A method for promoting the aggregation of quinoa protein isolate fibers and improving their gelation properties by germination

Abstract

The present invention relates to the field of food production, specifically to the field of protein gels, and more specifically to a method for promoting the aggregation of quinoa protein fibers and improving its gel property by germination. Using quinoa as the main raw material, through the following steps: 1) Germination: By germination, the properties of quinoa protein isolate are changed by the action of endogenous enzymes in quinoa; 2) Extract the protein in germinated quinoa by the alkali extraction and acid precipitation method, and obtain quinoa protein powder by freeze-drying; 3) Explore the effect of germination on the change of quinoa protein properties and the induction of fiber aggregation; 4) Gelation: Obtain quinoa protein gel by heating the quinoa protein isolate solution. The present invention can significantly improve the gel property of quinoa protein isolate by changing the properties of quinoa protein isolate through germination.

Description

Technical Field

[0001] The present invention relates to the field of food production, specifically to the field of protein gels, and more specifically to a method for promoting the aggregation of quinoa protein fibers and improving its gel property by germination. Background Art

[0002] Quinoa is a gluten-free pseudocereal with high nutritional value. It not only contains all essential amino acids required by the human body, but also is rich in nutrients such as unsaturated fatty acids and polysaccharides, and contains micronutrients such as minerals and vitamins. Quinoa protein is a high-quality protein with high in vitro digestibility. According to the FAO standard, the amino acid ratio of quinoa protein is balanced, which is a high-quality complete protein, and its quality can be comparable to that of skim milk powder and meat. In addition, quinoa protein is also a good source of bioactive peptides.

[0003] Seed germination is a green and clean processing method that can change the properties of proteins, including physical and chemical properties and structural properties. During the germination of quinoa seeds, endogenous proteases are activated, hydrolyzing some proteins and synthesizing new proteins. Germination causes changes in the composition, structure, and properties of proteins, which is more conducive to the fiber aggregation of proteins, thereby improving the gel property of quinoa protein.

[0004] Gel property is one of the important functional properties of proteins. However, the gel property of quinoa protein is weak, which limits its application in the field of food processing. Currently, the methods for improving the gel strength of proteins can be divided into three types: the first method is to add salt ions (such as NaCl and CaCl 2 ), but the intake of salt ions will cause potential health problems. The second method is restricted enzymatic hydrolysis, which exposes the protein active groups and is more conducive to the cross-linking between proteins. The third method is the fiber aggregation of proteins. Currently, there are a large number of studies on the formation of fiber aggregates by protein hydrolysis under the conditions of pH 2.0 and low ionic strength heating, which is beneficial to improving the gel strength. However, the aggregation of proteins into gels under strong acidic conditions has a negative impact on the sensory properties of foods. Therefore, the present invention introduces a method for promoting fiber aggregation under neutral conditions to improve the gel property. Summary of the Invention

[0005] The main technical problem to be solved by the present invention is to provide a method for promoting the aggregation of quinoa protein fibers and improving its gel property by germination.

[0006] The technical solution of the present invention is as follows:

[0007] A method for promoting the aggregation of quinoa protein fibers and improving its gel property by germination, characterized in that it comprises the following steps:

[0008] Step 1: Quinoa germination:

[0009] Soak quinoa in 1% sodium hypochlorite solution for 10 minutes for sterilization and disinfection, and then wash it thoroughly with sterile water or deionized water;

[0010] Soak the sterilized seeds in 1% hydrogen peroxide solution in the dark at 25°C for 24 hours, then rinse them thoroughly in deionized water. After that, spread the seeds on germination paper and culture them in a constant temperature and humidity incubator at 25°C, spraying 0.3% (w / v) hydrogen peroxide solution every day;

[0011] Dry the quinoa that has germinated for a certain period of time and grind it into powder;

[0012] Step 2: Extract quinoa protein:

[0013] Soak the quinoa powder in n-hexane for degreasing. After suction filtration, soak the solid in n-hexane again and repeat three times. After the last suction filtration, place the solid in a fume hood to volatilize naturally for 24 hours to completely volatilize the residual n-hexane.

[0014] Suspend the degreased quinoa powder in pure water, adjust the pH to alkaline, stir for 2 hours, take the supernatant after centrifugation, adjust the pH to acidic, centrifuge, and discard the supernatant to obtain the precipitate, which is quinoa protein;

[0015] Redissolve the obtained quinoa protein, adjust the pH to 7.0, and freeze-dry to obtain quinoa protein powder for standby.

[0016] Step 3: Preparation of quinoa protein fiber aggregates:

[0017] Suspend the obtained quinoa protein powder in pure water at a concentration of 2% (w / v), stir for 2 hours, then place it in a glass bottle, seal it with plastic wrap, and heat it in a water bath at 95°C for 30 minutes.

[0018] Step 4: Preparation of protein gel:

[0019] Suspend quinoa protein in deionized water and stir for 2 hours, store it in a 4°C refrigerator overnight for 12 hours, take it out and stir magnetically at room temperature for 30 minutes, place it in a specific container and heat it at 95°C for 30 minutes, cool it to room temperature and then store it in a 4°C refrigerator for 12 hours,

[0020] The following is the further optimization of the above technical solution by the present invention:

[0021] In step 1, the mass-volume ratio of quinoa to sodium hypochlorite solution is 1:2 - 3, and the mass-volume ratio of quinoa to hydrogen peroxide solution is 1:2 - 3;

[0022] In step 2, the mass-volume ratio of quinoa to n-hexane is 1:3 - 5, the specific value of adjusting the pH to alkaline is 8.0 - 11.0, and the specific value of adjusting the pH to acidic is 4.0 - 5.0;

[0023] In step 4, the protein content of quinoa protein suspended in deionized water is 16-22%.

[0024] In the present invention, the germination process is utilized to hydrolyze quinoa protein, changing the components, conformation and physicochemical properties of the protein. When quinoa protein is heated under neutral conditions, fiber aggregation occurs. The aggregation types of the protein thermogel include particle aggregation and fiber aggregation. The gel structure of particle aggregation is rough and has poor elasticity, while the gel network structure of fiber aggregation is dense, and its texture properties are significantly improved compared with the particle aggregation gel.

[0025] In order to further demonstrate the advantages of the present invention, the inventors conducted a comparative analysis on the fiber aggregates and gels prepared from quinoa proteins with different germination times, and the results are as follows:

[0026] 1. Sample preparation and treatment

[0027] Select quinoa with different germination times (0, 8, 16, 24 h) according to the germination process in step 1, extract quinoa protein according to the process in step 2, prepare thermal aggregates and quinoa protein gels respectively according to the processes in step 3 and step 4, and place the prepared gel samples in a 4°C refrigerator for standby.

[0028] 2. Transmission electron microscopy (TEM) to verify the protein aggregation type

[0029] Glow-discharge the 230-mesh carbon-coated copper grid for 45 seconds to improve the sample resolution. Incubate 10 μL aliquots of 0.05% (w / v) quinoa protein aggregate solution on the copper grid for 1 min, and then incubate with 4 μL of 2% (w / v) phosphotungstic acid for another 30 seconds. After each step, use a filter paper to drain the excess water along the periphery. Obtain the transmission electron micrograph of the quinoa protein aggregate using a 120 kV TEM (Talos L120CG2, ThermoFisher, USA).

[0030] 3. Effect of germination on the properties of quinoa protein

[0031] Solubility: Disperse the protein isolate at a concentration of 1% (w / v) in 50 mM phosphate (pH 7.0) buffer overnight. After centrifuging at 10000 g for 20 min, measure the protein concentration in the supernatant using the Bradford method. The solubility is expressed as the percentage of protein in the supernatant to the percentage of the original isolated protein.

[0032] Zeta potential: Zeta potential experiments and particle size measurements were carried out on a nanoparticle size analyzer. Quinoa protein isolate was dispersed in water (0.05%, w / v), adjusted to pH 7.0 with 1 M sodium hydroxide, and the surface charge was measured.

[0033] For each QPI sample, dispersions with concentrations of 25, 50, 100, 200, and 400 μg / mL were prepared using 10 mM Tris-HCl buffer at pH 7.0. Subsequently, 3 mL of each sample was taken and mixed with 50 μL of 8.0 mM ANS solution, and the reaction was carried out at room temperature in the dark for 20 min. The fluorescence intensities at 390 nm and 470 nm were recorded separately using a fluorescence spectrophotometer for excitation and emission, with a fixed slit width of 5 nm. The surface hydrophobicity was calculated as the initial slope of the linear regression analysis of the peak fluorescence intensity (minus the background) versus the QPI concentration.

[0034] 4. Texture determination of quinoa protein gels

[0035] The texture of the gel samples was determined using a food texture analyzer (TA.XTplus) with a probe model of P / 36. The parameters were set as follows: the pre-test speed was 1.5 mm / s, the test speed was 1 mm / s, the post-test speed was 1 mm / s, the probe penetration distance was 5 mm, the trigger force was 5 g, and the texture profile parameters (hardness, elasticity, adhesiveness, chewiness) were all recorded from the test. All tests were repeated 3 times at room temperature.

[0036] 5. Statistical analysis and graphing

[0037] Analysis of variance was performed using SPSS software, and the significant difference level between different samples was P < 0.05. Origin and Prism software were used for graphing.

[0038] 6. Results and analysis

[0039] 1) Effects of different germination times on quinoa protein aggregation

[0040] After heating in a 95 °C water bath, the aggregation behaviors of different quinoa proteins are as Figure 1 shown. It can be seen from the TEM images that the protein extracted from ungerminated quinoa in Figure a shows particulate aggregation, while the germinated quinoa protein shows fibrous aggregation. Further extending the germination time, the fibrous aggregation decreases, showing a phenomenon of a blend of fibrous aggregation and particulate aggregation.

[0041] 2) Effects of different germination times on quinoa protein properties

[0042] The effects of different germination times on the solubility, surface hydrophobicity, and potential of quinoa protein are as Figure 2 shown. As the germination time extends, the solubility of quinoa protein increases, the surface hydrophobicity gradually decreases, and the absolute value of the potential is higher at 8 h compared to 0 h, indicating that the hydrophobic interaction between proteins in the solution decreases.

[0043] 3) Effects of different germination times on the texture of quinoa protein gels

[0044] The influence of different germination times on the texture properties of quinoa protein gel is as Figure 3 shown. It can be found that with the extension of germination time, the gel hardness shows a trend of first increasing and then decreasing. The gel hardness, adhesiveness, chewiness and resilience of the gel prepared from the protein extracted after 8 h of germination are all significantly improved, and it will not have an adverse effect on elasticity. The gel hardness of the gels prepared from the proteins at 16 h and 24 h decreases, but it is higher than that of the protein gel without germination.

[0045] The beneficial effects of the present invention compared with the prior art are as follows:

[0046] The present invention uses the method of germination treatment to change the aggregation mode of quinoa protein during heating. After germination, the surface hydrophobicity of quinoa protein decreases, while the electronegativity increases, reducing the hydrophobic interaction between proteins and increasing the electrostatic repulsion force; under the synergistic action of repulsive force (electrostatic repulsion force) and attractive force (hydrophobic interaction), the aggregation rate of proteins slows down, which is beneficial for better orientation and arrangement of protein molecules, forming ordered fiber aggregates, and cross-linking of fibers with fibers occurs at high protein concentrations, forming a three-dimensional gel network structure with a dense structure and uniform pore size distribution, thereby improving the gelation of quinoa protein gel. Compared with the prior art, the present invention does not need to introduce salt ions, and the cost of treatment with the endogenous enzymes of quinoa itself under neutral conditions is lower, and it has a better taste. Description of the Drawings

[0047] Figure 1 , Influence of different germination times on the aggregation of quinoa protein.

[0048] Figure 2 , Influence of different germination times on the properties of quinoa protein

[0049] Figure 3 , Influence of different germination times on the texture of quinoa protein gel.

[0050] Figure 4 , Appearance diagram of quinoa protein gel with different germination times. Detailed Embodiments

[0051] Example 1:

[0052] Step 1, Quinoa germination:

[0053] Soak quinoa in 1% (w / v) sodium hypochlorite solution for 10 min for sterilization and disinfection, and then wash it thoroughly with sterile water or deionized water;

[0054] Sterilized seeds were soaked in a 1% hydrogen peroxide solution in the dark at 25 °C for 24 h, then thoroughly rinsed in deionized water. After that, the seeds were spread on germination paper and cultured in a constant temperature and humidity incubator at 25 °C, and 0.3% hydrogen peroxide was sprayed every day.

[0055] Quinoa that had germinated for a certain period was dried and ground into powder.

[0056] Step 2: Extract quinoa protein:

[0057] The quinoa powder was defatted three times in n-hexane at a ratio of 1:3, and then left to volatilize naturally for 24 h to completely volatilize the residual n-hexane.

[0058] The defatted quinoa powder was suspended in pure water, the pH was adjusted to alkaline, and it was stirred for 2 h. After centrifugation, the supernatant was taken, the pH was adjusted to acidic, and then centrifuged again. The supernatant was discarded, and the precipitate obtained was quinoa protein.

[0059] The obtained quinoa protein was redissolved, the pH was adjusted to 7.0, and then freeze-dried to obtain quinoa protein powder for standby.

[0060] Step 3: Preparation of quinoa protein fiber aggregates:

[0061] The obtained quinoa protein powder was suspended in pure water at a concentration of 2% (w / v), stirred for 2 h, and then placed in a glass bottle and heated in a water bath at 95 °C for 30 min.

[0062] Step 4: Preparation of protein gel:

[0063] The quinoa protein was suspended in deionized water and stirred for 2 h, then stored in a refrigerator at 4 °C overnight for 12 h. After taking it out, it was magnetically stirred at room temperature for 30 min, placed in a specific container and heated at 95 °C for 30 min, and then stored in a refrigerator at 4 °C after cooling to room temperature for 12 h.

[0064] Example 2

[0065] On the basis of Example 1, in Step 3, the quinoa protein powder was suspended in pure water at a ratio of 5% (w / v), stirred for 2 h, and then placed in a glass bottle and heated in a water bath at 95 °C for 30 min.

[0066] Example 3

[0067] On the basis of Example 1, in Step 4, the heating temperature was changed from 95 °C to 90 °C.

[0068] Example 4

[0069] On the basis of Example 1, in Step 4, the heating temperature was changed from 95 °C to 98 °C.

Claims

1. A method for promoting the aggregation of quinoa protein fibers and improving its gel property by germination, which is characterized in that: The specific production process includes the following steps: Step 1, Quinoa germination: Soak quinoa in 1% (w / v) sodium hypochlorite solution for 10 min for sterilization and disinfection, then thoroughly wash with sterile water or deionized water. Soak the sterilized seeds in 1% (w / v) hydrogen peroxide solution in the dark at 25 °C for 24 h, then thoroughly rinse in deionized water. After that, spread the seeds on germination paper and culture them in a constant temperature and humidity incubator at 25 °C, spraying 0.3% (w / v) hydrogen peroxide solution every day; Dry the quinoa germinated for a certain time and grind it into powder; Step 2, Extract quinoa protein: Soak the quinoa powder in n-hexane for defatting, after suction filtration, soak the solid in n-hexane again, repeat three times. After the last suction filtration, place the solid in a fume hood to volatilize naturally for 24 h to completely volatilize the residual n-hexane. Suspend the defatted quinoa powder in pure water, adjust the pH to alkaline, stir for 2 h, take the supernatant after centrifugation, adjust the pH to acidic, centrifuge, and discard the supernatant to obtain the precipitate, which is quinoa protein; Redissolve the obtained quinoa protein, adjust the pH to 7.0, freeze-dry to obtain quinoa protein powder for standby; Step 3, Preparation of quinoa protein fiber aggregates: Suspend the obtained quinoa protein powder in pure water at a concentration of 2% (w / v), stir for 2 hours, then place it in a glass bottle, seal it with plastic wrap, and heat it in a water bath at 95 °C for 30 min; Step 4, Preparation of protein gel: Suspend quinoa protein in deionized water and stir for 2 h to prepare a suspension with a protein content of 16 - 22%, and store it in a 4 °C refrigerator overnight for 12 h. Take it out, stir magnetically at room temperature for 30 min, place it in a specific container and heat it at 95 °C for 30 min, and store it in a 4 °C refrigerator after cooling to room temperature for 12 h.

2. A method for promoting the aggregation of quinoa protein fibers and improving its gel property by germination according to claim 1, which is characterized in that: The mass-to-volume ratio of quinoa to sodium hypochlorite solution in step 1 is 1:2 - 3.

3. A method for promoting the aggregation of quinoa protein fibers and improving its gel property by germination according to claim 1, which is characterized in that: The mass-to-volume ratio of quinoa to hydrogen peroxide solution in step 1 is 1:2 - 3.

4. A method for promoting the aggregation of quinoa protein fibers and improving its gel property by germination according to claim 1, which is characterized in that: The mass-to-volume ratio of quinoa to n-hexane in step 2 is 1:3 - 5.

5. A method for promoting the aggregation of quinoa protein fibers and improving its gel property by germination according to claim 1, which is characterized in that: The specific value of adjusting the pH to alkaline in step 2 is 8.0 - 11.0, and the specific value of adjusting the pH to acidic is 4.0 - 5.0.

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