and concentrated the spirulina protein, and promoted the conversion of free thiol groups between or within the protein molecules into disulfide bonds, making the network structure formed by the composite gel more stable. The results showed that the cross-linking of spirulina protein and sodium alginate polysaccharide was significantly enhanced when the sodium alginate concentration was 2.5%.

[0066] Example 6 Texture and morphology analysis of spirulina protein functional food

[0067] Texture measurement: Cut spirulina protein compound gel of uniform size and thickness onto the test bench, set the test speed to 60 mm / min, trigger force to 0.1 N, deformation to 80%, and interval between two presses to 1 s. Measure the elasticity, adhesiveness, chewiness, and hardness of each sample.

[0068] Scanning electron microscopy determination: The freeze-dried Spirulina protein composite gel sample was fixed on a cylindrical platform with conductive glue, gold was sprayed under vacuum with a sputtering coater, and placed on a Zeiss EVO MA field emission scanning electron microscope (500×) for morphological observation.

[0069] like Figure 7 , with the increase of sodium alginate polysaccharide concentration, the elasticity, viscosity, chewiness, hardness and other texture characteristics of the composite gel increased. When the sodium alginate concentration was 0.5%, the chewiness of the spirulina protein composite gel was 0.097±0.04mj, the viscosity was 0.357±0.05N, the elasticity was 0.213±0.03mm, and the hardness was 0.920±0.09N. When the sodium alginate concentration increased to 2.5%, the chewiness increased to 1.467±0.37mj, and the viscosity increased to 1.313±0.22N. At this time, the elasticity and hardness were 1.087±0.19mm and 5.367±0.86N, respectively. The results showed that when the sodium alginate concentration was 2.5%, the texture characteristics of spirulina protein were significantly (p<0.05). Figure 8When the concentration of sodium alginate polysaccharide is low, the three-dimensional network structure of spirulina protein composite gel is relatively irregular, loose, and has a large void diameter. With the increase of sodium alginate polysaccharide concentration, the structure of the composite gel shows a gradually enhanced honeycomb structure. When the concentration of sodium alginate is 2.5%, the three-dimensional honeycomb network structure of the gel becomes more obvious, the pores gradually decrease, and tend to be regular, orderly, and dense.

[0070] The above description is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements without departing from the principle of the present invention, and these improvements should also be regarded as within the protection scope of the present invention.

Claims

1. A novel spirulina protein, characterized in that: The preparation method of spirulina protein comprises the following steps: 1) adding spirulina into the solution for dispersion, then adding cellulase or pectinase, performing enzymolysis at 45-55° C., and then ultrasonically crushing the spirulina to obtain a wall-broken spirulina solution; 2) adding NaOH solution to the broken spirulina solution to adjust the pH to 8-10, adjusting the mass / volume ratio of the feed liquid to 1:30-1:45 at a treatment temperature of 20-40° C., and maintaining for 3-5 hours, and obtaining an extract after centrifugation; 3) adjusting the pH of the extract, separating by isoelectric precipitation method with ammonium sulfate salting out, standing at 4-10° C. for 0.5-4 h, and taking 40%-60% of the salted protein precipitate to obtain a precipitate; 4) using deionized water to dissolve the precipitate obtained in step 3) to obtain a primary protein solution, filtering with a 0.22 μm filter membrane, desalting with an ultrafiltration membrane with a molecular weight cutoff of 200, concentrating, sterilizing, and freeze-drying to obtain spirulina protein.

2. The novel Spirulina protein according to claim 1, characterized in that The solution in 1) is a PBS solution with a concentration of 0.001-0.1M and a pH of 6.8-7.

2.

3. The novel Spirulina protein according to claim 1, characterized in that The centrifugation in 2) is performed at 12000 rpm.

4. Use of the novel Spirulina protein according to claim 1 in the preparation of food or functional biological products.

5. The use according to claim 4, characterized in that The food or functional biological product is a gel food or a functional biological product.

6. A functional gel product that prevents choking, characterized in that: The product is prepared using the novel spirulina protein and sodium alginate as raw materials.

7. The anti-choking gel-type functional product according to claim 6, characterized in that: The anti-choking gel functional product is prepared by adding the spirulina protein described in claim 1 to a sodium alginate solution, adjusting the pH to 6.8-7.2, heating in a water bath at 75-90°C for 25-35 minutes, rapidly cooling in running water for 5-10 minutes, and storing at 4°C for 20-24 hours.

8. The anti-choking gel-type functional product according to claim 7, characterized in that: The concentration of the sodium alginate solution is 0.5% to 2.5%.

9. The anti-choking gel-type functional product according to claim 7, characterized in that: The final concentration of the spirulina protein added is 3-6%.

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