Pea complete protein component and preparation and identification method thereof

Abstract

The invention relates to the technical field of food, and provides a pea complete protein component and a preparation and identification method thereof.The preparation method of the complete protein comprises the steps that pea kernel powder and water are mixed and then subjected to first solid-liquid separation, and first clear liquid is obtained; adjusting the pH value of the first clear liquid to 5.8-6.2 to obtain a protein mixed liquid; adding lactic acid bacteria into the protein mixed solution, and standing for 1-3 hours at the temperature of 10 DEG C or below for layering; removing a bottom layer material of the layered material to obtain a residual material, and carrying out second solid-liquid separation on the residual material to collect a precipitate; performing first drying on the precipitate to obtain protein isolate; mixing the separated protein and water, and performing third solid-liquid separation to obtain a second clear liquid; and separating the second clear liquid through an ultrafiltration membrane, and performing secondary drying on the obtained percolate to obtain albumin, namely the complete protein. The method disclosed by the invention is green and environment-friendly, and the extracted complete protein is good in balance and meets nutritional requirements.

Description

Technical Field

[0001] The present invention relates to the field of food technology, and particularly to a pea full-value protein component and its preparation and identification methods. Background Art

[0002] As an important food crop, pea is the second largest edible legume crop in China and has important economic and agricultural value. The planting area and annual output of peas in China are both at a relatively high level, and its harvested area continues to grow. The extensive cultivation of peas effectively alleviates the resource bottleneck brought about by the soybean crisis and can reduce the risks brought about by international market fluctuations, providing important support for ensuring national food security and building a diversified food supply system.

[0003] Although peas contain rich nutritional components, their current application fields are relatively limited, mainly concentrated in the production of pea starch. Pea protein, as a by-product, belongs to a high-quality plant protein. Compared with animal-derived proteins, pea protein is more easily digested and absorbed by the human body, and does not contain lactose and gluten, making it particularly suitable for lactose-intolerant patients and people sensitive to gluten. However, currently pea protein is mainly used in feed processing and its high-value utilization has not been realized.

[0004] With the continuous development of the economy, people's demand for precise nutritional intake is increasing day by day, especially for special populations such as pregnant women, teenagers and the elderly, who pay more attention to the comprehensiveness and balance of protein intake. Animal protein can provide sufficient essential amino acids to meet the balance of nutritional needs, but long-term excessive intake of animal protein may increase the risk of chronic diseases. In contrast, although plant protein has various nutritional values, currently the only known full-value protein source is soy protein. Therefore, in order to provide a more abundant choice of plant protein, it is of great technical significance to study a new type of full-value protein from pea protein to meet the comprehensive and balanced nutritional needs of the human body. Summary of the Invention

[0005] After research, currently the protein extraction technology mainly uses the alkali dissolution and acid precipitation method, which is widely adopted because of its simple operation and low cost. However, this method has the risk of causing changes in the protein structure and thus denaturation due to long-term acid-base treatment, which affects its functional properties and further affects its application effect. Secondly, excessive acid-base emissions will have an adverse impact on the environment.

[0006] Therefore, the present invention provides a pea full-value protein component and its preparation and identification methods. By optimizing and regulating the protein structure through biological lactic acid bacteria fermentation, improving the amino acid composition, enriching sulfur-containing amino acids, and combining with controlling the water extraction and ultrafiltration technology to efficiently and precisely separate the albumin component, the efficient and precise preparation of the full-value protein from pea protein is realized.

[0007] Specifically, in a first aspect, the present invention provides a method for preparing a complete protein, comprising: Mixing pea kernel powder with water and then performing a first solid-liquid separation to obtain a first clear liquid; Adjusting the pH value of the first clear liquid to 5.8 - 6.2 to obtain a protein mixture; Adding lactic acid bacteria to the protein mixture and standing at a temperature below 10°C for 1 - 3 h for stratification; the number of lactic acid bacteria in every 100 mL of the protein mixture is 0.80×10 8 ~3.16×10 8 CFU; wherein, the stratification includes three layers, and the bottom layer is mainly composed of a mixture of starch and bacterial cells; Removing the bottom material of the stratified material to obtain a remaining material, and performing a second solid-liquid separation on the remaining material to collect the precipitate; the precipitate is dried for the first time to obtain a separated protein; Mixing the separated protein with water and then performing a third solid-liquid separation to obtain a second clear liquid; the second clear liquid is separated by an ultrafiltration membrane, and the obtained permeate is dried for the second time to obtain albumin, and the albumin is the complete protein.

[0008] With the gradual enrichment of pea varieties, the research on pea protein has gradually increased, and the content of sulfur-containing amino acids in some pea varieties has been significantly improved. However, from the perspective of variety optimization, there are deficiencies such as a long R & D cycle and high costs in regulating the content of sulfur-containing amino acids. In the research of the present invention, it is found that the acidic substances produced by lactic acid bacteria fermentation can not only be used to extract the separated protein from peas in the present invention, but also selectively precipitate proteins, and by strictly controlling the fermentation rate of lactic acid bacteria, the proportion of sulfur-containing amino acids in the obtained product can be increased.

[0009] Based on this, the present invention optimizes the extraction method of pea protein. By regulating the pH value of the above-mentioned protein mixture in stages, a complete protein derived from peas can be obtained, changing the industry status quo where the only source of complete protein is soybean protein. Among them, regulating the pH value of the protein mixture in stages plays a key role in the successful extraction of complete protein. The present invention first moderately adjusts the pH value to 5.8 - 6.2. Under this condition, there is less protein precipitation in the protein mixture. The main purpose is to make the initial fermentation rate of lactic acid bacteria small. With the addition of lactic acid bacteria, the pH value regulation enters the second stage. The core lies in adopting low-temperature fermentation. On the one hand, lactic acid bacteria will continue to slowly reduce the pH value of the system during fermentation, gradually inducing protein sedimentation, especially the enrichment of sulfur-containing amino acids. If the fermentation rate is too fast, the content of sulfur-containing amino acids in the obtained albumin will decrease. On the other hand, low-temperature fermentation can also inhibit the non-specific denaturation of proteins, maintain their functionality and structure, and promote precipitation formation and sedimentation; on the third hand, low-temperature fermentation can also enhance the hydrophobicity of bacteria, promote starch adsorption, and the bacteria-starch complex will naturally sediment due to the increase in density and volume, forming a more stable and easily separable precipitate, which is used as the bottom material of the stratified material and is easy to remove; on the fourth hand, low-temperature fermentation can also inhibit the growth of miscellaneous bacteria and reduce pollution.

[0010] Therefore, the present invention optimizes and regulates the protein structure through biological lactic acid bacteria fermentation, improves the amino acid composition, and further combines the control of water extraction and ultrafiltration technology to efficiently and accurately separate the albumin component, and uses membrane technology ultrafiltration to efficiently purify and enrich sulfur-containing amino acids. First, this method effectively avoids the damage and modification of the protein structure by traditional acid-base extraction. Second, the proportion of sulfur-containing amino acids is increased through microbial fermentation, significantly improving the amino acid balance of pea protein. Third, the pea complete protein is efficiently and accurately purified through membrane technology.

[0011] When adding lactic acid bacteria to the protein mixture, the lactic acid bacteria can be added in the form of freeze-dried powder or in the form of a bacterial suspension. From the perspective of economic benefits, the bacterial suspension is more economical and only needs to be cultivated.

[0012] Preferably, a pH value regulator is used to adjust the pH value of the first supernatant, and the pH value regulator uses citric acid.

[0013] In the present invention, the bottom material of the stratified material can be removed by conventional manual or automatic liquid separation devices.

[0014] According to the preparation method of the complete protein provided by the present invention, the cut-off molecular weight of the ultrafiltration membrane is 20 - 40 kDa.

[0015] In the present invention, the second supernatant is separated by an ultrafiltration membrane. The main component in the obtained filtrate is albumin. After being dissolved in ultrapure water and centrifuged to remove insoluble impurities, the protein components in the obtained supernatant are mainly albumin, but there may still be a certain amount of other small and medium molecular proteins, peptide segments, and a very small amount of unremoved high molecular complexes or antinutritional factors.

[0016] Therefore, the present invention further adopts membrane ultrafiltration treatment with a molecular cut-off limit of 20 - 40 kDa. Its main function is not simply "purification", but on the basis of fine fractionation of components, selectively retaining albumin and its relatively low molecular weight functional peptide segments, and effectively removing high molecular proteins (such as globulins, proteins or conjugates in polymer form, etc.) larger than the molecular cut-off limit, so as to achieve the following technical effects: Through the membrane ultrafiltration treatment with this molecular cut-off limit, the albumin component in the filtrate is enriched, and the protein molecular distribution becomes more concentrated. Furthermore, the amino acid composition balance of the extracted protein is significantly improved, especially showing a better ratio in the content of specific amino acids (such as leucine, threonine, etc.), enhancing its application potential as a complete plant protein.

[0017] As for the retained components, they may include globulins (usually with a molecular weight of 150 - 300 kDa), a small amount of natural protein complexes, or protein aggregates formed during the extraction process. These components may affect the solubility, stability or taste of the final product. Therefore, effectively removing them by membrane ultrafiltration has a positive effect on improving the functional properties of the final protein product.

[0018] The separated protein obtained in the present invention is mixed with water and then subjected to a third solid-liquid separation. The second supernatant still contains globulins, which are mainly located in the precipitate obtained from the third solid-liquid separation. The present invention can also further obtain globulins from peas by the following method: The precipitate obtained from the third solid-liquid separation is dissolved in a 2% NaCl solution at a mass ratio of 1:10, magnetically stirred for 1 - 3 h, centrifuged at a temperature below 40°C, dialyzed for 24 h using a dialysis membrane with a molecular weight of 3 kDa, and then freeze-dried to obtain globulins. Among them, the centrifugation speed is 2000 - 8000 r, and the centrifugation time is less than 60 min.

[0019] In the present invention, the NaCl salt solution with the above concentration is used as the extraction medium. The main purpose is to provide a salt environment with low concentration and weak ionic strength to promote the dissolution of legumin. Salt ions play a fundamental role in regulating the osmotic pressure of the system and stabilizing the protein dispersion state during this process, which helps the target globulin to be released from the raw material under mild conditions and avoid non-specific precipitation or aggregation of proteins. After this step is centrifuged, the insoluble impurities in the raw material are effectively removed, and the globulin component is enriched in the supernatant. It should be emphasized that the role of NaCl in this step is mainly to construct a suitable salt environment for extracting globulin at one time, and it does not affect the solubility or distribution of albumin.

[0020] According to the method for preparing the complete protein provided by the present invention, the separated protein and water are mixed at a mass ratio of 1:5 to 15 at a temperature below 40 °C, stirred for 1 to 3 h, and then the third solid-liquid separation is carried out to obtain the second clear liquid.

[0021] The present invention unexpectedly finds that the water-soluble protein in the separated protein is further extracted in a water-soluble manner. Among them, albumin, as a typical water-soluble protein, has characteristics such as a relatively small molecular weight (usually about 25 - 30 kDa) and good solubility. Under the above process, a complete protein with a higher extraction rate can be obtained.

[0022] According to the method for preparing the complete protein provided by the present invention, the pea kernel powder is derived from peas with a sulfur-containing amino acid content of more than 0.10 g / 100 g.

[0023] There are differences in the sulfur-containing amino acid content among different varieties. After testing various available pea varieties, the results are as follows: Table 1

[0024] As can be seen from the above table, it can be seen that the percentage of sulfur-containing amino acid content in Taiwan Changshouren peas is the highest, followed by Changshoudou No. 1.

[0025] Experiments have found that using peas with a higher sulfur-containing amino acid content is more conducive to obtaining a complete protein with good balance and high protein content.

[0026] According to the method for preparing the complete protein provided by the present invention, the second solid-liquid separation includes centrifugation at 2000 - 8000 r; and / or, the third solid-liquid separation includes centrifugation at 2000 - 8000 r.

[0027] According to the method for preparing the complete protein provided by the present invention, the first drying is freeze-drying; and / or, the second drying is freeze-drying.

[0028] According to the preparation method of the complete protein provided by the present invention, the pea kernel powder and water are mixed at a temperature below 40°C in a mass ratio of 1:2 to 5 and then centrifuged. The obtained supernatant is filtered through a filtering device with a pore size of 400 μm or less to obtain the first supernatant.

[0029] The extraction under the above process in the present invention helps to improve the extraction rate of the complete protein in the present invention and reduce the impurities therein.

[0030] According to the preparation method of the complete protein provided by the present invention, the pea kernel powder is obtained by drying pea kernels at a temperature below 50°C and then grinding.

[0031] In a second aspect, the present invention also provides a complete protein prepared by the preparation method of the complete protein as described above.

[0032] In a third aspect, the present invention also provides a method for identifying a complete protein, comprising: obtaining the amino acid contents of the complete protein to be tested and the standard complete protein; the standard complete protein is a complete protein of plant origin; the standard complete protein is soybean protein; the complete protein to be tested is prepared by the preparation method as described above; calculating the essential amino acid ratio, ratio coefficient and amino acid ratio coefficient score of the complete protein to be tested and the standard complete protein; comparing the amino acid ratio coefficient score of the complete protein to be tested with the amino acid ratio coefficient score of the standard complete protein.

[0033] A pea full - value protein component provided by the present invention, its preparation and identification methods. On the one hand, through a series of green extraction methods, the isolated protein and components are extracted from peas, reducing the negative impacts brought by the traditional alkali - dissolution and acid - precipitation method. These methods extract pea protein and its components through mild treatment without destroying the protein structure, thus maximizing the preservation of its nutritional and functional properties, avoiding the problems of protein denaturation and wastewater pollution caused by traditional extraction methods. At the same time, these green extraction technologies conform to the concept of sustainable development, helping to reduce energy consumption and environmental pollution during the production process, and providing more reliable technical support for eco - friendly agriculture and food production. On the other hand, the present invention effectively extracts the pea full - value protein component, obtaining a high - quality plant protein containing all essential amino acids. Compared with other plant proteins, the albumin prepared by the present invention, as the pea full - value protein, can meet the recommended ratios of essential amino acids for the human body by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO), ensuring the comprehensiveness and nutritional balance of the protein. In addition, the amino acid ratio of the pea full - value protein is relatively ideal, especially suitable for the nutritional needs of special groups such as pregnant women, teenagers, the elderly, and sports people, and can support healthy growth and development, maintain body functions, enhance immunity, promote metabolic functions, and prevent malnutrition and other problems, becoming a high - quality protein source in plant - based foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0035] Figure 1 It is the test result diagram of Test Example 1 provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention in conjunction with the drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0037] The following combines Figure 1 to describe a pea full - value protein component of the present invention, its preparation and identification methods.

[0038] For those where specific techniques or conditions are not indicated in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through regular channels. Among them, the varieties of peas used in the present invention and the content of sulfur-containing amino acids therein are as follows: Table 2

[0039] Freeze-dried lactic acid bacteria: Shanghai Collection of Biotechnology, strain name: Lactococcus lactis subsp. lactis, number: SHBCC D14463, specification: freeze-dried powder.

[0040] Example 1 A preparation method of pea full-price protein, the steps are as follows: (1) Soak Taiwan Changshouren peas in deionized water overnight, peel off the pea skins, and dry the remaining pea kernels at 45°C and grind them into powder to obtain pea kernel powder.

[0041] (2) Dissolve the pea kernel powder obtained in step (1) in ultrapure water at a temperature of 24°C at a mass ratio of 1:3, centrifuge at a temperature of 24°C, the centrifuge speed is 4000r, and the centrifuge time is 20min to obtain the supernatant (i.e., the protein mixture).

[0042] (3) Pass the supernatant obtained in step (2) through a 100-mesh sieve, adjust the pH of the supernatant to 5.8 - 6.2 with 0.1M citric acid aqueous solution. For every 100ml of supernatant, add 1ml of lactic acid bacteria suspension containing 1.58×10 8 CFU / ml, then refrigerate and precipitate for 2h to layer, set the refrigerator temperature to 4°C, separate the layered mixture by liquid separation to remove the bottom layer (mainly bacteria and starch and other substances), and centrifuge the remaining liquid at a centrifuge speed of 4000r for 20min, collect the precipitate, and freeze-dry to obtain pea protein isolate.

[0043] (4) Add the pea protein isolate obtained in step (3) to ultrapure water at a temperature of 24°C at a mass ratio of 1:10, stir magnetically for 2h, centrifuge at a temperature of 22°C, the centrifuge speed is 4000r, and the centrifuge time is 30min to obtain the supernatant and the precipitate.

[0044] (5) Ultrafilter the supernatant obtained in step (4) through a 30kDa membrane and freeze-dry to obtain albumin.

[0045] Dissolve the precipitate obtained in step (4) in a 2% NaCl solution at a mass ratio of 1:10, stir magnetically for 2 h, centrifuge at a temperature of 22 °C, with a centrifugation speed of 4000 r and a centrifugation time of 30 min, and dialyze for 24 h using a dialysis membrane with a molecular weight of 3 kDa, followed by lyophilization to obtain globulin.

[0046] Example 2 It is basically the same as Example 1, except that: replace the Taiwan Changshouren peas with Changshoudou No. 1 peas.

[0047] Example 3 It is basically the same as Example 1, except that: according to the proportion of 3.16×10 8 CFU lactic acid bacteria per 100 ml of supernatant, add freeze-dried lactic acid bacteria powder to it.

[0048] Comparative Example 1 It is basically the same as Example 1, except that: replace the Taiwan Changshouren peas with commercially available ordinary peas.

[0049] Comparative Example 2 It is basically the same as Example 1, except that: according to the proportion of 1.58×10 8 CFU lactic acid bacteria per 200 ml of supernatant, add freeze-dried lactic acid bacteria powder to it.

[0050] Comparative Example 3 It is basically the same as Example 1, except that: replace the refrigerator with an incubator, and set the temperature of the incubator to 25 °C.

[0051] Comparative Example 4 It is basically the same as Example 1, except that: use the method of alkali dissolution and acid precipitation to extract total protein, and the specific steps are as follows: (1) Soak the Taiwan Changshouren peas in deionized water overnight, peel off the pea skins, and dry the remaining pea kernels at 45 °C and grind them into powder to obtain pea kernel powder.

[0052] (2) Dissolve the pea kernel powder obtained in step (1) in ultrapure water at a temperature of 24 °C at a mass ratio of 1:10, adjust the pH to 9.5 with 0.1 M NaOH, stir for 2 h, centrifuge at 4000 r at a temperature of 24 °C for 20 min, adjust the pH of the supernatant to 4.5 with 0.1 M HCl, stir for 2 h, centrifuge at a temperature of 24 °C, with a centrifugation speed of 4000 r and a centrifugation time of 20 min, collect the precipitate, and freeze-dry to obtain pea protein isolate.

[0053] (3) Add the pea protein isolate obtained in step (2) to ultrapure water at 24 °C at a mass ratio of 1:10, stir magnetically for 2 h, centrifuge at 22 °C at a rotational speed of 4000 r for 30 min to obtain a supernatant and a precipitate.

[0054] (4) Ultrafilter the supernatant obtained in step (3) through a 30 kDa membrane, and freeze-dry to obtain albumin.

[0055] Dissolve the precipitate obtained in step (3) in a 2% NaCl solution at a mass ratio of 1:10, stir magnetically for 2 h, centrifuge at 22 °C at a rotational speed of 4000 r for 30 min, and dialyze for 24 h using a dialysis membrane with a molecular weight of 3 kDa, then freeze-dry to obtain globulin.

[0056] Test Example 1 Molecular Weight Use reducing SDS-PAGE to test the albumin and globulin prepared in Example 1. The test method is as follows: Prepare a protein mixture with a sample buffer ((1 mol / L Tris-HCl, pH 6.8), 50% glycerol, β-mercaptoethanol, 10% SDS, 1% bromophenol blue), heat in a water bath at 95 °C for 10 min to obtain an electrophoresis sample solution. Prepare a stacking gel with a concentration of 5% and a separating gel with a concentration of 12%. Respectively take 10 μL of albumin and globulin samples and add them to the lanes, and perform gel electrophoresis under constant current conditions using an electrophoresis power supply. Set the voltage to 80 V before the sample enters the separating gel, and increase the voltage to 120 V after it enters the separating gel. After electrophoresis, wash the gel in a petri dish and stain it with Coomassie Brilliant Blue R250. Use a protein marker with a molecular weight of 11 - 245 kDa as a standard.

[0057] The test results are as Figure 1 shown. As can be seen from the figure, albumin and globulin are composed of different subunits. Albumin is mainly composed of subunits around 10 kDa, and globulin is mainly composed of subunits of 15 - 71 kDa. It can be seen that the albumin and globulin separated and extracted in Example 1 are effective.

[0058] Test Example 2 Protein Content Determination method: Refer to the Kjeldahl method in "GB 5009.5-2016 National Food Safety Standard Determination of Protein in Foods".

[0059] The test results of the albumin and globulin prepared in the above Examples 1 - 2 and Comparative Example 1 are shown in Table 3 below.

[0060] Table 3

[0061] As can be seen from the above test results, compared with commercially available ordinary peas, the crude protein content in albumin and globulin prepared from Taiwan Changshouren peas and Changshoudou No. 1 peas with higher sulfur-containing amino acid content is higher.

[0062] Test Example 3 Amino Acid Content Determination method: Refer to GB 5009.124-2016.

[0063] The test results of the albumin and globulin prepared in the above Examples 1-2 and Comparative Example 1 are shown in Table 4 below.

[0064] Table 4

[0065] As can be seen from the above table, albumin is rich in glutamic acid, arginine, glycine, and the contents of the remaining amino acids are relatively balanced; globulin is rich in glutamic acid, arginine, phenylalanine, alanine, leucine, etc.

[0066] Among them, the contents of the corresponding sulfur-containing amino acids (methionine and cystine) and the albumin extraction rate in the albumin prepared in Examples 1-3 and Comparative Examples 1-4 are shown in Table 5 below. Albumin extraction rate = mass of extracted albumin / total protein mass in peas × 100%.

[0067] Table 5

[0068] As can be seen from the above table, using peas with high sulfur-containing amino acid content as raw materials helps to obtain albumin with high sulfur-containing amino acid content. On this basis, it can also be seen that the conditions of cold precipitation by lactic acid bacteria and the dosage of lactic acid bacteria play a key role in improving the sulfur-containing amino acid content and extraction rate of albumin. The sulfur-containing amino acid content and extraction rate of the albumin prepared by the method of the present invention are much higher than those of the conventional alkali dissolution and acid precipitation method.

[0069] Test Example 4 Essential Amino Acid Ratio (RAA) Test method: The essential amino acid composition of pea protein and its components was compared with the amino acid pattern of the reference protein (in this invention, the reference amino acid content of the ideal protein proposed by WHO / FAO was adopted, referring to He Y, Qin H, Wen J, et al. Characterization of amino acid composition, nutritional value, and taste of fruits from different Actinidia arguta resources[J]. Journal of Food Quality, 2024, 2024(1): 1005194.). RAA = content of a certain essential amino acid in the sample / content of the same essential amino acid in the reference pattern. It reflects whether a certain essential amino acid in the protein to be tested is sufficient and quickly identifies the limiting amino acid of the protein. The closer the value of RAA is to 1, the closer its content is to the content of the corresponding amino acid in the reference protein amino acid pattern.

[0070] Among them, the reference amino acid content of the ideal protein proposed by WHO / FAO is shown in Table 6 below: Table 6

[0071] In the above table, represents essential amino acids.

[0072] The albumin and globulin prepared in Examples 1 - 2 and Comparative Example 1 above were tested, and the test results are shown in Table 7 below.

[0073] Table 7

[0074] It can be seen from the above results that the first limiting amino acid of albumin in the two pea varieties with higher sulfur - containing amino acids is leucine; the first limiting amino acid of globulin is sulfur - containing amino acids, and the limiting amino acids of albumin and globulin in the commercially available pea protein powder are sulfur - containing amino acids.

[0075] Test Example 5 Amino Acid Ratio Coefficient (RC) Test method: The ratio of the amino acids in pea protein and its components to the equivalent amount of amino acids in a portion of food with the pattern amino acids. RC = ratio of a certain amino acid RAA in the sample / average value of the ratios of essential amino acids in the sample. Its geometric mean is sensitive to low values and can highlight the influence of limiting amino acids.

[0076] The albumin and globulin prepared in Examples 1 - 2 and Comparative Example 1 above were tested, and the test results are shown in Table 8 below.

[0077] Table 8

[0078] As can be seen from the above results, compared with RAA, the RC value eliminates the influence of the differences in the absolute contents of different essential amino acids on the calculation, and reflects the contribution of essential amino acids to amino acid balance.

[0079] From the analysis of single amino acid comparison (essential amino acid ratio RAA, ratio coefficient RC), the balance of albumin is higher than that of globulin.

[0080] Test Example 6 Score of Amino Acid Ratio Coefficient (SRC) of Essential Amino Acids Determination method: SRC = 100 - CV×100, where CV is the coefficient of variation of the amino acid ratio coefficient (RC), which is the ratio of the standard deviation of the amino acid ratio coefficient to the average value.

[0081] The albumin and globulin prepared in the above Examples 1 to 2 and Comparative Example 1 were tested, and the test results are shown in Table 9 below.

[0082] Table 9

[0083] As can be seen from the above results, the SRC of albumin obtained from the three kinds of peas is significantly higher than that of globulin. The amino acid balance of albumin is good, and its protein quality is close to that of the reference protein.

[0084] After research, the SRC value of the full-price protein soy protein is 67.39. The SRC of albumin in two pea varieties with higher sulfur-containing amino acids is higher than that of the existing full-price protein soy protein, while the SRC of albumin in the commercially available ordinary pea protein powder is lower than that of the full-price protein soy protein.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a complete protein, characterized in that, Comprising: Mixing pea kernel powder with water and then performing first solid-liquid separation to obtain a first clear liquid; Adjusting the pH value of the first clear liquid to 5.8 - 6.2 to obtain a protein mixture; Add lactic acid bacteria to the protein mixture and let it stand for 1 - 3 h at a temperature below 10 °C for stratification; the number of lactic acid bacteria in every 100 mL of the protein mixture is 0.80×10 8 ~3.16×10 8 CFU; Removing the bottom material of the stratified material to obtain a remaining material, performing second solid-liquid separation on the remaining material to collect a precipitate; drying the precipitate by first drying to obtain separated protein; Mixing the separated protein with water and then performing third solid-liquid separation to obtain a second clear liquid; Subjecting the second clear liquid to ultrafiltration membrane separation, and drying the obtained permeate by second drying to obtain albumin, and the albumin is the complete protein; 2. The preparation method of the complete protein according to claim 1, characterized in that, The cut-off molecular weight of the ultrafiltration membrane is 20 - 40 kDa.

3. The preparation method of the complete protein according to claim 1, characterized in that, The separated protein and water are mixed at a temperature below 40°C in a mass ratio of 1:5 - 15, stirred for 1 - 3 h, and then subjected to third solid-liquid separation to obtain the second clear liquid.

4. The preparation method of the complete protein according to claim 1, characterized in that, The pea kernel powder is derived from peas with a sulfur-containing amino acid content of more than 0.10 g / 100 g.

5. The preparation method of the complete protein according to claim 1, characterized in that, The second solid-liquid separation includes centrifuging at 2000 - 8000 r; and / or, the third solid-liquid separation includes centrifuging at 2000 - 8000 r.

6. The preparation method of the complete protein according to claim 1, characterized in that, The first drying is freeze-drying; and / or, the second drying is freeze-drying.

7. The preparation method of the complete protein according to claim 1, wherein The pea kernel powder and water are mixed at a temperature below 40°C in a mass ratio of 1:2 - 5, centrifuged, and the obtained clear liquid is further filtered through a filtering device with a pore size of 400 μm or less to obtain the first clear liquid.

8. The preparation method of the complete protein according to claim 1, characterized in that, The pea kernel powder is obtained by drying pea kernels at a temperature below 50°C and grinding.

9. The complete protein prepared by the preparation method of the complete protein according to any one of claims 1 - 8.

10. A method for identifying a complete protein, characterized in that, Comprising: Obtaining the amino acid contents of the to-be-tested complete protein and the standard complete protein; the standard complete protein is a plant-derived complete protein; the standard complete protein is soy protein; the to-be-tested complete protein is prepared by the preparation method according to any one of claims 1 - 8; Calculating the essential amino acid ratio, ratio coefficient, and amino acid ratio coefficient score of the to-be-tested complete protein and the standard complete protein; Comparing the amino acid ratio coefficient score of the to-be-tested complete protein with the amino acid ratio coefficient score of the standard complete protein.

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