Polypeptide FADHPG and its application in the preparation of antifreeze and antioxidant preparations

Abstract

The present invention discloses a polypeptide FADHPG and its application in the preparation of antifreeze and antioxidant preparations, belonging to the technical field of bioactive peptides. The amino acid sequence of the polypeptide FADHPG is FADHPG, as shown in SEQ ID NO.1. The present invention also discloses the application of the polypeptide FADHPG in the preparation of antifreeze and antioxidant preparations. The present invention mines new antifreeze and antioxidant peptides from the protein of Penaeus vannamei by a dual deep learning method. Experimental analysis and verification show that the average particle size of ice crystals treated with the polypeptide FADHPG is reduced by more than 50%, and the free radical scavenging ability reaches 47%, with excellent effect. The present invention provides a new raw material basis for the development of new, efficient, composite and multifunctional antifreeze and antioxidant agents, and has good application prospects and important application value.

Description

Technical Field

[0001] The invention relates to a polypeptide FADHPG and application thereof in the preparation of an antifreeze and antioxidant preparation, belonging to the technical field of bioactive peptides. Background Art

[0002] During the frozen storage of food, ice crystal damage and oxidation reactions often occur simultaneously, and the two synergistically aggravate the quality deterioration. Specifically, ice crystal damage destroys the cell membrane structure, releases pro-oxidant enzymes (such as lipoxygenase), and accelerates lipid oxidation; free radicals produced by oxidation reactions further damage cell components and reduce the nutritional and sensory properties of food. In the prior art, antifreeze and antioxidant functions are mostly achieved by adding different preparations separately, but composite additives may cause compatibility problems and increase production costs.

[0003] Antifreeze peptides can regulate the growth of ice crystals by adsorbing on their surfaces, significantly improving the quality of frozen foods. Antioxidant peptides delay oxidation reactions by scavenging free radicals, inhibiting lipid peroxidation, and chelating metal ions. In recent years, multifunctional peptides (i.e., multifunctional peptides) have become a research hotspot due to their synergistic effects and high efficiency. Multifunctional peptides with both antifreeze and antioxidant activities can solve the problem of quality deterioration caused by ice crystal formation and oxidation reactions during the freezing process. Therefore, the development of a single peptide with both antifreeze and antioxidant functions has important application value.

[0004] Litopenaeus vannamei ( Litopenaeus vannamei ) is one of the most productive farmed shrimp species in the world. Its muscle is rich in protein and nutritional value, and has great potential for the development of multifunctional bioactive peptides. So far, there has been no report on the isolation of peptides with both antifreeze and antioxidant functions from Litopenaeus vannamei. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a polypeptide FADHPG and its application in the preparation of antifreeze and antioxidant preparations, belonging to the technical field of bioactive peptides.

[0006] The present invention is achieved through the following technical solutions:

[0007] A polypeptide FADHPG, whose amino acid sequence is FADHPG, as shown in SEQ ID NO.2.

[0008] The polypeptide FADHPG is used in the preparation of antifreeze and antioxidant preparations.

[0009] The present invention combines virtual enzymatic hydrolysis, deep learning and multiple screening technologies to mine antifreeze antioxidant peptides from Penaeus vannamei. First, myosin (sequence ID: LOC113807016), which has the highest content in Penaeus vannamei, is selected from the NCBI database as the enzymatic hydrolysis object. Then, the theoretical antioxidant peptide sequence is obtained using the AnOxPePred online program, and the amino acid sequence of the above myosin is input into the program for virtual enzymatic hydrolysis. Since short peptides usually have better stability, biological activity and solubility, only peptides with a sequence length of 2 to 10 amino acids are selected, and then PeptideRanker is used to score the peptides to screen for peptides that are more likely to have biological activity. Then, the antifreeze peptide prediction model based on the bidirectional long short-term memory network (Bidirectional LSTM, Bi-LSTM) deep learning algorithm trained by the inventor's team is used to screen the potential antioxidant peptide model to obtain potential antifreeze antioxidant peptides. Then, the toxicity of the screened peptides was tested by the ToxinPred online program, and the water solubility of the screened peptides was tested by the Innovagentool online program to obtain candidate antifreeze and antioxidant peptides. Finally, potential antifreeze and antioxidant peptides were synthesized in the solid phase and verified by experiments to screen out multifunctional peptides with both antifreeze and antioxidant properties.

[0010] The present invention uses dual deep learning to compositely screen antifreeze antioxidant peptides in Litopenaeus vannamei, and as a result, four potential antifreeze antioxidant peptides are screened out, namely, polypeptide FADHPGQ, polypeptide FADHPG, polypeptide DHPG and polypeptide RNEVPPHLF. Experimental analysis and verification show that compared with the control, the average particle size of ice crystals treated with polypeptide FADHPG is reduced by more than 50%, and the free radical scavenging ability reaches 47%, with excellent results. The present invention provides a new raw material basis for the development of new, efficient, composite and multifunctional antifreeze antioxidants, and has good application prospects and important application value.

[0011] The present invention mines new antifreeze and antioxidant peptides from the protein of Penaeus vannamei through a dual deep learning method, avoiding the time-consuming and labor-intensive disadvantages caused by step-by-step separation and purification, and avoiding the loss of polypeptides caused by cumbersome experimental steps. The polypeptide can simultaneously inhibit ice crystal growth and scavenge free radicals, effectively solving the complex quality problems of frozen foods.

[0012] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Schematic diagram of ice crystal size detection results.

[0014] Figure 2 : Schematic diagram of free radical scavenging rate test results.

[0015] Figure 3 : Schematic diagram of thawing loss rate detection results. DETAILED DESCRIPTION

[0016] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0017] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0018] Example 1 Protein determination of Litopenaeus vannamei

[0019] Myosin is the protein with the highest content in Penaeus vannamei. Myosin with sequence ID LOC113807016 was selected from the NCBI database as the enzymatic hydrolysis object, and potential antifreeze and antioxidant peptides were obtained through a deep learning model.

[0020] Example 2: Initial screening of potential antioxidant peptides using deep learning

[0021] The AnOxPePred antioxidant peptide screening model was used to screen potential antioxidant peptides from Litopenaeus vannamei myosin (https: / / services.healthtech.dtu.dk / services / AnOxPePred-1.0 / ). The myosin amino acid sequence in Example 1 was pasted into the window, "Minimum peptide length" was set to 2, "Maximum peptide length" was set to 10, and "Submit" was clicked to obtain peptide sequences with a length between 2 and 10. Potential antioxidant peptides were then screened based on "Scavenger>0.5".

[0022] Results: A total of 303 potential antioxidant peptide sequences were screened from myosin from Litopenaeus vannamei using the AnOxPePred antioxidant peptide prediction model.

[0023] Example 3 Deep learning further screens highly active antioxidant peptides

[0024] Potential antioxidant peptides were further screened by PeptideRanker (distilldeep.ucd.ie / PeptideRanker / ). The 303 potential antioxidant peptide sequences obtained in Example 2 were pasted into the window, and then "Predict" was clicked to obtain the PeptideRanker score of the above sequence. The higher the score, the greater the possibility that the peptide has biological activity. Finally, the peptides were screened again with a PeptideRanker score greater than 0.5.

[0025] Results: 150 potential antioxidant peptides with high bioactivity were screened out from 303 potential antioxidant peptide sequences using the PeptideRanker peptide scoring model.

[0026] Example 4 Deep learning screening of antifreeze and antioxidant peptides

[0027] Antifreeze peptides and non-antifreeze peptides were collected from existing Chinese and English papers, and training and test sets were constructed respectively. A position-encoded antifreeze peptide prediction model AFPepPredictor was developed using data enhancement and bidirectional long short-term memory network algorithms, and the code was encapsulated as an h5 model file. AFPepPredictor was used to further screen antioxidant peptides with potential antifreeze activity from highly bioactive antioxidant peptides. The 150 potential highly bioactive antioxidant peptides obtained in Example 3 were saved as a txt file, and the encapsulated h5 antifreeze peptide prediction model file was opened in PyCharm, and the peptides were screened again with a score greater than 0.7.

[0028] Results: AFPepPredictor antifreeze peptide prediction model was used to screen 21 potential antifreeze antioxidant peptides from 150 potential high bioactive antioxidant peptide sequences.

[0029] Example 5 Verification of the toxicity and water solubility of potential antifreeze antioxidant peptides

[0030] The toxicity and water solubility of the 21 peptides obtained in Example 4 were tested by two online programs, ToxinPred (https: / / crdd.osdd.net / raghava / toxinpred / index.html) and Innovagentool (https: / / www.innovagen.com / proteomicstools). Non-toxic and water-soluble peptides were selected as potential antifreeze and antioxidant peptides for subsequent analysis.

[0031] Results: The four peptides, namely, polypeptide FADHPGQ, polypeptide FADHPG, polypeptide DHPG and polypeptide RNEVPPHLF, were non-toxic and well soluble in water. Among them, the amino acid sequence of polypeptide FADHPGQ is shown in SEQ ID NO.1, the amino acid sequence of polypeptide FADHPG is shown in SEQ ID NO.2, the amino acid sequence of polypeptide DHPG is shown in SEQ ID NO.3, and the amino acid sequence of polypeptide RNEVPPHLF is shown in SEQ ID NO.4.

[0032] Example 6 Solid Phase Synthesis of Potential Antifreeze and Antioxidant Peptides

[0033] Since the sequences of peptide FADHPGQ, peptide FADHPG, and peptide DHPG are obviously similar, and the peptide FADHPG has a higher PeptideRanker score, this sequence was selected for testing among the three. Sangon Biotech Co., Ltd. (Shanghai, China) was commissioned to synthesize peptide FADHPG and peptide RNEVPPHLF using the Fmoc solid phase method, with a purity of not less than 95%.

[0034] Example 7 Determination of ice recrystallization inhibition ability of potential antifreeze antioxidant peptides

[0035] The peptides FADHPG and RNEVPPHLF were dissolved in 20% sucrose solution (unit: g / ml) to prepare 20 mg / mL sample solutions. The experiment was divided into three groups: FADHPG group and RNEVPPHLF group, and the sucrose solution without peptide was used as the control group. 2 μL of sample was titrated on a glass slide and covered with a glass coverslip. The slide was quickly frozen to -20°C at a rate of 20°C / min and kept for 1 min. The sample temperature was then raised to -6°C at a rate of 10°C / min and then kept for 15 min. ImageJ was used to analyze the changes in ice crystal size.

[0036] The ice crystal size test results of each group are as follows Figure 1 As shown, after the same recrystallization time, the ice crystal particle area of ​​the control group was larger than that of the FADHPG group and the RNEVPPHLF group, and the ice crystal particle area of ​​the FADHPG group was also smaller than that of the RNEVPPHLF group, indicating that the polypeptide FADHPG has good ice recrystallization inhibition performance and exhibits good antifreeze activity.

[0037] Example 8 Determination of free radical scavenging ability of potential antifreeze antioxidant peptides

[0038] Prepare 0.1 mmol / L 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) solution with anhydrous ethanol, mix 100 μL of the solution with 100 μL of antifreeze antioxidant peptide sample solution, and measure the absorbance value A at 517 nm.1 ; Mix 100 μL of anhydrous ethanol with 100 μL of antifreeze antioxidant peptide sample solution and measure the absorbance value A 2 ; Mix 100 μL of DPPH-anhydrous ethanol solution with 100 μL of ultrapure water and measure the absorbance value A 3 The DPPH free radical scavenging rate was calculated according to the following formula: .

[0039] The free radical scavenging rate test results of peptide FADHPG and peptide RNEVPPHLF are as follows Figure 2 As shown, from Figure 2 It can be found that the free radical scavenging rate of polypeptide FADHPG is as high as 47%, and the free radical scavenging rate of polypeptide RNEVPPHLF is 22%, so polypeptide FADHPG has good antioxidant capacity.

[0040] Example 9 Antifreeze application of potential antifreeze antioxidant peptides in shrimp paste

[0041] Remove the shrimp heads and shells, take out the shrimp meat, rinse it clean to make the moisture content about 50%, use a meat grinder to chop it until it becomes a viscous shrimp paste, and then divide it into three groups, among which the control group adds 1% salt (weight percentage, the same below), the FADHPG group adds 1% salt and 1% polypeptide FADHPG, and the RNEVPPHLF group adds 1% salt and 1% polypeptide RNEVPPHLF. After mixing, weigh the mass before freezing, recorded as m 1 Freeze at −18℃ for 18 h and thaw at 4℃ for 6 h as one freeze-thaw cycle. After 4 freeze-thaw cycles, thaw on a slope at room temperature for 1 h and weigh the mass again, recording it as m. 2 The thawing loss rate is calculated according to the following formula: .

[0042] The results of thawing loss rate test of each group are as follows Figure 3 As shown, from Figure 3 It can be found that compared with the control group, the addition of polypeptide FADHPG significantly reduced the thawing loss of shrimp paste, indicating that polypeptide FADHPG reduced the recrystallization of ice crystals in shrimp paste during frozen storage, alleviated the damage to the shrimp paste structure caused by ice crystal growth, and played an antifreeze protection role. Therefore, polypeptide FADHPG has potential application value in the frozen storage of shrimp paste foods.

[0043] In summary, through the above experimental results, it can be found that the polypeptide FADHPG has good antifreeze and antioxidant abilities, and is a new type of antifreeze and antioxidant peptide with high potential.

[0044] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A polypeptide FADHPG, characterized in that: The amino acid sequence is FADHPG, as shown in SEQ ID NO.

2.

2. Use of the polypeptide FADHPG according to claim 1 in the preparation of antifreeze and antioxidant preparations.

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