Method for modifying wheat gluten protein by microencapsulation of fresh spirulina

Abstract

The invention discloses a method for modifying wheat gluten protein through fresh spirulina microencapsulation, and belongs to the technical field of food wheat protein processing. According to the method, the composition and process parameters of microcapsule materials (sodium alginate, chitosan and the like) are finely controlled, fresh spirulina with biological activity is microencapsulated, and wheat gluten protein is modified. The invention aims to optimize the network structure of gluten protein and improve the nutritional value and functional performance of the gluten protein by regulating and controlling the adding proportion of fresh spirulina microcapsules (SP-MICs) and adopting a specific modification treatment process. The problem of unbalanced amino acid composition of traditional gluten protein is solved, and essential nutrients are increased. Subunit distribution in the mucedin and the proportion of glutenin and prolamin are adjusted, so that the surface of the mucedin is finer and smoother. The problems of poor gluten protein structure and poor solubility are solved. The method is easy and convenient to operate, efficient, green and environmentally friendly, and the functional characteristics and the application value of the wheat gluten protein can be fully improved.

Description

Technical Field

[0001] The invention relates to the field of food processing technology development, and in particular to a method for modifying wheat gluten protein by microencapsulation of fresh spirulina. The modification method can significantly improve the quality of wheat gluten protein and expand its application range in the food industry. Background Art

[0002] Wheat flour is the main ingredient of many foods, especially baked goods and pasta, because it is rich in gluten protein. The quality of wheat products depends largely on the composition and structural properties of gluten protein, which plays a vital role in the elasticity and overall texture of the dough. Although gluten protein is a plant-based protein, its amino acid composition is not balanced and lacks essential amino acids such as lysine. Therefore, it is difficult to meet the body's demand for comprehensive protein by relying solely on gluten protein. Due to the unique amino acid composition and molecular structure of gluten protein, its water solubility is low and its foaming ability is weak, which limits its application in certain food areas. Under conditions of high fat, high sugar or high water content, the stability of the gluten network is easily disturbed or destroyed, thereby weakening its functional performance in the dough and affecting the texture and taste of the product.

[0003] Spirulina is a nutrient-rich blue algae that has attracted attention as a potential food additive due to its high protein content of 60-70% and the availability of essential amino acids, pigments, vitamins and minerals. The safety of spirulina has been widely confirmed through numerous scientific studies and experiments and is considered a safe superfood. In addition, the antioxidants and essential fatty acids in spirulina have anti-inflammatory and immunomodulatory effects. The addition of spirulina to wheat flour is expected to improve the nutritional content and functional properties of the food, modify the gluten protein network and enhance the sensory properties of the product.

[0004] Currently, spirulina powder is the main product on the market because it is easy to store and transport. However, compared with spirulina powder, fresh spirulina has many advantages. Without high-temperature drying treatment, it can avoid nutritional loss caused by processing, so the active ingredients it contains (such as protein, vitamins, antioxidants and enzymes, etc.) are richer and more complete. This makes fresh spirulina superior to spirulina powder in terms of nutritional value and biological activity. Studies have shown that the antioxidant activity of fresh spirulina is significantly higher than that of dried spirulina powder. Fresh spirulina is a low-processed product that retains its original natural state and is suitable for the needs of the organic and natural food market.

[0005] Despite these advantages, fresh spirulina has a short storage and shelf life and requires cold chain transportation and preservation, which limits its widespread promotion. This complex storage requirement not only increases costs, but also limits the widespread promotion and application scope of fresh spirulina. To overcome these challenges, microencapsulation of fresh spirulina has become a potential solution. Encapsulating spirulina with materials such as sodium alginate and chitosan can effectively extend its shelf life while avoiding the loss of nutrients and active ingredients. The microencapsulation process can protect spirulina from adverse external environments such as oxidation, light or heat damage, thereby maintaining its biological activity. In addition, microcapsules also have the function of masking the unique odor of spirulina, enhancing its sensory properties, and making it more widely used in food and health products. The application of this technology can solve the problems of storage and transportation, and also provide technical support for the development and promotion of fresh spirulina in functional foods, which will help promote the diversification of its market applications.

[0006] However, the application of fresh spirulina microcapsules (SP-MICs) in wheat flour products is little known, and there is a gap in understanding the exact mechanism by which SP-MICs affect the gluten protein network and the overall physicochemical properties of wheat flour products (dough rheology, water absorption and baking performance). There is also a lack of comprehensive research on the optimal addition ratio of SP-MICs required to significantly improve the characteristics of gluten proteins without affecting the sensory qualities of fortified foods such as taste, aroma and mouthfeel. Addressing these gaps is crucial to fully realize the potential of SP-MICs as an innovative additive for wheat flour products. Therefore, based on the above situation, this field urgently needs to develop a modification method that can not only optimize the gluten protein network structure and significantly improve the nutritional value and functional properties of wheat gluten proteins, but also be simple to operate, green and efficient. This method has important practical application value and research significance. Summary of the invention

[0007] In view of this, the present invention provides a method for modifying wheat gluten protein by microencapsulation of fresh spirulina, the purpose of which is to optimize the network structure of gluten protein and improve its nutritional value and functional performance by regulating the addition ratio of fresh spirulina microcapsules (SP-MICs) and adopting a specific modification process. Improve the imbalance of amino acid composition of traditional gluten protein and increase essential nutrients. Regulate the subunit distribution, glutenin and alcohol-soluble protein ratio in gluten protein to make the surface of gluten protein more delicate. Solve the problems of poor protein structure and poor solubility of gluten protein. The method is simple to operate, efficient and environmentally friendly, and can fully improve the functional characteristics and application value of wheat gluten protein.

[0008] Based on the above purpose, the present invention adopts the following technical solution:

[0009] The method for modifying wheat gluten protein by microencapsulation of fresh spirulina comprises the following steps:

[0010] (1) Fresh spirulina concentrated algae liquid or algae mud is mixed with sodium alginate (SA) in a certain weight ratio, and then atomized and sprayed / microfluidically dripped into a mixed solution of calcium chloride and chitosan (CS) to form spirulina microcapsules.

[0011] (2) adding distilled water to flour at room temperature to form flour flocs; placing the fresh spirulina microcapsules and flour flocs obtained in step (1) in a mechanical stirrer for stirring, and then standing in a hot and humid environment to let the flour rise, so as to promote the combination of fresh spirulina, alginate and chitosan in the spirulina microcapsules with wheat gluten protein.

[0012] (3) Soak the dough in distilled water and adjust the pH of the water with glacial acetic acid. Knead the dough gently by hand and change the water every 3 minutes. Repeat 5 times to ensure that the water-soluble components and gluten protein are washed away.

[0013] (4) Collecting the gluten protein in step (3), and vacuum freeze-drying to obtain fresh spirulina microcapsule-modified wheat gluten protein.

[0014] In one embodiment, the steps for preparing the Spirulina algae liquid or algae mud in step (1) are as follows:

[0015] (5) After the fresh Spirulina was harvested, it was separated from the culture medium using a 450-mesh nylon filter and washed three times with distilled water;

[0016] (6) Seal the spirulina with plastic wrap and sterilize under ultraviolet light for 10 min to ensure that the physiological structure and state of Spirulina are not destroyed;

[0017] (7) After the ultraviolet sterilization in step (6), add a small amount of ultrapure water and mix well. Use an ELISA reader to detect its ultraviolet absorption value at 680nm to obtain its OD 680 Between 30-40, it is spirulina concentrate;

[0018] (8) After the ultraviolet sterilization in step (6), the spirulina is dehydrated to a water content of 40-50%, thereby obtaining the spirulina mud.

[0019] In one embodiment, the fresh spirulina concentrate or algae mud prepared in steps (7) and (8) is added together with 0.5%-1% vitamin C to a 0.5-2% sodium alginate solution, stirred at 300 rpm for 10 min until the mixture is uniformly mixed, and the mixed solution is sprayed / dropped into a cross-linking agent 0.5-2% calcium chloride and chitosan 1:1 (v / v) mixed solution in an atomized state through an atomizer / microfluidic device to form fresh spirulina microcapsules with a particle size of 50-100 / 150-200 μm, which are named SP-MICs.

[0020] In one embodiment, in the step (1), the content of SA is 0.5-2%, the content of is 0.5-2%, the content of CS is 1-2%, and the volume ratio of CaCl2 to CS is 1:1 or 1:2.

[0021] In one embodiment, in step (2), the mass of water added to the mixture is 35-40% of the total mass after mixing, and the weight ratio of spirulina microcapsules added to the flour mixture is 1%-4%. The two are mixed and stirred for 30 minutes at a stirring speed of 200 rpm; the dough is allowed to rest for 30 minutes in an environment with a temperature of 35-38°C and a humidity of 65%-70%.

[0022] In one embodiment, the temperature of the distilled water in step (3) is 25° C., and the pH value of the water is adjusted to 6.0, showing weak acidity.

[0023] The present invention has the following beneficial advantages:

[0024] (1) After the wheat gluten protein and SP-MICs are mixed and treated in the present invention, the protein content of the wheat gluten is significantly increased, the imbalance of the amino acid composition is improved, and the essential amino acids required by the human body are supplemented. The microcapsules fully retain the biological activity of fresh spirulina and mask the unique smell of spirulina. The pigments contained therein can significantly improve the sensory properties of gluten protein, and substances such as vitamins, carotenoids, and γ-linolenic acid can improve the antioxidant capacity of gluten protein.

[0025] (2) The modification method of the present invention significantly enhances the texture characteristics of gluten protein, can bind to the amino acid residues on the surface of gluten protein through SP-MICs, improve its surface hydrophobic sites, reduce its surface hydrophobicity; reduce the free thiol content, increase the internal disulfide bond content, promote the cross-linking of glutenin and alcohol-soluble protein in gluten protein, and through Ca 2+ The stability of the gluten network is strengthened through ionic bonds and hydrophobic interactions.

[0026] (3) The modification method of the present invention is easy to operate, directly utilizes fresh spirulina, reduces the cost of spray drying or freeze drying, reduces energy consumption, facilitates storage and transportation, saves costs, and is green and environmentally friendly, and is suitable for large-scale industrial production.

[0027] In summary, the modified gluten protein of the present invention significantly enhances its application prospects in noodle products and functional foods while maintaining its excellent processing properties. The modified protein has shown wide applicability in the fields of food processing, health foods and nutritional supplements, and can effectively meet consumers' demand for high-quality healthy foods. Its unique advantages give it important industrial value and promotion potential, and provide new ideas and development directions for food processing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 The reverse phase high performance liquid chromatograms of gluten protein with different SP-MICs added ratios as described in the examples (Figure A, Figure B).

[0030] Figure 2 This is the effect of different SP-MICs addition ratios on the SDS extraction rate and surface hydrophobicity of gluten protein as described in the examples.

[0031] Figure 3 This is the effect of different SP-MICs addition ratios on the free thiol and disulfide bond contents of gluten protein as described in the examples.

[0032] Figure 4 This is the effect of different SP-MICs addition ratios on the non-covalent interactions of gluten protein as described in the examples.

[0033] Figure 5 Fourier transform infrared spectra of gluten protein with different SP-MICs addition ratios as described in the examples (Figure A, Figure B) and their effects on the secondary structure content of gluten protein (Figure C).

[0034] Figure 6 Electron microscopic images of the microstructure of gluten protein at different SP-MICs addition ratios as described in the examples. DETAILED DESCRIPTION

[0035] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0036] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased from the market.

[0037] All experiments in this embodiment were repeated three times, and the data are expressed as the mean ± standard deviation (SD) of three repetitions. Data were analyzed using one-way analysis of variance (ANOVA) and SPSS, and Duncan's multiple extreme variance test was used to compare the mean and significance (P value was 0.05). The figures drawn in the full text were drawn by Origin software.

[0038] Embodiment 1

[0039] Preparation of SP-MICs:

[0040] After the fresh spirulina is harvested, a 450-mesh nylon filter is used to separate the spirulina from the culture medium to ensure the integrity and physiological activity of the fresh spirulina. Then, the fresh spirulina is filtered and washed three times with distilled water to remove the salt and alkali on the surface of the filaments and the impurities on the surface to maintain its nutrients. 680 30-40% fresh spirulina liquid or 40-50% fresh spirulina mud with water content was added to 30mL sodium alginate (SA) solution (0.5-2% w / v) and stirred at 300r / min for 10min using a magnetic stirrer. 1-2% chitosan (CS) was dissolved in 1% acetic acid and mixed with 0.5-2% (v / v) calcium chloride solution in a ratio of 6:4. Subsequently, the mixed solution of Spriulina and SA was titrated in a mixed solution of calcium chloride and CS. Fresh spirulina microcapsules (SP-MICs) were collected, wrapped in tin foil, and stored at 4°C in the dark. The particle size and distribution of SP-MICs were evaluated using a laser particle size analyzer.

[0041] Embodiment 2

[0042] Preparation of SP-MICs modified wheat gluten protein:

[0043] A control group sample was set up: 100 g of traditional wheat flour was the 0% group (M0).

[0044] Four groups of gradient implementation samples were set up: Spirulina powder was mixed with traditional wheat flour in weight ratios of 1% to 4% and 96% to 99%, 1g of SP-MICs in Example 1 and 99g of traditional wheat flour were the 1% group (M1), and the remaining additions were the 2% group (M2), 3% group (M3) and 4% group (M4).

[0045] The five groups of samples (M0, M1, M2, M3, M4) were fully mixed and uniformly mixed. At room temperature, distilled water was gradually added to the mixture, and the moisture content was 35-40%. Then, the dough mixture was placed in a mechanical stirrer for stirring for 30 minutes at a stirring speed of 200 rpm until the gluten network structure was formed to obtain a uniform spirulina-wheat dough. In a hot and humid environment, the temperature was 35-38°C, the humidity was 65%-70%, and the dough was proofed for 30 minutes to promote the combination of fresh spirulina, alginate and chitosan in the spirulina microcapsule with wheat gluten protein; then the dough was immersed in 25°C distilled water, and the pH value of the water was adjusted to 6.0 with glacial acetic acid. The slightly acidic environment improved the solubility of gluten protein. The dough was gently kneaded by hand, and the water was changed every 3 minutes, and repeated 5 times to ensure that the water-soluble components and gluten protein were washed clean; the gluten protein after washing was collected and vacuum freeze-dried to obtain fresh spirulina microcapsule modified wheat gluten protein.

[0046] Embodiment 3

[0047] Area protein subunit distribution determination experiment:

[0048] Test method: 100 mg of sample was dissolved in 3 mL of phosphate buffer (0.05 mol / L, pH 7.6, containing 0.4 mol / L NaCl), vortexed, magnetically stirred for 10 min and centrifuged (20°C, 10,000×g, 15 min). The sample was then extracted twice with 3 ml of deionized water, and the supernatant was discarded after centrifugation. The precipitate was extracted three times with 3 ml of 60% ethanol (20°C, 20 min), and then centrifuged (20°C, 10,000×g, 20 min), and the supernatant was collected as the gluten protein extract. The obtained precipitate was continuously extracted three times at 60°C with 3 mL of solution (50% isopropanol, containing 0.05 mol / L, pH7.5 Tris-HCL, 2.0 mol / L urea, 1.0% DTT), and the supernatant was collected as the gluten protein extract after centrifugation (20°C, 10,000×g, 20 min).

[0049] Chromatographic conditions were as follows: injection volume 10 μL, mobile phases of water containing 0.1% (v / v) trifluoroacetic acid (A) and acetonitrile containing 0.1% (v / v) trifluoroacetic acid (B), column temperature 60°C, flow rate 0.6 mL / min, gradient elution (mobile phase B increased from 24% to 56% in 50 min). UV detection wavelength was 214 nm.

[0050] Embodiment 4

[0051] SDS gluten extraction and surface hydrophobicity experiment:

[0052] Determination method: The sample (300 mg) was added to 10 mL of 0.5% (w / v) SDS solution (containing 8 mol / L urea; 4 mol / L sodium ethylenediamine tetraacetate; 0.09 mol / L glycine, 0.086 mol / L Tris, pH 9.1). The experimental bottle was incubated in a rotary shaker (200 r / min, 25°C) for 30 min and then centrifuged for 20 min (15,000 r / min). The fluorescence intensity of the supernatant was measured by a multimode microplate reader. The experimental parameters were an excitation wavelength of 295 nm, an emission spectrum of 300-450 nm, and an emission spectrum slit width of 5 nm.

[0053] Determination method: Mix the sample (50 mg) with 1 mL of 20 mmol / L sodium phosphate buffer (pH 7.0) and 200 μL of 1 mg / mL bromophenol blue (BPB). Shake at room temperature (200 r / min, 25°C) for 1 hour, then centrifuge (4000 r / min, 10 min). Take 100 μL of the supernatant and mix it with 900 μL of sodium phosphate buffer, and measure the absorbance at 595 nm using a Multimode microplate reader. Phosphate buffer was used as a blank control. The BPB binding amount was calculated as follows:

[0054] BPB binding amount (μg) = 200 μg × (blank absorbance value - sample absorbance value) / blank absorbance value

[0055] Embodiment 5

[0056] Free thiol and disulfide bond content determination experiment:

[0057] Determination method: Use Tris-gly buffer (5.2g Tris, 3.45g glycine, 0.6g ethylenediaminetetraacetic acid, pH8.0) as solvent to prepare 8mol / L urea, 5mol / L guanidine hydrochloride mixed solution, 4mg / mL Ellman reagent and 8mol / L urea solution. Dissolve the sample (75mg) in 1.0mL Tris-Gly buffer. Then add guanidine hydrochloride (4.7g) and add Tris-Gly buffer to 10mL. Incubate on a constant temperature shaker at 25℃ and 200r / min for 3h. Collect the supernatant and centrifuge at 10000r / min for 10min.

[0058] The mixed solution for determining free thiol groups is: 1 ml of supernatant, 4 ml of urea guanidine hydrochloride solution, and 0.05 mL of Ellman's reagent. After reacting for 10 min, the absorbance of the mixture at 412 nm is measured, and the SH content is calculated by the following formula. These measurements are repeated three times.

[0059] Free SH content (μmol / g) = 73.53 × A 412 ×5.02 / 7.5

[0060] When determining the total SH content, take 1 mL of the supernatant and mix it with 4 mL of urea guanidine hydrochloride and 0.05% β-mercaptoethanol solution, and stir at room temperature for 3 hours. Then add 10 mL (13%) of trichloroacetic acid and let it stand in a 37°C incubator for 1 hour. After centrifugation (10000 r / min, 10 min), discard the supernatant and wash twice with 5 mL of trichlorourea acetic acid solution. The precipitate is dissolved in 10 mL of urea (8 mol / L), 0.04 mL of Ellman's reagent (DTNB) is added, and the absorbance is measured at 412 nm.

[0061] Total SH content (μmol / g) = 73.53 × A 412 ×10 / 7.5

[0062] Disulfide bond (SS) content (μmol / g) = (total SH content - free SH content) / 2

[0063] Embodiment 6

[0064] Intermolecular force experiment:

[0065] Test method: First, prepare 4 different reagents in phosphate buffer PBS (0.05M, pH 7.0): PBS+0.05M NaCl (PA); PBS+0.6M NaCl (PB); PBS+0.6M NaCl+1.5M urea (PC); PBS+0.6M NaCl+8M urea (PD). Then, evenly disperse the freeze-dried pasta flakes sample (0.2-0.6g) in 5mL of the above reagents, shake at 25℃ for 1h, and centrifuge at 10000g for 20min. Extract 0.1mL of supernatant from the test tube, add 5mL of Coomassie Brilliant Blue G-250, mix well, and measure the absorbance at 595nm with a UV spectrophotometer within 30min. Determine the supernatant protein content by Bradford method, and the soluble protein content is expressed as mg / g sample, with bovine serum albumin as the standard.

[0066] Embodiment 7

[0067] Secondary structure detection:

[0068] Test method: The changes in the secondary structure of gluten protein were analyzed using FT-IR spectrometer (Vetex70, Bruker). 1.0 mg of powdered freeze-dried pasta sample was mixed with KBr at a ratio of 1:100, thoroughly ground in an agate mortar and pressed into thin slices, which were then placed in a Fourier transform infrared spectrometer for measurement. The data were processed by OMNIC (Version 9.2) and Peak Fit (4.12) software with a resolution of 2 cm -1 , at 400-4000cm-1 The amide I band (1600-1700 cm -1 The absorption peaks of the amide I were baseline corrected, Gaussian smoothed, and deconvoluted, and the second-order derivatives were taken for analysis. The relative amounts of α-helix, β-sheet, β-turn, and random coil were determined by calculating the ratio of the peak area corresponding to each secondary structure to the total amide I peak area.

[0069] Embodiment 8

[0070] SEM microstructure determination:

[0071] Determination method: The microstructural changes of gluten protein were determined using a Zeiss Gemini Sigma 300VP SEM at an accelerating voltage of 3.0 kV. According to the method of Li et al.27, the sample was ion sputtered for 5 minutes and the cross-sectional structure was observed at 100x, 200x, and 500x magnifications.

[0072] Experimental results analysis

[0073] 1. Analysis of gluten protein subunit distribution experimental results:

[0074] Using reverse phase high performance liquid chromatography (RP-HPLC) technology, the distribution, aggregation behavior and structural changes of glutenin subunits in SP-MICs wheat flour products can be analyzed based on the differences in polarity and retention time of glutenin subunits. Alcohol-soluble protein subunits include (ω-5-, ω-1, 2-, α / β-, γ-) and wheat glutenin subunits (D-LMS, HMS, B / C LMS). The retention time of each subunit can be determined from Figure 1As seen in A and B. In the alcohol-soluble protein subunit part, with the addition of SP-MICs, the content of ω-5- and ω-1,2-subunits in M1, M2 and M3 samples increased slightly compared with M0, but the content of α / β- and γ-subunits decreased. The content of ω-5- and α / β-subunits in M4 sample was higher than that in M0, and the content of ω-1,2- and γ-subunits was lower than that in M0. The content of γ-subunit was higher than that in M3. α / β- and γ- are sulfur-rich glutenins, and the improvement of α / β- and γ-subunits may be due to the binding with gluten through SS bonds, indicating that the addition of SP-MICs promotes the aggregation of gluten protein in dough. The high molecular weight glutenin (HMW-GS) subunit in glutenin showed an upward trend. Compared with M0 and other added groups, the HMW-GS value in M3 sample was the highest. Studies have shown that high molecular weight glutenin subunits play a key role in gluten formation, and an increase in their content tends to enhance the elasticity and toughness of dough, thereby improving the texture and processing properties of pasta products. Overall, the content and ratio of alcohol-soluble proteins and glutenins in gluten protein changed significantly after the addition of SP-MICs. Compared with M0, M3 reduced the content of alcohol-soluble proteins and increased the content of glutenins. This indicates that the addition of SP-MICs helps to regulate the ratio of alcohol-soluble proteins to glutenins in gluten protein, increase the gluten content, and improve the rheological properties of gluten.

[0075] 2. SDS extraction and surface hydrophobicity analysis:

[0076] Effect of SP-MICs on the extraction rate of wheat flour gluten Figure 2 ) showed that the fluorescence intensity of M2 and M3 was lower than that of M0, while the fluorescence intensity of M4 was slightly higher than that of M3. This indicates that the addition of SP-MICs affects the aggregation of wheat gluten subunits, and moderate addition (2%-3% SP-MICs) helps to compact the gluten network and reduce solubility. The surface hydrophobicity results showed that compared with M0, the binding amount of BPB to M2 and M3 was lower, and the fixation effect of BPB on gluten protein decreased with the increase of SP-MICs content. The addition of SP-MICs reduced the surface hydrophobicity of gluten protein. This suggests that SP-MICs can directly bind to and shield the hydrophobic points on the surface of gluten. In addition, conformational changes in proteins may cause the originally exposed hydrophobic side chains to be wrapped inside the molecule, thereby reducing contact with water and reducing surface hydrophobicity.

[0077] 3. Analysis of free thiol and disulfide bond results:

[0078] The addition of SP-MICs has an effect on the content of free sulfhydryl groups and disulfide bonds in gluten ( Figure 3). At different addition ratios, the free thiol content gradually decreased, and the disulfide bond content gradually increased. When 3% was added, the free thiol content was the lowest, and the disulfide bond content was the highest. Compared with M3, the disulfide bond content of M4 decreased slightly. This trend shows that an appropriate amount of SP-MICs helps to increase the formation of disulfide bonds and enhance the cross-linking and stability of gluten protein.

[0079] 4. Analysis of intermolecular forces:

[0080] The solubility of gluten in different solvents, such as Figure 4 As shown, it was used to evaluate the dynamic changes of the interaction between Spirulina and gluten molecules. Compared with M0, the soluble protein content in each solvent system changed after the addition of SP-MICs, and the solubility of protein in the four solutions was PD>PC>PA>PB. The strength of hydrogen bonds and ionic bonds is lower than that of hydrophobic interactions. The relatively low solubility in PA and PB solutions indicates that gluten proteins contain fewer ionized amino acid residues, resulting in weaker ionic interactions. The presence of PC and / or PD solutions increased the solubility of proteins, indicating that the non-covalent intermolecular interactions (hydrogen bonds and / or hydrophobic interactions) that stabilize the structure of gluten proteins are significantly enhanced. This may be because SP-MICs can moderately expand the protein structure, change the composition of hydrogen bonds in the peptide chain, and promote the formation of hydrogen bonds. The higher ion content in SP-MICs enhances the hydrophobic interactions between proteins.

[0081] 5. FTIR detection of gluten protein secondary structure analysis

[0082] The results showed that the secondary structure of gluten protein changed significantly after adding 1%-3% SP-MICs ( Figure 5 ). The relative content of the secondary structure of gluten protein is β-fold > β-turn > random coil > α-helix. Among them, β-fold is the main structure of gluten protein, and its relative content increases significantly after adding SP-MICs. During the extrusion process, gluten protein usually undergoes denaturation and cross-linking polymerization by forming intermolecular disulfide bonds. In particular, after adding an appropriate amount of SP-MICs, the content of disulfide bonds and high molecular weight proteins will increase, which helps to form a tighter protein network. This structural change further affects the secondary structure of gluten protein, which is manifested as an increase in the β-fold content.

[0083] 6. SEM microstructure analysis

[0084] Microstructure observations showed that regardless of whether gluten was added or not, a sponge-like structure was formed inside, including many complete pores. The network structure of gluten protein after adding SP-MICs was relatively stable, and the pore size was relatively uniform. The pore size of the control group without adding SP-MICs was larger and unevenly distributed. The addition of an appropriate amount of SP-MICs (M3) made the pore structure of gluten protein more uniform and dense, and increased the resistance of gluten protein ( Figure 6 ), the starch granules are more complete and the surface is more delicate. Due to the high content of protein and dietary fiber in SP-MICs, or the interaction between the anionic alginate in SP-MICs and the positively charged amino acid residues in the gluten molecules, the microstructure changes, thereby promoting the interaction between polypeptide chains. A relatively dense gel composite structure is formed, and its properties are similar to those of gluten proteins that form the gluten network.

[0085] As mentioned above, the above examples are only used to explain the technical solutions of the present invention, rather than to limit the present invention; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some technical features equivalently; and such changes or substitutions will not cause the essence of the respective technical solutions to deviate from the spirit and scope of each implementation method in the present invention.

Claims

1. A method for modifying wheat gluten protein by microencapsulation of fresh spirulina, characterized in that: The following steps are involved: (1) fresh spirulina concentrated algae liquid or algae mud is mixed with 0.5-2% sodium alginate (SA) in a certain weight ratio, and sprayed / microfluidically dripped into a 0.5-2% calcium chloride and chitosan (CS) mixed solution to form spirulina microcapsules; (2) adding distilled water to flour at room temperature with a water content of 35-40% to form flour flocs; placing the mixture of fresh spirulina microcapsules and flour flocs obtained in step (1) in a mechanical stirrer for 30 minutes at a stirring speed of 200 rpm and resting the flour for 30 minutes to promote the combination of fresh spirulina, alginate and chitosan in the spirulina microcapsules with wheat gluten protein; (3) Soak the dough in 25°C distilled water to reduce the thermal denaturation of the protein. Adjust the pH of the water to 6.0 with glacial acetic acid. The slightly acidic environment improves the solubility of gluten protein. Knead the dough gently by hand and change the water every 3 minutes. Repeat 5 times to ensure that the water-soluble components and gluten protein are washed away; (4) Collecting the gluten protein in step (3), and vacuum freeze-drying to obtain fresh spirulina microcapsule-modified wheat gluten protein.

2. The method according to claim 1, characterized in that The steps for preparing fresh Spirulina algae liquid or algae mud in step (1) are as follows: (5) After the fresh Spirulina was harvested, it was separated from the culture medium using a 450-mesh nylon filter and washed three times with distilled water; (6) Seal the spirulina with plastic wrap and sterilize under ultraviolet light for 10 min to ensure that the physiological structure and state of Spirulina are not destroyed; (7) After the ultraviolet sterilization in step (6), add a small amount of ultrapure water and mix well. Use an ELISA reader to detect its ultraviolet absorption value at 680nm to obtain its OD 680 Between 30-40, it is spirulina concentrate; (8) After the ultraviolet sterilization in step (6), the spirulina is dehydrated to a water content of 40%, thereby obtaining the spirulina mud.

3. The method according to claim 1, characterized in that The steps for preparing fresh spirulina microcapsules in step (1) are as follows: The fresh spirulina concentrate or algae mud prepared in claim 2 and 0.5%-1% vitamin C are added to a 0.5-2% sodium alginate solution, stirred at 300 rpm for 10 minutes until the mixture is uniformly mixed, and the mixed solution is sprayed / dropped into a cross-linking agent 0.5-2% calcium chloride and chitosan 1:1 (v / v) mixed solution through an atomizer / microfluidic device in an atomized state to form fresh spirulina microcapsules with a particle size of 50-100 / 150-200 μm, which are named SP-MICs.

4. The method according to claim 1, characterized in that In the step (3), the weight ratio of spirulina microcapsules added to the flour-flour mixture is 1%-4%.

5. The wheat gluten protein modified by Spirulina microencapsulation is prepared according to the method according to any one of claims 1 to 4.

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