Soybean protein isolate modified by organic acid as well as preparation method and application thereof

Abstract

The invention discloses soybean protein isolate modified by organic acid as well as a preparation method and application of the soybean protein isolate, and belongs to the technical field of functional food. The preparation method comprises the following steps: (1) dissolving soybean protein isolate powder in a solvent to prepare a soybean protein isolate stock solution; (2) dissolving organic acid in a solvent to prepare an organic acid stock solution; (3) mixing the soybean protein isolate stock solution prepared in the step (1) and the organic acid stock solution prepared in the step (2) to obtain a soybean protein isolate-organic acid mixed solution; and carrying out pH adjustment, water bath heating and homogenization on the soybean protein isolate-organic acid mixed solution to obtain the organic acid modified soybean protein isolate. According to the invention, the edible organic acid is added into the soybean protein isolate solution to prepare the soybean protein isolate emulsion of which the emulsifying characteristic and the solubility are remarkably improved, so that the processing performance of the soybean protein isolate is improved, and a new direction is provided for the application of the organic acid in food processing.

Description

Technical Field

[0001] The present invention relates to the technical field of functional foods, and in particular to a soybean protein isolate modified by organic acid, and a preparation method and application thereof. Background Art

[0002] Soybean has a history of more than 3,000 years of cultivation in China. It is one of the top ten food crops and one of the four major oil crops in China. China's current annual soybean production has reached 20 million tons. The main components of soybeans are protein and oil, accounting for 40% (w / w) and 20% (w / w) of soybean dry matter, respectively. It has a full range of amino acids, covering the 10 essential amino acids required by the human body and rich in content, and is a high-quality protein. In industry and production and consumption, soy protein is widely used in a variety of food systems. Protein is a long-chain polymer formed by the dehydration condensation of amino acids. It contains both hydrophilic and lipophilic groups. In theory, it can be used as a good emulsifier for emulsified processed foods, but the stability of soy protein isolate emulsions is easily affected by environmental factors and its own properties. In addition, since the stability of protein emulsions is related to the storage characteristics and shelf life of actual products, maintaining or extending the stability of emulsions has always been a concern for researchers. Generally, freshly prepared emulsions will experience unstable phenomena such as flocculation, aggregation, creaming, sedimentation, and inversion. Therefore, its widespread use in the food industry is limited. Emulsification properties are important functional properties of food proteins. The emulsification properties and solubility of proteins are the decisive factors in forming the unique sensory and flavor of certain foods. In-depth research on the emulsification properties of soy protein isolate can promote the development of the soy protein industry and more effectively improve the utilization value of soybean resources. Summary of the invention

[0003] Purpose of the invention: In order to solve the technical problems existing in the prior art, the present invention aims to provide a soy protein isolate modified with organic acid having excellent processing function and nutritional function, and the present invention also provides a preparation method and application of the above-mentioned soy protein isolate modified with organic acid.

[0004] Technical solution: The method for preparing the soy protein isolate modified by organic acid of the present invention comprises the following steps:

[0005] (1) dissolving soy protein isolate powder in a solvent to prepare a soy protein isolate stock solution;

[0006] (2) dissolving an organic acid in a solvent to prepare an organic acid stock solution;

[0007] (3) mixing the soy protein isolate stock solution prepared in step (1) and the organic acid stock solution prepared in step (2) to obtain a soy protein isolate-organic acid mixed solution, and adjusting the pH of the soy protein isolate-organic acid mixed solution, heating in a water bath, and homogenizing to obtain an organic acid-modified soy protein isolate.

[0008] Furthermore, in step (1), the preparation method of the soy protein isolate powder is: crushing and sieving dried soybeans to obtain soy powder; mixing the soy powder with a n-hexane-ethanol mixed solvent in proportion, and defatting to obtain defatted soy powder; extracting soy protein isolate from the defatted soy powder by an alkali dissolution and acid precipitation method, and freeze-drying to obtain the soy protein isolate powder.

[0009] Furthermore, in step (1), the mass concentration of the soy protein isolate in the soy protein isolate stock solution is 20-40 g / L.

[0010] Furthermore, in step (2), the mass concentration of the organic acid in the organic acid stock solution is 10-20 g / L; the organic acid is a food organic acid, and the solvent is water.

[0011] Furthermore, the food organic acid is one or more of citric acid, acetic acid, succinic acid, malic acid or tartaric acid.

[0012] Preferably, the organic acid is succinic acid, malic acid or tartaric acid.

[0013] Furthermore, in step (3), in the soy protein isolate-organic acid mixed solution, the ratio of soy protein isolate to organic acid is 10g / L:0.1-10.0g / L, preferably 10g / L:5.0g / L; the pH of the mixed solution is adjusted to 7.0±0.1 and then heated in a water bath at 50-55°C for 6-8h, cooled after heating, and soybean oil is added after cooling, and homogenized at 13000-15000rpm for 2-4min using a high shear homogenizer.

[0014] The present invention also provides soybean protein isolate modified by organic acid obtained by the preparation method.

[0015] The above-mentioned application of soy protein isolate modified by organic acid in functional food processing.

[0016] Principle of the invention: Organic acids are mainly extracted and separated from plants or agricultural and sideline products in nature to obtain organic compounds with certain physiological activities. Organic acids have simple structures, small molecular weights, excellent structural flexibility, and contain active groups such as -OH and -COOH on the molecular chain, which are easy to be chemically modified. The modified organic acid derivatives can form emulsions through various crosslinking forms such as chemical crosslinking, photocrosslinking, and enzyme crosslinking, and can give emulsions specific properties according to specific application requirements, such as enhancing emulsification activity, emulsification stability, and improving solubility. In addition, organic acids are non-toxic, tasteless, easily degradable, pollution-free to the environment, and inexpensive, and are widely used in food processing and pharmaceutical fields. Because organic acids contain polyhydroxyl groups, they have strong hydrophilicity, good solubility in water, and certain moisture retention, which is conducive to improving emulsification properties. Organic acids can combine with soy protein isolate through hydrophobic interactions, electrostatic interactions, and hydrogen bonds, thereby improving the emulsification properties and microstructure of soy protein isolate emulsions, and with the increase of organic acid concentration, the interaction between soy protein isolate and organic acids is enhanced, and the functional properties of protein emulsions are also improved. Succinic acid, malic acid and tartaric acid are all dicarboxylic acids with similar structures. The difference between them lies in the number of hydroxyl groups they contain.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention confirms that succinic acid, malic acid and tartaric acid can significantly improve the functionality of soy protein isolate emulsion, enhance the emulsification activity, emulsification stability and protein solubility of the emulsion, and can be used as a functional food for development; (2) The present invention has found the optimal addition amount of succinic acid, malic acid and tartaric acid, and their concentrations are all 5.0 g / L. By selecting organic acids of different types and concentrations, a variety of emulsions can be prepared, which is beneficial to improving the application of soy protein isolate in food; (3) The preparation method of the present invention can further understand the organic acid modification of soy protein isolate emulsion, and provide a new idea for the application of soy protein isolate, organic acid and modified protein emulsion in the food industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The solubility and turbidity graphs of proteins obtained in Examples 1 to 15 of the present invention and Comparative Example 1;

[0019] Figure 2 The ultraviolet absorption spectra obtained in Examples 1 to 15 of the present invention and Comparative Example 1 are shown;

[0020] Figure 3 The infrared absorption spectra obtained from Examples 1 to 15 of the present invention and Comparative Example 1 are shown;

[0021] Figure 4 The sodium dodecyl sulfate-polyacrylamide gel electrophoresis diagrams obtained in Examples 1 to 15 of the present invention and Comparative Example 1;

[0022] Figure 5 The emulsification activity and emulsification stability diagram of the emulsions prepared in Examples 1 to 15 of the present invention and Comparative Example 1;

[0023] Figure 6 The microstructure diagram of the emulsions prepared in Examples 1 to 15 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with specific embodiments and drawings.

[0025] Embodiment 1: The method for preparing the organic acid modified soybean protein isolate emulsion of the present invention comprises the following steps:

[0026] (1) preparing a soy protein isolate stock solution: weighing soy protein isolate powder and dissolving it in ultrapure water to prepare a soy protein isolate solution, stirring it magnetically for 3 h and then storing it at 4° C. for 12 h to completely hydrate it to a concentration of 20 g / L;

[0027] Among them, the preparation method of soy protein isolate powder is as follows: select dry soybeans, grind the soybeans through a mill, and pass through an 80-mesh sieve. Then sieve to obtain soybean fine powder, and then use a mixed solvent of n-hexane and ethanol to defatted (soybean powder: 100% ethanol: n-hexane = 5:2:18, w / v / v), stir at low speed in a fume hood for 1h and then filter, and repeat defatting 2-3 times depending on the color of the filtrate. Then the obtained defatted powder is dried overnight in a fume hood to fully evaporate the residual organic solvent. The dried defatted powder is fully mixed with deionized water at a solid-liquid ratio of 1:10 (w / v), and the solution is adjusted to pH = 8.0 with 2mol / LNaOH, stirred at room temperature for 1h, and then centrifuged at 13500g for 30min at 4°C to obtain a supernatant. The pH of the supernatant was adjusted to 4.5 with 2 mol / L HCl, and then centrifuged at 3300 g for 30 min at 4°C to obtain a precipitate. The precipitate was dispersed in water and the pH was adjusted to 7.0 with 2 mol / L NaOH. After freeze-drying, it was stored at 4°C to obtain soy protein isolate powder.

[0028] (2) Prepare succinic acid stock solution: weigh succinic acid and dissolve it in water, stir magnetically for 2-3 hours, and then store it at 4°C for 12 hours, with a concentration of 20 g / L;

[0029] (3) The succinic acid stock solution was diluted to 0.2 g / L and then mixed with the soy protein isolate stock solution at a volume ratio of 1:1. After mixing, the concentration of soy protein isolate was 10 g / L and the concentration of succinic acid was 0.1 g / L. The pH was adjusted to 7.0. After magnetic stirring for 0.5 h, the mixed solution was heated in a water bath at 50°C for 6 h. After cooling to room temperature, soybean oil was added and homogenized at 13000 rpm for 2 min, 40 s each time for a total of 3 times. After completion, the resulting emulsion was stored at 4-6°C for use.

[0030] Example 2: The difference from Example 1 is that in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of succinic acid is 0.5 g / L.

[0031] Example 3: The difference from Example 1 is that in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of succinic acid is 1.0 g / L.

[0032] Example 4: The difference from Example 1 is that in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of succinic acid is 5.0 g / L.

[0033] Example 5: The difference from Example 1 is that in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of succinic acid is 10.0 g / L.

[0034] Example 6: The difference from Example 1 is that in step (2), a malic acid stock solution is prepared, and in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of malic acid is 0.1 g / L.

[0035] Example 7: The difference from Example 6 is that in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of malic acid is 0.5 g / L.

[0036] Example 8: The difference from Example 6 is that in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of malic acid is 1.0 g / L.

[0037] Example 9: The difference from Example 6 is that in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of malic acid is 5.0 g / L.

[0038] Example 10: The difference from Example 6 is that in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of malic acid is 10.0 g / L.

[0039] Example 11: The difference from Example 6 is that in step (2), a tartaric acid stock solution is prepared, and in step (3), the concentration of soy protein isolate in the mixed solution is 10 g / L, and the concentration of tartaric acid is 0.1 g / L.

[0040] Example 12: The difference from Example 11 is that in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of tartaric acid is 0.5 g / L.

[0041] Example 13: The difference from Example 11 is that in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of tartaric acid is 1.0 g / L.

[0042] Example 14: The difference from Example 11 is that in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of tartaric acid is 5.0 g / L.

[0043] Example 15: The difference from Example 11 is that in step (3), in the mixed solution, the concentration of soy protein isolate is 10 g / L, and the concentration of tartaric acid is 10.0 g / L.

[0044] Comparative Example 1: The difference from Example 1 is that in step (3), no organic acid stock solution is added to the soy protein isolate solution, and an equal volume of water is added, and the concentration of the soy protein isolate is 10 g / L.

[0045] Example 16: The mixed solutions prepared in Examples 1 to 15 and Comparative Example 1 were subjected to performance testing, including:

[0046] (1) Determination of protein solubility and turbidity

[0047] The prepared mixed protein solution was centrifuged at 20°C and 5000g for 10 min, and the supernatant was taken to determine the protein content using the biuret method. The protein solubility was:

[0048]

[0049] The prepared mixed protein solution was diluted to 1.0 g / L with 50 mM citric acid-phosphate buffer (pH 7.0), and the absorbance of the diluted solution at 600 nm was measured using a UV spectrophotometer as its turbidity.

[0050] (2) Determination of UV absorption spectrum

[0051] The protein concentration of the mixed solution was diluted to 1.0 g / L with 50 mM citric acid-phosphate buffer (pH 7.0), and the UV absorption spectrum of the diluted sample in the range of 200-400 nm was scanned with a UV-2600i spectrophotometer.

[0052] (3) Determination of infrared absorption spectrum

[0053] The prepared mixed protein solution was freeze-dried. An appropriate amount of potassium bromide and a small amount of freeze-dried sample were crushed in an agate mortar, pressed into a transparent tablet, and loaded into a mold. The infrared absorption spectrum of the sample was scanned using an FT-IR spectrometer with a scanning wave number range of 4000-400 cm -1 , resolution 2cm -1 . Scan the background before measuring the sample spectrum.

[0054] (4) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0055] The protein concentration of the mixed solution was diluted to 2.0 g / L with 50 mM citric acid-phosphate buffer (pH 7.0). Before electrophoresis, the diluted mixed solution was mixed with equal volumes of sample buffer (4% SDS, 20% glycerol, 0.125 MTris, pH 6.8), with or without the addition of 5% β-mercaptoethanol (βME) to obtain a final protein concentration of 1.0 g / L, and boiled at 100°C for 3 min. For samples without βME, 1 mM N-ethylmaleimide (NEM) was added to prevent the formation of disulfide bonds during the heating process. Subsequently, 15 μL of each boiled sample was loaded into a 5% polyacrylamide concentrating gel and separated with a 12.5% ​​separation gel. The gel was concentrated and separated with a constant current of 50 mA and a constant current of 90 mA, respectively. The gel was stained with Coomassie Brilliant Blue R250 solution for 3 h, and then destained with 7.5% (v / v) glacial acetic acid and 5% (v / v) methanol until the background was clear.

[0056] (5) Determination of emulsifying activity (EAI) and emulsifying stability (ESI)

[0057] Preparation of emulsion: Mix the prepared soy protein isolate mixed solution with soybean oil in a ratio of 3:1, and use a high shear homogenizer to homogenize and emulsify at a speed of 13000rpm for 2min (40s once, three times in total, with an interval of 10s each time). The emulsified solution is quickly transferred to a 25mL beaker.

[0058] For the determination of emulsifying activity, take 25 μL from the bottom of the cup, dilute it in 5 mL of 0.1% SDS, and immediately detect its absorbance at 500 nm. After the emulsion is allowed to stand in the beaker for 30 minutes, dilute it in the same way as above, and measure the absorbance at 500 nm. The calculation formulas for emulsifying activity (EAI) and emulsifying stability (ESI) are as follows:

[0059]

[0060] Where C is the protein concentration before emulsification (g / mL), is the volume fraction of oil in the emulsion (v / v), with a dilution factor of 201.

[0061]

[0062] Among them A t and A 0 Represent the absorbance values ​​at 30min and 0min respectively.

[0063] (6) Microstructure of emulsion

[0064] Analyze the microstructure of freshly prepared emulsions using an optical microscope. Place a small drop of the freshly prepared emulsion on a clean glass slide and cover it with a coverslip. Immediately after preparation, use a 40x objective to observe the surface of the emulsion droplet.

[0065] The above test results are as follows Figure 1-Figure 6 shown.

[0066] like Figure 1 As shown in the figure, the solubility of the soy protein isolate solution with organic acid added is significantly improved compared with the control without organic acid. The amount of organic acid added has a great influence on the solubility of protein. With the increase of succinic acid, malic acid and tartaric acid concentration, the solubility of soy protein isolate gradually decreases. This result may be due to the interaction between organic acid and protein, which leads to the change of soy protein isolate structure and the formation of complex aggregates, and the cross-linked products with larger molecular weight lead to the gradual decrease of solubility. When the concentration of succinic acid, malic acid and tartaric acid is 10.0g / L, the solubility of protein is the lowest, and at this time, some insoluble polymers are produced.

[0067] In general, turbidity showed an opposite trend to solubility. Turbidity increased with increasing organic acid concentration, which may be due to the formation of insoluble protein aggregates.

[0068] like Figure 2As shown, amino acids with benzene ring structures have characteristic absorption peaks at 260-280nm. Compared with the control group, the addition of three organic acids can significantly increase the absorbance of soy protein isolate in the range of 260-280nm. With the increase of organic acid concentration, the maximum ultraviolet absorption intensity of soy protein isolate solution increases continuously, indicating that the chromophore increases and more hydrophobic groups are exposed, thereby increasing the ultraviolet absorption intensity. However, the effect is more obvious when the organic acid concentration is 5.0g / L. This may be because after the soy protein isolate is hydrolyzed by organic acid, the protein structure unfolds, causing the tryptophan and tyrosine hidden inside the molecule to be exposed on the surface of the protein molecule.

[0069] like Figure 3 As shown in the figure, FT-IR spectroscopy can be used to characterize the interaction between organic acids and soy protein isolate and the changes in the secondary structure of proteins. Compared with untreated soy protein isolate, the peak near the amide A band shifts to a higher wave number with the increase of organic acid, which is related to the formation of hydrogen bonds. This may be due to the hydrogen bond interaction between the carboxyl group of organic acid and the amino acid of protein, which affects the structure and vibration frequency of protein, resulting in the destruction or change of the original hydrogen bond structure of protein. In general, the amide I band and amide II band are more sensitive to changes in the secondary structure of proteins. The absorption peak of the amide I band of soy protein isolate treated with organic acid shifted. The reason for this phenomenon may be that the carbonyl group in the protein was modified by organic acid and changed, which promoted the covalent cross-linking between soy protein isolate and organic acid. In addition, under high concentration malic acid and tartaric acid treatment, the amide I or II absorption peak of soy protein isolate disappeared, which may be due to the complexation between soy protein isolate and organic acid hydroxyl group, resulting in the rearrangement of protein peptide chain.

[0070] like Figure 4 As shown in Figure 2, the SDS-PAGE diagram can reflect the aggregation degree of proteins. Under non-reducing conditions, compared with the control group, more macromolecular aggregates were gradually observed with the addition of organic acids. At the same time, with the increase of succinic acid, malic acid and tartaric acid concentrations, the content of aggregates decreased significantly. When the concentrations of malic acid and tartaric acid were 0.1g / L and 0.5g / L, thicker bands were formed, and large molecular weight aggregates were more abundant, but slightly less at high concentrations. This may be that organic acids hindered the aggregation of soybean globulin through electrostatic repulsion. Under reducing conditions, after adding β-mercaptoethanol, all disulfide bonds were cleaved and aggregates disappeared. Therefore, it is inferred that macromolecular protein aggregates are the result of cross-linking of soybean protein isolate through disulfide bonds.

[0071] like Figure 5As shown in the results, the addition of organic acid significantly improved the emulsification activity and emulsification stability of soy protein isolate. As the concentration of succinic acid and malic acid increased from 0 g / L to 5.0 g / L, the emulsification activity increased significantly, and the maximum emulsification activity was shown at 5.0 g / L. The improvement of the emulsification activity of soy protein isolate can be attributed to the increase in protein solubility, because insoluble protein cannot be adsorbed on the oil / water interface. When the concentration of organic acid increased to 10.0 g / L, the emulsification activity decreased. This is because with the increase in the degree of cross-linking, larger molecular proteins were produced, which led to a decrease in their solubility and an adverse effect on their emulsification activity. Organic acid significantly improved the emulsification stability at a low concentration of 0.1-1.0 g / L. This may be because after the organic acid cross-linked with soy protein isolate, the cationic amino groups on the protein were converted into anionic residues, and the net negative charge increased, which was conducive to the formation of a charged layer of protein around the oil, resulting in mutual repulsion of proteins, reducing interfacial energy and delaying aggregation. This process improved the stability of the emulsion. When the organic acid reaches a high concentration, the emulsion stability decreases due to the formation of insoluble aggregates at high concentrations.

[0072] like Figure 6 As shown, the emulsion system is composed of oil droplets of varying sizes and continuous phase proteins, and the proteins are dispersed around the oil droplets. In the blank control group, oil droplets with larger diameters can be clearly observed, and the oil droplet sizes are significantly different, indicating that the oil droplets may have aggregated. When the organic acid concentration reaches 1.0g / L and 5.0g / L, the large particle oil droplets in the emulsion system disappear, and the oil droplets are smaller and more evenly distributed, indicating that there is a strong electrostatic repulsion between proteins, which further hinders the aggregation of oil droplets and maintains the stability of the emulsion. When the organic acid is at a high concentration, the volume of the oil droplets in the emulsion system becomes larger, which may be due to the phenomenon of oil droplet aggregation, which is consistent with Figure 5 The emulsifying activity results were consistent with those in the

Claims

1. A method for preparing soybean protein isolate modified by organic acid, characterized in that: The following steps are involved: (1) dissolving soy protein isolate powder in a solvent to prepare a soy protein isolate stock solution; (2) dissolving an organic acid in a solvent to prepare an organic acid stock solution; (3) mixing the soy protein isolate stock solution prepared in step (1) and the organic acid stock solution prepared in step (2) to obtain a soy protein isolate-organic acid mixed solution, and adjusting the pH of the soy protein isolate-organic acid mixed solution, heating in a water bath, and homogenizing to obtain an organic acid-modified soy protein isolate.

2. The preparation method according to claim 1, characterized in that: In step (1), the preparation method of the soy protein isolate powder is as follows: crushing and sieving dried soybeans to obtain soy powder; mixing the soy powder with a n-hexane-ethanol mixed solvent in proportion, and performing a defatting treatment to obtain defatted soy powder; extracting soy protein isolate from the defatted soy powder by an alkali dissolution and acid precipitation method, and freeze-drying to obtain the soy protein isolate powder.

3. The preparation method according to claim 1, characterized in that: In step (1), the mass concentration of the soy protein isolate in the soy protein isolate stock solution is 20-40 g / L.

4. The preparation method according to claim 1, characterized in that: In step (2), the mass concentration of the organic acid in the organic acid stock solution is 10-20 g / L.

5. The preparation method according to claim 1, characterized in that: In step (2), the organic acid is a food organic acid.

6. The preparation method according to claim 5, characterized in that: The food organic acid is one or more of citric acid, acetic acid, succinic acid, malic acid or tartaric acid.

7. The preparation method according to claim 1, characterized in that: In step (3), in the soy protein isolate-organic acid mixed solution, the ratio of soy protein isolate to organic acid is 10 g / L: 0.1-10.0 g / L.

8. The preparation method according to claim 7, characterized in that: In step (3), the pH of the mixed solution is adjusted to 7.0±0.1 and then heated in a water bath at 50-55°C for 6-8h. After the heating is completed, the mixed solution is cooled. After cooling, soybean oil is added and homogenized at 13000-15000 rpm for 2-4min using a high shear homogenizer.

9. Soy protein isolate modified with organic acid obtained according to the preparation method according to any one of claims 1 to 8.

10. Use of the soy protein isolate modified with organic acid according to claim 9 in functional processing of food.

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