A method for preparing plant-derived antifreeze peptides by ultrasound-assisted enzymatic hydrolysis and its application

Abstract

This invention relates to a method for preparing plant-derived antifreeze peptides using ultrasound-assisted enzymatic hydrolysis and its applications, comprising the following steps: pretreating a protein suspension with ultrasound; then enzymatically hydrolyzing the suspension with Alcalase, collecting the supernatant, dialysis to remove salt, and freeze-drying to obtain the antifreeze peptides. This invention utilizes ultrasound pretreatment and, by controlling the enzymatic hydrolysis conditions, the prepared antifreeze peptides exhibit 1.10–2.40 times higher thermal hysteresis activity, improved ice recrystallization inhibition activity, and 1.94–3.68 times higher surface hydrophobicity. α ~spiral and β The folding efficiency was increased by 1.17–1.35 times and 1.01–1.12 times, respectively. This invention utilizes ultrasound to expose the antifreeze active sites in proteins and employs a limited enzymatic hydrolysis method to directionally prepare highly active plant-derived antifreeze peptides, saving enzymatic hydrolysis time. The resulting antifreeze peptides exhibit excellent cryoprotective effects on frozen dough and yeast.

Description

Technical Field

[0001] This invention belongs to the field of plant protein deep processing technology, specifically relating to a method for preparing plant-derived antifreeze peptides by ultrasound-assisted enzymatic hydrolysis and the application of plant-derived antifreeze peptides. Background Technology

[0002] Antifreeze proteins (AFPs) have attracted widespread attention due to their ability to inhibit ice crystal growth and recrystallization, thus maintaining the quality of frozen products. Recent research indicates that the expression of antifreeze protein activity is not a function of the entire protein, but rather related to specific polypeptide chain domains in localized areas. Obtaining safe, compact, and highly active antifreeze peptides through enzymatic hydrolysis of protein byproducts has become an emerging research hotspot. However, protein byproducts generated during agricultural product processing undergo significant deformation due to physical factors such as heat, high pressure, mechanical extrusion, and shearing during processing. Furthermore, their low solubility, high susceptibility to water absorption and swelling, and poor dispersibility severely affect the release of active antifreeze peptide fragments and the activity of the hydrolysate during enzymatic hydrolysis. Targeted regulation of the substrate protein molecular structure to obtain highly active antifreeze peptide fragments is key to overcoming the limitations of antifreeze peptide applications and significantly improving the quality of frozen foods. Summary of the Invention

[0003] To address the problems existing in the prior art, this invention provides a method for preparing plant-derived antifreeze peptides by ultrasound-assisted enzymatic hydrolysis. This method is a novel physical modification technique. Using this method to treat plant-derived proteins can significantly improve the functional properties of the proteins, enhance their antifreeze activity, and efficiently prepare antifreeze peptides.

[0004] To solve the above problems, the present invention adopts the following technical solution:

[0005] A method for preparing antifreeze peptides by ultrasound-assisted enzymatic hydrolysis involves preparing a protein suspension at a mass fraction of 1-5%, followed by ultrasonic pretreatment to improve the protein molecular structure and target and expose antifreeze active sites (hydrophobic amino acids of the protein, mainly Val, Met, Leu, Pro and Glu, as well as antifreeze active sites such as α-helices and β-sheets). The protein is then subjected to limited enzymatic hydrolysis to obtain highly active plant-derived antifreeze peptides.

[0006] Furthermore, in the above technical solution, the protein is gluten protein, flaxseed meal protein, or barley bran protein.

[0007] Furthermore, in the above technical solution, the ultrasonic frequency of the ultrasonic pretreatment is 20-60 kHz, the ultrasonic power density is 50-200 W / L, and the treatment time is 5-20 min.

[0008] Furthermore, in the above technical solution, the ultrasonic working mode is a flat panel type, a probe type, or a combined type, wherein the combined type is a flat panel type combined with a probe type.

[0009] Furthermore, in the above technical solution, the flat-plate ultrasonic time is 5-20 min, the ultrasonic power density is 50-200 W / L, the ultrasonic pretreatment temperature is 30-50℃, and the ultrasonic frequency is 20-60 kHz.

[0010] Furthermore, in the above technical solution, the probe-type ultrasonic working time is 10-15 min, the ultrasonic power density is 50-150 W / L, the ultrasonic processing temperature is 30-40℃, and the ultrasonic frequency is 20-40 kHz.

[0011] Furthermore, in the above technical solution, the collaborative ultrasonic working time is 10-15 min, the ultrasonic power density is 100-150 W / L, the ultrasonic treatment temperature is 35-40℃, and the ultrasonic frequency is 20-35 kHz.

[0012] Furthermore, in the above technical solution, the specific operation of the probe-type ultrasonic pretreatment is as follows: the ultrasonic probe is inserted into the protein suspension and the ultrasonic wave is turned on to treat it. The insertion depth of the ultrasonic probe in the protein suspension is 2-4 cm, and the diameter of the ultrasonic probe is 0.2-0.5 cm.

[0013] Furthermore, in the above technical solution, the protein suspension after ultrasonic pretreatment is enzymatically hydrolyzed using Alcalase protease. The specific conditions for enzymatic hydrolysis are: pH 8.0–9.0, temperature 45–55℃, Alcalase protease added at a ratio of 6080 U / g, and limited enzymatic hydrolysis time of 30–120 min, preferably 60–90 min.

[0014] Furthermore, the above technical solution also includes purifying the protein suspension after removing the polysaccharides from the plant fiber tissue by enzymatic hydrolysis. The specific purification process is as follows: adjusting the pH of the protein suspension to 8.0–9.0, stirring at 48–52°C for 30–120 min, inactivating the enzyme by bathing in a 100°C water bath for 10 min, cooling, centrifuging to collect the supernatant, and adjusting the pH of the supernatant to 6.8–7.2.

[0015] Furthermore, in the above technical solution, the supernatant collected by centrifugation is dialyzed in deionized water at 4°C for 24 hours to remove salt, freeze-dry, and grind through an 80-100 mesh sieve to obtain plant-derived antifreeze peptides.

[0016] The present invention also provides plant-derived antifreeze peptides prepared using the method described above.

[0017] This invention also provides the application of the plant-derived antifreeze peptides as cryoprotectants in frozen dough, yeast, and minced meat, which have good cryoprotection effects.

[0018] The beneficial effects of this invention are as follows: The cavitation effect generated by ultrasound exposes hydrophobic groups inside protein molecules to the molecular surface, and ultrasound pretreatment for a certain period of time can effectively regulate the molecular structure of proteins. This invention uses ultrasound technology to pretreat byproduct proteins such as gluten, flaxseed meal protein, and barley bran protein to regulate the protein molecular structure. Limited enzymatic hydrolysis technology is then used for targeted enzymatic hydrolysis, significantly shortening the hydrolysis time and obtaining highly active antifreeze peptides.

[0019] (1) This invention uses gluten protein, flaxseed protein, and highland barley protein as raw materials. Differential thermal scanning and optical microscopy were used to study the effects of different ultrasonic pretreatments on the thermal hysteresis activity, ice recrystallization inhibition activity, molecular weight distribution, amino acid composition, and surface hydrophobicity of antifreeze peptides. The results showed that the thermal hysteresis activity of the prepared antifreeze peptides was increased by 1.10–2.40 times, the ice recrystallization inhibition activity was improved, the surface hydrophobicity was increased by 1.94–3.68 times, and the α-helix and β-sheet were increased by 1.17–1.35 times and 1.01–1.12 times, respectively. Furthermore, the synergistic ultrasonic working mode was superior to the single working mode. This invention uses ultrasound to expose the antifreeze active sites (hydrophobic amino acids and α-helices) in proteins and employs a limited enzymatic hydrolysis method to directionally prepare highly active plant-derived antifreeze peptides, saving enzymatic hydrolysis time and providing a theoretical basis for the efficient preparation of antifreeze peptides.

[0020] (2) The ultrasonic pretreatment method provided by the present invention has a short action time, is simple to operate and easy to control, and the resulting antifreeze peptides have excellent functional properties.

[0021] (3) The antifreeze peptides prepared in this invention have good cryoprotective effects on frozen dough and yeast. Attached Figure Description

[0022] Figure 1The following are the results of the effect of ultrasound-assisted enzymatic hydrolysis on the activity of flaxseed meal peptide IRI in the embodiments of the present invention (Note: A1, sucrose solution; A2, sucrose + plate-type ultrasound antifreeze peptide; A3, sucrose + probe-type ultrasound antifreeze peptide; A4, sucrose + synergistic ultrasound antifreeze peptide; B1, sucrose + ultrasound antifreeze peptide for 5 min; B2, sucrose + ultrasound antifreeze peptide for 10 min; B3, sucrose + ultrasound antifreeze peptide for 15 min; B4, sucrose + ultrasound antifreeze peptide for 20 min; C1, sucrose + ultrasound power density of 50 W / L; C2, sucrose + ultrasound power density of 100 W / L; C3, sucrose + ultrasound power density of 150 W / L; C4, sucrose + ultrasound power density of 200 W / L; magnification, 100x).

[0023] Figure 2 The results of the effect of ultrasound-assisted enzymatic hydrolysis on the size distribution of flaxseed meal peptide ice crystals are shown in the embodiments of the present invention. Figure 3 The results of ultrasound-assisted enzymatic hydrolysis preparation of antifreeze peptides and its effect on surface hydrophobicity are shown in the embodiments of the present invention.

[0024] Figure 4 The results show the effect of antifreeze peptides on yeast survival rate. Detailed Implementation

[0025] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of protection of the present invention, which is defined by the claims.

[0026] Unless otherwise specified, all experimental reagents and materials used in the embodiments of this invention are commercially available.

[0027] Unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0028] Example 1

[0029] A method for preparing antifreeze peptides by ultrasound-assisted enzymatic hydrolysis includes the following steps:

[0030] (1) Prepare three protein suspensions with a mass fraction of 3% each: gluten protein, flaxseed meal protein and highland barley bran protein. Stir the prepared solutions at 50°C for 10 minutes and pre-treat them with probe-type ultrasound, flat-plate ultrasound and synergistic ultrasound respectively.

[0031] (2) Probe-type ultrasonic, flat-panel ultrasonic and synergistic ultrasonic treatment, with power density uniformly set to 150W / L, ultrasonic treatment time 15min, and treatment temperature 30℃.

[0032] (3) After the protein suspension is treated with ultrasound, Alcalase alkaline protease is added at a ratio of 6080 U / g. The enzymatic hydrolysis time is 90 min. Immediately after the enzymatic hydrolysis is completed, the protease hydrolysate is placed in a 100℃ water bath for 10 min to inactivate the enzyme. After cooling to room temperature, the pH value is adjusted to 7.0. The protein is then dialyzed in deionized water at 4℃ for 24 h, freeze-dried, ground and sieved to obtain protein powder.

[0033] Comparative Example 1

[0034] The comparative method of this invention is similar to that of Example 1, except that the ultrasonic pretreatment step in Example 1 is omitted, while the other process conditions and parameters are the same as those in Example 1.

[0035] Results Analysis

[0036] The ultrasonic working modes in the examples—flat plate, probe, and synergistic—were measured respectively; the ultrasonic power density was 150 W / L, the ultrasonic treatment time was 15 min, and the thermal hysteresis activity, ice-inhibiting recrystallization activity, molecular weight distribution, surface hydrophobicity, and secondary structure of the samples obtained in the comparative examples were also measured. Specific results are as follows.

[0037] Table 1. Ultrasonic-assisted enzymatic hydrolysis preparation of antifreeze peptides and their effect on antifreeze activity.

[0038]

[0039] Note: THA stands for thermal hysteresis activity. Results are expressed as the mean ± standard deviation of three trials. Different superscript letters in the same column indicate significant differences (p < 0.05).

[0040] Table 2 Effects of ultrasound-assisted enzymatic hydrolysis on the molecular weight distribution of plant-derived antifreeze peptides

[0041]

[0042]

[0043] Note: M1, >2000Da; M2, 1000~2000Da; M3, 200~1000Da; M4, <200Da; M5<1000Da.

[0044] Table 3 Effects of ultrasound-assisted enzymatic hydrolysis on the secondary structure of antifrozen peptides

[0045]

[0046] Table 4. Effects of ultrasound-assisted enzymatic hydrolysis on the amino acid composition of gluten antifreeze peptides

[0047]

[0048] Analysis of the above results shows that, compared with the control sample, as shown in Tables 1 and 2, the thermal hysteresis activity of the enzymatic hydrolysate after ultrasonic pretreatment of the protein increased by 1.1 to 2.4 times, and to some extent, it caused a shift in the molecular weight distribution of the peptides towards smaller peptides, with the molecular weight mainly concentrated around 200–1000 Da; Figure 1 , 2 The results show that the ice recrystallization inhibition activity of the samples pretreated with ultrasound was improved and the proportion of large ice crystals was reduced. According to Table 3, the α-helix and β-sheet structures were increased by 1.17–1.35 times and 1.01–1.12 times, respectively, compared with the control sample. Table 4 shows that ultrasound treatment exposed the active sites of amino acids. According to the results, the antifreeze peptides are rich in several amino acids such as Glu, Pro, Leu, Val, and Phe. Among them, the relative contents of amino acids such as Val, Met, Pro, and Ser were significantly increased compared with the control sample after ultrasound treatment.

[0049] Comparative Example 2: Taking gluten antifreeze peptides as an example, following the steps of Example 1, probe-type ultrasonic treatment was used. Then, Alcalase alkaline protease was added at an enzyme-to-protein ratio of 6080 U / g. The enzymatic hydrolysis times were 30 min, 60 min, 90 min, and 120 min. Immediately after the hydrolysis was completed, the protease hydrolysate was placed in a 100℃ water bath for 10 min to inactivate the enzyme. After cooling to room temperature, the pH was adjusted to 7.0, and the solution was dialyzed in deionized water at 4℃ for 24 h. After freeze-drying, the solution was ground and sieved to obtain protein powder. The thermal hysteresis activity was examined, and the results are shown in Table 5.

[0050] Table 5. Effect of enzymatic hydrolysis time on the thermal hysteresis activity of gluten hydrolysate.

[0051]

[0052] As shown in Table 5, the thermal hysteresis activity of gluten peptides first increases and then decreases with the extension of enzymatic hydrolysis time and the deepening of enzymatic hydrolysis. The thermal hysteresis activity is strongest when the enzymatic hydrolysis time is 60-90 min. This proves that moderate hydrolysis can maximize the acquisition of antifreeze peptide active fragments and avoid the secondary decomposition of active fragments due to excessive enzymatic hydrolysis.

[0053] Example 2

[0054] The applications of plant-derived antifreeze peptides as cryoprotectants are as follows:

[0055] (1) The dough preparation formula is as follows: flour (100%), water (50%), dry yeast (1.5%), antifreeze peptides (0.5%);

[0056] (2) First, mix the yeast and antifreeze peptides evenly in some deionized water. Then, add all the ingredients to the mixer and mix for 5 minutes. Divide the dough, shape it by hand, and seal it in a polyethylene bag.

[0057] (3) Quickly place the prepared dough into a -40℃ ultra-low temperature freezer and let it stand for 1 hour to allow the center temperature of the dough to reach -18℃. Then transfer it to a -18℃ freezer for further freezing.

[0058] (4) The dough is then subjected to repeated freeze-thaw cycles (8 times). Each freeze-thaw cycle consists of a 23-hour freezing process at -18°C and a 1-hour thawing process. The dough is thawed in a constant temperature and humidity chamber at 25°C and 80% humidity.

[0059] Comparative Example 2 (Control Sample)

[0060] The only difference from Example 2 is that the step of adding antifreeze peptides in Example 2 is omitted; all other process conditions and parameters are the same as in Example 2.

[0061] Results Analysis

[0062] The effects of gluten antifreeze peptides, flaxseed meal antifreeze protein, highland barley bran antifreeze peptides, and dough in the examples and the comparative examples on fermentation characteristics, moisture distribution, texture characteristics, and yeast cell survival rate were determined respectively. The specific results are as follows.

[0063] Table 6. Effects of antifreeze peptides on the fermentation properties of frozen dough.

[0064]

[0065] Note: H m The maximum height the dough reached; h, the final height of the dough at the end of the test; T1, the time required for the dough to reach its maximum height; T x Vt is the time when the dough begins to cavitate, i.e., when the dough begins to leak CO2; Vr is the total volume of gas released; Vt is the volume of CO2 remaining in the dough at the end of the experiment.

[0066] Table 7. Effects of antifreeze peptides on moisture distribution in frozen dough.

[0067]

[0068]

[0069] Table 8. Effects of antifreeze peptides on the textural properties of frozen dough.

[0070]

[0071] Analysis of the results in Tables 6 and 7 shows that, compared to the control sample, the H content of the dough with 0.5% antifreeze peptides was significantly higher.m The levels of antifreeze peptides and h increased by 1.18–1.24 and 1.14–1.52 times, respectively, and the time required for the dough to reach its maximum height also decreased significantly. According to Table 7, compared with the control dough, the addition of antifreeze peptides increased the A content in the dough. 21 Significantly improved, A 22 Significantly reduced, A 23 Improvements were observed when adding flaxseed meal antifreeze peptides; according to Figure 1 The yeast cell survival rate results show that adding antifreeze peptides can effectively inhibit the damage to yeast cells caused by freezing. These findings effectively demonstrate that adding antifreeze peptides to dough can effectively reduce the destructive effects of freezing on yeast and gluten protein systems, thereby shortening the dough's proofing time and improving the yeast's gas production and the dough's gas-holding capacity. Furthermore, a stable moisture state and uniform moisture distribution are more beneficial for dough fermentation, resulting in fermented pasta with a large specific volume and a soft, elastic texture.

[0072] Finally, the above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing plant-derived antifreezing polypeptides by ultrasonic-assisted enzymatic hydrolysis, characterized in that, The method comprises the following steps: The protein is prepared into a protein suspension for ultrasonic pretreatment, and then the protein suspension after ultrasonic pretreatment is subjected to limited enzymolysis to obtain a high-activity plant source anti-freezing polypeptide; The ultrasonic pretreatment has an ultrasonic frequency of 20-35 kHz, an ultrasonic power density of 100-150 W / L, and a treatment time of 10-15 min; The ultrasonic working mode is a cooperative mode, and the cooperative mode is a flat plate combined with a probe type; The protein suspension after ultrasonic pretreatment is subjected to enzymolysis by using Alcalase protease, and the enzymolysis conditions are as follows: pH 8.0-9.0, temperature 45-55 ℃, Alcalase protease is added in a ratio of 6080 U / g of enzyme to substrate, and the limited enzymolysis time is 60-90 min; The mass fraction of the protein suspension is 1-5%, and the protein is glutenin, flaxseed meal protein or barley bran protein.

2. The method for preparing plant-derived antifreeze polypeptides by ultrasonic-assisted enzymatic hydrolysis according to claim 1, characterized in that: The flat plate ultrasonic time is 5-20 min, the ultrasonic power density is 50-200 W / L, the ultrasonic pretreatment temperature is 30-50 ℃, and the ultrasonic frequency is 20-60 kHz; The probe type ultrasonic working time is 10-15 min, the ultrasonic power density is 50-150 W / L, the ultrasonic treatment temperature is 30-40 ℃, and the ultrasonic frequency is 20-40 kHz.

3. The method for preparing plant-derived antifreeze peptides by ultrasound-assisted enzymatic hydrolysis according to claim 1, characterized in that: Further, the protein suspension after enzymolysis is subjected to purification treatment, and the purification treatment is specifically operated as follows: the pH value of the protein suspension is adjusted to 8.0-9.0, and stirring is performed at 48-52 ℃ for 30-120 min, enzyme is inactivated by water bath at 100 ℃ for 10 min, after cooling, the supernatant is collected by centrifugation, and the pH value of the supernatant is adjusted to 6.8-7.

2.

4. The method for preparing plant-derived antifreeze polypeptides by ultrasonic-assisted enzymatic hydrolysis according to claim 3, characterized in that: The centrifugally collected supernatant is dialyzed in 4 ℃ deionized water for 24 h, desalted, freeze-dried, ground through an 80-100 mesh sieve, and the plant source anti-freezing polypeptide is obtained.

5. The plant source anti-freezing polypeptide prepared by the method of any one of claims 1-4.

6. Application of the plant source anti-freezing polypeptide of claim 5 as a freezing protective agent in frozen dough, yeast and meat paste.

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