Modified purple perilla cake protein modified by high-speed dispersion-assisted microfluidization technology and phosphorylation and preparation method thereof

Abstract

The invention belongs to the technical field of plant protein modification, and discloses a high-speed dispersion-assisted microfluidization technology and phosphorylation modification combined modified purple perilla cake protein and a preparation method thereof. The preparation method comprises the following steps: mixing purple perilla cake meal protein with water, and carrying out homogenization treatment by utilizing a high-speed dispersion assisted microfluidization technology to obtain a purple perilla cake meal protein dispersion liquid; and mixing the purple perilla cake protein dispersion liquid with phosphate, performing phosphorylation modification, and sequentially performing dialysis and freeze drying to obtain the modified purple perilla cake protein. The solubility, emulsifying capacity, foaming performance, oil holding capacity and thermal stability of the modified purple perilla cake protein prepared by combining a high-speed dispersion-assisted microfluidization technology with phosphorylation modification are all remarkably improved, and reference is provided for value-added utilization of the purple perilla cake protein in preparation of functional active factor delivery systems and food and health care products.

Description

Technical Field

[0001] The invention relates to the technical field of plant protein modification, and in particular to a high-speed dispersion-assisted microfluidization technology combined with phosphorylation-modified modified perilla cake protein and a preparation method thereof. Background Art

[0002] In recent years, plant protein has attracted great attention as a good substitute for animal protein due to its advantages such as rich nutrition, low price and renewable nature. In particular, the use of agricultural by-products to obtain plant protein is a sustainable method, which will enhance the commercial value of agricultural by-products and reduce environmental pollution. Perilla seeds, as a new type of health-care plant oil resource, have attracted much attention for their rich linolenic acid content (about 50-75%) and important physiological functions. Perilla cake is a by-product of perilla seed oil extraction, with aromatic odor and good palatability. In addition, the protein content of perilla cake can reach 28-45%, with superior protein titer and high nutritional value, and can be used as a good source of plant protein. However, the water solubility of perilla cake protein is low and its functional properties are poor, which greatly limits its application in the preparation of functional active factor delivery systems and food and health products. Therefore, how to modify the structure of natural perilla cake protein and improve its functional properties is of great significance to enhance the added value of perilla cake and extend the perilla industry chain.

[0003] At present, a variety of methods have been used to improve the functional properties of plant proteins, mainly including enzymatic, physical and chemical methods. Sometimes, in order to achieve better modification effects or limited by certain modification conditions, it is often necessary to use two or more methods in combination to achieve a synergistic effect and maximize the improvement of the functional properties of proteins. Among these modification methods, the enzymatic method has good safety and high efficiency, but its relatively high cost limits its wide application. Physical modification mainly improves the functional properties of proteins by means of heat treatment, mechanical treatment, magnetic field, ultrasound and low-dose irradiation. Among them, microfluidization technology is a new processing technology developed in recent years. It has been widely used in non-thermal treatment of food processing. It integrates multiple unit operations such as transportation, mixing, ultrafine grinding, pressurization, high-speed impact, pulse vibration, homogenization, etc. After the protein is affected by this, it will cause changes in the tissue structure, thereby improving its functional properties. Although this method is simple to operate, safe and low-cost, its ability to act is very limited. Compared with enzymatic and physical methods, chemical modification is more effective in improving the molecular structure and functional properties of proteins. The methods that are currently used more are mainly phosphorylation, acylation, glycosylation, etc. Among them, phosphorylation modification increases the electronegativity and electrostatic repulsion between protein molecules by introducing phosphate groups into the protein molecular structure, making it easier to disperse in water, thereby increasing the solubility, emulsification and foaming properties of the protein. Compared with other chemical modification methods, phosphorylation modification is safer and will not cause chemical reagent residues. However, the introduction of excessive phosphates may affect the taste of food and bring food safety risks. Summary of the invention

[0004] The purpose of the present invention is to provide a modified perilla cake protein combined with phosphorylation modification by high-speed dispersion-assisted microfluidization technology and a preparation method thereof, so as to solve the problem that the existing plant protein modification methods cannot effectively improve the functional properties of perilla cake protein.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a modified perilla cake protein by high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification, comprising the following steps:

[0007] (1) hydraulic defatting and n-hexane defatting are performed on perilla seeds in sequence; the organic solvent in the defatted product is removed, and the perilla cake protein in the defatted product is extracted by alkali dissolution and acid precipitation method;

[0008] (2) mixing the perilla cake protein with water, and performing a homogenization process using a high-speed dispersion-assisted microfluidization technique to obtain a perilla cake protein dispersion;

[0009] (3) mixing the perilla cake protein dispersion with phosphate for phosphorylation modification; and dialyzing and freeze-drying the modified dispersion in sequence to obtain modified perilla cake protein.

[0010] Preferably, in the preparation method, in step (1), the perilla seeds are pre-treated by baking before hydraulic defatting;

[0011] The baking pretreatment conditions include: a temperature of 120 to 150° C. and a time of 20 to 40 minutes.

[0012] Preferably, in the preparation method, in step (1), the pressure of the hydraulic degreasing is 30 to 100 MPa.

[0013] Preferably, in the preparation method, in step (1), the conditions for n-hexane degreasing include: a solid-liquid ratio of 1:1 to 1:5 g / mL, a single degreasing time of 2 to 4 hours, and a degreasing number of times of 1 to 4 times.

[0014] Preferably, in the preparation method, in step (1), the alkali dissolution and acid precipitation method specifically comprises the following steps:

[0015] The defatted product after the organic solvent is removed is mixed with water, the pH is adjusted to 8.5-10, and an alkaline dissolution reaction is performed to obtain an extract; the extract is centrifuged for the first time, and the supernatant is retained; the supernatant is adjusted to a pH of 4-5, and then allowed to stand, and centrifuged for the second time to obtain perilla cake protein;

[0016] The liquid ratio of the defatted product after removing the organic solvent to the water is 1:5 to 1:30 g / mL;

[0017] The conditions of the alkaline dissolution reaction include: temperature of 40 to 55° C. and time of 1 to 3 hours;

[0018] The conditions of the first centrifugation include: a rotation speed of 6000-10000 rpm and a time of 10-30 min;

[0019] The standing time is 1 to 3 hours;

[0020] The conditions for the second centrifugation include: a rotation speed of 5000-8000 rpm and a time of 5-15 min.

[0021] Preferably, in the preparation method, in step (2), the concentration of the perilla cake protein after mixing with water is 2 to 10 g / 100 mL;

[0022] In step (2), the homogenization process using high-speed dispersion-assisted microfluidization technology specifically includes:

[0023] The solution obtained by mixing perilla cake protein and water is subjected to high-speed shear dispersion, and the shear-dispersed solution is subjected to microfluidization homogenization to obtain a perilla cake protein dispersion;

[0024] The conditions of the high-speed shear dispersion include: a rotation speed of 10000 to 16000 rpm and a time of 5 to 20 min;

[0025] The conditions of the microfluidic homogenization treatment include: a homogenization pressure of 30 to 150 MPa, and a homogenization number of 1 to 4 times.

[0026] Preferably, in the preparation method, in step (3), the phosphate includes sodium tripolyphosphate or sodium trimetaphosphate;

[0027] In step (3), the mass of the phosphate is 1 to 4% of the mass of the perilla cake protein in the perilla cake protein dispersion.

[0028] Preferably, in the preparation method, in step (3), the phosphorylation modification conditions include: in a water bath, pH 8.5-10, temperature 35-55° C., and time 1-5 h.

[0029] Preferably, in the preparation method, in step (3), the dialysis conditions include: molecular weight cutoff of 8000 to 14000 kDa, temperature of 4°C, and time of 12 to 36 h.

[0030] The invention also provides a modified perilla cake protein.

[0031] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention utilizes perilla cake as a raw material to extract and separate protein, which is beneficial to improving the added value of perilla cake, alleviating environmental pollution, reducing the huge waste of beneficial components, and providing a technical reference for the resource utilization of perilla cake protein.

[0033] (2) The present invention uses high-speed dispersion-assisted microfluidization technology to process the perilla cake protein dispersion, disaggregating large perilla cake protein aggregates into small particles, fully exposing the internal hydrophobic groups, which is beneficial to reducing the particle size of the perilla cake protein, increasing its solubility, and further promoting the subsequent phosphorylation reaction.

[0034] (3) The present invention adopts phosphorylation reaction to modify the perilla cake protein dispersion. The introduction of phosphate groups increases the electronegativity of the protein surface and further increases its hydration capacity. At the same time, the phosphorylation modification reagents used, sodium tripolyphosphate or sodium trimetaphosphate, are permitted food additives, thereby ensuring the safety of the obtained modified plant protein product.

[0035] (4) The present invention utilizes high-speed dispersion-assisted microfluidization technology in combination with phosphorylation modification of perilla cake protein. Compared with single microfluidization treatment and phosphorylation modification technology, the solubility, emulsification ability, foaming performance, oil holding capacity and thermal stability of the perilla cake protein obtained by the present invention are significantly improved, thereby broadening the application of perilla cake protein in the food industry, especially in the field of food colloids, and providing a reference for increasing the added value of perilla cake and the value-added utilization of perilla cake protein in the preparation of functional active factor delivery systems and food and health products.

[0036] (5) The preparation method of the present invention has simple process, low cost, green and safe, no chemical reagent residue, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the description of the embodiments or the prior art are briefly introduced below.

[0038] Figure 1 The particle size and zeta potential of the perilla cake protein dispersion obtained in Examples 1 to 4 and Comparative Examples 1 to 2, wherein A is the average particle size, B is the particle size distribution diagram, and C is the absolute zeta potential value;

[0039] Figure 2 The surface hydrophobicity and free thiol content of the perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2, wherein A is the surface hydrophobicity result and B is the free thiol content;

[0040] Figure 3 The molecular weight distribution of the perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2;

[0041] Figure 4 The SEM images of the perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2 are shown;

[0042] Figure 5 The secondary structure and tertiary conformation of the perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2, wherein A is the circular dichroism spectrum, B is the ultraviolet spectrum, and C is the fluorescence spectrum;

[0043] Figure 6 The results of solubility, emulsifying ability, foaming properties, and oil holding capacity of the perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2, wherein A is solubility, B is emulsifying ability, C is foaming properties, and D is oil holding capacity;

[0044] Figure 7 The thermal stability results of the perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2 are shown, wherein A is the mass loss and B is the weight loss rate. DETAILED DESCRIPTION

[0045] The present invention provides a method for preparing a modified perilla cake protein by high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification, comprising the following steps:

[0046] (1) hydraulic defatting and n-hexane defatting are performed on perilla seeds in sequence; the organic solvent in the defatted product is removed, and the perilla cake protein in the defatted product is extracted by alkali dissolution and acid precipitation method;

[0047] (2) mixing the perilla cake protein with water, and performing a homogenization process using a high-speed dispersion-assisted microfluidization technique to obtain a perilla cake protein dispersion;

[0048] (3) mixing the perilla cake protein dispersion with phosphate for phosphorylation modification; and dialyzing and freeze-drying the modified dispersion in sequence to obtain modified perilla cake protein.

[0049] In the present invention, in step (1), the perilla seeds are pre-treated by baking before hydraulic defatting.

[0050] In the present invention, the baking pretreatment conditions include: the temperature is preferably 120-150°C, more preferably 130-145°C, more preferably 140°C; the time is preferably 20-40 min, more preferably 25-35 min, more preferably 30 min.

[0051] In the present invention, in step (1), the pressure of the hydraulic degreasing is preferably 30 to 100 MPa, more preferably 60 to 90 MPa, and even more preferably 80 MPa.

[0052] In the present invention, in step (1), between the hydraulic degreasing and the n-hexane degreasing, the step further includes: pulverizing the defatted cake obtained by the hydraulic degreasing.

[0053] In the present invention, the conditions for the pulverization are not limited, and the mesh size of the defatted cake after pulverization can be 40 to 80 meshes.

[0054] In the present invention, in step (1), the conditions for n-hexane degreasing include: the solid-liquid ratio is preferably 1:1-1:5 g / mL, more preferably 1:2-1:4 g / mL, and more preferably 1:3 g / mL; the single degreasing time is preferably 2-4 hours, more preferably 2-3 hours, and more preferably 2 hours; the number of degreasing times is preferably 1-4 times, more preferably 2-4 times, and more preferably 3 times.

[0055] In the present invention, in step (1), the alkali dissolution and acid precipitation method preferably comprises the following steps:

[0056] The defatted product after removing the organic solvent is mixed with water, the pH is adjusted to 8.5-10, and an alkaline dissolution reaction is performed to obtain an extract; the extract is centrifuged for the first time, and the supernatant is retained; the supernatant is adjusted to a pH of 4-5, then allowed to stand, and centrifuged for a second time to obtain perilla cake protein.

[0057] In the present invention, the particle size of the defatted product after the organic solvent is removed is preferably 40 to 80 meshes, more preferably 50 to 70 meshes, and more preferably 60 meshes.

[0058] In the present invention, the material-liquid ratio of the defatted product after removal of the organic solvent to the water is preferably 1:5 to 1:30 g / mL, more preferably 1:10 to 1:25 g / mL, and more preferably 1:20 g / mL.

[0059] In the present invention, the pH of the defatted product after the organic solvent is removed is preferably adjusted to 8.5 to 10, more preferably to 8.5 to 9.5, and even more preferably to 9 after being mixed with water.

[0060] In the present invention, the reagent for adjusting the pH to 8.5-10 is not limited, and an inorganic alkaline solution well known to those skilled in the art can be used. Specifically in the embodiment of the present invention, the reagent is preferably a 3 mol / L NaOH solution.

[0061] In the present invention, the conditions for the alkaline dissolution reaction include: the temperature is preferably 40-55°C, more preferably 40-50°C, and more preferably 45°C; the time is preferably 1-3h, more preferably 1.5-2.5h, and more preferably 2h; and the alkaline dissolution reaction is preferably carried out in a water bath.

[0062] In the present invention, the conditions for the first centrifugation include: the rotation speed is preferably 6000-10000 rpm, more preferably 7000-9000 rpm, more preferably 8000 rpm; the time is preferably 10-30 min, more preferably 15-25 min, more preferably 15 min.

[0063] In the present invention, the pH of the supernatant is preferably adjusted to 4 to 5, more preferably 4.2 to 4.8, and even more preferably 4.5.

[0064] In the present invention, the reagent for adjusting the pH to 4-5 is not limited, and an inorganic acid solution well known to those skilled in the art can be used. Specifically in the embodiment of the present invention, the reagent is preferably a 3 mol / L HCl solution.

[0065] In the present invention, the standing time is preferably 1 to 3 hours, more preferably 1.5 to 2.5 hours, and more preferably 2 hours.

[0066] In the present invention, the conditions for the second centrifugation include: the rotation speed is preferably 5000-8000 rpm, more preferably 7000-8000 rpm, more preferably 8000 rpm; the time is preferably 5-15 min, more preferably 8-12 min, more preferably 10 min.

[0067] In the present invention, after the second centrifugation, the process preferably further comprises: dissolving the precipitate obtained from the second centrifugation with water, adjusting the pH of the system to neutral with 1 mol / L HCl solution, and then freeze-drying.

[0068] In the present invention, the freeze-drying conditions include: the drying time is preferably 48 to 72 hours, more preferably 55 to 70 hours, and more preferably 60 hours; the vacuum degree is preferably 5 to 30 Pa, more preferably 10 to 20 Pa, and more preferably 10 Pa.

[0069] In the present invention, in step (2), the concentration of the perilla cake protein after mixing with water is preferably 2-10 g / 100 mL, more preferably 5-8 g / 100 mL, and more preferably 6 g / 100 mL.

[0070] In the present invention, in step (2), the homogenization treatment using high-speed dispersion-assisted microfluidization technology preferably includes:

[0071] The solution obtained by mixing perilla cake protein and water is subjected to high-speed shear dispersion, and the shear-dispersed solution is subjected to microfluidization homogenization to obtain a perilla cake protein dispersion.

[0072] In the present invention, the high-speed shearing and dispersing device is preferably a T18 high-speed disperser.

[0073] In the present invention, the conditions for high-speed shear dispersion include: the rotation speed is preferably 10000-16000 rpm, more preferably 12600-15000 rpm, and more preferably 13400 rpm; the time is preferably 5-20 min, more preferably 10-15 min, and more preferably 10 min.

[0074] In the present invention, the conditions of the microfluidic homogenization treatment include: the homogenization pressure is preferably 30-150 MPa, more preferably 60-120 MPa, and more preferably 90 MPa; the number of homogenization times is preferably 1-4 times, more preferably 2-4 times, and more preferably 3 times.

[0075] In the present invention, in step (3), the phosphate preferably includes sodium tripolyphosphate or sodium trimetaphosphate, and more preferably sodium tripolyphosphate.

[0076] In the present invention, in step (3), the mass of the phosphate is preferably 1 to 4% of the mass of the perilla cake protein in the perilla cake protein dispersion, more preferably 2.5 to 4%, and even more preferably 4%.

[0077] In the present invention, in step (3), the conditions for phosphorylation modification include: preferably in a water bath; pH is preferably 8.5-10, more preferably 8.5-9, more preferably 9; temperature is preferably 35-55°C, more preferably 40-50°C, more preferably 45°C; time is preferably 1-5h, more preferably 1.5-3.5h, more preferably 2h.

[0078] In the present invention, in step (3), the dialysis conditions include: the molecular weight cutoff is preferably 8000-14000 kDa; the temperature is preferably 4°C; the time is preferably 12-36 h, more preferably 26-36 h, and more preferably 36 h; the dialysis reagent is preferably water.

[0079] In the present invention, in step (3), the freeze-drying conditions include: the drying time is preferably 48 to 72 hours, more preferably 55 to 70 hours, and more preferably 60 hours; the vacuum degree is preferably 5 to 30 Pa, more preferably 10 to 20 Pa, and more preferably 10 Pa.

[0080] The invention also provides a modified perilla cake protein.

[0081] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0082] Example 1

[0083] This embodiment provides a method for preparing a modified perilla cake protein by high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification, comprising the following steps:

[0084] (1) The perilla seeds were infrared-baked at 150°C for 30 min, and then hydraulically defatted at 80 MPa; the cake after hydraulic defatting was crushed at high speed to a size of 60 mesh, and then secondary defatting was performed using n-hexane. The conditions for secondary defatting were: solid-liquid ratio 1:3 g / mL, single degreasing time 2 h, degreasing times 3 times, and then the organic solvent was evaporated and sieved to obtain 60-mesh perilla cake powder; 50 g of perilla cake powder was taken and mixed with 1000 mL of distilled water, and the pH of the system was adjusted to 9.0 using 3.0 mol / L NaOH solution, and the mixture was stirred and extracted in a constant temperature water bath at 45°C for 1.5 h to obtain an extract; the extract was then centrifuged at 8000 rpm for 15 min, the supernatant was collected, the pH of the supernatant was adjusted to 4.5 using 3 mol / L HCl solution, and the precipitate was collected and dissolved with a small amount of distilled water and 1 mol / L The pH of the system was adjusted to neutral with HCl solution, and the mixture was freeze-dried for 60 h under a vacuum degree of 10 Pa to obtain perilla cake protein.

[0085] (2) preparing a 6 g / 100 mL perilla cake protein aqueous solution, treating it with a T18 high-speed disperser at a shear rate of 13400 rpm for 10 min, and then homogenizing the aqueous solution under a microfluidization pressure of 90 MPa for 3 times to obtain a perilla cake protein dispersion;

[0086] (3) Add sodium tripolyphosphate with a protein content of 4% to the perilla cake protein dispersion, adjust the pH of the system to 9.0, and stir the mixture in a water bath at 45°C for 2 h. After the reaction, transfer the mixed solution to a 8000-14000 kDa molecular weight dialysis bag, and stir and dialyze with distilled water at 4°C for 36 h. Collect the dialyzate, and freeze-dry it at a vacuum degree of 10 Pa for 60 h to obtain modified perilla cake protein, which is recorded as MP-PPI-90MPa.

[0087] Example 2

[0088] This embodiment provides a method for preparing modified perilla cake protein by high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification. The difference from Example 1 is that the microjet pressure in step (2) is modified to 60 MPa, and the other parameter conditions are the same as those in Example 1, which are recorded as MP-PPI-60 MPa.

[0089] Example 3

[0090] This embodiment provides a method for preparing modified perilla cake protein by high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification. The difference from Example 1 is that the microjet pressure in step (2) is modified to 30 MPa, and the other parameter conditions are the same as those in Example 1, which are recorded as MP-PPI-30 MPa.

[0091] Example 4

[0092] This embodiment provides a method for preparing modified perilla cake protein by high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification. The difference from Example 1 is that the microjet pressure in step (2) is modified to 120 MPa, and the other parameter conditions are the same as those in Example 1, which are recorded as MP-PPI-120 MPa.

[0093] Comparative Example 1

[0094] This comparative example provides a method for preparing natural perilla cake protein, which differs from Example 1 in that step (2) and step (3) are deleted, and other parameter conditions are the same as those in Example 1, and are denoted as PPI.

[0095] Comparative Example 2

[0096] This comparative example provides a method for preparing phosphorylated modified perilla cake protein, which differs from Example 1 in that step (2) is deleted, and other parameter conditions are the same as those in Example 1, and is denoted as P-PPI.

[0097] The particle size and zeta potential of the perilla cake protein dispersions of Examples 1 to 4 and Comparative Examples 1 to 2 were measured using a Malvern nanoparticle size and zeta potential analyzer. The results are as follows: Figure 1 shown.

[0098] Figure 1 The results of A in the figure showed that compared with natural perilla cake protein (PPI) and phosphorylated perilla cake protein (P-PPI), the average particle size of modified perilla cake protein (MP-PPI) combined with high-speed dispersion-assisted microfluidization technology was significantly reduced (the minimum was 309.57 nm). This may be because the non-covalent bonds in the protein were destroyed during the modification process, resulting in the disaggregation of larger perilla cake protein aggregates into small particles with a narrow distribution. Figure 1 The results of B in Figure 3 show that the particle size distributions of PPI, P-PPI and MP-PPI are all unimodal, and with the increase of microjet pressure, the maximum peak position of MP-PPI gradually moves to the left. Figure 1 The results of C in show that the absolute zeta potential value of MP-PPI is slightly increased compared with PPI and P-PPI, which may be due to the microfluidization treatment causing the protein molecular structure to unfold and expose more electronegative groups; secondly, the introduction of phosphate groups further increases the surface electronegativity of the protein.

[0099] The surface hydrophobicity, free thiol content and molecular weight distribution of the perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2 were analyzed to explore the effect of microfluidic pressure on the basic properties of perilla cake protein. The surface hydrophobicity was analyzed by ANS as a fluorescent probe and measured by a fluorescence spectrophotometer; the free thiol content was measured by DTNB colorimetry; the molecular weight distribution was characterized by SDS-PAGE gel electrophoresis, and the specific results are shown in Figure 2 and Figure 3 shown.

[0100] Figure 2 The results showed that the surface hydrophobicity of MP-PPI was higher than that of PPI ( Figure 2 A) and free thiol content ( Figure 2 B) in the figure were significantly increased, indicating that in the process of high-speed dispersion-assisted microfluidization combined with phosphorylation modification, the molecular structure of perilla cake protein unfolded and the hidden hydrophobic groups and thiol groups were exposed. However, compared with P-PPI, the surface hydrophobicity and free thiol content of modified perilla cake protein treated with 30-120MPa microfluidization pressure were slightly reduced, but still significantly higher than PPI. This may be due to the high-speed dispersion-assisted microfluidization combined with phosphorylation modification, which caused some proteins to aggregate, thereby re-embedding the exposed hydrophobic groups and thiol groups.

[0101] Depend on Figure 3 It can be seen that 5 different bands were observed in the molecular weight range of 10-60 kDa for PPI, P-PPI and MP-PPI. However, the subunit composition of all samples did not show obvious differences, indicating that high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification did not cause significant changes in the molecular weight of perilla cake protein, and its primary structure was still intact.

[0102] The microscopic morphology of the perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2 was analyzed by scanning electron microscope (SEM). Figure 4 shown.

[0103] Figure 4 The results showed that natural perilla cake protein was aggregated by different sizes and had a rough surface. After high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification, the larger and irregular protein aggregates were broken into small fragments, and the surface structure became flat and smooth with holes. This may be due to the microfluidization treatment and the introduction of electronegative groups, which led to the transformation of the protein aggregation structure into a network structure, which is beneficial to improve the functional properties of the protein.

[0104] The secondary structure and tertiary conformation of the perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2 were analyzed by circular dichroism, ultraviolet and fluorescence spectroscopy. Figure 5, as shown in Table 1.

[0105] Figure 5 The results of A in and Table 1 show that the percentages of α-helix, β-fold, β-turn and random coil structures of natural perilla cake protein are 13.50%, 18.40%, 36.30% and 32.70%, respectively. After high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification, the α-helix ratio of the modified perilla cake protein decreased (from 13.50% to 12.00%), while the β-turn and random coil ratios showed an upward trend. This may be because high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification destroyed the interaction between different parts of the perilla cake protein molecules. Generally, the α-helix is ​​the most tightly connected structure in the protein molecule. Therefore, the reduction in the α-helix ratio indicates that high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification may cause the structure of perilla cake protein to become looser, which further clarifies Figure 4 The reason why holes appear on the surface of modified perilla cake protein. Figure 5 As shown in B, PPI, P-PPI and MP-PPI all have characteristic peaks at 280nm. However, compared with PPI, the absorbance intensity of MP-PPI at the maximum peak is enhanced and reaches the highest value at 120MPa, indicating that the high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification promotes the exposure of chromophores embedded in protein molecules, further increases the ultraviolet absorption intensity, and then affects the microenvironment where the perilla cake protein is located, causing changes in the protein conformation. Figure 5 From the C in Figure 2, we can see that the maximum emission wavelength of natural perilla cake protein can be observed at 333nm. After high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification, the maximum emission wavelength of modified perilla cake protein blue-shifted from 333nm to 331nm, indicating that the polar microenvironment of tryptophan residues became more hydrophobic. In addition, after high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification, the relative fluorescence intensity of modified perilla cake protein increased significantly, which may be due to the change in the conformation of modified perilla cake protein, resulting in more chromophores exposed to the surrounding microenvironment.

[0106] Table 1. Figure 5 The table in A

[0107]

[0108] The solubility, emulsifying ability, foaming properties, oil holding capacity and thermal stability of the perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2 were analyzed to explore the effect of microfluidization pressure on the functional properties and thermal stability of perilla cake protein. The specific results are shown in Figure 6 and Figure 7 shown.

[0109] Solubility determination: Weigh 0.1 g of PPI, P-PPI and MP-PPI samples respectively in 10 mL of deionized water, stir magnetically at room temperature for 0.5 h, then centrifuge at 5000 rpm for 10 min, collect the supernatant, and determine its protein content using the Coomassie brilliant blue colorimetric method. The protein solubility was calculated according to formula (1).

[0110]

[0111] Determination of emulsification activity and emulsification stability: The perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2 was dissolved in distilled water to prepare 1 wt% sample solutions. 5 mL of soybean oil was added to 15 mL of the sample solution and homogenized at 13400 rpm for 2 min using a high-speed homogenizer. 100 μL of the sample was immediately drawn from the bottom of the tube after the homogenization and after standing for 10 min, and diluted with 10 mL of 0.1 wt% SDS. The absorbance at 500 nm was measured and marked as A0 and A1. 10 The calculation formulas for the emulsification activity index (EAI) and the emulsification stability index (ESI) are as shown in formula (2) and formula (3).

[0112]

[0113] In formula (2), N is the dilution factor, is the fraction of oil phase in the system, C is the protein concentration, and L is the light path of the colorimetric cell.

[0114]

[0115] In formula (3), A0 is the absorbance at 0 min, A 10 is the absorbance after standing for 10 min, and ΔT is the time interval between two emulsification tests.

[0116] Determination of foaming property and foam stability: Prepare 100 mL of the solution of perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2 (1%, pH 7.0), place in a 250 mL graduated cylinder, and process with a high-speed homogenizer at 15000 rpm for 2 min, and record the liquid level before and after foaming. The calculation of foaming capacity (FC) and foam stability (FS) refers to formula (4).

[0117]

[0118] In formula (4), V1 is the height of the liquid level after homogenization, and V2 is the height of the liquid level after standing for 30 minutes.

[0119] Determination of oil holding capacity: 1.0 g of the perilla cake protein obtained in Examples 1 to 4 and Comparative Examples 1 to 2 was placed in a pre-weighed 50 mL centrifuge tube, 30 mL of soybean oil was added, the mixture was shaken at room temperature for 1.5 h, and then centrifuged at 5000 rpm for 10 min. After discarding the unabsorbed oil layer, the centrifuge tube and the precipitate were weighed. The oil holding capacity was calculated according to formula (5).

[0120]

[0121] In formula (5), m is the sample mass, m1 is the weight of the centrifuge tube, and m2 is the weight of the sample and centrifuge tube after oil absorption.

[0122] Thermal stability analysis: The perilla cake protein powder (3 mg) obtained in Examples 1 to 4 and Comparative Examples 1 to 2 was weighed in a ceramic crucible and heated from 40°C to 600°C at a heating rate of 10°C / min in a nitrogen environment. The weight loss of the sample was analyzed and calculated by the software provided by the TGA Q500 thermogravimetric analyzer.

[0123] Figure 6 The results of A in the experiment showed that the solubility of natural perilla protein was 23.08%, and the solubility of phosphorylated perilla protein was 54.72%. After high-speed dispersion-assisted microfluidization combined with phosphorylation modification, the solubility of modified perilla protein increased significantly and reached a maximum value of 81.64% at 90 MPa. This may be because high-speed dispersion-assisted microfluidization combined with phosphorylation modification caused conformational changes or structural unfolding of perilla protein, which in turn led to more hydrophilic groups being exposed on the surface of protein molecules. In addition, some hydrophobic interactions and hydrogen bonds between natural perilla protein molecules may be destroyed during the high-speed dispersion-assisted microfluidization combined with phosphorylation modification, further increasing the solubility of perilla protein. In addition, compared with PPI, the particle size of modified perilla protein was significantly reduced, which helped to increase its interaction with water molecules, thereby improving the solubility of perilla protein. Figure 6 The results of B in the experiment show that the emulsifying activity of natural perilla cake protein is low. After high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification, the emulsifying activity and emulsifying stability of the modified perilla cake protein are significantly improved, and the relationship is pressure-dependent. This may be because the high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification reduces the particle size of perilla cake protein, improves the surface hydrophobicity, promotes the adsorption of protein at the oil-water interface, improves the stability of the interfacial film, and thus reduces the interfacial tension. In addition, the increase in solubility promotes the diffusion rate of protein at the oil-water interface, further improving the emulsifying properties of perilla cake protein. Figure 6From the C in Figure 2, we can see that compared with natural perilla protein, high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification can significantly improve the foaming property of perilla protein, which is attributed to the partial unfolding of the protein during the modification process, which improves its molecular flexibility and surface hydrophobicity, thereby facilitating the formation of an air-water interface. In addition, the reduction in the particle size of perilla protein helps to improve its solubility, further promoting the adsorption of protein at the air-water interface. Figure 6 As shown in D, with the increase of microfluidization pressure, the oil holding capacity of perilla cake protein showed a gradual upward trend and reached a maximum value at 120 MPa. Generally, proteins with higher surface hydrophobicity tend to have higher fat absorption rate. Therefore, the improvement of oil holding capacity of modified perilla cake protein after high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification may be attributed to the exposure of buried hydrophobic groups in perilla cake protein.

[0124] Depend on Figure 7 It can be seen that PPI, P-PPI and MP-PPI experienced three thermal degradation steps in the temperature range of 40 to 600°C. The temperature range of the first stage was 40 to 120°C, which was related to the removal of adsorbed water. The temperature range of the second stage was 150 to 500°C, which was the stage with the largest weight loss. This stage was related to the structural decomposition of the protein. The change in the temperature range of 500 to 600°C was the third thermal degradation stage. The slope of the curve in this stage gradually slowed down, which may be due to the oxidation of partially decomposed perilla cake protein. In addition, within the entire temperature change range, there was no significant difference in the maximum pyrolysis temperature of PPI and MP-PPI. However, compared with PPI, the thermal degradation rate of MP-PPI treated with different pressures was greatly reduced, indicating that the thermal stability of the modified perilla cake protein modified by high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification was significantly improved. This may be because the structure of the modified perilla cake protein became more compact due to the high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification. Figure 4 This was also confirmed by the microscopic morphology observation of perilla cake protein.

[0125] In summary, high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification is an effective protein modification method, which can significantly improve the functional properties of perilla cake protein and thus expand its application in food systems.

[0126] Example 5

[0127] This embodiment provides a method for preparing a modified perilla cake protein by high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification, comprising the following steps:

[0128] (1) The perilla seeds were infrared-baked at 120°C for 40 min, and then hydraulically degreased at 100 MPa; the cake after hydraulic degreasing was crushed at high speed to a crushing mesh of 80 mesh, and then secondary degreasing was performed using n-hexane. The conditions for secondary degreasing were: solid-liquid ratio 1:3 g / mL, single degreasing time 4 h, degreasing times 2 times, and then the organic solvent was evaporated and sieved to obtain 80-mesh perilla cake powder; 50 g of perilla cake powder was taken and mixed with 750 mL of distilled water, and the pH of the system was adjusted to 9.0 with 3.0 mol / L NaOH solution, and extracted with stirring in a constant temperature water bath at 45°C for 2 h to obtain an extract; then the extract was centrifuged at 8000 rpm for 15 min, the supernatant was collected, and the pH of the supernatant was adjusted to 4.5 with 3 mol / L HCl solution. After standing at room temperature for 2 h, it was centrifuged at 8000 rpm for 10 min, the precipitate was collected, dissolved with a small amount of distilled water, and extracted with 1 mol / L The pH of the system was adjusted to neutral with HCl solution, and the mixture was freeze-dried for 65 h under a vacuum degree of 10 Pa to obtain perilla cake protein;

[0129] (2) preparing a 5 g / 100 mL perilla cake protein aqueous solution, treating it with a T18 high-speed disperser at a shear rate of 12,600 rpm for 15 min, and then homogenizing the aqueous solution under a microfluidization pressure of 120 MPa for 3 times to obtain a perilla cake protein dispersion;

[0130] (3) Adding sodium trimetaphosphate with a protein content of 4% to the perilla cake protein dispersion, adjusting the pH of the system to 9.0, and stirring the mixture in a water bath at 45°C for 2 h; after the reaction, transferring the mixture to a 8000-14000 kDa molecular weight dialysis bag, and dialyzing it with distilled water at 4°C for 26 h; collecting the dialyzate, and freeze-drying it at a vacuum degree of 10 Pa for 65 h to obtain modified perilla cake protein.

[0131] Example 6

[0132] This embodiment provides a method for preparing a modified perilla cake protein by high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification, comprising the following steps:

[0133] (1) The perilla seeds were infrared-baked at 120°C for 40 min, and then hydraulically degreased at 90 MPa; the cake after hydraulic degreasing was crushed at high speed to a crushing mesh of 80 mesh, and then secondary degreasing was performed using n-hexane. The conditions for secondary degreasing were: solid-liquid ratio 1:3 g / mL, single degreasing time 3 h, degreasing times 3 times, and then the organic solvent was evaporated and sieved to obtain 80-mesh perilla cake powder; 50 g of perilla cake powder was taken and mixed with 850 mL of distilled water, and the pH of the system was adjusted to 8.5 with 3.0 mol / L NaOH solution, and extracted with stirring in a constant temperature water bath at 40°C for 2 h to obtain an extract; then the extract was centrifuged at 8000 rpm for 15 min, the supernatant was collected, the pH of the supernatant was adjusted to 4.5 with 3 mol / L HCl solution, and the precipitate was collected and dissolved with a small amount of distilled water and 1 mol / L The pH of the system was adjusted to neutral with HCl solution, and the mixture was freeze-dried for 70 h under a vacuum degree of 15 Pa to obtain perilla cake protein.

[0134] (2) preparing an 8 g / 100 mL perilla cake protein aqueous solution, treating it with a T18 high-speed disperser at a shear rate of 15,000 rpm for 10 min, and then homogenizing the aqueous solution under a microfluidization pressure of 60 MPa for 3 times to obtain a perilla cake protein dispersion;

[0135] (3) Adding sodium tripolyphosphate with a protein content of 4% to the perilla cake protein dispersion, adjusting the pH of the system to 9.0, and stirring the mixture in a water bath at 45°C for 2 h; after the reaction, transferring the mixed solution to a 8000-14000 kDa molecular weight dialysis bag, and dialyzing it with distilled water at 4°C for 30 h; collecting the dialyzate, and freeze-drying it at a vacuum degree of 15 Pa for 70 h to obtain modified perilla cake protein.

[0136] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing modified perilla cake protein by high-speed dispersion-assisted microfluidization technology combined with phosphorylation modification, characterized in that: The following steps are involved: (1) hydraulic defatting and n-hexane defatting are performed on perilla seeds in sequence; the organic solvent in the defatted product is removed, and the perilla cake protein in the defatted product is extracted by alkali dissolution and acid precipitation method; (2) mixing the perilla cake protein with water, and performing a homogenization process using a high-speed dispersion-assisted microfluidization technique to obtain a perilla cake protein dispersion; (3) mixing the perilla cake protein dispersion with phosphate for phosphorylation modification; and dialyzing and freeze-drying the modified dispersion in sequence to obtain modified perilla cake protein.

2. The preparation method according to claim 1, characterized in that: In step (1), the perilla seeds are pre-treated by baking before hydraulic defatting; The baking pretreatment conditions include: a temperature of 120 to 150° C. and a time of 20 to 40 minutes.

3. The preparation method according to claim 1 or 2, characterized in that: In step (1), the pressure of the hydraulic degreasing is 30 to 100 MPa.

4. The preparation method according to claim 3, characterized in that: In step (1), the conditions for n-hexane degreasing include: a solid-liquid ratio of 1:1 to 1:5 g / mL, a single degreasing time of 2 to 4 hours, and a degreasing number of 1 to 4 times.

5. The preparation method according to claim 1 or 4, characterized in that: In step (1), the alkali dissolution and acid precipitation method specifically comprises the following steps: The defatted product after the organic solvent is removed is mixed with water, the pH is adjusted to 8.5-10, and an alkaline dissolution reaction is performed to obtain an extract; the extract is centrifuged for the first time, and the supernatant is retained; the supernatant is adjusted to a pH of 4-5, and then allowed to stand, and centrifuged for the second time to obtain perilla cake protein; The liquid ratio of the defatted product after removing the organic solvent to the water is 1:5 to 1:30 g / mL; The conditions of the alkaline dissolution reaction include: temperature of 40 to 55° C. and time of 1 to 3 hours; The conditions of the first centrifugation include: a rotation speed of 6000-10000 rpm and a time of 10-30 min; The standing time is 1 to 3 hours; The conditions for the second centrifugation include: a rotation speed of 5000-8000 rpm and a time of 5-15 min.

6. The preparation method according to claim 1, characterized in that: In step (2), the concentration of the perilla cake protein after mixing with water is 2 to 10 g / 100 mL; In step (2), the homogenization process using high-speed dispersion-assisted microfluidization technology specifically includes: The solution obtained by mixing perilla cake protein and water is subjected to high-speed shear dispersion, and the shear-dispersed solution is subjected to microfluidization homogenization to obtain a perilla cake protein dispersion; The conditions of the high-speed shear dispersion include: a rotation speed of 10000 to 16000 rpm and a time of 5 to 20 min; The conditions of the microfluidic homogenization treatment include: a homogenization pressure of 30 to 150 MPa, and a homogenization number of 1 to 4 times.

7. The preparation method according to claim 1, characterized in that: In step (3), the phosphate includes sodium tripolyphosphate or sodium trimetaphosphate; In step (3), the mass of the phosphate is 1 to 4% of the mass of the perilla cake protein in the perilla cake protein dispersion.

8. The preparation method according to claim 1 or 7, characterized in that: In step (3), the phosphorylation modification conditions include: in a water bath, pH 8.5-10, temperature 35-55° C., and time 1-5 h.

9. The preparation method according to claim 8, characterized in that: In step (3), the dialysis conditions include: molecular weight cut-off of 8000 to 14000 kDa, temperature of 4°C, and time of 12 to 36 hours.

10. The modified perilla cake protein obtained by the preparation method according to any one of claims 1 to 9.

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