A green and efficient preparation method for high-yield, high-quality oil protein isolate

Abstract

The present invention belongs to the technical field of plant protein extraction, and provides a green and efficient preparation method for high-quality oilseed protein isolate with high yield. The method of the present invention comprises the following steps: low-temperature pressing, crushing, screening and removing residual oil from oilseeds in sequence to obtain defatted oilseed meal; mixing the defatted oilseed meal with water, adjusting the pH value to 8-10, and obtaining a mixed solution; centrifuging the mixed solution, collecting the supernatant, and concentrating and purifying the supernatant by using a weak base synchronous ultrafiltration method with precise scale control to obtain a highly concentrated solution; redissolving the highly concentrated solution with water in a volume ratio of 1:1-3, stirring, and obtaining a reconstituted solution; freeze-drying the reconstituted solution to obtain high-quality oilseed protein isolate. The oilseed protein isolate prepared by the process of the present invention has the advantages of short extraction cycle, high protein yield, and environmental friendliness, and the oilseed protein prepared by the present invention has high functional properties, high nutritional value, and good flavor.

Description

Technical Field

[0001] The present invention relates to the technical field of plant protein extraction, and in particular to a green and efficient preparation method of high-quality oil protein isolate with high yield. Background Art

[0002] Protein is one of the three major nutrients required by the human body. Due to the rapid global population growth, projected to reach 10 billion by 2050, protein supply will not be able to meet this rapidly increasing demand. Currently, the main sources of protein are animal protein and plant protein. Animal protein relies heavily on animal husbandry, which poses serious environmental pollution and resource consumption issues. Furthermore, its high levels of saturated fatty acids and cholesterol can easily lead to various chronic diseases. Plant protein is not only widely available, environmentally friendly, and consistent with sustainable development, but also boasts a comprehensive amino acid profile, giving it a higher nutritional value. Therefore, tapping into more plant protein resources is a crucial way to ensure a balanced protein supply in my country.

[0003] The global oilseed industry holds enormous development potential, and my country possesses abundant resources. In recent years, the cultivated area and yield of oilseeds such as soybeans and flaxseed have been on an upward trend. For example, by 2020, my country's soybean planting area reached 9.87 million hectares, with an annual output of 19.6 million tons. Oilseed meal, such as soybeans and flaxseed, is a major byproduct of the oilseed extraction industry, and its production is also increasing annually. However, currently, oilseed meal in my country is primarily used as feed and fertilizer, and its potential for high-value utilization remains largely untapped. Oilseed meal, such as soybeans and flaxseed, typically contains 30-55% high-quality protein. Therefore, the efficient utilization of oilseed meal protein will contribute to my country's efficient supply of edible protein. As a new alternative to animal protein, it will facilitate the transition to large-scale, low-cost, and sustainable protein production.

[0004] The main extracting method of oilseed meal protein at present is the alkali-soluble acid precipitation method, basic principle is to make oilseed meal protein effectively dissolve and remove the impurity that is insoluble in alkaline solution in alkaline environment, adjust to its precipitation near the isoelectric point of protein with acid again, but this method makes protein show the aggregation behavior of height owing to approaching the isoelectric point of protein in the acid precipitation process, thereby cause the oilseed protein functional characteristic poor that prepares, the defects such as digestibility are low, and this just makes traditional extraction technology can not well meet the fast development demand of food industry.If only adopt alkali-soluble step, there is the problem that impurity is many (being mainly compositions such as polyphenols, monosaccharide, inorganic salts), protein content is low (general<60%) in the protein that enrichment obtains, therefore, be badly in need of breakthrough after alkali-soluble step and do not use the method for acid precipitation, avoid the gathering of protein near the isoelectric point, thereby prepare the high-quality oilseed protein with high yield. Because the molecular weight of components such as polyphenols, monosaccharides, and salt ions in impurities is much smaller than that of oilseed proteins (mainly globulin and albumin), ultrafiltration is basically completely permeable to water, minerals, organic acids, non-protein nitrogen, and substances of relatively small molecular weight, and its production conditions are mild, and equipment cost and running cost are low. Therefore, by controlling the solubility and scale of protein under alkaline conditions, and then synchronously adopting ultrafiltration to accurately control the scale method, small molecular weight impurities are removed, protein is retained and prevented from aggregation, and it is expected to achieve the preparation of high-quality oilseed protein. The present invention is to upgrade and transform the existing industrialized alkali dissolution and acid precipitation method for preparing oilseed protein, and has potential industrial application and promotion potential. Summary of the Invention

[0005] In order to overcome the above-mentioned defects in the prior art, the present invention provides a green and efficient preparation method of high-quality oil protein isolate with high yield.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a green and efficient method for preparing high-quality oil protein isolate with high yield, comprising the following steps:

[0008] (1) The oil is subjected to low-temperature pressing, crushing, screening and residual oil removal in sequence to obtain defatted oil meal;

[0009] (2) mixing the defatted oilseed meal with water, adjusting and maintaining the pH value to 8 to 10, and stirring to obtain a mixed solution;

[0010] (3) centrifuging the mixed solution, collecting the supernatant, and concentrating and purifying the supernatant to 1 / 6 to 1 / 8 of the original volume using a weak base synchronous ultrafiltration precision control scale method to obtain a highly concentrated solution;

[0011] (4) adding water to the highly concentrated solution at a volume ratio of 1:1 to 3 at a pH of 8 to 10 for redissolution, stirring to obtain a redissolution;

[0012] (5) freeze-drying the reconstituted solution to obtain high-quality oil protein isolate.

[0013] Preferably, the types of oil in step (1) include soybean, linseed and perilla, and the temperature of the low-temperature pressing is 60-90°C.

[0014] Preferably, the aperture of the sieve used for the crushing and screening in step (1) is 0.25-0.35 mm, and the method for removing residual oil includes any one of supercritical CO2 extraction, subcritical fluid extraction and Soxhlet extraction.

[0015] Preferably, in step (2), the mixing ratio of the defatted oil meal and water is 1 g: 8-15 mL, the reagent for adjusting the pH value is NaOH, and the stirring time is 1.5-2.5 h.

[0016] Preferably, the centrifugal speed in step (3) is 8000-12000 g, the centrifugal time is 25-35 min, the centrifugal temperature is 4-25° C., and the pH value of the supernatant is 8-10.

[0017] Preferably, in the weak base synchronous ultrafiltration precise scale control method in step (3), the pH of the weak base is 8 to 10, and the ultrafiltration uses a membrane with a molecular weight range of 5 to 30 KDa to precisely control the scale. The types of the membrane include membrane or PES membrane.

[0018] Preferably, the flow rate during the concentration and purification process in step (3) is 80 to 120 mL / min.

[0019] Preferably, the stirring time in step (4) is 25 to 35 minutes.

[0020] Preferably, the freeze-drying temperature in step (5) is -65 to -85°C, and the freeze-drying time is 12 to 72 hours.

[0021] The present invention also provides high-quality oil protein isolate prepared by the above preparation method.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. Extraction advantages:

[0024] (1) The extraction process of the present invention is relatively simple, green and efficient. Compared with the alkali dissolution and acid precipitation method and the salting-out method, the weak base synchronous ultrafiltration precise control scale technology covered by the present invention avoids the use of strong acids and strong bases to a great extent, and is more in line with the green and sustainable development concept.

[0025] (2) High protein yield. That is, the protein purity of the soybean protein isolate (MS-SPI) of Examples 1 to 3 of the present invention is as high as 83.82%, and the protein yield (protein recovery rate) is 1.5 to 2.8 times that of traditional protein extraction technologies (alkali dissolution and acid precipitation method, salting out method); the protein purity of the flaxseed protein isolate (MS-FPI) of Examples 4 to 6 is as high as 94.06%, and the protein yield is 2 to 3 times that of the alkali dissolution and acid precipitation method.

[0026] 2. Advantages of functional characteristics:

[0027] (1) The oil protein isolate of the present invention has excellent emulsification properties. The emulsification stability of soybean protein isolate (MS-SPI) of Examples 1 to 3 of the present invention is as high as 252.41 min, which is 2.43 times that of comparative example 1 (AE-SPI) and 3.82 times that of comparative example 2 (SE-SPI). The emulsification stability of Examples 4 to 6 of the present invention (MS-FPI) is as high as 164.42 min, which is 1.66 times that of comparative example 3 (AE-SPI). Compared with comparative example 4 (AE-PPI), Example 7 (MS-PPI) also has the best emulsification properties.

[0028] (2) The oil protein isolate of the present invention has excellent foaming properties. The foaming properties of the soybean protein isolate (MS-SPI) of Examples 1 to 3 of the present invention are as high as 203.33%, the foaming properties of the flaxseed protein isolate (MS-FPI) of Examples 4 to 6 of the present invention are as high as 202.22%, and the foaming properties of the perilla seed protein isolate (MS-PPI) of Example 7 are as high as 101.67%, which are significantly better than the oil protein isolates obtained in Comparative Examples 1 to 4. In addition, the MS-FPI of the present invention has better thermo-gelling properties than the AE-FPI obtained by conventional extraction methods, and a better gelling effect can be achieved using a lower protein concentration.

[0029] (3) The oil protein isolate of the present invention has excellent solubility. The soy protein isolate (MS-SPI) of Examples 1 to 3 of the present invention has the best solubility of 83.06%. The solubility of the flaxseed protein isolate (MS-FPI) of Examples 4 to 6 of the present invention is as high as 89.59%, and the solubility of Example 7 (MS-PPI) is as high as 93.71%, which is 1.5 to 2.5 times the solubility of the protein of Comparative Examples 1 to 4.

[0030] (4) Advantages of nutritional properties:

[0031] The oil protein isolates extracted by the present invention have excellent protein digestibility. The digestibility of the three oil protein isolates obtained in Examples 1 to 7 is significantly better than that of the same type of oil protein isolates obtained in Comparative Examples 1 to 4, up to 1.2 to 4.3 times.

[0032] (5) Advantages in flavor:

[0033] The oil protein isolate extracted by the present invention exhibits excellent flavor characteristics. Compared with Comparative Examples 1 and 2, the soy protein isolate (MS-SPI-1) prepared in Example 1 significantly reduces odorous volatile compounds, significantly improves beany and other irritating odors, and has relatively high levels of aromatic hydrocarbons and esters, giving it a good aromatic flavor profile. The flaxseed protein isolate (MS-FPI-1) prepared in Example 4 also significantly reduces odorous volatile components compared to Comparative Example 3, and has a relatively high content of esters, giving MS-FPI-1 a unique aroma and significantly improving unpleasant mushroom and earthy odors. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0035] Figure 1 is a flow chart of the preparation process of the present invention;

[0036] Figure 2 The gel effect of flaxseed protein isolate products obtained by different methods. DETAILED DESCRIPTION

[0037] The present invention provides a green and efficient method for preparing high-quality oil protein isolate with high yield, comprising the following steps:

[0038] (1) The oil is subjected to low-temperature pressing, crushing, screening and residual oil removal in sequence to obtain defatted oil meal;

[0039] (2) mixing the defatted oilseed meal with water, adjusting and maintaining the pH value to 8 to 10, and stirring to obtain a mixed solution;

[0040] (3) centrifuging the mixed solution, collecting the supernatant, and concentrating and purifying the supernatant to 1 / 6 to 1 / 8 of the original volume using a weak base synchronous ultrafiltration precision control scale method to obtain a highly concentrated solution;

[0041] (4) adding water to the highly concentrated solution at a volume ratio of 1:1 to 3 at a pH of 8 to 10 for redissolution, stirring to obtain a redissolution;

[0042] (5) freeze-drying the reconstituted solution to obtain high-quality oil protein isolate.

[0043] In the present invention, the types of oil in step (1) preferably include soybean, linseed and perilla, and the temperature of the low-temperature pressing is preferably 60-90°C, more preferably 90°C.

[0044] In the present invention, the aperture of the sieve used for the crushing and screening in step (1) is preferably 0.25-0.35 mm, more preferably 0.28-0.32 mm, and even more preferably 0.3 mm; the method for removing residual oil preferably includes any one of supercritical CO2 extraction, subcritical fluid extraction and Soxhlet extraction, and is more preferably Soxhlet extraction.

[0045] In the present invention, in step (2), the defatted oilseed meal is mixed with water, the pH value is adjusted and maintained at 8 to 10, and the mixture is stirred to obtain a mixed solution. More preferably, the defatted oilseed meal is mixed with water, the pH value is adjusted and maintained at 9, and the mixture is stirred to obtain a mixed solution. The purpose of adjusting the pH value in this step is to ensure sufficient dissolution of protein in the defatted oilseed meal.

[0046] In the present invention, the mixing ratio of the defatted oil meal and water in step (2) is preferably 1 g:8-15 mL, more preferably 1 g:9-12 mL, and even more preferably 1 g:10 mL; the stirring time is preferably 1.5-2.5 h, more preferably 1.8-2.2 h, and even more preferably 2 h; and the reagent for adjusting the pH value is preferably NaOH.

[0047] In the present invention, the mixed solution is centrifuged in step (3), the supernatant is collected, and the supernatant is concentrated and purified by a weak base synchronous ultrafiltration precise scale control method, and concentrated to 1 / 6 to 1 / 8 of the original volume to obtain a highly concentrated solution. It is further preferred that the mixed solution is centrifuged, the supernatant is collected, and the supernatant is concentrated and purified by a weak base synchronous ultrafiltration precise scale control method, and concentrated to 1 / 7 of the original volume to obtain a highly concentrated solution.

[0048] In the present invention, the centrifugal speed in step (3) is preferably 8000-12000g, more preferably 9000-11000g, and even more preferably 10000g; the centrifugal time is preferably 25-35min, more preferably 28-32min, and even more preferably 30min; the centrifugal temperature is preferably 3-25°C, and even more preferably 4°C; the pH value of the supernatant is preferably 8-10, and even more preferably 9.

[0049] In the present invention, the pH of the weak base in the weak base synchronous ultrafiltration precise scale control method in step (3) is preferably 8 to 10, more preferably 9, and the ultrafiltration preferably uses a membrane with a molecular weight range of 5 to 30 KDa to precisely control the scale, and the types of the membrane preferably include membrane or PES membrane, more preferably membrane.

[0050] In the present invention, the flow rate in the concentration and purification process in step (3) is preferably 80 to 120 mL / min, more preferably 90 to 110 mL / min, and even more preferably 100 mL / min.

[0051] In the present invention, in step (4), the highly concentrated solution is redissolved in water at a volume ratio of 1:1 under the condition of a pH value of 8 to 10, and stirred to obtain a reconstituted solution. More preferably, the highly concentrated solution is redissolved in water at a volume ratio of 1:1 under the condition of a pH value of 9, and stirred to obtain a reconstituted solution.

[0052] In the present invention, the stirring time in step (4) is preferably 25 to 35 minutes, more preferably 28 to 32 minutes, and even more preferably 30 minutes.

[0053] In the present invention, the freeze-drying temperature in step (5) is preferably -65 to -85°C, more preferably -70 to 80°C, and even more preferably -75°C; the freeze-drying time is 12 to 72 hours, more preferably 24 to 60 hours, and even more preferably 48 hours.

[0054] The present invention also provides the oil protein isolate obtained by the extraction method.

[0055] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] The oilseed varieties used in the following examples and comparative examples are: southern soybean, variety: You 6019#; flaxseed, variety: Ningya 21#; perilla, variety: white perilla seed.

[0057] The experimental methods used in the following examples and comparative examples are as follows:

[0058] Determination of protein content:

[0059] The protein content of the examples and comparative examples was determined by the Kjeldahl method, and the nitrogen conversion coefficient was determined according to the national standard GB5009.5-2016.

[0060] The calculation formula for protein yield is:

[0061] (1) Protein extraction rate (%) = (protein mass / cake mass) × 100%

[0062] (2) Protein yield (%) = [(protein mass × protein content) / (meal mass × protein content)] × 100%

[0063] Functional characterization of proteins:

[0064] (1) Solubility determination

[0065] Dilute the protein sample with ultrapure water to a 1 mg / mL test solution and centrifuge at 4000 rpm for 10 min. Collect the supernatant and determine the protein concentration using the Bradford assay with BSA as the standard protein.

[0066] Solubility (%) = [(supernatant protein content × 100) / total protein content of sample before centrifugation] × 100%

[0067] (2) Determination of emulsification and emulsion stability

[0068] 1% (w / v) protein solutions were prepared using the different treatments. 15 mL of sample was homogenized with 5 mL of walnut oil at 14,000 rpm for 2 min. The freshly prepared emulsion (50 μL) was mixed with 5 mL of 0.1% SDS at 500 nm and stored at room temperature for 10 min. The absorbance (A10) was then measured again.

[0069] The calculation formulas for emulsification (EAI) and emulsion stability (ESI) are as follows:

[0070] EAI(m 2 / g) = (2 × 2.303 × A0 × DF) / [θ × C × 10000]

[0071] ESI(min)=(A0×ΔT) / ΔA×100%

[0072] Where DF is the dilution factor; C is the protein concentration, mg / mL; θ is the oil volume fraction; ΔT = 10 min, ΔA is the ratio of A0 to A 10 difference.

[0073] (3) Determination of foaming properties and foam stability

[0074] 15 mL of the prepared protein solution was stirred at 14,000 rpm for 2 minutes using an IKA high-speed homogenizer. Immediately after stirring, the foam was poured into a 50 mL graduated cylinder, and the foam volume at 2 and 60 minutes was recorded to calculate the foaming activity (FA) and foam stability (FS).

[0075] FA (%) = V2 / 15 × 100

[0076] FS(%)=V 60 / V2×100

[0077] Where V2 is the foam volume in 2 minutes, V 60 is the foam volume at 60 min.

[0078] (4) Gel property determination

[0079] The protein concentrations were 1%, 3%, 5%, 7%, and 10%. The protein dispersions in the test tubes were heated in a 95°C water bath for 30 min, immediately cooled in an ice water bath, and stored at 4°C for approximately 24 h before being taken out for photographic observation.

[0080] (5) Determination of digestibility

[0081] The digestibility was determined by referring to the method reported by Brodkorb et al. in the report “INFOGEST static in vitro simulation of gastrointestinal food digestion”.

[0082] (6) Determination of volatile compounds

[0083] The volatile components were determined according to the method reported by Liao et al. in the report "Effects of "nine steaming nine sun-drying" on proximate composition, protein structure and volatile compounds of black soybeans".

[0084] Example 1

[0085] First, the soybeans were low-temperature pressed (CA59G, Gomet, Germany) and crushed and sieved (standard sieve, pore size 0.3 mm) to obtain low-temperature pressed soybean meal; then, the residue was further removed by Soxhlet extraction for 8 h (the extraction solvent was n-hexane / petroleum ether) to obtain defatted soybean meal. The defatted soybean meal was dispersed in ultrapure water at a ratio of 1:10 (w / v), stirred at room temperature (25 ° C) for 2 h, and the pH value was adjusted to 9.0 with NaOH to ensure sufficient dissolution of the protein in the defatted soybean meal. Next, it was centrifuged at a speed of 10000g for 30 min to remove insoluble impurities and collect the protein-rich supernatant. It was then transferred to the sample chamber of the membrane separation device and the weak base synchronous ultrafiltration method (MWCO 5KDa) was used to precisely control the scale. The supernatant was concentrated and purified using a filtration membrane at a flow rate of 100 mL / min to 1 / 8 of the total volume. Subsequently, the concentrate was reconstituted at a pH of 9.0 at a volume ratio of 1:1 (stirring for 30 minutes until complete dissolution). Finally, the resulting solution was freeze-dried to obtain soy protein isolate (MS-SPI-1).

[0086] Example 2

[0087] First, soybeans were cryogenically pressed (CA59G, Komet, Germany) and pulverized and sieved (standard sieve, 0.3 mm pore size) to obtain cryogenically pressed soybean meal. Defatted soybean meal was then extracted using a Soxhlet apparatus (with n-hexane / petroleum ether) for 8 h to further remove residual oil. The defatted soybean meal was then dispersed in ultrapure water at a ratio of 1:15 (w / v), stirred at room temperature (25°C) for 2 h, and the pH was adjusted to 8.0 with NaOH to ensure sufficient dissolution of the defatted soybean meal protein. Next, the mixture was centrifuged at 8000 g for 30 min to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation apparatus and concentrated and purified using a weak base simultaneous ultrafiltration method (PES membrane with a MWCO of 10 kDa) with a precise scale control method at a flow rate of 100 mL / min to a concentration of 1 / 7 of the total volume. Subsequently, the concentrate was redissolved at a volume ratio of 1:1 under a pH of 9.0 (stirred for 30 minutes until completely dissolved). Finally, the resulting solution was freeze-dried to obtain soy protein isolate (MS-SPI-2).

[0088] Example 3

[0089] First, soybeans were cryogenically pressed (CA59G, Komet, Germany) and pulverized and sieved (standard sieve, 0.3 mm pore size) to obtain cryogenically pressed soybean meal. Defatted soybean meal was then extracted using a Soxhlet apparatus (n-hexane / petroleum ether) for 8 h to further remove residual oil. The defatted soybean meal was then dispersed in ultrapure water at a ratio of 1:8 (w / v), stirred at room temperature (25°C) for 2 h, and the pH was adjusted to 10.0 with NaOH to ensure sufficient dissolution of the defatted soybean meal protein. Next, the mixture was centrifuged at 12,000 g for 30 min to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation apparatus and concentrated and purified using a weak base simultaneous ultrafiltration method (PES membrane with a MWCO of 30 kDa) with a precise scale control method at a flow rate of 100 mL / min to a concentration of 1 / 6 of the total volume. Subsequently, the concentrate was redissolved at a volume ratio of 1:3 under a pH of 9.0 (stirred for 30 minutes until completely dissolved). Finally, the resulting solution was freeze-dried to obtain soy protein isolate (MS-SPI-3).

[0090] Example 4

[0091] First, the flax seeds were washed in a 50°C water bath to remove linseed gum and then dried at 50°C. The flax seeds were then cryogenically pressed, crushed, and sieved to obtain cryogenically pressed flaxseed meal. Supercritical CO2 extraction (SCCO2 extraction: cryogenically pressed meal was placed in an extraction tank. The extraction temperature and pressure were set at 42°C and 30 MPa, the extraction time was 1 hour, and the CO2 flow rate was 18 L / h (separation tank I: 10 MPa, 60°C, separation tank II: the same pressure as the CO2 storage tank, 35°C)) was then used to further remove residual oil and obtain defatted flaxseed meal. The defatted flaxseed meal was then dispersed in ultrapure water at a ratio of 1:15 (w / v), stirred at room temperature (25°C) for 2 hours, and the pH was adjusted to 9.0 with NaOH to ensure sufficient dissolution of the protein in the defatted flaxseed meal. Next, the mixture was centrifuged at 8000 g for 30 minutes to remove insoluble impurities and collect the protein-rich supernatant. Then, it was transferred to the sample chamber of the membrane separation device and the weak base synchronous ultrafiltration method (MWCO 5KDa) was used to precisely control the scale. The supernatant was concentrated and purified using a filtration membrane at a flow rate of 100 mL / min to 1 / 8 of the total volume. Subsequently, the concentrate was reconstituted at a pH of 9.0 at a 1:1 volume ratio (stirring for 30 minutes until complete dissolution). Finally, the resulting solution was freeze-dried to obtain flaxseed protein isolate (MS-FPI-1).

[0092] Example 5

[0093] First, the flax seeds were washed in a 50°C water bath to remove linseed gum and then dried at 50°C. The flax seeds were then cryogenically pressed, crushed, and sieved to obtain cryogenically pressed flaxseed meal. Supercritical CO2 extraction (SCCO2 extraction: cryogenically pressed meal was placed in an extraction tank. The extraction temperature and pressure were set at 42°C and 30 MPa, the extraction time was 1 hour, and the CO2 flow rate was 18 L / h (separation tank I: 10 MPa, 60°C, separation tank II: the same pressure as the CO2 storage tank, 35°C)) was then used to further remove residual oil and obtain defatted flaxseed meal. The defatted flaxseed meal was then dispersed in ultrapure water at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2 hours, and the pH was adjusted to 8.0 with NaOH to ensure sufficient dissolution of the protein in the defatted flaxseed meal. Next, the mixture was centrifuged at 10,000 g for 30 minutes to remove insoluble impurities and collect the protein-rich supernatant. The supernatant was then transferred to the sample chamber of a membrane separation device and concentrated and purified using a weak base simultaneous ultrafiltration precision control scale method (PES membrane with a MWCO of 10 kDa) at a flow rate of 100 mL / min to a concentration of 1 / 7 of the total volume. Subsequently, the concentrate was reconstituted at a pH of 9.0 at a volume ratio of 1:1 (stirring for 30 minutes until completely dissolved). Finally, the resulting solution was freeze-dried to obtain flaxseed protein isolate (MS-FPI-2).

[0094] Example 6

[0095] First, the flax seeds were washed in a 50°C water bath to remove linseed gum and then dried at 50°C. The flax seeds were then cryogenically pressed, crushed, and sieved to obtain cryogenically pressed flaxseed meal. Supercritical CO2 extraction (SCCO2 extraction: cryogenically pressed meal was placed in an extraction tank. The extraction temperature and pressure were set at 42°C and 30 MPa, the extraction time was 1 hour, and the CO2 flow rate was 18 L / h (separator tank I: 10 MPa, 60°C, separator tank II: the same pressure as the CO2 storage tank, 35°C)) was then used to further remove residual oil and obtain defatted flaxseed meal. The defatted flaxseed meal was then dispersed in ultrapure water at a ratio of 1:8 (w / v), stirred at room temperature (25°C) for 2 hours, and the pH was adjusted to 10.0 with NaOH to ensure sufficient dissolution of the protein in the defatted flaxseed meal. Next, the mixture was centrifuged at 12,000 g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. Then, it was transferred to the sample chamber of the membrane separation device, and the supernatant was concentrated and purified using a weak base synchronous ultrafiltration precision control scale method (PES membrane with MWCO 30KDa) with a flow rate of 100mL / min and concentrated to 1 / 6 of the total volume. Subsequently, the concentrate was redissolved at a volume ratio of 1:3 under pH 9.0 (stirred for 30 minutes until completely dissolved). Finally, the resulting solution was freeze-dried to obtain flaxseed protein isolate (MS-FPI-3).

[0096] Example 7

[0097] First, the perilla seeds were low-temperature pressed and crushed and sieved to obtain low-temperature pressed perilla seed meal; then, subcritical fluid extraction was used (subcritical fluid extraction: the low-temperature pressed meal was placed in an extraction tank. The material-solvent ratio was 1:3, the extraction solvent was n-butane, and the extraction temperature was set at 45°C. Soak for 15 minutes to drain the mixed oil into a tank. After seven soaks, the compressor and vacuum pump were used to desolventize to a vacuum below -0.08MPa, and then the tank was opened to take out the meal) to further remove the residual oil and obtain defatted perilla seed meal. The defatted perilla seed meal was dispersed in ultrapure water at a ratio of 1:15 (w / v), stirred at room temperature (25°C) for 2h, and the pH value was adjusted to 9.0 with NaOH to ensure sufficient dissolution of the protein in the defatted perilla seed meal. Next, centrifuge at a speed of 10,000g for 30 minutes to remove insoluble impurities and collect the protein-rich supernatant. Then, it was transferred to the sample chamber of the membrane separation device, and the weak base synchronous ultrafiltration method (MWCO 5KDa) was used to precisely control the scale. The supernatant was concentrated and purified using a membrane (filtered with a flow rate of 100 mL / min) to 1 / 8 of the total volume. Subsequently, the concentrate was redissolved at a pH of 9.0 at a volume ratio of 1:1 (stirring for 30 minutes until complete dissolution). Finally, the resulting solution was freeze-dried to obtain perilla protein isolate (MS-PPI).

[0098] Comparative Example 1

[0099] Soy protein isolate (AE-SPI) was obtained by alkali dissolution and acid precipitation. First, soybeans were cryo-pressed (CA59G, Komet, Germany) and pulverized and sieved (standard sieve, 0.3 mm pore size) to obtain cryo-pressed soybean meal. Defatted soybean meal was then extracted using a Soxhlet apparatus (n-hexane / petroleum ether) for 8 h to further remove residual oil. The defatted soybean meal was then dispersed in ultrapure water at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2 h, and the pH was adjusted to 9.0 with 2 M NaOH. The mixture was then centrifuged at 10,000 g for 30 min to remove insoluble impurities, and the supernatant was collected. The pH was then adjusted to 4.5. Centrifugation was repeated under the same conditions, and the precipitate was collected and reconstituted in ultrapure water at a ratio of 1:5 (w / v) at pH 7.0. Finally, the resulting solution was freeze-dried to obtain soy protein isolate (AE-SPI).

[0100] Comparative Example 2

[0101] Soy protein isolate (SE-SPI) was obtained by the salting-out method. First, soybeans were cryo-pressed (CA59G, Komet, Germany) and pulverized and sieved (standard sieve, 0.3 mm pore size) to obtain cryo-pressed soybean meal. Defatted soybean meal was then extracted for 8 h using a Soxhlet apparatus (n-hexane / petroleum ether) to further remove residual oil. The defatted soybean meal was dissolved in a 0.3 M CaCl₂ solution at a ratio of 1:10 (w / v), and the pH was adjusted to 5.6 with 2 M NaOH. The solution was then centrifuged at 10,000 g for 30 min to remove insoluble impurities, and the supernatant was collected. The solution was then dialyzed against ultrapure water at 4°C for 48 h (conductivity: ~30 μS / cm). Finally, the resulting solution was freeze-dried to obtain the soy protein isolate (SE-SPI).

[0102] Comparative Example 3

[0103] Flaxseed protein isolate (AE-FPI) was obtained by alkali dissolution and acid precipitation. First, the flaxseeds were washed in a 50°C water bath to remove linseed gum and then dried at 50°C. The flaxseeds were then cryogenically pressed, crushed, and sieved to obtain cryogenically pressed flaxseed meal. Supercritical CO2 extraction (SCCO2 extraction: cryogenically pressed meal was placed in an extraction tank. The extraction temperature and pressure were set at 42°C and 30 MPa, the extraction time was 1 hour, and the CO2 flow rate was 18 L / h (separator tank I: 10 MPa, 60°C; separator tank II: the same pressure as the CO2 storage tank, 35°C)) was used to further remove residual oil and obtain defatted flaxseed meal. Defatted flaxseed meal was then dispersed in ultrapure water at a ratio of 1:15 (w / v), stirred at room temperature (25°C) for 2 hours, and the pH was adjusted to 9.0 with 2 M NaOH. The mixture was then centrifuged at 10,000 g for 30 minutes to remove insoluble impurities, and the supernatant was collected. Subsequently, the pH was adjusted to 4.2. Centrifugation was repeated under the same conditions, and the precipitate was collected and reconstituted in ultrapure water at a pH of 7.0 at a ratio of 1:5 (w / v). Finally, the resulting solution was freeze-dried to obtain flaxseed protein isolate (AE-FPI).

[0104] Comparative Example 4

[0105] Perilla seed protein isolate (AE-PPI) was obtained by alkali dissolution and acid precipitation. First, perilla seeds were cryogenically pressed, crushed, and sieved to obtain cryogenically pressed perilla seed meal. Subcritical fluid extraction (SFE) was then used to further remove residual oil and obtain defatted perilla seed meal. (Subcritical fluid extraction: The cryogenically pressed meal was placed in an extraction tank with a material-to-solvent ratio of 1:3, n-butane as the extraction solvent, and the extraction temperature set at 45°C. The mixture was soaked for 15 minutes, and the mixed oil was drained into the tank. After seven soaks, the mixture was desolventized using a compressor and a vacuum pump to a vacuum below -0.08 MPa, and the tank was opened and the meal was removed.) The defatted perilla seed meal was dispersed in ultrapure water at a ratio of 1:15 (w / v), stirred at room temperature (25°C) for 2 hours, and the pH was adjusted to 9.0 with 2M NaOH. The mixture was then centrifuged at 10,000 g for 30 minutes to remove insoluble impurities, and the supernatant was collected. The pH was then adjusted to 4.5. The mixture was centrifuged again under the same conditions, and the precipitate was collected and reconstituted in ultrapure water with a pH of 7.0 at a ratio of 1:5 (w / v). Finally, the resulting solution was freeze-dried to obtain perilla seed protein isolate (AE-PPI).

[0106] Experimental Example 1

[0107] Taking the soy protein obtained in Examples 1 to 3 and Comparative Examples 1 to 2 as examples, the functional effects, soy protein content, protein extraction rate, and protein yield of the protein products obtained by different methods were studied. The results are shown in Tables 1, 2, and 3.

[0108] Table 1 Content, protein extraction rate and recovery rate of soy protein isolate obtained by different methods

[0109]

[0110] Note: Different letters (ae) in the same column indicate significant differences (P<0.05).

[0111] As shown in Table 1, the protein content of the soy protein isolate prepared by Examples 1 to 3 of the present invention is at a relatively high level, which meets the requirements of protein concentrate or protein isolate in domestic commercial protein products. The protein conversion coefficient for soy protein products in China is 6.25, and the protein conversion coefficient used in other countries is also mostly 6.25. The protein content data in Table 1 are converted by N×6.25. The protein content of Examples 1 to 3 is 80.00~83.82%, which can be used for comparison of the protein content of soy protein related products at home and abroad. The protein yield in Comparative Example 1 obtained by alkali dissolution and acid precipitation is 37.63%, the protein yield in Comparative Example 2 obtained by salting out is 27.43%, and the protein yield in Examples 1 to 3 can reach up to 77.57%, which is 2.06 times and 2.8 times that of alkali dissolution and acid precipitation and salting out, respectively, much higher than traditional methods and has higher promotion value.

[0112] Table 2 Functional effects of soy protein isolate products obtained by different methods

[0113]

[0114]

[0115] Note: Different letters (ae) in the same column indicate significant differences (P<0.05).

[0116] As shown in Table 2, Examples 1 to 3 have good functional properties and nutritional values:

[0117] (1) The proteins in Examples 1 to 3 of the present invention have the best foaming properties. The emulsifying properties of Examples 1 to 3 are slightly lower than those of Comparative Example 1, but significantly higher than those of Comparative Example 2. In addition, the emulsifying stability is significantly better than that of the Comparative Example, reaching a maximum of 252.41 min, which is 2.43 times and 3.82 times that of Comparative Example 1 and Comparative Example 2, respectively.

[0118] (2) The proteins in Examples 1 to 3 of the present invention have the best foaming properties. The foaming properties of Examples 1 to 3 are as high as 232.22%, which is 1.60 times that of the comparative example, and have relatively good foam stability.

[0119] (3) The proteins in Examples 1 to 3 of the present invention have the best solubility. The solubility of Examples 1 to 3 is as high as 83.06%, which is higher than that of Comparative Example 1 and 1.56 times that of Comparative Example 2.

[0120] (4) The proteins in Examples 1 to 3 of the present invention had the best digestibility. The digestibility of Examples 1 to 3 was as high as 86.16%, which was 1.29 times that of Comparative Example 1 and 2.81 times that of Comparative Example 2.

[0121] The flavor profile of soy plays a crucial role in the acceptance and preference of soy protein products. SPME-GCMS analysis identified 43, 69, and 38 volatile compounds in Comparative Example 1 (AE-SPI), Comparative Example 2 (SE-SPI), and Example 1 (MS-SPI-1), respectively. The classification of these volatile components is detailed in Tables 3 and 4.

[0122] Table 3 Volatile compounds of soy protein isolate products obtained by different methods

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] Note: “-” indicates that the volatile compound was not detected, the same below.

[0131] Table 4 Categories of volatile compounds of soy protein isolate products obtained by different methods

[0132]

[0133] As shown in Tables 3 and 4, compared to Comparative Example 1 (AE-SPI) or Comparative Example 2 (SE-SPI), Example 1 (MS-SPI-1) significantly reduced the volatile components by 5 or 31 respectively. The primary volatile compounds in Comparative Example 1 (AE-SPI) and Example 1 (MS-SPI-1) were aldehydes, alcohols, and esters, while the primary volatile compounds in Comparative Example 2 (SE-SPI) were alcohols and acids. Compared to Comparative Example 1 (AE-SPI), the relative content of aldehydes in Example 1 (MS-SPI-1) was significantly reduced by 14.97%, significantly reducing beany and other irritating odors. Meanwhile, the relatively high content of aromatic hydrocarbons and esters imparted a unique aroma to Example 1 (MS-SPI-1). Compared to Comparative Example 2 (SE-SPI), the relative content of acids in Example 1 (MS-SPI-1) was reduced by 53.91%, and the relative content of alcohols was reduced by 73.04%, significantly reducing the unpleasant mushroom and earthy odors. Therefore, compared with Comparative Examples 1 and 2, the beany smell and other unpleasant odors of Example 1 are significantly improved, and the Example 1 has good aromatic flavor characteristics.

[0134] Experimental Example 2

[0135] Taking the flaxseed protein isolate obtained in Examples 4 to 6 and Comparative Example 3 as an example, the functional effects, flaxseed protein content, protein extraction rate and recovery rate of the protein products obtained by different methods were studied. The results are shown in Tables 5, 6 and Figure 2 shown.

[0136] Table 5 Content, protein extraction rate and recovery rate of flaxseed protein isolate obtained by different methods

[0137]

[0138] Note: Different letters (ac) in the same column indicate significant differences (P<0.05).

[0139] Table 6 Functional effects of flaxseed protein isolate products obtained by different methods

[0140]

[0141] Note: Different letters (ad) in the same column indicate significant differences (P<0.05).

[0142] As shown in Table 6, Examples 4 to 6 have the best functional properties and nutritional value. The emulsification, foaming properties, solubility and digestibility of Examples 4 to 6 are significantly better than the flaxseed protein in Comparative Example 3.

[0143] Depend on Figure 2It can be seen that the flaxseed protein MS-FPI of Examples 4 to 6 of the present invention can achieve better gelling effect by using lower protein concentration, which is far superior to the gelling effect of the comparative example AE-FPI.

[0144] The flavor profile of flax plays a crucial role in the acceptance and preference of flax protein products. SPME-GCMS analysis identified 41 and 44 volatile compounds in Comparative Example 3 (AE-FPI) and Example 4 (MS-FPI-1), respectively. The classification of these volatile components is detailed in Table 7.

[0145] Table 7 Volatile components of flaxseed protein isolate products obtained by different methods

[0146]

[0147]

[0148]

[0149] Note: “-” means the volatile compound was not detected.

[0150] Table 8 Categories of volatile compounds of soy protein isolate products obtained by different methods

[0151] Compound (%) Comparative Example 3 (AE-FPI) Example 4 (MS-FPI-1) hydrocarbon compounds 7.02(8) 5.24(6) Ester compounds 17.11(8) 41.94(4) Alcohol compounds 58.98(12) 39.1(14) Aldehyde compounds 0(0) 4.12(5) Ketone compounds 1.35(1) 5.45(6) Acid compounds 11.59(10) 13.46(7) Phenols 10.06(2) 8.31(2) total 41 44

[0152] As shown in Tables 7 and 8, Example 4 (MS-FPI-1) significantly reduced the volatile components by three compared to Comparative Example 3 (AE-FPI). The primary volatile compounds in Comparative Examples 3 and 4 were alcohols and acids. Compared to Comparative Example 3, the flaxseed protein isolate obtained in Example 4 had a relatively high content of esters, imparting a distinctive aroma to MS-FPI-1. The reduced relative content of alcohols significantly reduced the unpleasant mushroom and earthy odors. Therefore, compared to Comparative Example 1, Example 1 exhibited significantly improved odor and possessed a favorable aromatic flavor profile.

[0153] Experimental Example 3

[0154] Taking the perilla protein obtained in Example 7 and Comparative Example 4 as examples, the functional effects, perilla protein content, protein extraction rate, and recovery rate of the protein products obtained by different methods were studied. The results are shown in Tables 9 and 10.

[0155] Table 9 Content, protein extraction rate and recovery rate of perilla protein isolate obtained by different methods

[0156]

[0157] Note: Different letters (ab) in the same column indicate significant differences (P<0.05).

[0158] Table 10 Functional effects of perilla seed protein isolate products obtained by different methods

[0159]

[0160] Note: Different letters (ab) in the same column indicate significant differences (P<0.05).

[0161] As shown in Tables 9 and 10, the perilla protein isolate obtained in Example 7 has the highest protein yield and the best functional properties and nutritional value. The emulsification, foaming properties, solubility and digestibility of Example 7 are significantly better than those of the perilla protein isolate in Comparative Example 4.

[0162] In summary, the weak base synchronous ultrafiltration precise size control technology encompassed by the present invention has broken through the method of not using acid precipitation after the alkali dissolution step, avoided the aggregation of proteins near the isoelectric point, and endowed the oil plant isolate protein with relatively flexible structural characteristics. It also effectively removed small molecules such as polyphenols, soluble dietary fiber, and inorganic salts, thereby achieving the concentration and purification of the oil plant isolate protein. Compared with oil plant isolate protein extraction technologies such as traditional alkali dissolution, acid precipitation, and salting out, the oil plant isolate protein extracted using the process of the present invention has the advantages of high protein yield, environmental friendliness, high functional properties, high digestibility, and good flavor.

[0163] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A green and efficient method for preparing high-quality oil protein isolate with high yield, characterized in that: The steps include: (1) The oil is subjected to low-temperature pressing, crushing, screening and residual oil removal in sequence to obtain defatted oil meal; (2) mixing the defatted oilseed meal with water, adjusting and maintaining the pH value to 8 to 10, and stirring to obtain a mixed solution; (3) centrifuging the mixed solution, collecting the supernatant, and concentrating and purifying the supernatant to 1 / 6 to 1 / 8 of the original volume using a weak base synchronous ultrafiltration precision control scale method to obtain a highly concentrated solution; (4) adding water to the highly concentrated solution at a volume ratio of 1:1 to 3 at a pH of 8 to 10 for redissolution, stirring to obtain a redissolution; (5) freeze-drying the reconstituted solution to obtain high-quality oil protein isolate; In step (1), the temperature of the low-temperature pressing is 60 to 90° C.; In step (1), the method for removing residual oil includes any one of supercritical CO2 extraction, subcritical fluid extraction and Soxhlet extraction; In step (3), the pH of the weak base in the weak base synchronous ultrafiltration precision control scale method is 8-10, and the ultrafiltration uses a membrane with a molecular weight of 5KDa, 10KDa or 30KDa to precisely control the scale. The types of the membrane include Hydrosart ® membrane or PES membrane; The freeze-drying temperature in step (5) is -65 to -85°C, and the freeze-drying time is 12 to 72 hours; The types of oils in step (1) include soybeans, flaxseeds and perilla; In step (2), the mixing ratio of the defatted oil meal and water is 1 g: 8-15 mL, the reagent for adjusting and maintaining the pH value is NaOH, and the stirring time is 1.5-2.5 h; The stirring time in step (4) is 25 to 35 minutes.

2. The green and efficient method for preparing high-quality oil protein isolate with high yield according to claim 1, characterized in that: The aperture of the sieve used for the crushing and screening in step (1) is 0.25 to 0.35 mm.

3. The green and efficient method for preparing high-quality oil protein isolate with high yield according to claim 1, characterized in that: The centrifugal speed in step (3) is 8000-12000 g, the centrifugal time is 25-35 min, the centrifugal temperature is 4-25° C., and the pH value of the supernatant is 8-10.

4. The green and efficient method for preparing high-quality oil protein isolate with high yield according to claim 1, characterized in that: The flow rate during the concentration and purification process in step (3) is 80 to 120 mL / min.

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