Camelina sativa protein peptide with high biological activity and functional property as well as preparation method and application of Camelina sativa protein peptide

Abstract

The invention discloses camelina sativa protein peptide with high biological activity and functional property as well as a preparation method and application thereof, and belongs to the field of plant protein processing. The method comprises the following steps: (1) crushing camelina sativa, sieving to obtain camelina sativa powder, putting the camelina sativa powder into a normal hexane solution, and stirring for degreasing; (2) soaking the degreased camelina sativa powder with water, and centrifuging to remove soluble polysaccharide in the camelina sativa; and (3) putting the camelina sativa powder into water, and extracting camelina sativa protein powder in an alkali-solution and acid-isolation manner. And (4) performing ultrasonic treatment on the camelina sativa protein powder, adding alkaline protease for enzymolysis, and performing enzyme deactivation to obtain the camelina sativa protein peptide. The amino acid sequence of the protein active peptide is as shown in any one or more of SEQ ID NO.1-SEQ ID NO.6. The invention further discloses a preparation method of the protein active peptide. The camelina sativa protein peptide with high biological activity and functional properties is prepared through an ultrasonic-assisted enzymolysis means, and wide application of the camelina sativa protein peptide in the field of food processing can be achieved.

Description

Technical Field

[0001] The invention relates to a camelina protein peptide having high biological activity and functional properties and a preparation method and application thereof, belonging to the field of plant protein processing. Background Art

[0002] Camelina is a cruciferous herb with a short life cycle and an annual yield of 1500-3000 kg / hectare. It has low requirements for the planting environment and is less threatened by diseases during its growth. As a new, low-cost and economical oil crop resource, it has attracted widespread attention in recent years. Camelina is rich in nutrients, including 30-40% protein and 30-40% oil. It also contains rich trace elements and antioxidant active ingredients, and is a new source of plant protein and oil with great potential. At present, camelina resources are only used in the food field in a few countries such as Canada and the United States. In China, there are outstanding problems in the deep processing and utilization of camelina protein resources, such as lack of processing and utilization technology and unclear functional activity research, which to a certain extent restricts the industrial application of camelina protein resources.

[0003] Camelina protein has a high content, but the solubility, functional properties and biological activity of natural camelina protein are not ideal, which limits its application in the food industry. At present, the traditional processing technology of plant protein refers to the use of acid-base hydrolysis, acylation, phosphorylation and other means to destroy the interaction between protein and starch, dietary fiber and other components and the internal structure of protein molecules, release protein, and improve the solubility and functional properties of protein; traditional processing methods are simple and easy, but they are prone to the formation of harmful products such as lysine and alanine, reducing the nutritional properties of the product.

[0004] Compared with traditional treatment methods, the bio-enzymatic technology has mild treatment conditions, strong specificity, and high efficiency. It can effectively avoid the introduction of harmful chemicals, retain more of the nutritional value of the protein, improve the biological activity of the protein, and produce products with better functional properties.

[0005] In addition, ultrasound is a green, safe, and short-acting eco-innovative technology. As an auxiliary extraction technology, it can effectively improve the enzymatic hydrolysis efficiency of proteins by destroying the structure of protein aggregates through the cavitation effect and mechanical action.

[0006] Based on this, developing ultrasound-combined bio-enzyme green biomanufacturing technology for camelina protein to improve the biological activity and functional properties of camelina protein has important scientific value and practical significance for promoting the green and sustainable development of the plant protein industry and developing green, nutritious and healthy foods. Summary of the invention

[0007] [Technical issues]

[0008] Currently, the utilization rate of camelina protein resources is low and the processing performance is poor.

[0009] [Technical solution]

[0010] The first object of the invention is to provide an active peptide from camelina protein, the amino acid sequence of which is shown in any one of SEQ ID NO.1 to SEQ ID NO.6.

[0011] In one embodiment of the present invention, under neutral pH conditions, the protein peptide has a protein solubility higher than 50%, a foaming property higher than 150%, a foam stability higher than 49%, an ABTS free radical scavenging rate higher than 230 μmol TE / gprotein, and a DPPH free radical scavenging rate higher than 82%.

[0012] Furthermore, in one embodiment of the present invention, the protein peptide has a protein solubility higher than 52%, a foaming property higher than 160%, a foam stability higher than 50%, an ABTS free radical scavenging rate higher than 240 μmol TE / g protein, and a DPPH free radical scavenging rate higher than 85%.

[0013] The second object of the present invention is to provide a method for preparing camelina protein peptide having both high antioxidant activity and functional properties, comprising the following steps:

[0014] Step 1: Grind camelina and sieve to obtain camelina powder, and place the camelina powder in a n-hexane solution and stir to defat;

[0015] Step 2: Soaking the defatted camelina powder in water, and removing soluble polysaccharides in the camelina powder after centrifugation;

[0016] Step 3: placing camelina powder in water, extracting camelina protein by alkali dissolution and acid precipitation, centrifuging the obtained dispersion, redissolving the precipitate in water, adjusting the pH to 6-8, dialyzing and drying to obtain camelina protein powder;

[0017] Step 4: After the camelina protein powder is treated with ultrasound, protease is added to hydrolyze and inactivate the enzyme to obtain camelina protein peptides.

[0018] In one embodiment of the present invention, in step 1, the sieving is through a 60-80 mesh sieve.

[0019] In one embodiment of the present invention, the solid-liquid ratio of the camelina powder to the n-hexane solution in step 1 is 1:10-1:30 (w / v).

[0020] In one embodiment of the present invention, the degreasing time in step 1 is 12-24 hours, preferably 12 hours.

[0021] In one embodiment of the present invention, the solid-liquid ratio of the camelina powder to the aqueous solution in step 2 is 1:10-1:30 (w / v), preferably 1:15 (w / v).

[0022] In one embodiment of the present invention, the soaking time in step 2 is 1-2 hours, preferably 2 hours.

[0023] In one embodiment of the present invention, the centrifugal speed parameter in step 2 is 5000-10000 r / min, preferably 5000 r / min, and the time is 10-30 min, preferably 15 min.

[0024] In one embodiment of the present invention, the solid-liquid ratio of the camelina powder to the aqueous solution in step 3 is 1:10-1:30 (w / v), preferably 1:15 (w / v).

[0025] In one embodiment of the present invention, the soaking time in step 3 is 2-4 hours, preferably 2 hours.

[0026] In one embodiment of the present invention, the alkaline solution in step 3 is adjusted to pH 8-10, the solution for adjusting the pH is 2-4 mol / L NaOH solution, preferably 4 mol / L NaOH solution; the temperature parameter is 40-50°C, preferably 40°C.

[0027] In one embodiment of the present invention, the pH of the acid precipitation in step 3 is adjusted to 3-5, preferably 4.5, the solution for adjusting the pH is 2-4 mol / L HCl solution, preferably 4 mol / L HCl solution; the temperature parameter is 40-50°C, preferably 40°C.

[0028] In one embodiment of the present invention, after the dispersion obtained in step 3 is centrifuged, the precipitate is redissolved in water and the pH is adjusted to 6-8, preferably pH 7.

[0029] In one embodiment of the present invention, the power of the ultrasonic treatment in step 4 is 120-360W; preferably, the power of the ultrasonic treatment is 240-360W; more preferably, the power of the ultrasonic treatment is 360W.

[0030] In one embodiment of the present invention, the ultrasonic treatment time in step 4 is 5-35 min. Preferably, the ultrasonic treatment time is 10-35 min; preferably, the ultrasonic treatment time is 10-25 min; preferably, the ultrasonic treatment time is 15 min.

[0031] In one embodiment of the present invention, the protease described in step 4 is alkaline protease, and the ratio of enzyme to substrate is 1:30-1:50 (w / w), preferably 1:50 (w / w).

[0032] In one embodiment of the present invention, the enzymatic hydrolysis time in step 4 is 10 min-120 min; preferably, the enzymatic hydrolysis time is 10 min-90 min; more preferably, the enzymatic hydrolysis time is 30 min-90 min; more preferably, the enzymatic hydrolysis time is 40 min-80 min; more preferably, the enzymatic hydrolysis time is 50 min-70 min.

[0033] In one embodiment of the present invention, the pH of the enzymatic hydrolysis in step 4 is 8-10, preferably 8; the temperature is 40-50°C, preferably 40°C.

[0034] In one embodiment of the present invention, the enzyme inactivation in step 4 includes inactivation in a boiling water bath, with the parameter of heating at 100° C. for 10 min.

[0035] The third object of the present invention is to provide a composition comprising the protein peptide as described in any one of the above or the camelina protein peptide prepared by the method described in any one of the above.

[0036] The fourth object of the present invention is the use of the protein peptide as described above, or the camelina protein peptide having both biological activity and functional properties obtained by the method described above, or the composition as described above in food or medicine.

[0037] The fifth objective of the present invention is to obtain a novel camelina active peptide sequence by combining virtual screening means, as shown in any one or more of SEQ ID NO.1-SEQ ID NO.6.

[0038] The sixth object of the present invention is to provide a medicine comprising the protein peptide as described in any one of the above or the camelina protein peptide prepared by the method as described in any one of the above, or comprising the composition as described in the above.

[0039] The seventh object of the present invention is to provide a food comprising the protein peptide as described in any one of the above or the camelina protein peptide prepared by the method described in any one of the above, or comprising the composition as described in the above.

[0040] [Beneficial Effects]

[0041] (1) The present invention uses high-purity camelina protein from which soluble polysaccharides have been removed as raw material, and prepares camelina protein peptides by ultrasound-coupled enzyme method. The protein solubility under pH 7 conditions is increased from 28.56% to 65.07%.

[0042] (2) The present invention uses high-purity camelina protein with soluble polysaccharides removed as raw material, and prepares camelina protein peptides by ultrasound-coupled enzyme method. The functional properties of the protein are significantly improved under pH 7 conditions, wherein the foaming property is 179.76%, which is 28.28% higher than that of unmodified camelina protein, and the foam stability is 56.59%, which is 31.60% higher than that of unmodified camelina protein.

[0043] (3) The present invention uses high-purity camelina protein from which soluble polysaccharides have been removed as raw material, and prepares camelina protein peptides by ultrasound-coupled enzyme method. The ABTS free radical scavenging rate under pH 7 conditions is increased from 130.29 μmol TE / gprotein to 307.94 μmol TE / gprotein, and the DPPH free radical scavenging rate under pH 7 conditions is increased from 74.32% to 90.12%.

[0044] (4) The present invention adopts the method of first removing soluble polysaccharides and preparing high-purity camelina protein by alkali dissolution and acid precipitation, with a protein purity of >90%, and then preparing camelina protein peptide by ultrasound-assisted enzymatic hydrolysis. The processing process is simple, easy to operate, and has low production cost. The prepared camelina protein peptide has high antioxidant activity and functional properties, has a wide range of applications in the field of food processing, and provides a direction for the high-value processing and utilization of camelina. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a scanning electron micrograph of camelina protein.

[0046] Figure 2 This is a scanning electron micrograph of camelina protein after ultrasonic treatment.

[0047] Figure 3 This is a scanning electron micrograph of camelina protein after ultrasound-assisted enzymatic hydrolysis.

[0048] Figure 4 The hydrolysis curves of camelina protein before and after ultrasonic treatment by alkaline protease. DETAILED DESCRIPTION

[0049] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0050] Test method:

[0051] 1.1 Determination method of hydrolysis degree:

[0052] The degree of hydrolysis was determined according to the pH-stat method. Camelina protein powder before and after ultrasonic treatment was dispersed in deionized water to prepare a camelina protein solution (10 mg / mL). The suspension was stirred at room temperature for 1 h to ensure sufficient hydration of the protein. The camelina protein solution was placed in a 50°C water bath and the pH was adjusted to 8.0, and alkaline protease was added for enzymatic hydrolysis. The pH was kept stable by continuously adding 1M NaOH solution, and the degree of hydrolysis (DH) value was calculated using the volume of NaOH consumed.

[0053]

[0054] Where B: volume of NaOH consumed (mL); N b : NaOH consumption concentration; α: α-NH 2 The dissociation degree of the group; M p : total amount of protein in the substrate; H tot : Millimoles of peptide bonds per gram of substrate (mmol / g).

[0055] 1.2 Solubility determination method:

[0056] After centrifuging the camelina protein solution after ultrasound-assisted enzymatic hydrolysis, the supernatant was diluted 50 times and reacted with the soluble protein using a BCA protein kit. The absorbance was detected at 562 nm and the protein solubility was calculated according to formula (2):

[0057]

[0058] Among them C 1 is the protein concentration in the supernatant, and C2 is the total protein concentration.

[0059] 1.3 Foaming performance determination method:

[0060] The CPI suspension after enzymatic hydrolysis (10 mg / mL, 10 mL) was added to a centrifuge tube and homogenized for 1 min using a high-speed disperser at 20,000 rpm. The foam volume at 0 min and 30 min after homogenization was recorded as V 1 , V 2 The foamability (FC) and foam stability (FS) were calculated using the following formula.

[0061]

[0062] Where V is the volume of protein solution before homogenization; V 1 V is the volume of the homogenized foam; 2 It is the foam volume after homogenization and leaving for 30 minutes.

[0063] 1.4 ABTS free radical scavenging rate determination method:

[0064] T-AOCAssay Kit was used for ABTS detection. ABTS solution and oxidant solution were mixed in a ratio of 1:1 to prepare ABTS stock solution, and placed in the dark for 12 hours. ABTS stock solution was diluted with 80% ethanol to prepare ABTS working solution, so that its A734 was 0.70±0.05. 10mM Trolox solution was diluted to 0, 15, 0.30, 0.60, 0.90, 1.20 and 1.50mM solutions. 200μLABTS working solution was added to each well, 10μL distilled water was added to the blank control, 10μL Trolox standard solution of various concentrations was added to the standard curve detection well, 10μL sample (2mg / mL) was added to the sample detection well, and incubated at room temperature for 6min in the dark, the absorbance was measured at 734nm, and the ABTS free radical scavenging rate was calculated by comparing with the standard curve.

[0065] 1.5 DPPH free radical scavenging rate determination method:

[0066] A 0.1 mM DPPH solution was prepared with anhydrous ethanol, 500 μL DPPH was mixed with 500 μL sample (2 mg / mL), and incubated at room temperature in the dark for 30 min, and then the absorbance was measured at 517 nm. The DPPH free radical scavenging activity was calculated using formula (5).

[0067]

[0068] A S : Absorbance of DPPH solution and sample solvent, A c : Absorbance of sample solution and ethanol solution, A b : Absorbance of DPPH solution and ethanol solution.

[0069] 1.5 Protein purity determination method:

[0070] The purity of camelina protein was determined by Kjeldahl method according to GB 5009.5-2016. Weigh 0.1g protein powder, 3g K 2 SO 4 , 0.2 g CuSO 4 , 20mL concentrated H 2 SO 4 Place the digestion tube in a digestion apparatus for digestion. The digestion program is set to 100°C for 20 min, 200°C for 20 min, 300°C for 20 min, and 400°C for 1 h. Automatic liquid addition, distillation, titration, and recording of titration data are performed on a fully automatic Kjeldahl nitrogen analyzer (add sodium hydroxide solution, hydrochloric acid or sulfuric acid standard solution, and boric acid solution containing mixed indicator A or B before use). The content of camelina protein is calculated according to the following formula (6).

[0071]

[0072] in:

[0073] X——protein content in the sample, in %;

[0074] V 1 ——The volume of sulfuric acid or hydrochloric acid standard titrant consumed by the test solution, in milliliters (mL);

[0075] V 2 ——The volume of sulfuric acid or hydrochloric acid standard titrant consumed by the reagent blank, in milliliters (mL);

[0076] c——concentration of sulfuric acid or hydrochloric acid standard titration solution, in moles per liter (mol / L);

[0077] m——mass of the sample, in grams (g);

[0078] V 3 ——The volume of digestive fluid absorbed, in milliliters (mL);

[0079] F——coefficient of nitrogen conversion to protein;

[0080] 100——Conversion factor.

[0081] 1.6 Determination method of soluble sugar:

[0082] Take 5g camelina protein powder, add 100mL75% ethanol as extraction solvent, shake and extract in a 45℃ water bath for 60min, evaporate to remove ethanol, add 1mL each of 15% potassium ferrocyanide solution and 30% zinc sulfate solution as protein precipitant, shake well. Wash with distilled water into a 100mL volumetric flask, filter to volume, take 40mL of filtrate into a 50mL volumetric flask, add 1mL 1moL / L hydrochloric acid solution, hydrolyze in a boiling water bath for 10min, cool, add 2 drops of phenolphthalein indicator, neutralize with about 0.5mol / L sodium hydroxide to red, then adjust with 1moL / L dilute hydrochloric acid until the red color just disappears, adjust to volume, filter and set aside. Use a pipette to draw 0-5.0mL of glucose standard solution into a 100mL conical flask, add water to a total volume of 5.0mL, add 5.00mL of copper reagent, and heat in a boiling water bath for 15min. Cool to room temperature, add 2mL of sulfuric acid + oxalic acid mixture along the wall, and shake immediately to completely dissolve the cuprous oxide precipitate. When titrated to light yellow-green with 0.005mol / L sodium thiosulfate working solution, add about 0.5mL of starch indicator and titrate until the blue disappears as the end point. Use the difference between the titration value of the blank test and the standard glucose solution as the ordinate to draw a standard curve. Use a pipette to draw 1-5mL of sample filtrate into a conical flask, and then operate in the same way as the standard curve, record the titration value V, and use water instead of the sample as a blank. Substitute the difference between the blank test and the sample titration into the regression equation to obtain the soluble sugar content in the sample.

[0083]

[0084] in:

[0085] X represents the content of soluble sugar in the sample, %; A represents the mass of glucose in the sample solution obtained by substituting the difference between the blank and sample titration values ​​into the standard curve, mg; m represents the mass of the sample, g; V represents the volume of the sample solution absorbed during the measurement, mL; H represents the water content of the sample, %.

[0086] The present invention is further described in detail below by way of examples, but the scope of the present invention is not limited in any way.

[0087] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0088] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0089] Example 1

[0090] The preparation route of camelina protein comprises the following steps:

[0091] (1) The camelina was crushed in a grinder and passed through a 60-80 mesh sieve to obtain camelina powder, and the camelina powder was placed in a n-hexane solution and stirred for degreasing, the solid-liquid ratio was 1:15 (w / v), and the degreasing time was 24 hours.

[0092] (2) Soaking defatted camelina powder in deionized water at a solid-liquid ratio of 1:15 (w / v) for 2 h; removing soluble polysaccharides from the camelina powder after centrifugation at a speed of 5000 r / min for 15 min.

[0093] (3) The camelina powder was placed in an appropriate amount of deionized water with a solid-liquid ratio of 1:15 (w / v), and the camelina protein was extracted by alkali dissolution and acid precipitation. The pH of the alkali dissolution was adjusted to 10, and the solution for adjusting the pH was a 4 mol / L NaOH solution. The pH of the acid precipitation was adjusted to 4.5, and the solution for adjusting the pH was a 4 mol / L HCl solution. The temperature parameter was 40°C. After the obtained dispersion was centrifuged, the precipitate was redissolved in deionized water, the pH was adjusted to 7, and the camelina protein powder was obtained after dialysis and freeze-drying (Scanning electron microscopy image as shown in FIG. Figure 1 ).

[0094] Example 2

[0095] The preparation route of camelina protein peptide comprises the following steps:

[0096] (1) Camelina protein powder was ultrasonically treated at a power of 360 W for 15 min (Scanning electron microscopy image as shown in Fig. Figure 2 ).

[0097] (2) Add alkaline protease for enzymatic hydrolysis, the ratio of enzyme to substrate is 1:50 (w / w), the enzymatic hydrolysis time is 120 min, the enzymatic hydrolysis pH is 8, and the temperature is 40°C. After the enzymatic hydrolysis is completed, the enzyme is inactivated by heating in a water bath at 100°C for 10 min to obtain camelina protein peptide (Scanning electron microscopy image as shown in FIG. Figure 3 ).

[0098] The hydrolysis curve of camelina protein before and after ultrasound is as follows Figure 4 As shown, with the extension of hydrolysis, the hydrolysis curve of camelina protein gradually tends to equilibrium, and ultrasonic pretreatment helps to improve the hydrolysis rate of camelina protein. Compared with the untreated group, the hydrolysis rate increased by 13.48%.

[0099] Example 3 Optimization of Ultrasonic Power

[0100] The ultrasonic treatment power in step (1) of Example 2 was adjusted to 120, 360, 240, and 480 W, and the rest was kept the same as in Example 2 to obtain camelina protein peptide.

[0101] Comparative Example 1

[0102] The ultrasonic and enzymatic hydrolysis steps were omitted, and the other steps were kept the same as in Example 2 to obtain unmodified camelina protein.

[0103] In order to study the effects of ultrasound and enzymatic hydrolysis steps on the protein extraction effect, the present invention performed performance tests on the modified camelina proteins obtained in Examples 2 and 3 and the unmodified camelina protein obtained in Comparative Example 1, including solubility, foaming property, foam stability, ABTS clearance rate and DPPH clearance rate. The results are shown in Table 1.

[0104] Table 1 Test results of protein performance of Example 2 and Example 3

[0105]

[0106] It can be seen from Table 1 that: as the ultrasonic power increases from 120W to 360W, the solubility of camelina protein increases significantly from 50.52% to 65.07%, but when the ultrasonic power is 480W, the solubility of camelina protein decreases to 60.21%, indicating that when the ultrasonic treatment power is 360W, the solubility of camelina protein is optimal, and the solubility is increased by 1.27 times compared with the unmodified camelina protein in Comparative Example 1. Correspondingly, the foaming property, foam stability, ABTS clearance rate, and DPPH clearance rate of camelina protein also reach the highest value when the ultrasonic treatment power is 360W, which are increased by 28.28%, 21.34%, 1.36 times, and 21.26% respectively compared with the unmodified camelina protein in Comparative Example 1. This indicates that ultrasound-assisted enzymatic hydrolysis can improve the functional properties and antioxidant activity of the protein while improving the solubility of camelina protein. According to Figure 1-4 It can be found that the unmodified camelina protein presents a block-like aggregate structure. After ultrasonic treatment, the protein is broken and the pores increase, which helps to improve the protein hydrolysis rate. Therefore, ultrasound-assisted enzymatic hydrolysis helps to improve the functional properties and activity of the protein.

[0107] Comparative Example 2

[0108] The steps of soaking the defatted camelina powder in water and removing the soluble polysaccharides in the camelina after centrifugation were omitted, and the rest were the same as in Example 2 to obtain modified camelina protein peptides.

[0109] In order to study the effect of the water soaking step to remove soluble polysaccharides on the protein extraction effect, the present invention carried out performance tests on the modified camelina protein obtained in Example 2 and the modified camelina protein obtained in Comparative Example 2, including the protein purity and soluble sugar content shown in Table 2, and the solubility, foaming property, foam stability, ABTS clearance rate and DPPH clearance rate shown in Table 3.

[0110] Table 2 Test results of protein purity and soluble sugar content of comparative example 2

[0111] example Protein purity (%) Soluble sugar content (%) Comparative Example 2 81.25 7.91 Example 2 91.31 1.85

[0112] Table 3 Test results of protein performance of comparative example 2

[0113]

[0114] As can be seen from Table 2, the purity of camelina protein from which soluble polysaccharides were not removed in Comparative Example 2 was 81.25%, and the soluble sugar content was 7.91%. After the step of removing soluble polysaccharides, the soluble sugar content in camelina protein was reduced to 1.85%, and the protein purity was greatly increased to 91.31%. Since camelina contains a large amount of soluble polysaccharides, the step of removing soluble polysaccharides in camelina after centrifugation without soaking defatted camelina powder in water, camelina protein and polysaccharides are cross-linked, resulting in a decrease in the purity of camelina protein. As can be seen from Table 3, due to the decrease in the purity of camelina in Comparative Example 2, the enzymolysis efficiency of protease is reduced, and compared with Example 2, the functional properties and antioxidant activity are reduced to a certain extent.

[0115] Example 4 Optimization of ultrasonic time

[0116] The ultrasonic treatment time in step (1) of Example 2 was adjusted to 5, 25, and 35 min, and the other steps were kept the same as in Example 2 to obtain camelina protein peptide.

[0117] Comparative Example 3

[0118] Step (1) in Example 2 was omitted, and the rest of the steps were kept the same as in Example 2 to obtain unsonicated camelina protein peptide.

[0119] In order to study the effects of different process parameters of the ultrasonic step on the protein extraction effect, the present invention conducted performance tests on the modified camelina proteins obtained in Examples 2 and 4 and the modified camelina proteins obtained in Comparative Example 3, including solubility, foaming property, foam stability, ABTS clearance rate and DPPH clearance rate. The results are shown in Table 4.

[0120] Table 4 Test results of protein performance of Example 4

[0121]

[0122]

[0123] It can be seen from Table 4 that with the increase of ultrasonic treatment time, the solubility, foaming property, foam stability and ABTS clearance rate of camelina protein peptide are significantly improved. Compared with the camelina protein peptide without ultrasonic treatment in Comparative Example 3, when the ultrasonic time is 15min, the solubility of camelina protein peptide is increased by 42.76%, the foaming property is increased by 19.08%, the foam stability is increased by 7.89%, the ABTS clearance rate is increased by 39.42%, and the DPPH clearance rate is increased by 12.38%. However, when the ultrasonic time is extended to 25 and 35min, the functional properties and antioxidant activity of camelina protein peptide decrease, indicating that excessive ultrasound may cause partial aggregation of protein, which is not conducive to improving the proteolysis effect.

[0124] Example 5 Optimization of enzymolysis time

[0125] The enzymatic hydrolysis time in step (2) of Example 2 was adjusted to 10, 30, 60, and 90 min, and the rest was kept consistent with Example 2 to obtain camelina protein peptide.

[0126] Comparative Example 4

[0127] Step (2) in Example 2 was omitted, and the rest of the steps were kept the same as in Example 2 to obtain unenzymatically treated camelina protein peptide.

[0128] In order to study the effects of different process parameters in the enzymatic hydrolysis step on the protein extraction effect, the present invention conducted performance tests on the modified camelina proteins obtained in Examples 2 and 5 and the modified camelina proteins obtained in Comparative Example 4, including solubility, foaming property, foam stability, ABTS clearance rate and DPPH clearance rate. The results are shown in Table 5.

[0129] Table 5 Test results of protein performance of Example 5 and Comparative Example 3

[0130]

[0131] It can be seen from Table 5 that: with the increase of enzymatic hydrolysis time, the solubility and antioxidant activity of camelina protein peptides are significantly improved. Compared with the unenzymatically hydrolyzed camelina protein in Comparative Example 4, when the enzymatic hydrolysis time reaches 120 minutes, the solubility and ABTS clearance rate are increased by 77.73% and 109.06%, respectively, and the DPPH clearance rate is increased by 14.13%. This shows that the extension of the enzymatic hydrolysis time is conducive to the release of soluble peptides, and at the same time helps to increase the release of small molecule peptides, which helps to improve the antioxidant activity of the system. With the extension of the enzymatic hydrolysis time, the foaming property of camelina protein peptides also increases significantly, reaching the highest value when the enzymatic hydrolysis time is 120 minutes. However, the foam stability of camelina protein peptides is best when the enzymatic hydrolysis time is 60 minutes, reaching 56.59%. Figure 3It can be seen that under the conditions of 120 minutes of enzymatic hydrolysis, protein peptides intertwined to form a network structure, indicating that although long-term enzymatic hydrolysis helps to improve solubility and antioxidant activity, the formed peptide network structure aggregates are not conducive to the stability of the air-water interface, thereby reducing the foam stability.

[0132] Example 6 Determination of Camelina protein peptide sequence

[0133] The camelina protein obtained in Example 1 was subjected to protein peptide sequence determination to obtain a protein sequence. Specifically, the amino acid sequence of camelina protein (coded as J7I9N7) was obtained through the UniProt database, and the camelina protein was subjected to simulated alkaline protease (EC3.4.21.62) enzymolysis using the "enzyme action" program in the BIOPEP-UWM database. The peptides obtained by virtual enzymolysis were compared with the antioxidant peptides reported in the BIOPEP-UWM database, and the unreported novel peptides were screened for potential biological activity scores in the PeptideRanker prediction website. The higher the biological activity score, the greater the biological activity of the peptide. The toxicity of the peptide was predicted using the online website ToxinPred. Finally, a camelina protein active peptide with a score higher than the biological activity score>0.5 and no toxicity was obtained, and the amino acid sequence is shown in SEQ ID NO.1-SEQ ID NO.6 in Table 6.

[0134] Table 6 Amino acid sequence of active peptides from camelina protein

[0135] Serial number Peptide sequence Peptide amino acid composition Isoelectric point Molecular weight SEQ ID NO.1 GRPR Gly-Arg-Pro-Arg 12.01 484.6 SEQ ID NO.2 VPQ Val-Pro-Gln-Gly-Phe 5.88 546.69 SEQ ID NO.3 GPMS Gly-Pro-Met-Ser 5.88 390.5 SEQ ID NO.4 VKF Val-Lys-Gly-Pro-Phe 9.11 546.73 SEQ ID NO.5 AQAF Ala-Gln-Ala-Phe 5.88 435.52 SEQ ID NO.6 VIRPPL Val-ILe-Arg-Pro-Pro-Lys 10.11 693.97

[0136] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A camelina protein active peptide, characterized in that: The amino acid sequence of the protein active peptide is shown in any one of SEQ ID NO.1 to SEQ ID NO.

6.

2. The protein peptide according to claim 1, characterized in that Under neutral pH conditions, the protein peptide has a protein solubility higher than 50%, a foaming property higher than 150%, a foam stability higher than 49%, an ABTS free radical scavenging rate higher than 230 μmol TE / g protein, and a DPPH free radical scavenging rate higher than 82%.

3. The protein peptide according to claim 2, characterized in that The protein peptide has a protein solubility higher than 52%, a foaming property higher than 160%, a foam stability higher than 50%, an ABTS free radical scavenging rate higher than 240 μmol TE / g protein, and a DPPH free radical scavenging rate higher than 85%.

4. A method for preparing camelina protein peptide, characterized in that: The steps include: Step 1: Grind camelina and sieve to obtain camelina powder, and place the camelina powder in a n-hexane solution and stir to defat; Step 2: Soaking defatted camelina powder in water, and removing soluble polysaccharides in camelina after centrifugation; Step 3: placing camelina powder in water, extracting camelina protein by alkali dissolution and acid precipitation, centrifuging the obtained dispersion, redissolving the precipitate in water, adjusting the pH to 6-8, dialyzing and drying to obtain camelina protein powder; Step 4: After the camelina protein powder is ultrasonically treated, protease is added to hydrolyze and inactivate the enzyme to obtain camelina protein peptides.

5. The method according to claim 4, characterized in that In step 1, the sieving is through a 60-80 mesh sieve; and / or Preferably, the solid-liquid ratio of the camelina powder to the n-hexane solution is 1:10-1:30 w / v; and / or Preferably, the degreasing time is 12-24 hours.

6. The method according to claim 4, characterized in that In step 2, the solid-liquid ratio of camelina powder to water is 1:10-1:30 w / v, and the soaking time is 1-2 h; and / or Preferably, in step 2, the centrifugal parameters are 5000-10000 r / min and the time is 10-30 min.

7. The method according to claim 1, characterized in that In step 3, the camelina powder is placed in water, wherein the ratio of the camelina powder to water is 1:10-1:30 w / v, and the soaking time is 2-4 hours; and / or Preferably, in step 3, in the alkali dissolution and acid precipitation, the alkali dissolution pH is adjusted to 8-10, the solution for adjusting the pH is a 2-4 mol / L NaOH solution, and the temperature parameter is 40-50° C.; and / or Preferably, in step 3, in the alkali dissolution and acid precipitation, the pH of the acid precipitation is adjusted to 3-5, the solution for adjusting the pH is 2-4 mol / L HCl solution, and the temperature parameter is 40-50°C.

8. The method according to claim 4, characterized in that In step 4, the power of the ultrasonic treatment is 120-360W; preferably, the power of the ultrasonic treatment is 240-360W; preferably, the power of the ultrasonic treatment is 360W; and / or Preferably, the ultrasonic treatment time is 5-35 min; preferably, the ultrasonic treatment time is 10-35 min; preferably, the ultrasonic treatment time is 10-25 min; preferably, the ultrasonic treatment time is 15 min; and / or Preferably, the protease is alkaline protease, the ratio of enzyme to substrate is 1:30-1:50 w / w, and / or Preferably, the enzymolysis time is 10min-120min; preferably, the enzymolysis time is 10min-90min; preferably, the enzymolysis time is 30min-90min; preferably, the enzymolysis time is 40min-80min; preferably, the enzymolysis time is 50min-70min; and / or Preferably, the pH of the enzymatic hydrolysis is 8-10 and the temperature is 40-50°C.

9. A composition comprising the protein peptide according to claims 1 to 3 or the camelina protein peptide prepared by the method according to any one of claims 4 to 8.

10. Use of the protein peptide according to claims 1 to 3, or the camelina protein peptide prepared by the method according to any one of claims 4 to 8, or the composition according to claim 9 in food or medicine.

11. A medicine comprising the protein peptide according to any one of claims 1 to 3 or the camelina protein peptide prepared by the method according to any one of claims 4 to 8, or comprising the composition according to claim 9.

12. A food comprising the protein peptide according to any one of claims 1 to 3 or the camelina protein peptide prepared by the method according to any one of claims 4 to 8, or comprising the composition according to claim 9.

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