Method for improving emulsibility and thermal stability of ginkgo nut protein isolate through Maillard reaction

Abstract

The invention discloses a method for improving emulsibility and thermal stability of ginkgo nut protein isolate through Maillard reaction, and belongs to the field of protein modification. The method comprises the following steps: (1) dissolving ginkgo nut protein isolate powder in a solvent, and fully hydrating to obtain a ginkgo nut protein isolate solution; (2) dissolving polysaccharide in a solvent, and fully hydrating to obtain a polysaccharide solution; (3) mixing the polysaccharide solution and the ginkgo nut protein isolate solution to obtain a protein-polysaccharide mixed solution; (4) adjusting the pH value of the protein-polysaccharide mixed solution, and stirring to obtain a composite solution; and (5) carrying out Maillard reaction on the composite solution, mixing the obtained solution with an oil phase, and homogenizing to form the emulsion containing the ginkgo nut protein isolate-polysaccharide copolymer. The emulsion prepared on the basis of the Maillard reaction has good emulsibility and thermal stability, a practical method is provided for improving the function of the ginkgo nut protein isolate, and meanwhile the application range of the ginkgo nut protein isolate in the food field is widened.

Description

Technical Field

[0001] The invention relates to the technical field of protein modification, and in particular to a method for improving the emulsification and thermal stability of ginkgo fruit protein isolate by using a Maillard reaction. Background Art

[0002] Ginkgo biloba protein isolate is a natural plant food ingredient with rich functional properties and physiological activities. Ginkgo biloba is widely planted, and China's annual output of ginkgo biloba can reach 60,000 tons. It is a plant protein with great development potential. However, the functional properties of ginkgo biloba protein isolate, such as solubility and emulsification, are lower than those of other plant proteins, which limits its application in the food industry.

[0003] In addition, existing protein modifications cannot simultaneously improve the emulsifying activity and emulsifying thermal stability of proteins. Summary of the invention

[0004] Purpose of the invention: The present invention aims to provide a method for improving the emulsification and thermal stability of ginkgo biloba protein isolate through Maillard reaction.

[0005] Technical solution: The method for improving the emulsification and thermal stability of ginkgo biloba protein isolate by Maillard reaction of the present invention comprises the following steps:

[0006] (1) dissolving the ginkgo biloba protein isolate powder in a solvent and obtaining a ginkgo biloba protein isolate solution after sufficient hydration;

[0007] (2) dissolving the polysaccharide in a solvent and obtaining a polysaccharide solution after sufficient hydration;

[0008] (3) mixing the polysaccharide solution and the ginkgo fruit protein isolate solution to obtain a protein-polysaccharide mixed solution;

[0009] (4) adjusting the pH of the protein-polysaccharide mixed solution and stirring to obtain a composite solution;

[0010] (5) subjecting the composite solution to a Maillard reaction, and mixing the resulting solution with an oil phase, and homogenizing to form an emulsion containing a Ginkgo biloba protein isolate-polysaccharide copolymer.

[0011] Furthermore, in step (1), the mass concentration of the ginkgo biloba protein isolate solution is 15-20 g / L, and the solvent is water.

[0012] Furthermore, in step (2), the mass concentration of the polysaccharide solution is 50-60 g / L; the polysaccharide is pullulan, which can simultaneously improve the emulsifying activity and emulsifying stability of ginkgo nut protein isolate, while other sugars cannot achieve the simultaneous improvement of the emulsifying activity and emulsifying stability of the protein.

[0013] Furthermore, in step (3), in the protein-polysaccharide mixed solution, the mass ratio of polysaccharide to ginkgo fruit protein isolate is 1:4-4:1.

[0014] Furthermore, in step (4), the pH is 7±0.1.

[0015] Furthermore, in step (5), the conditions of the Maillard reaction are: the reaction temperature is 70±5°C, the reaction time is less than 30h, preferably 12-24h; the oil phase is soybean oil; the volume fraction of the oil phase in the polysaccharide-modified ginkgo fruit protein isolate emulsion is 25-30%; the homogenization speed is 13500-15000rpm, and the homogenization treatment time is 120-150s.

[0016] Principle of the invention: Due to its good emulsifying ability, protein forms a viscoelastic adsorption layer on the surface of oil droplets, effectively preventing the aggregation of droplets, thereby improving the emulsifying properties of the emulsion. Polysaccharides further improve the emulsifying properties by adjusting the rheological properties of the system. Therefore, combining polysaccharides and proteins is a feasible way to enhance the emulsifying properties of emulsions.

[0017] Pullulan is a linear polysaccharide formed by polymerizing maltotriose repeating units connected by α-1,4 glycosidic bonds through α-1,6 glycosidic bonds. It is an edible polymer with potential applications in the food industry. In the present invention, hydrophilic pullulan undergoes a Maillard reaction with protein. On the one hand, the Maillard reaction can increase the concentration and fluidity of protein in aqueous solution, while enhancing the emulsification ability of the interface around the oil droplets; on the other hand, the pullulan grafted onto the protein can generate a certain spatial repulsion between the oil droplets, promote the adsorption of protein on the surface of the oil droplets and form a dense and porous structure, thereby inhibiting the aggregation and flocculation between the droplets, thereby improving the emulsification performance of the emulsion.

[0018] The invention discloses a method for preparing a pullulan and ginkgo fruit protein isolate emulsion by using a Maillard reaction, wherein a hydrated solution of ginkgo fruit protein isolate and a hydrated solution of pullulan are mixed in proportion, and a Maillard reaction is performed, and homogenization and emulsification are performed to prepare a copolymer emulsion. Compared with a single ginkgo fruit protein isolate or pullulan emulsion, the pullulan and ginkgo fruit protein isolate copolymer emulsion has significantly improved emulsification activity and emulsification stability, and the thermal stability of the emulsion is also significantly improved.

[0019] The present invention is a promising method for improving the functional properties of proteins by coupling proteins with polysaccharides through the Maillard reaction. The reaction is formed by covalent bonding of the free amino groups of proteins with the carbonyl groups of reducing sugars. It does not require the addition of any chemical reagents and has the characteristics of being mild, green and efficient. In addition, the modification of ginkgo fruit protein isolate is conducive to reducing resource waste and reducing dependence on animal protein, thus forming sustainable development of agriculture and food.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention uses pullulan and ginkgo biloba protein isolate from natural sources to prepare an emulsion by compounding through a natural green Maillard reaction. The emulsion has good rheological properties, emulsification activity and emulsification stability. Therefore, it can be used as a functional matrix to promote the application of plant protein in the food industry; (2) The present invention adopts a green and safe method to regulate the Maillard reaction process. By accurately controlling the concentration of reactants and the reaction time of the reaction system, pullulan and ginkgo biloba protein isolate can fully contact and cross-link during the Maillard reaction to form a structurally stable and uniform composite emulsion. This regulation method not only ensures the high efficiency of the reaction process, but also improves the biological activity and functionality of the composite product, providing strong technical support for the development of functional foods; (3) The method of the present invention can further understand the ginkgo biloba protein isolate composite emulsion, and provide a new idea for the application of ginkgo biloba protein isolate, pullulan and modified protein emulsion in the food industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The SDS graphs of the emulsions prepared at different ratios of pullulan solution (PULL) to ginkgo biloba protein isolate (GSPI) solution and different Maillard reaction times in Examples 1 to 6 of the present invention and Comparative Examples 1 to 6;

[0022] Figure 2 The particle size potential diagrams of the emulsions prepared at different ratios of pullulan solution to ginkgo biloba protein isolate solution and different Maillard reaction times in Examples 1 to 6 of the present invention and Comparative Examples 1 to 6, the left figure shows different ratios of pullulan solution to ginkgo biloba protein isolate solution after Maillard reaction for 24 hours, and the right figure shows different reaction times at a PULL to protein mass ratio of 4:1;

[0023] Figure 3 The emulsification activity and emulsification stability of the emulsions prepared at different ratios of pullulan solution to ginkgo fruit protein isolate solution and after Maillard reaction for 24 hours in Example 2, Example 4, Example 6 and Comparative Example 6 of the present invention are shown;

[0024] Figure 4 The storage modulus, loss modulus and apparent viscosity graphs of the emulsions prepared at different ratios of pullulan solution to ginkgo fruit protein isolate solution and different Maillard reaction times in Examples 1 to 6 of the present invention and Comparative Examples 1 to 6;

[0025] Figure 5The microstructure diagrams of the emulsions prepared at different ratios of pullulan solution to ginkgo fruit protein isolate solution and different Maillard reaction times in Examples 1 to 6 of the present invention and Comparative Examples 1 to 6;

[0026] Figure 6 The stability diagrams of different ratios of pullulan solution to ginkgo biloba protein isolate solution in Example 2, Example 4, Example 6 and Comparative Example 6 of the present invention under pasteurization at different temperatures after Maillard reaction for 24 hours. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific embodiments and drawings.

[0028] Example 1: The method provided in this example for improving the emulsification and thermal stability of ginkgo biloba protein isolate by Maillard reaction comprises the following steps:

[0029] (1) Dissolve 2 g of Ginkgo biloba protein isolate powder in 100 mL of deionized water, stir magnetically for 2 hours, and place in a 4° C. refrigerator for 12 hours to ensure that the Ginkgo biloba protein isolate solution is fully hydrated to obtain a Ginkgo biloba protein isolate solution with a mass concentration of 20 g / L;

[0030] (2) Dissolve 5 g of pullulan in 100 mL of deionized water, stir magnetically for 2 hours, and place in a 4°C refrigerator for 12 hours to ensure that the pullulan is fully hydrated to obtain a pullulan solution with a mass concentration of 50 g / L;

[0031] (3) mixing a pullulan solution having a mass concentration of 50 g / L and a ginkgo biloba protein isolate solution having a mass concentration of 20 g / L so that the mass ratio of pullulan to ginkgo biloba protein isolate in the protein-polysaccharide mixed solution is 0.25:1, and magnetically stirring for 2 hours to obtain a protein-polysaccharide mixed solution;

[0032] (4) adjusting the pH of the protein-polysaccharide mixed solution to 7.0, mixing and stirring to obtain a composite solution;

[0033] (5) The composite solution was subjected to Maillard reaction at 70° C. for 12 hours, and then immediately cooled. The resulting solution was mixed with soybean oil having a volume fraction of 25%. An emulsion containing a copolymer of ginkgo biloba protein isolate and pullulan was formed at a homogenization speed of 13,500 rpm and a homogenization time of 120 s.

[0034] Example 2: The difference from Example 1 is that in step (5), the constant temperature reaction time is 24 hours.

[0035] Comparative Example 1: The difference from Example 1 is that in step (5), no Maillard reaction is performed (ie, the reaction is performed for 0 hours).

[0036] Example 3: The method provided in this example for improving the emulsification and thermal stability of ginkgo biloba protein isolate by Maillard reaction comprises the following steps:

[0037] (1) Dissolve 2 g of Ginkgo biloba protein isolate powder in 100 mL of deionized water, stir magnetically for 2 hours, and place in a 4° C. refrigerator for 12 hours to ensure that the Ginkgo biloba protein isolate solution is fully hydrated to obtain a Ginkgo biloba protein isolate solution with a mass concentration of 20 g / L;

[0038] (2) Dissolve 5 g of pullulan in 100 mL of deionized water, stir magnetically for 2 hours, and place in a 4°C refrigerator for 12 hours to ensure that the pullulan is fully hydrated to obtain a pullulan solution with a mass concentration of 50 g / L;

[0039] (3) mixing a pullulan solution having a mass concentration of 50 g / L and a ginkgo biloba protein isolate solution having a mass concentration of 20 g / L so that the mass ratio of pullulan to ginkgo biloba protein isolate in the protein-polysaccharide mixed solution is 1:1, and magnetically stirring for 2 hours to obtain a protein-polysaccharide mixed solution;

[0040] (4) adjusting the pH of the protein-polysaccharide mixed solution to 7.0, mixing and stirring to obtain a composite solution;

[0041] (5) The composite solution was subjected to Maillard reaction at 70° C. for 12 hours, and then immediately cooled. The resulting solution was mixed with soybean oil having a volume fraction of 25%. An emulsion containing a copolymer of ginkgo biloba protein isolate and pullulan was formed at a homogenization speed of 13,500 rpm and a homogenization time of 120 s.

[0042] Example 4: The difference from Example 3 is that in step (5), the constant temperature reaction time is 24 hours.

[0043] Comparative Example 2: The difference from Example 3 is that in step (5), no Maillard reaction is performed (ie, the reaction is performed for 0 hours).

[0044] Example 5: The method provided in this example for improving the emulsification and thermal stability of ginkgo biloba protein isolate by Maillard reaction comprises the following steps:

[0045] (1) Dissolve 2 g of Ginkgo biloba protein isolate powder in 100 mL of deionized water, stir magnetically for 2 hours, and place in a 4° C. refrigerator for 12 hours to ensure that the Ginkgo biloba protein isolate solution is fully hydrated to obtain a Ginkgo biloba protein isolate solution with a mass concentration of 20 g / L;

[0046] (2) Dissolve 5 g of pullulan in 100 mL of deionized water, stir magnetically for 2 hours, and place in a 4°C refrigerator for 12 hours to ensure that the pullulan is fully hydrated to obtain a pullulan solution with a mass concentration of 50 g / L;

[0047] (3) mixing a pullulan solution having a mass concentration of 50 g / L and a ginkgo biloba protein isolate solution having a mass concentration of 20 g / L so that the mass ratio of pullulan to ginkgo biloba protein isolate in the protein-polysaccharide mixed solution is 4:1, and stirring the mixture under magnetic stirring for 2 hours to obtain a protein-polysaccharide mixed solution;

[0048] (4) adjusting the pH of the protein-polysaccharide mixed solution to 7.0, mixing and stirring to obtain a composite solution;

[0049] (5) The composite solution was subjected to Maillard reaction at 70° C. for 12 hours, and then immediately cooled. The resulting solution was mixed with soybean oil having a volume fraction of 25%. An emulsion containing a copolymer of ginkgo biloba protein isolate and pullulan was formed at a homogenization speed of 13,500 rpm and a homogenization time of 120 s.

[0050] Example 6: The difference from Example 5 is that in step (5), the constant temperature reaction time is 24 hours.

[0051] Comparative Example 3: The difference from Example 5 is that in step (5), no Maillard reaction is performed (ie, the reaction is performed for 0 hours).

[0052] Comparative Example 4: The preparation method of the ginkgo biloba protein isolate emulsion provided in this comparative example comprises the following steps:

[0053] (1) Dissolve 2 g of Ginkgo biloba protein isolate powder in 100 mL of deionized water, stir magnetically for 2 hours, and place in a 4° C. refrigerator for 12 hours to ensure that the Ginkgo biloba protein isolate solution is fully hydrated to obtain a Ginkgo biloba protein isolate solution with a mass concentration of 20 g / L;

[0054] (2) adjusting the pH of the ginkgo fruit protein isolate solution to 7.0, and mixing and stirring to obtain a solution;

[0055] (3) After the solution was kept at 70° C. without reacting (i.e., reacting for 0 hours), it was immediately cooled to obtain a Ginkgo biloba protein isolate composite solution.

[0056] Comparative Example 5: The difference from Comparative Example 4 is that in step (3), the constant temperature reaction time is 12 hours.

[0057] Comparative Example 6: The difference from Comparative Example 4 is that in step (3), the constant temperature reaction time is 24 hours.

[0058] The properties of the emulsions prepared in Examples 1 to 6 and Comparative Examples 1 to 6 were measured, including:

[0059] (1) Polyacrylamide gel electrophoresis (SDS-PAGE): The complex solution was diluted with citric acid-phosphate buffer to a mass concentration of 2 g / L. The diluted solution was mixed with equal volumes of a non-reducing buffer (containing: 40 g / L SDS, 252.26 g / L glycerol, 15.14 g / L Tris-HCl (pH = 6.8), 0.05 g / L bromophenol blue, 0.125 g / L NEM) and a reducing buffer (containing: 40 g / L SDS, 252.26 g / L glycerol, 15.14 g / L Tris-HCl (pH = 6.8), 0.05 g / L bromophenol blue, 55.5 g / L βME), and boiled at 100°C for 3 min. The sample volume per well was 15 μL. The 50 g / L concentrating gel and the 125 g / L separating gel were electrophoresed. Then, the sample was stained with a staining solution (Coomassie Brilliant Blue R250) for 3 hours, and destained with a destaining solution (5.0% methanol by volume and 7.5% glacial acetic acid by volume) until the bands were clear and there was no background color.

[0060] (2) Particle size and ζ-potential determination: The composite solution was diluted to 2 g / L with citric acid-phosphate buffer (50 mM, pH 5.0, 6.0, 7.0). The particle size and ζ-potential were measured using a nanoparticle size potential analyzer.

[0061] (3) Take about 1 mg of the freeze-dried sample and mix it with potassium bromide in a ratio of 1:99, grind it and press it into tablets. Scan and analyze in the wave number range of 4000-400 cm-1, set the resolution to 0.2 cm-1, and accumulate the scanning signal 64 times.

[0062] (4) Determination of emulsification activity and emulsification stability: 15 mL of sample solution with different Maillard reaction degrees was mixed with 5 mL of soybean oil and emulsified using a homogenizer at a speed of 13500 r / min. The entire emulsification process lasted for 2 min. The specific operation was: each emulsification was 40 s, for a total of three times, with an interval of 5 s each time.

[0063] (5) Observation of emulsion microstructure: Take an appropriate amount of emulsion sample and drop it on a glass slide and cover it with a cover glass, taking care to remove bubbles. Use an optical microscope to observe the microstructure of the sample and record the microscopic image.

[0064] (6) The emulsion was placed in a test tube and heated at 70°C and 90°C for 30 min and 3 min, respectively, to simulate pasteurization conditions and evaluate the thermal stability of the emulsion. The heated sample was then naturally cooled to 25°C at room temperature and the changes in the sample were observed and recorded.

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

[0066] like Figure 1 As shown, under different Maillard reaction times, under non-reducing conditions, the molecular weight of the control ginkgo fruit protein isolate was observed to be 49kDa, and under reducing conditions, the molecular weights of the acidic and basic subunits of the control ginkgo fruit protein isolate were 28kDa and 20kDa, respectively. In addition, under non-reducing conditions (-βME), the glycosylated ginkgo fruit protein isolate showed obvious protein aggregation, resulting in the formation of higher molecular weight bands (>180kDa). This shows that the degree of grafting is enhanced at higher polysaccharide concentrations and longer reaction times. SDS-PAGE confirmed the formation of a copolymer of ginkgo fruit protein isolate-pullulan. Under reducing conditions (+βME), the high molecular weight bands in the gel were dissociated, indicating that the Maillard reaction process involves cross-linking of disulfide bonds.

[0067] like Figure 2 As shown, under different Maillard reaction times, after the Maillard reaction, the particle sizes of all Ginkgo biloba protein isolate-pullulan copolymers are smaller than the control comparative example group, because the Maillard reaction of Ginkgo biloba protein isolate-pullulan promotes the unfolding of protein peptide chains, enhances the intermolecular forces, and the soluble aggregates of proteins become smaller, thereby causing the particle size to decrease. In addition, the absolute value of the ζ-potential of the sample treated by the Maillard reaction increases. The Maillard reaction can induce the reduction of charged groups, thereby consuming positively charged amino acids. Simultaneous heating causes the protein peptide chains to unfold, exposing more charged groups, resulting in an increase in the absolute value of the ζ-potential.

[0068] like Figure 3 As shown, the infrared spectra of natural GSPI and GSPI-PULL freeze-dried samples with different concentrations, which are the addition reaction products of PULL Maillard reaction for 24 hours, are shown in Figure 3 As shown, in the GSPI-PULL copolymer, the peak at 3700–3200 cm -1 The amide A band and 1700-1600 cm -1 The tensile strength of the amide I band increased, which can be attributed to the covalent reaction between GSPI and PULL to form Maillard products, such as Amadori compounds.

[0069] like Figure 4 As shown in the figure, compared with Comparative Examples 4 to 6, the emulsification activity of adding a small amount of pullulan was not significantly improved. However, when the polysaccharide concentration increased, the emulsification activity of the Ginkgo biloba protein isolate-pullulan copolymer was significantly improved, and the emulsification activity of the sample with a polysaccharide to protein ratio of 4:1 and a reaction time of 24 hours was the largest, reaching 7.11 m 2 / g. This indicates that the copolymerization of protein and polysaccharide exhibits complementary and synergistic effects in modifying the emulsifying properties of protein. Covalent copolymerization of pullulan and protein makes the protein more amphiphilic, surface active and absorbable at the water-in-oil interface.

[0070] The changing trend of emulsification stability is similar to that of emulsification activity, and the emulsification stability of protein after Maillard reaction is higher than that of the control. After the Maillard reaction, the exposure of hydrophilic and hydrophobic groups gives the protein a more flexible structure and can quickly migrate to the oil-water interface. When the ratio of polysaccharide to protein is 4:1, the emulsification stability of the protein decreases, and the high ratio of ginkgo fruit protein isolate-pullulan combination leads to insufficient interface coverage, thereby promoting the aggregation of the dispersed phase. When the pullulan ratio is too high, although the emulsification activity of the protein is enhanced, the protein binding sites at the oil-water interface will also be lost.

[0071] like Figure 5 As shown in the figure, with the addition of pullulan, the emulsion droplets are reduced, and the high concentration of polysaccharide completely covers the surface of the droplets and stabilizes the emulsion interface, thereby preventing the aggregation of the droplets and promoting the stability of the emulsion. At the same time, with the increase of reaction time, the droplet size of the emulsion is significantly reduced and more uniform, the droplet density is high, and the droplets are closely arranged to form a continuous droplet network.

[0072] like Figure 6 As shown in the figure, pasteurization at different temperatures was simulated under the conditions of 70℃ water bath for 30min and 90℃ water bath for 3min. As a result, at the heating temperature of 70℃, there was no significant difference between the samples before and after heating, and the emulsion structure was significant and relatively uniform. When the heating temperature was 90℃, the emulsion structure of natural GSPI was completely destroyed after heating, and gelation occurred. This may be because the high temperature destroyed the structure of the protein adsorbed on the oil-water interface, and GSPI underwent irreversible denaturation, thereby preventing the stability of the emulsion. The emulsion that underwent the Maillard reaction still maintained the emulsion structure. This result shows that the emulsion that underwent the Maillard reaction has a certain thermal stability. The Maillard reaction can enhance the thermal stability of GSPI through differential interactions and the effects of excluded volume.

Claims

1. A method for improving the emulsification and thermal stability of ginkgo biloba protein isolate by Maillard reaction, characterized in that: The following steps are involved: (1) dissolving the ginkgo biloba protein isolate powder in a solvent and obtaining a ginkgo biloba protein isolate solution after sufficient hydration; (2) dissolving the polysaccharide in a solvent and obtaining a polysaccharide solution after sufficient hydration; (3) mixing the polysaccharide solution and the ginkgo fruit protein isolate solution to obtain a protein-polysaccharide mixed solution; (4) adjusting the pH of the protein-polysaccharide mixed solution and stirring to obtain a composite solution; (5) subjecting the composite solution to a Maillard reaction, and mixing the resulting solution with an oil phase, and homogenizing to form an emulsion containing a Ginkgo biloba protein isolate-polysaccharide copolymer.

2. The method according to claim 1, characterized in that In step (1), the mass concentration of the ginkgo biloba protein isolate solution is 15-20 g / L.

3. The method according to claim 1, characterized in that In step (2), the mass concentration of the polysaccharide solution is 50-60 g / L.

4. The method according to claim 1, characterized in that: In step (2), the polysaccharide is pullulan.

5. The method according to claim 1, characterized in that In step (3), in the protein-polysaccharide mixed solution, the mass ratio of polysaccharide to ginkgo fruit protein isolate is 1:4-4:

1.

6. The method according to claim 1, characterized in that In step (4), the pH is 7±0.

1.

7. The method according to claim 1, characterized in that In step (5), the conditions of the Maillard reaction are: reaction temperature is 70±5° C., and reaction time is less than 30 h.

8. The method according to claim 1, characterized in that In step (5), the oil phase is soybean oil.

9. The method according to claim 1, characterized in that: In step (5), the volume fraction of the oil phase in the polysaccharide-modified ginkgo fruit protein isolate emulsion is 25-30%.

10. The method according to claim 1, characterized in that In step (5), the homogenization speed is 13500-15000 rpm, and the homogenization treatment time is 120-150 s.

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