Antifreeze glycopeptide as well as preparation method and application thereof

Abstract

The invention discloses an anti-freezing glycopeptide as well as a preparation method and application thereof, and relates to the technical field of freezing resistance. The amino acid sequence of the antifreeze glycopeptide is AFP1: DWWDPGFFGFPPH, and the amino acid sequence of the antifreeze glycopeptide is as shown in SEQ ID No. 1; aFP2: YGGPKPSPVDIDDW, and the AFP2 is as shown in SEQ ID No. 2. According to the preparation method disclosed by the invention, the crab shell enzymolysis product is creatively used as a basis, a non-enzymatic glycosylation method is adopted, and the crab shell polypeptide is combined with the glucan, so that the stability and the flavor of the polypeptide are obviously improved, and the anti-freezing protection performance is improved.

Description

Technical Field

[0001] The present invention relates to the field of antifreeze technology, and more particularly to an antifreeze glycopeptide and a preparation method and application thereof. Background Art

[0002] At present, people have long used commercial antifreeze to reduce freezing damage of cold chain products and have achieved certain results. Currently commercial antifreezes include polyphosphates, sugars, alcohols and their compounds. Polyphosphates can increase the bitterness of food and affect the body's calcium absorption, and can aggravate the symptoms of patients with hypertension and chronic kidney disease. Sugars are high in sweetness and calories, which goes against the trend of a healthy, low-sugar diet. In addition, although antifreezes such as sugars, alcohols, and polyphosphates can effectively reduce the formation of ice crystals in refrigerated foods, temperature fluctuations will inevitably occur during low-temperature transportation, causing the recrystallization of small ice crystals. Further enlargement of ice crystals will still cause damage to the tissue structure of the food.

[0003] In recent years, antifreeze peptides (AFPs) have attracted extensive attention due to their antifreeze activity and the ability to maintain the quality of frozen products. They have been widely used in various foods, such as livestock and poultry meat, aquatic products, vegetables, fruits, pasta and dairy products. However, most studies on AFPs have focused on their isolation from natural organisms or their production through genetic engineering, and it has been shown that peptides extracted from aquatic organisms have problems such as bitterness and low stability. The limited availability and safety of AFPs restrict their application.

[0004] Therefore, providing a safe, healthy and efficient new antifreeze agent for application in the food industry and other fields is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides an antifreeze glycopeptide and a preparation method and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for preparing an antifreeze glycopeptide comprises the following steps:

[0008] 1) Preparation of crab shell powder:

[0009] The crab shells were cleaned, heated in an oven at 100°C for 2 h, crushed, and sieved to obtain crab shell powder;

[0010] 2) Alcalase2.4+ irradiation enzymolysis:

[0011] Temperature: 60°C, substrate concentration 40 mg / ml, enzyme dosage 12000 / U, irradiation intensity 2000 Gy;

[0012] 3) Preparation of glycopeptides:

[0013] The weight ratio of lyophilized crab shell polypeptides (CSPs) to dextran was 1:2, dissolved in deionized water to a final concentration of 100 mg / mL, and the reaction solution was heated and stirred in a water bath at pH 8.0 and 50°C for 60 min;

[0014] 4) Filtering:

[0015] The CSPs and dextran were removed by ultrafiltration and vacuum drying to obtain antifreeze glycopeptides (GCSPs).

[0016] An antifreeze glycopeptide, the amino acid sequence of the antifreeze glycopeptide comprising:

[0017] AFP1: DWWDPGFFGFPPH, as shown in SEQ ID No. 1;

[0018] AFP2: YGGPKPSPVDIDDW, as shown in SEQ ID No.2.

[0019] Furthermore, the mesh size of the sieve used in step 1) is 100 meshes.

[0020] Furthermore, the molecular weight cut-off of the ultrafiltration device is 3kDa and 15kDa.

[0021] Application of an antifreeze glycopeptide in the preparation of food antifreeze agents.

[0022] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0023] The present invention proposes for the first time to hydrolyze crab shells by irradiation + enzymolysis, and verify the antifreeze protection performance of the enzymolysis product, and based on this, the polypeptide is combined with glucan by Maillard reaction to finally form an antifreeze glycopeptide. The glycopeptide provided by the present invention is an effective antifreeze protective agent. The addition of polysaccharides effectively improves the stability and flavor of the polypeptide. Its effect is better than commercial antifreeze agents such as phosphate / sucrose on the market, and it is greener and harmless to the human body.

[0024] The present invention creatively adds an irradiation method to the enzymatic hydrolysis process, making the enzymatic hydrolysis of crab shells more stable and efficient, solving the current problem of environmental pollution caused by crab shell waste and achieving high-value utilization.

[0025] The present invention creatively combines crab shell polypeptide with glucan based on crab shell enzymatic hydrolysate by a non-enzymatic glycosylation method, thereby significantly improving the stability and flavor of the polypeptide and increasing the antifreeze protection performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0027] Figure 1 is the yeast survival rate of different treatment groups of the present invention;

[0028] Figure 2 The antifreeze protection effect of different groups of polypeptides of the present invention on yeast; Figure 2 a. Figure 2 b is the normal group; Figure 2 c. Figure 2 d is the single freeze-thaw group; Figure 2 e. Figure 2 f is the secondary freeze-thaw group; Figure 2 b, Figure 2 d, Figure 2 fAdditional 5% crab shell peptides;

[0029] Figure 3 It is a total ion current chromatogram of the polypeptide liquid chromatography-tandem mass spectrometry of the present invention;

[0030] Figure 4 is the molecular weight distribution of the polypeptide of the present invention;

[0031] Figure 5 The absorbance (294 and 420 nm) of the sample and the survival rate of Saccharomyces cerevisiae during the reaction of the polypeptide and glucan under different reaction conditions of the present invention are shown; Figure 5 a is the ratio of polypeptide to glucan (w / w); Figure 5 b is the reaction temperature; Figure 5 c is the reaction time; Figure 5 d is the reaction pH;

[0032] Figure 6 The thawing loss and cooking loss of the different treatment groups of the present invention; Figure 6 a is the thawing loss of different treatment groups; Figure 6 b is the cooking loss of different treatment groups;

[0033] Figure 7 It is the T2 transverse relaxation time spectrum of different treatment groups of the present invention;

[0034] Figure 8 Spectral analysis of CSPs and GCSPs of the present invention; Figure 8 a is the fluorescence emission spectrum excited at 350 nm; Figure 8 b is Fourier transform infrared spectrum;

[0035] Fig. 9 The differential scanning calorimetry (DSC) curve and glass transition temperature diagram of the present invention; Fig. 9 a is the differential scanning calorimetry (DSC) curve of the freezing and melting process of GCSPs at different concentrations; Fig. 9 b is the glass transition temperature of CSPs and GCSPs; Fig. 9 c is the differential scanning calorimetry (DSC) curves of CSPs and BSA during freezing and melting;

[0036] Fig.10 The scanning electron microscope (SEM) images of the surface structures of the glucan, CSPs and GCSPs of the present invention; Fig.10 a. Fig.10 b is dextran; Fig.10 c. Fig.10 d is CSPs; Fig.10 e. Fig.10 f is GCSPs;

[0037] Fig.11 This is the flavor wheel diagram of the differential metabolites of GCSPs and CSPs of the present invention;

[0038] Fig.12 Thermogravimetric analysis weight change of the present invention; Fig.12 a is the weight change diagram of thermogravimetric analysis; Fig.10 b is the weight change rate diagram. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Example 1

[0041] Extraction of Crab Shell Peptides

[0042] Crab shell → wash the remaining meat → dry → crush → sieve → protease + irradiation → enzymatic hydrolysis product

[0043] 1. Take the crab shell from the swimming crab, collect the byproducts such as the exoskeleton, gastropod, and crab shell, wash it with running water and drain the surface water. Then heat it in an oven at 100℃ for 2h until the crab shell becomes crisp. Use an ultrafine grinder to process the crab shell, then filter it with a 100-mesh sieve and place it in deionized water as an enzymatic substrate for later use.

[0044] 2. Use Alcalase2.4 (manufacturer: Novozymes, trade name: Alcalase 2.4L FG, product number: JXDBM) + irradiation method to enzymolyze the treated crab shells, and use the orthogonal method to screen the optimal enzymolysis conditions.

[0045] Table 1 Orthogonal experiment of crab shell irradiation + bio-enzymatic hydrolysis conditions

[0046]

[0047]

[0048] The enzymatic hydrolysis conditions finally obtained through orthogonal experiments were: temperature: 60°C, substrate concentration 40 mg / ml, enzyme addition amount 12000 / U, irradiation intensity 2000Gy, and the final protein extraction rate was 69.3%.

[0049] Verification of the antifreeze performance of crab shell polypeptides

[0050] After yeast activation, different masses of crab shell polypeptides were added to prepare a bacterial suspension with a concentration of 0% to 8% (m / V) of crab shell polypeptide. The suspension was immediately subjected to two freeze-thaw cycles. Each freeze-thaw cycle included freezing at -(20±2)°C for 24 hours and thawing at (4±2)°C for 4 hours. A yeast suspension with a glycerol concentration of 8% (m / V) was used as a positive control. The unfrozen and thawed suspension was placed in a 32°C incubator as a blank group, and each group was repeated 3 times. The final experimental results are shown in Figure 1 , Figure 2 :

[0051] Figure 1 is the survival rate of yeast in different treatment groups;

[0052] Figure 2 The antifreeze protection effect of different groups on yeast (a, c, e on the left are normal group, one freeze-thaw group, and two freeze-thaw groups, and b, d, and f on the right are 5% crab shell polypeptide added)

[0053] After freeze-thaw treatment, the growth status and survival rate of yeast in each group were affected as follows Figure 3As shown. In the normal group without freeze-thaw treatment, a considerable number of yeast colonies grew on the PDA plate. The yeast used in this study was extremely intolerant to cold treatment. After a 24-hour freeze-thaw cycle, the control group had very few colonies growing on the PDA plate, and its survival rate was only 12.5%. However, the addition of crab shell polypeptide and glycerol significantly increased the survival rate of yeast after freeze-thaw cycles (P<0.05). And the yeast survival rate of 5% shrimp shell polypeptide added reached 88.5%, which was better than the positive control group of yeast suspension with a glycerol concentration of 8% (P<0.05). Compared with other AFPs isolated from silver carp muscle and sericin, the crab polypeptide obtained after irradiation enzymatic hydrolysis has a more significant antifreeze protection effect. This is because the enzymatic hydrolysis of crab shell polypeptide is more thorough, and the molecular weight is concentrated in a smaller range. Studies have shown that the change of large molecular AFP into small molecular active fragments can further improve its antifreeze effect. However, when the amount of peptide added exceeded 5%, the survival rate of yeast began to show a downward trend (P<0.05), indicating that the antifreeze protection of crab shell peptide on yeast was not linearly dose-dependent, which was consistent with the property that antifreeze peptide could modify ice crystals in a non-colligative form to reduce the freezing point of the solution. From the transmission electron microscope, it can be seen that the yeast cells after freezing and thawing have obvious cavities and damage, and the peptide can well protect the morphology and budding characteristics of the cells, and the peptide is aggregated on the cell surface.

[0054] Peptideomics analysis

[0055] Reductive alkylation

[0056] Take an appropriate amount of sample and dissolve it in 50mM NH4HCO3. On this basis, add dithiothreitol (DTT) to make its concentration 10mmol / L, and then reduce it in a 56℃ water bath for 1h; add iodoacetamide (IAM) solution to adjust the concentration to 50mmol / L, and treat it in the dark for 40min; desalt it with a self-filled desalting column, and finally remove the solvent at 45℃ in a vacuum centrifugal concentrator.

[0057] Liquid chromatography-tandem mass spectrometry detection

[0058] Liquid chromatography conditions: injection volume 5mL; chromatographic column: Acclaim PepMap RPLC C18 (150mm×150μm, 1.9μm); mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% acetonitrile; flow rate: 600nL / min; single component analysis time: 66min. Gradient elution: 0-2min 4%-8% B phase, 2-45min 8%-28% B phase, 45-55min 28%-40% B phase, 55-56min 40%-95% B phase, 56-66min 95% B phase.

[0059] Mass spectrometry conditions: primary mass spectrometry, resolution: 70000, automatic gain control target: 3e6, Maximu-mIT: 100 ms, scanning range: 100-1500 m / z: secondary mass spectrometry, resolution: 17500, automatic gain control target: 1e5, MaximumIT: 50 ms, TopN: 20, normalized fragmentation energy: 28.

[0060] Peptide sequence analysis

[0061] The mass spectrometry raw files were analyzed for peptide sequences using the software PEAKS Studio 10.6, and the parameters were set as follows: fixed modification: cysteine ​​carbamidomethylation; variable modification: oxidation, acetylation (N-term); enzyme: non-specific; database: crab; missed restriction sites: 3; primary mass spectrometry error: 20 ppm;

[0062] Secondary mass spectrometry error: 0.02Da; peptide / fragment ion mass: monoisotope; significance threshold: 0.01. Based on the UniProt protein library (https: / / www.uniprot.org / ), high-confidence peptides were selected for downstream protein identification analysis.

[0063] Peptideomics analysis

[0064] Total ion chromatogram of LC-MS / MS analysis ( Figure 3 ) shows the typical characteristic peaks of specific peptides. A total of 1004 peptides were identified, of which 47.4% were peptides with a molecular weight of 700-1000 Da, and 47.1% were peptides with a molecular weight of 1000-2000 Da. Figure 4 ). About 89.9% of the peptides in CSPs are composed of 6 to 14 amino acids. Compared with these fish-derived antifreeze peptides, the peptides obtained from crab shells by irradiation and enzymatic hydrolysis have smaller molecular weights and shorter lengths. For example, Cao et al. found that the molecular weight of squid skin hydrolysates below 5000Da accounted for 82.34% of the hydrolysates, among which the short peptides with a molecular weight of 1000 to 2000Da accounted for the largest content, accounting for 22.69%, mainly composed of 7 to 15 amino acids. Due to their higher biological activity, small molecular weight antifreeze peptides have better thermal hysteresis activity than large molecular weight antifreeze peptides.

[0065] AFPs were screened using the antifreeze prediction platform according to the confidence level from high to low. The top 20 peptides identified as AFPs were selected and further bioinformatics prediction was performed. The test results are shown in Table 2.

[0066] Table 2 Physicochemical properties of antifreeze peptides

[0067]

[0068] The hydrophilicity of a peptide depends on the proportion of polar amino acids in the peptide, which are usually neutral amino acids with polar groups on the side chains, such as serine (Ser), threonine (Thr), cysteine ​​(Cys), or polar amino acids, such as charged aspartic acid (Asp), histidine (His), and lysine (Lys), which belong to the hydrophilic end and can make the peptide have a higher overall average hydrophilicity (GRAVY value). Become more hydrophilic and form more hydrogen bonds. Non-polar amino acids such as phenylalanine (Phe), leucine (Leu), and tryptophan (Trp) are usually folded and hidden in the peptide, making them less accessible to the outside world. With the assistance of these amino acids, the peptide can enhance the stability of water during freezing and reduce the formation of large ice crystals, thereby avoiding open-chain denaturation of the peptide due to changes in spatial conformation. At the same time, peptides with an instability index > 40 have poor in vitro stability and are generally not considered to be effective antifreeze peptides. Based on this, 9 potential antifreeze peptides were finally obtained through the antifreeze test platform. Based on the instability index, hydrophilicity and confidence, two polypeptide sequences were selected as research objects and named AFP1 and AFP2 respectively:

[0069] AFP1: DWWDPGFFGFPPH, as shown in SEQ ID No. 1;

[0070] AFP2: YGGPKPSPVDIDDW, as shown in SEQ ID No. 2;

[0071] Among them, AFP2 also conforms to the Gly-Pro-X tripeptide repeat sequence and GPPG-structural fingerprint commonly found in antifreeze peptides.

[0072] Glycosylation of antifreeze glycopeptides

[0073] The lyophilized crab shell polypeptides (CSPs) and dextran were dissolved in deionized water at a mass ratio of 3:1, 2:1, 1:1, 1:2, and 1:3, respectively, to a final concentration of 100 mg / mL, and the reaction solution was heated and stirred in a water bath at pH 8.0 and 50°C for 60 min.

[0074] To investigate the effect of reaction time on the freezing survival rate of yeast and the degree of Maillard reaction, the ratio of CSPs to dextran was 1:1 (w / w), and the reaction was carried out at pH 8.0 and 50℃ for different times (0.5, 1.0, 2.0, 3.0, and 4.0 h, respectively). The effect of reaction temperature on the survival rate of yeast and the degree of Maillard reaction was studied. The reaction was carried out in a shaker at pH 8.0 and different temperatures (40, 50, 60, 70, and 80℃) for 60 min. The effect of reaction pH was studied when the ratio of CSPs to dextran was 1:1 (w / w), and the reaction was carried out at 50℃ and different pH (6.0, 7.0, 8.0, 9.0, and 10.0) for 60 min. The sample (4 mL) was cooled in an ice bath for 30 min to terminate the reaction. Under the corresponding conditions, its MR degree was evaluated at 294 nm and 420 nm using a UV spectrophotometer.

[0075] The results obtained from the different reaction ratios of CSPs and glucan (w / w) showed that when the ratio of CSPs to glucan was 1:2 (w / w), the survival rate of yeast after freeze-thaw treatment was higher ( Figure 5 a The ratio of polypeptide to glucan (w / w)). Figure 5 b reaction temperature), reaction time 1h ( Figure 5 c reaction time), pH 8.0 ( Figure 5 a reaction pH), and the best experimental results were obtained. On the basis of the single factor experiment, the optimal conditions for the Maillard reaction were obtained by orthogonal experimental design. The results showed that the optimal conditions for the Maillard reaction were 40℃, 1h, pH 7.0, and the ratio of antifreeze peptide to glucan was 1:2 (w / w) (data not shown). The yeast prepared under the optimal reaction conditions was frozen at -20℃ with 10mg / mL CSPs for 24h, and its survival rate could reach 91.4%.

[0076] The change trends of the absorbance values ​​of glycopeptide solutions at 294nm and 420nm under different reaction conditions are as follows Figure 5As shown in , the absorbance at 294nm is significantly higher than that at 420nm. At the beginning of the Maillard reaction, the survival rate of yeast increases with the increase of the absorbance value at 294nm. But when the absorbance at 420nm increases significantly, the viability of yeast decreases. The Maillard reaction process is divided into three main stages (early, middle and late). In the initial stage, sugar amines condense and Amadori rearrangement occurs. A colorless product is formed in this stage. In the middle stage, the reaction color weakens or turns yellow, with strong absorption in the near-ultraviolet region. In the final stage, aldehyde-amide polymerization and aldol condensation form highly colored compounds, accompanied by the formation of heterocyclic nitrogen compounds. The absorbance at 294nm is often used to monitor the initiation of the Maillard reaction. Generally, a higher absorbance indicates a faster reaction rate. Previous studies have shown that ultraviolet absorbance at 294nm usually represents the intermediate of the Maillard reaction product, while the absorbance at 420nm can be used to detect the later stage. In summary, combined with Figure 5 It can be seen that the activity of yeast after freeze-thaw is related to the degree of Maillard reaction.

[0077] When the absorbance at 420nm increased significantly, the survival rate of yeast decreased, which may be because the Maillard intermediates were formed into final products through aldol condensation. Previous studies have shown that the final products of the Maillard reaction have both antibacterial and bactericidal functions. Based on this, it is inferred that the intermediates of the Maillard reaction are mainly responsible for the cryoprotection of yeast. The cell protection mechanism of antifreeze peptides is believed to be the result of hydrogen bonds between cell membrane lipids and water around cells. During the freezing process, free water freezes first, and hydroxyl groups can combine with phospholipid molecules on the cell membrane to form hydrogen bonds, replacing water molecules to form a hydrated membrane and protect the integrity of the cell membrane. After the glycosylation reaction, glucan and antifreeze peptides are covalently bonded to form antifreeze glycopeptides. Therefore, the cell protection mechanism of antifreeze glycopeptides may be its potential combination with the hydrophilic groups of the cell membrane, and the protective effect of antifreeze glycopeptides on the cell membrane is better than that of antifreeze peptides, which may be due to the fact that the antifreeze glycopeptides formed after non-enzymatic modification of glucan contain more -OH.

[0078] On the basis of the single factor experimental data, orthogonal experimental design was used to further optimize the reaction conditions of peptide glycosylation. The product obtained under the optimal conditions (mass ratio of CSPs to dextran was 1:2, reaction temperature was 50℃, reaction time was 1h, pH 8.0) was filtered by ultrafiltration device (3kDa and 15kDa molecular weight cutoff) to remove CSPs and dextran, and then vacuum dried. The antifreeze glycopeptide analogs prepared by Maillard reaction were identified as GCSPs.

[0079] Protective effect of glycopeptides and polypeptides on frozen pork

[0080] Processing of fresh pork samples

[0081] In a low-temperature cold storage, the connective tissue, fat, and excess tissue such as pig skin in the pork were removed with a scalpel, and then each piece of tenderloin was divided into 6 to 10 portions, each of which was about 30±0.5 grams. The meat samples were randomly divided into 6 groups: 1) meat samples without any treatment, namely the control group; 2) meat samples injected with 1% w / w phosphate commercial antifreeze, namely the phosphate group; 3) meat samples injected with 1% w / w sucrose, namely the sucrose group; 4) meat samples injected with saline only, namely the saline group; 5) meat samples injected with 1% w / w CSPs, namely the peptide group; 6) meat samples injected with 1% w / w GCSPs, namely the glycopeptide group. Except for the control group, the solvents of the other 5 groups were all saline. Each sample was injected with a 5mL medical grade syringe, and the blank group was not injected. The volume ratio of the liquid to the meat sample for each injection is 1:10 (V / w), and when injecting, five positions should be selected at the four corners and the middle of the meat block to keep the injection operation of each meat sample basically consistent. All injection processes are completed in a low-temperature cold storage. Each meat sample is packaged in a self-sealing bag, and then the meat sample is placed flat in a refrigerator at 4°C for 6 hours to allow the injected solution to evenly penetrate the meat sample. Then, all meat samples are placed in a -20°C refrigerator for 24 hours and then thawed in a 4°C refrigerator for 12 hours.

[0082] Thawing loss

[0083] The thawing loss was calculated based on the ratio of the difference in the mass of pork before and after freezing to the mass before freezing. The mass of the meat was weighed before freezing, and then the meat was placed in a ziplock bag and marked accordingly. The thawed meat sample was taken out, the moisture on the surface of the meat was wiped dry with kitchen paper, and then re-weighed and recorded. The thawing loss of the frozen meat sample was measured at room temperature throughout the process and was calculated using the following formula (1):

[0084] Thawing loss (%) = "1-"2 / "1×100(1)

[0085] Among them, m1 refers to the mass of the fresh meat sample before freezing (g), and m2 refers to the mass of the meat sample after thawing (g).

[0086] Cooking loss

[0087] The cooking loss determination method is to weigh the meat before freezing and put it in a ziplock bag. Heat the thawed meat sample in a hot water bath at 80°C. When the average temperature inside the meat sample reaches 75°C using an infrared thermometer, take it out of the water bath and cool it to room temperature. Finally, take each meat sample out of the ziplock bag, wipe the surface of the meat with kitchen paper, and then weigh it again and record it. The cooking loss of the sample is calculated using the following formula (2):

[0088] Cooking loss (%) = "1-"3 / "1×100(2)

[0089] Among them, m1 refers to the mass of the fresh meat sample before freezing (g), and m3 refers to the mass of the meat sample after cooking (g).

[0090] Figure 6 ( Figure 6 a: Thawing loss in different treatment groups; Figure 6 b: Cooking loss of different treatment groups;) shows that after freezing, the water loss of the control group and the normal saline group samples was significantly higher than that of the other treatment groups, whether thawing or cooking (P<0.05). This is similar to the results of Chen's study on the effect of antifreeze peptides on chicken breast quality (Chen et al., 2023). Among them, the water retention effect of the added polypeptide group and glycopeptide group was better than that of the sucrose group but weaker than that of the complex phosphate group (P<0.05). Although salt antifreeze protectants prevent freezing denaturation by changing the microenvironment of proteins, and are also effective in improving water retention, antifreeze glycopeptides have significant advantages in practical applications due to the limitations of the amount added and the impact on food flavor.

[0091] Moisture distribution measurement

[0092] The water distribution state in the pork samples treated differently was analyzed by low-field nuclear magnetic resonance using LF-NMR image analyzer. Before testing, the thawed pork was cut into 10mm×10mm×20mm pieces to facilitate placement in a 15mm nuclear magnetic resonance glass tube. Before testing, the temperature of each meat sample was kept as close to room temperature as possible. The nuclear magnetic resonance tube was placed in the sample slot, and the transverse relaxation time (T2) of the sample was measured using Niumag N MR software and CPMG sequence. The attenuation curve of each sample was converted into a relaxation time model for merging and inversion. After further iteration and optimization, relaxation information such as relaxation time and signal component of different samples was obtained.

[0093] Low-field nuclear magnetic resonance (LF-NMR) is a non-destructive, non-invasive spectroscopic technique that can visually display water changes in muscle tissue. Figure 7 The results showed that the T21 relaxation time of the peptide group and phosphate was the longest, both of which were 49.77ms. The corresponding results of P21 showed that among all the treatment groups, the non-mobile water content of the meat samples in the peptide and phosphate treatment groups was the highest (P<0.05). This reflects that the non-mobile water in the meat samples in the peptide and phosphate treatment groups is most tightly bound to the muscle after freezing. At the same time, it can be seen that in terms of the T22 relaxation ratio (P22), the peptide and phosphate treatment groups have the longest T22 relaxation time and the smallest P22 ratio, indicating that the proportion of free water in their water distribution is relatively small (P<0.05). It can be seen that peptides and phosphates significantly reduce the mobility of various forms of water in pork during the freezing process and reduce the thawing loss of pork, which is consistent with the thawing loss results.

[0094] Texture characteristics

[0095] The thawed meat samples were cut into small pieces of 1cm×1cm×1cm, and the texture characteristics of the samples were tested using a texture analyzer. The P36R probe was selected to perform compression tests on each sample, and the measurements were performed under the following conditions: the pre-test rate, test rate, and post-test rate were all 1mm / s, the trigger force was 20g, the test mode was set to "compression mode", the compression ratio was 50%, and the time interval between two consecutive tests was 3s. The results are shown in Table 3.

[0096] Table 3 Changes in texture characteristics of pork in different treatment groups

[0097]

[0098]

[0099] As shown in Table 3, when the texture properties of meat samples were determined, it was found that during the freezing process, the hardness, resilience and chewiness of the group with commercial antifreeze agents such as phosphate and sucrose and the glycopeptide group increased significantly (P<0.05). At the same time, there was no significant effect on the elasticity and cohesion of the frozen pork samples. There were no significant differences in the various indicators between the antifreeze peptide and saline groups. Overall, the glycopeptide and phosphate treatment groups showed better texture properties than the blank group and other treatment groups. According to the research findings of Cai et al. (Cai et al., 2019), the increase in hardness and resilience of samples in the glycopeptide and phosphate treatment groups may be related to their detection of lower free water content in meat samples in low-field nuclear magnetic resonance (Table 3) and less thawing loss after freezing ( Figure 6 a). Therefore, in our study, the good textural properties of the glycopeptide-treated samples can be attributed to the fact that the addition of glycopeptides improved the water retention of pork samples and better preserved the permeability and integrity of cell membranes.

[0100] Color determination

[0101] The color of the meat samples was measured using a CR-10Plus colorimeter. When measuring the color, A10 light was used as the light source, an observation angle of 10° was used, and the diameter of the measured area was 20 mm. The L* value (brightness), a* value (red-green color), and b* value (yellow-blue color) of each meat sample were recorded. The results are shown in Table 4.

[0102] Table 4 Color changes of pork samples in different treatment groups

[0103] Control group Phosphate group Sucrose group Normal saline group Peptide Group Glycopeptide group L* <![CDATA[49.55±0.21 a ]]> <![CDATA[44.33±0.32 e ]]> <![CDATA[48.58±0.28 b ]]> <![CDATA[50.08±0.26 a ]]> <![CDATA[46.15±0.15 d ]]> <![CDATA[47.45±0.18 c ]]> a* <![CDATA[6.55±0.08 c ]]> <![CDATA[5.85±0.10 d ]]> <![CDATA[5.80±0.09 d ]]> <![CDATA[6.38±0.05 c ]]> <![CDATA[7.55±0.12 a ]]> <![CDATA[7.35±0.06 b ]]> b* <![CDATA[22.60±0.11 b ]]> <![CDATA[21.15±0.08 c ]]> <![CDATA[22.45±0.14 b ]]> <![CDATA[23.38±0.09 a ]]> <![CDATA[23.70±0.15 a ]]> <![CDATA[23.37±0.24 a ]]>

[0104] As can be seen from Table 4, the L* values ​​of the blank group and the saline group were the highest, close to or exceeding 50, and there was no significant difference between the two. In addition, the phosphate group had the lowest L* value (P<0.05), followed by the polypeptide and glycopeptide groups. This is mainly because after the addition of the above-mentioned salts or other antifreeze agents during the freezing process, the ice crystals produced inside the muscle were more delicate and uniform, so the condensed water attached to the surface of the meat was also reduced accordingly, resulting in a lower light reflection intensity formed on the surface of the meat sample. In this case, the brightness of the meat sample was also reduced accordingly. In terms of redness (a*) and yellowness (b*), the polypeptide and glycopeptide groups were significantly higher than the other groups (P<0.05), which may be because AFP effectively controlled the recrystallization of ice crystals during growth and freezing, thereby maintaining the integrity of the cells and reducing protein oxidation (Du et al., 2020). Therefore, the degree of myoglobin denaturation was reduced, reducing the reduction in redness. In general, the addition of glycopeptides can better maintain the quality of frozen meat products.

[0105] Characterization of glycopeptides (GCSPs)

[0106] Table 5 Molecular weight of dextran and GCSPs determined by gel permeation chromatography

[0107]

[0108]

[0109] Figure 8 Spectral analysis of CSPs and GCSPs. Figure 8 a is the fluorescence emission spectrum excited at 350 nm; Figure 8 b is Fourier transform infrared spectrum;

[0110] Improved performance of glycopeptides (GCSPs) compared to peptides (CSPs)

[0111] Improved antifreeze performance

[0112] CSPs have been previously shown to have significant thermal hysteresis activity (THA) and also show significant cryoprotective effects on yeast in low temperature environments. DSC was used to further test the THA of GCSPs. The DSC curves of the freezing and melting processes of GCSPs solutions are shown in Figure 2. Fig. 9 The THA and ice content (Φ) calculated from the DSC curve at the same insulation temperature (Th) are shown in Table 6.

[0113] Compared with the DSC curve of the BSA control group ( Fig. 9 c), CSPs( Fig. 9 c) and GCSPs( Fig. 9a) The exothermic peak was significantly delayed, indicating that CSPs and GCSPs caused thermal hysteresis. In addition, compared with the exothermic peak of CSPs, the delay time of GCSPs was significantly longer than that of CSPs at the same insulation temperature. This indicates that the GCSPs solution (20 mg / mL) obtained after non-enzymatic glycosylation exhibits stronger thermal hysteresis activity. Studies have shown that THA is negatively correlated with the ice crystal fraction (Φ) in the equilibrium sample. From the comparison in Table 4, the THA value (1.23℃) in the GCSPs equilibrium sample is significantly higher than the THA value of BSA and CSPs at the same Th (0.3℃); correspondingly, the number of ice nuclei (Φ) in the GCSPs equilibrium sample is significantly lower than that of BSA and CSPs detected at the same Th (0.3℃).

[0114] The antifreeze mechanism of glycopeptides is an adsorption inhibition process, in which GCSPs bind to the surfaces of growing ice crystals, which grow with high surface curvature. Since the energy required to add water molecules to the protruding surface is high, a non-equilibrium freezing point depression is observed when the melting point remains unchanged, resulting in a difference between the freezing point and the melting point, namely thermal hysteresis activity. In addition, GCSPs can inhibit the transformation of water into an ice state by affecting the self-organization of water, structuring nearby water molecules, and reducing the ice content of the sample. The reason why GCSPs exhibit strong thermal hysteresis activity may be speculated that GCSPs can increase the viscosity of the solution and reduce the ice content of the solution after glycosylation. This result is consistent with the DSC data. At the same Th, GCSPs have a lower freezing rate (Φ) and a lower crystallization onset temperature (T0) than CSPs (Table 6). Therefore, we can speculate that the reason why GCSPs exhibit better thermal hysteresis activity than CSPs is that GCSPs can reduce T0 and reduce the proportion of ice crystal particles.

[0115] Table 6 Differential scanning calorimetry results of CSPs, BSA and GCSPs

[0116]

[0117]

[0118] Glycopeptide morphology

[0119] Fig.10The scanning electron microscope (SEM) images of the surface structures of dextran (a, b), CSPs (c, d) and GCSPs (e, f). The structure of dextran is relatively rough, showing a relatively three-dimensional layered structure with irregular shapes and multiple structures such as holes, particles and blocks. For untreated CSPs, many spherical and smooth particles were observed on the surface of the sample. This shape conforms to the typical spherical structure of proteins and polypeptides. It can be observed that the GCSPs sheet structure shows the formation of some round particles of different sizes, the surface is not smooth, there are wrinkles, and an aggregated sheet structure is formed. However, the surface structure is tighter than that of dextran, without large gaps and cracks. Compared with the relatively regular morphology of CSPs, the changes in the morphology of GCSPs after modification can be attributed to the glycosylation cross-linking between CSPs and dextran.

[0120] Glycopeptides enhance flavor

[0121] Aroma components can objectively reflect the flavor characteristics of different samples and are important indicators for evaluating flavor quality. The types and main components of volatile flavor components in peptide and glycopeptide samples determined by GC-MS are shown in the flavor wheel (see Fig.11 : Flavor wheel of differential metabolites between GCSPs and CSPs; Note: The second circle is the top 10 sensory flavor characteristics annotated by the differential metabolites in this comparison group, and the outermost circle represents the differential metabolites. )

[0122] The differences in volatile flavor components between glycopeptides and polypeptides showed that the number of metabolites related to sweetness (35), fruity aroma (37), and floral aroma (34) was the largest, mainly pyran derivatives, terpenes, and aldehyde compounds.

[0123] Pyran compounds themselves do not have very clear odor characteristics, but many pyran derivatives (such as furanones and coumarins) have complex aromas and play an important role in a variety of natural and synthetic fragrances. Terpenoids are usually the source of floral, fruity, grassy, ​​woody and other odors. For example, 2,6,10,10-tetramethyl-1-oxaspiro[4.5]decene (H.Wang et al., 2020) and nerol (as one of the terpenoids) have these characteristics. As shown in Table 5, the upregulation of 2,6,10,10-tetramethyl-1-oxaspiro[4.5]decene was 8.04 times, indicating that the fragrance in glycopeptides has been significantly improved. In addition, benzene derivatives (p-anisaldehyde diethyl acetal) (Seo et al., 2024) with a vanilla aroma also showed an upregulation. In contrast, benzoic acid, a benzene derivative associated with pungent odors and bitter almond flavors, was significantly downregulated, 3.63 times that of the peptide sample. At the same time, the characteristic compound octen-3-ol that causes odor was reduced by 6.09 times, indicating that the flavor of the glycopeptide synthesized after the Maillard reaction is more inclined to sweet fragrance, effectively eliminating the original fishy smell of the peptide extract.

[0124] Table 7 Significant metabolite changes in some glycopeptides and polypeptides

[0125]

[0126] Improved stability of glycopeptides

[0127] The thermal properties of CSPs and GCSPs were studied by TG and DTG. Fig.12 It can be seen that the weight loss of all samples in the initial stage in the temperature range of about 25℃~100℃ is mainly caused by the evaporation of water adsorbed on the surface of the material. Fig.10 a It can be seen that CSPs began to decompose at 156.95℃, and the weight loss rate was the largest at 297.59℃. Fig.10 (a), the thermal degradation of the polymer has three steps in the temperature range of 30-600℃. The first stage of the TG curve, from room temperature to about 140℃, is related to the loss of adsorbed and bound water. Comparing the TG results of GCSPs and their components, some differences can be observed. Compared with CSPs (peak at 70.37℃), GCSPs seem to lose more weight of water at lower temperatures (peak 40-110℃), which may be due to the combination of polypeptides and glucans in GCSPs forming more hydrogen bonds and adsorbing more water molecules, resulting in a higher water content in GCSPs, which indicates that GCSPs are more hydrophilic. The temperature range of the second step of thermal degradation is 275-350℃, which corresponds to the gradual decomposition of polypeptides and sugar chains from GCSPs. In contrast to CSPs, the third step of thermal degradation in the temperature range of 350-500℃ may be due to the oxidation of partially decomposed CSPs under the action of airflow. In the temperature range of 110-275℃ and 350-500℃, the maximum mass loss rate of GCSPs is lower than that of CSPs. It is worth noting that the mass of GCSPs remains stable in a certain temperature range after losing water, and the decomposition temperature is significantly delayed compared with CSPs. It may be that CSPs and dextran are covalently bonded, which is consistent with the analysis of FT-IR spectrum results, thereby enhancing the thermal stability of GCSPs.

[0128] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing an antifreeze glycopeptide, characterized in that: The following steps are involved: 1) Preparation of crab shell powder: The crab shells were cleaned, heated in an oven at 100°C for 2 h, crushed, and sieved to obtain crab shell powder; 2) Alcalase2.4+ irradiation enzymolysis: Temperature: 60°C, substrate concentration 40 mg / ml, enzyme dosage 12000 / U, irradiation intensity 2000 Gy; 3) Preparation of glycopeptides: The weight ratio of lyophilized crab shell polypeptide to glucan was 1:2, dissolved in deionized water to a final concentration of 100 mg / mL, and the reaction solution was heated and stirred in a water bath at pH 8.0 and 50°C for 60 min; 4) Filtering: The crab shell polypeptide and glucan are removed by filtering through an ultrafiltration device, and then vacuum dried to obtain antifreeze glycopeptide.

2. The antifreeze glycopeptide prepared according to claim 1, characterized in that The amino acid sequence of the antifreeze glycopeptide comprises: AFP1: DWWDPGFFGFPPH, as shown in SEQ ID No. 1; AFP2: YGGPKPSPVDIDDW, as shown in SEQ ID No.

2.

3. The method for preparing the antifreeze glycopeptide according to claim 1, characterized in that: The mesh number of the sieve used in step 1) is 100 meshes.

4. The method for preparing the antifreeze glycopeptide according to claim 1, characterized in that: The molecular weight cut-off of the ultrafiltration device is 3 kDa and 15 kDa.

5. Use of the antifreeze glycopeptide according to claim 2 in preparing food antifreeze agents.

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