Identification of novel antifreeze peptides from yak skin gelatin ultrasound-assisted enzymatic hydrolysate

Graphical abstract

Abstract

In this paper, a new type of antifreeze peptide was identified from the ultrasonic-assisted enzymolysis product of yak skin. First, the antifreeze peptides were obtained by ultrasonic-assisted enzymolysis of yak skin gelatin. The obtained peptides with 150 W ultrasound treatment could increase the survival rate of Lactobacillus plantarum from 16 % to 60 % after four freeze–thaw cycles. Then, the component with the highest antifreeze activity in the peptides’ ultrafiltration product was analyzed by LC-MS/MS to obtain 961 peptides. After screening of antifreeze activity and physical properties, three antifreeze peptide sequences were obtained (S1: GERGGPGGPGPQ, S2: PGGAEGPGRDAQQP, S3: VAPPGAPKKEH). DSC analysis, Lactobacillus plantarum cryoprotection experiments and molecular docking showed that the S1 sequence had the best antifreeze activity. This study provides a new idea for the high-value utilization of yak by-products and a potential candidate for food antifreeze agents.

1. Introduction

Freezing can provide a suitable temperature environment for the production, processing and storage of meat products. Therefore, freezing can effectively reduce the growth and reproduction of microorganisms and slow down the spoilage of meat products [1]. However, at low temperatures, the freezing of water in fresh meat products will lead to the formation of ice nuclei and the growth of ice crystals [2], [3]. Large ice crystals will destroy the cell structure of meat products, thereby causing the quality of meat products to deteriorate [4].

Adding antifreeze agents is one of the most effective ways to reduce the quality deterioration of meat products during the freezing process. Carbohydrate antifreeze agents are currently the most widely used antifreeze agents in food [5]. Antifreeze peptides (AFPs) are a class of protein hydrolysates that can lower the freezing point of the solution, inhibit the growth and recrystallization of ice crystals, and thus prevent meat product tissues from being damaged by freezing [6]. In addition, compared with carbohydrate antifreeze, antifreeze peptides are rich in amino acids. Also, they do not introduce unnecessary sweetness to meat products. This facilitates a low-sugar, healthy diet. Therefore, antifreeze peptides, as a new type of antifreeze agent, have received widespread attention in recent years [1], [7].

Since antifreeze peptides were first discovered in Arctic fish in 1969 [8], studies have shown that antifreeze peptides are widely present in fish [6], [9], [10], pigs [11], cattle [12], insects [13] and plants [14], [15] in cold habitats. The application of antifreeze proteins or antifreeze peptides in food has also attracted attention. Cao found that bovine collagen antifreeze peptides could improve the quality of steamed bread made from frozen dough [12]. Kashyap found that antifreeze peptides could reduce the drip loss of frozen beans [16]. Wang found that antifreeze proteins could slow down the quality deterioration of frozen pork patties during freeze–thaw cycles [17]. Tian found that antifreeze peptides could prevent the denaturation of the three-dimensional structure of fish mince and the loss of juice after freeze–thaw cycles [18]. However, there are few reports on antifreeze peptides from yak skin.

Ultrasound-assisted extraction, as an environmentally friendly and efficient pretreatment method, has been widely used in the preparation of bioactive peptides or hydrolysates in food in recent years [19], [20], [21], [22]. Ultrasonic waves can make enzymes more accessible to peptide bonds, thereby enhancing the production of bioactive peptides [23], [24]. However, ultrasonic-assisted enzymolysis has not been reported in the preparation of antifreeze peptides.

Therefore, this study aims to use ultrasound-assisted hydrolysis to efficiently obtain peptides with high antifreeze activity from yak skin and verify its mechanism of action at the molecular level. First, yak skin gelatin hydrolysate was obtained by ultrasonic-assisted enzymolysis, and the effect of ultrasound power on antifreeze activity was investigated to determine the optimal conditions for ultrasonic-assisted enzymolysis. Then, the ultrasonic-assisted enzymolysis products were further separated and subjected to peptidomic analysis. Finally, the cryoprotectivity and interaction mechanism of the chemically synthesized antifreeze peptides were verified and explained by both experiments and molecular simulation. Therefore, this paper provides a new idea for the high-value and efficient utilization of yak skin and a potential new antifreeze agent.

2. Materials and Methods

2.1. Materials

Yak (Qinghai Plateau Yak) skins were collected from Changgui Breeding Cooperative in Tianzhu Tibetan Autonomous County, Wuwei City, Gansu Province and divided into 20 × 20 cm² pieces and stored under −20 °C for use. Alkaline protease (200 U/mg) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The screened peptide sequences (95 % purity) were provided by Hefei Xingtai Biotechnology Co., Ltd., China.

2.2. Preparation of yak skin hydrolysates (YSHs)

The yak skin gelatin was prepared according to the method in the laboratory [25]. Yak skin gelatin was enzymatically hydrolyzed with alkaline protease, with an enzyme addition amount of 4 % and a solid–liquid ratio of 1:15. For ultrasonic-assisted enzymolysis, the sample was treated with an ultrasonic cell disruptor (SCIENTZ-11D, Ningbo Xinzhi Biotechnology Co. Ltd., China) with different powers (50 W, 100 W, 150 W, 200 W) for 10 min at room temperature at the beginning of hydrolysis. The hydrolysates were heated to 90 °C for 10 min to inactivate the enzyme after hydrolysis. Then the supernatant was collected after centrifugation of 6000 r/min for 15 min and freeze-dried for further use. The yield of antifreeze hydrolysates was calculated by the following formula:

Yield = m/m_c*100 % (1)

where the m means the mass of freeze-dried hydrolyzed peptides, m_c means the mass of gelatin.

2.3. Antifreeze activity of peptides

2.3.1. Thermal hysteresis activity of peptides

The thermal hysteresis activity was determined by the method of Cao [26] with modifications. 5 μL of 10 mg/mL sample was pipetted into a liquid crucible. Using the empty crucible as a reference, the sample was cooled in a differential scanning calorimeter (DSC25, TA Waters Technologies Inc., US) to −40 °C at a rate of 5 °C/min, held for 1 min, and then heated to 10 °C at a rate of 2 °C/min. Immediately, it was cooled to −40 °C again at 5 °C/min and held for 1 min. And then the sample was heated at a rate of 2 °C/min to a partially melted state. The temperature was recorded as the holding temperature T_h. And the sample was maintained at T_h for 2 min. Then the sample was cooled to −25 °C at a rate of 2 °C/min, and the onset crystallization temperature was recorded as T_o. The thermal hysteresis activity (THA) was calculated as follows:

THA = T_h-T_o (2)

2.3.2. Cryoprotection activity of Lactobacillus plantarum

Cryoprotection activity of Lactobacillus plantarum was determined according to Zeng’s method [27] with modifications. The activated Lactobacillus plantarum suspension was subjected to freeze–thaw cycles (freezing at −18 °C for 24 h and thawing at 37 °C for 15 min as one cycle), and the absorbance of Lactobacillus plantarum at 600 nm after each freeze–thaw cycle was measured by a UV–Vis spectrometer (T6 New Century, Beijing Puxi General Instrument Co. Ltd., China). The control group was done by replacing the antifreeze peptides with solvents. The relative survival rate (R) of Lactobacillus plantarum was expressed as the percentage of the cell concentration before and after freezing.

R = OD_after/OD_before × 100 % (3)

where OD_before and OD_after represents the absorbance of Lactobacillus plantarum at 600 nm before and after freeze–thaw treatment.

2.4. Effect of ultrasonic-assisted enzymolysis on YSHs

2.4.1. Degree of hydrolysis

15 mL of 37 % formaldehyde was mixed with 7.8 mL of acetylacetone and diluted to 100 mL in a volumetric flask to make the color development solution. 1 mL of sample was mixed with 2 mL of sodium acetate-acetic acid buffer solution (pH 4.8) and 2 mL color developer solution and heated in boiling water for 10 min. The absorbance of the mixed solution at 400 nm was measured. The standard curve was prepared by performing the same reaction using ammonium sulfate instead of the sample. The amino nitrogen content was calculated according to the absorbance results. And the degree of hydrolysis was calculated as follows

DH = amino nitrogen content/total nitrogen content × 100 % (4)

2.4.2. Particle size

200 mg of antifreeze peptides were added in 20 mL of PBS buffer (0.05 mol/L, pH 7.0) and centrifuged for 10 min. The supernatant was using to measure the particle size by a laser particle size analyzer (Bettersize2600, Dandong Baite Instrument Co., Ltd. China).

2.4.3. X-ray diffraction

X-ray Diffractometer (Shimadzu XRD-6100, Japan) was used to measure the X-ray diffraction of antifreeze peptides. The scanning parameters were as follows: speed 2°/min, diffraction angle range 2 ∼ 40° and step width 0.02.

2.4.4. Thermogravimetric analysis

Simultaneous thermal analyzer (NETZSCH 449F3, Germany) was used to measure the thermogravimetric of antifreeze peptides. The measuring parameters were as follows: initial temperature 30 °C, final tempreture 600 °C, heating rate 20 °C/min, nitrogen.

2.5. Ultrafiltration of YSHs

The YSHs with highest antifreeze activity prepared by ultrasonication were further separated using the ultrafiltration centrifuge tubes (1 kDa, Pall Corporation, US; 3 kDa, Merck Millipore, US) under 6000 r/min for 15 min at 4 °C. The separated components were freeze-dried and recorded as F1, F2 and F3 according to their molecular weight from small to large.

2.6. LC- MS/MS tests

Ultrafiltration components with highest antifreeze activity were analyzed by a high-resolution mass spectrometry (Q-Exactive HF, Thermo Fisher Scientific, US) coupled to a nano upgraded high performance liquid chromatograph (EASY-nLC 1200, Thermo Fisher Scientific, US). The positive-ion mass spectrometry was used and the analysis time was 120 min. The mass-to-charge ratios of peptides and peptide fragments were collected as follows: 20 fragment spectra were collected after each full scan. The scan range was 200–1800, the primary resolution was 60000, the secondary resolution was 15000, and the collision energy was CE28 eV.

2.7. Screening of antifreeze peptide sequence

The peptide sequences were collected from the mass spectrometry results analyzed by Proteome Discoverer 2.5. The sequences with high abundance were then screened by ToxinPred database (https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php) and Cryoprotect (https://codes.bio/cryoprotect/) database.

2.8. Molecular docking

Molecular docking was performed according to Yang’s method [6]. The ice crystal structure model was built using GenIce1.0.10 and used as the receptor molecule. The peptides’ structure models were modeled by AlphaFold2 and used as the ligand molecule. The docking was performed by Hex 8.0.0 software. The ice crystal-peptide interaction was analyzed by Discovery Studio 2019 and PyMOL software.

2.9. Statistical analysis

All experiments were conducted in 3 parallel groups, and the data were expressed as mean ± standard deviation. The data was analyzed by SPSS 26.0 software and the graphs were made by GraphPad Prism 10 software.

3. Results and Discussion

3.1. Effect of ultrasonic-assisted enzymolysis on YSHs

3.1.1. Thermal hysteresis activity and yield

Fig. 1 shows the results of thermal hysteresis activity and yields of YSHs treated with different ultrasound powers. When treated with 150 W ultrasound, the thermal hysteresis activity and yield were 4.21 ± 0.03 °C and 54.10 ± 0.26 %. The thermal hysteresis activity and yield increased when the ultrasound power increased from 0 to 150 W (p < 0.05) and decreased when the ultrasound power increased from 150 W to 200 W (p < 0.05). On one hand, ultrasound treatment could make the peptide bonds more accessible for the enzyme by cavitating of protein structures [28]. From 0 to 150 W, ultrasound waves improved the enzymatic hydrolysis of yak skin gelatin and produced more lower molecular weight peptides. As can be seen in Table 1, the amount of peptides under 3 kDa increased as the ultrasound power increased from 0 to 150w. According to previous studies, peptides under 3 kDa had higher antifreeze activity [29]. On the other hand, high intensity ultrasound could make the proteins to aggregate. And the bubble clusters generated by the cavitation effect may in turn weaken the cavitation effect [28], thus causing the decreasing of thermal hysteresis and yield when ultrasound power increased from 150 W to 200 W. Although the effect of ultrasound for antifreeze peptides have rarely been reported, studies have shown that ultrasound could improve the hydrolysis and activity of bioactive peptides [30], [31], [32].

Fig. 1.

Thermal hysteresis activity and yields of yak skin antifreeze peptides treated with different ultrasoundpowers.

Table 1.

Molecular distribution of YSHs prepared by different ultrasound powers.

Ultrasound power/W	Mw < 3000 Da/%	Mw3000-10000 Da/%	Mw > 10000 Da/%
0	7.32	56.81	35.88
50	27.96	69.79	2.26
100	10.25	85.56	4.19
150	69.3	26.14	4.56
200	40.74	52.42	6.84

3.1.2. Cryoprotection activity of Lactobacillus plantarum

As shown in Fig. 2, after 4 freeze–thaw cycles, the relative survival rate of control group was 16 %. The relative survival rates of Lactobacillus plantarum treated with YSHs prepared by non-ultrasound, 50 W, 100 W, 150 W, and 200 W ultrasound after four freeze–thaw cycles were 39 %, 45 %, 51 %, 60 %, and 54 %, respectively. The relative survival rates of Lactobacillus plantarum added with YSHs were higher than that of in the control group. Among them, the relative survival rate of Lactobacillus plantarum in the 150 W treatment group was the highest. The results were consistent with the thermal hysteresis activity results. This may indicate that 150 W ultrasonic power could better hydrolyze yak skin gelatin to obtain small molecule antifreeze peptides. But it was not too high to cause protein aggregation.

Fig. 2.

Relative survival rates of yak skin antifreeze peptides prepared by different ultrasound powers for the Lactobacillus plantarum in different freeze–thaw cycles.

3.1.3. Degree of hydrolysis

Fig. 3 shows the degree of hydrolysis (DH) of yak skin antifreeze peptides prepared by different ultrasound powers. Compared with non-ultrasound group, the ultrasound treatment could significantly improve the degree of hydrolysis of yak skin gelatin. With the power of ultrasound increasing from 50 W to 150 W, the DH of yak skin gelatin increased. It might be that the ultrasound has a cavitation effect. When it passed through yak skin gelatin proteins, the pressure changes would cause changes in the protein’s conformation. Thus, more enzymatic sites were exposed, and the degree of hydrolysis increased [30], [33]. As the ultrasound power increased from 150 W to 200 W, the DH of yak skin gelatin decreased. Ultrasound has the heat effect, ultrasound with excessive powers might cause the proteins to aggregate or destruct thus reducing the degree of hydrolysis [28], [34].

Fig. 3.

Degree of hydrolysis of yak skin antifreeze peptides prepared by different ultrasound powers.

3.1.4. Particle size

As shown in Fig. 4, the average particle size of 50 W, 100 W, 150 W and 200 W ultrasound-treated antifreeze peptides were 597 nm, 570 nm, 417 nm and 551 nm, respectively. They were significantly smaller than that of non-ultrasound treatment group (722 nm). And the particle sizes of different groups were in line with the results of degree of hydrolysis. The higher the degree of hydrolysis, the more complete the hydrolysis, resulting in a smaller molecular weight and smaller particle size of the hydrolysates [34]. The results indicated proper ultrasound treatment could improve the hydrolysis of YSHs while excessive power of ultrasound treatment might lead to peptides aggregation.

Fig. 4.

Partical size of yak skin antifreeze peptides prepared by different ultrasound powers.

3.1.5. X-ray diffraction

As shown in Fig. 5, all YSHs had broad diffraction peaks at 2θ of 7° and 21°, which might be attributed to their amorphous structure [35]. YSHs also had two sharp characteristic diffraction peaks at around 27.5° and 32°. Among them, the YSH treated with 200 W ultrasound had a higher diffraction peak at 21° and a lower diffraction peak at 32°compared with other experimental groups. This indicated that 200 W treatment made the yak skin antifreeze peptides more amorphous. This might be due to excessive energy causing protein aggregation or destruction, which was consistent with the results of DH and particle size.

Fig. 5.

XRD of yak skin antifreeze peptides prepared by different ultrasound powers.

3.1.6. Thermogravimetric analysis

Fig. 6 shows the thermogravimetric analysis of yak skin antifreeze peptides. As can be seen from Fig. 6, as the temperature increased, the YSHs experienced three weight loss stages. First, between 26–140 °C, the mass of the YSHs decreased slightly, which might be due to the evaporation of water [36]. Between 140–260 °C, the mass of YSH decreased steadily, among which the antifreeze peptides without ultrasound treatment having the highest mass loss (11.03 %). This suggested that sonication could improve the thermal stability of antifreeze peptides. Finally, between 260–600 °C, the mass of the YSHs decreased significantly. When the mass tended to be stable, the YSHs treated with 150 W ultrasonic treatment had the largest mass remaining, with 47.9 % remaining at 598.4 °C. This showed that the YSHs treated with 150 W ultrasound had better thermal stability. The TGA results indicated the ultrasound treatment improved the thermal stability of antifreeze peptides, which is in line with Qu’s findings [37]. Their research found that ultrasound-assisted extraction treatment improved the thermal stability of walnut protein. And it might be due to the reason that ultrasound could lead to more ordered secondary structures of peptides [38].

Fig. 6.

Thermogravimetric analysis and DTG of yak skin antifreeze peptides prepared by different ultrasound powers.

3.2. Antifreeze activity of different peptides fractions after ultrafiltration

The YSHs were further separated by ultrafiltration. Using 1 kDa and 3 kDa ultrafiltration centrifuge tubes, the antifreeze peptides were divided into three components: <1kDa, 1–3 kDa and > 3 kDa, which were recorded as F1, F2 and F3, respectively. The antifreeze properties of F1, F2 and F3 were characterized by thermal hysteresis activity and cryoprotection for Lactobacillus plantarum. As shown in Fig. 7A, the thermal hysteresis activity of F2 (3.87 °C) was significantly higher than that of F1(3.46 °C) and F3 (3.19 °C), which was consistent with the results of Cao [39]. Their study found that short peptides with a molecular weight below 2.5 kDa had higher antifreeze activity. Studies have also shown that small molecule peptides below 3 kDa have better antifreeze properties [30]. As can be seen from Fig. 7B, F2 had the best cryoprotective property against Lactobacillus plantarum, and the relative survival rate was 67 % after 4 freeze–thaw cycles. Therefore, the F2 component was further subjected to LC-MS/MS analysis.

Fig. 7.

Thermal hysteresis activity of F1, F2 and F3(A); relative survival rates of F1, F2 and F3 for the Lactobacillus plantarum in different freeze–thaw cycles (B).

3.3. Peptidomic analysis

Fig. 8 shows the total ion chromatography of F2. 961 sequences were identified in the LC-MS/MS test. High-abundance sequences were used to screen small peptides that were non-toxic, hydrophilic, and had high antifreeze activity using the ToxinPred database and the Cryoprotect database. Three sequences were finally screened as potential research objectives: GERGGPGGPGPQ (S1), PGGAEGPGRDAQQP (S2), and VAPPGAPKKEH (S3). The screened sequences contained abundant glycine, alanine and proline, which was consistent with the literature report that antifreeze activity was closely related to the presence of certain specific amino acids in the peptide [7].

Fig. 8.

Total ion chromatography of F2 by LC-MS/MS.

3.4. Comparison of antifreeze activity of S1 and other antifreeze agents

The antifreeze activity of the selected sequences was further investigated using thermal hysteresis activity and Lactobacillus plantarum cryoprotection experiments. The results in Fig. 9A showed that the thermal hysteresis activities of S1, S2, and S3 were 4.64 ± 0.03, 4.39 ± 0.02, and 4.36 ± 0.03, respectively, which were higher than those of the control groups of sucrose (3.06 ± 0.04), sorbitol (3.50 ± 0.05), and the mixture of sucrose and sorbitol (4.1 ± 0.04). As can be seen from Fig. 9B, after 4 freeze–thaw cycles, the survival rates of Lactobacillus plantarum with the addition of S1, S2 and S3 were 68 %, 65 % and 62 %, respectively, which were much higher than the 16 % of the negative control group and higher than the positive control group of sucrose (45 %), sorbitol (50 %) and a mixture of sucrose and sorbitol (57 %). This showed that the screened peptides had the potential to become novel antifreeze agents.

Fig. 9.

Thermal hysteresis activity of different antifreeze agents (A); relative survival rates of different antifreeze agents for the Lactobacillus plantarum in different freeze–thaw cycles (B).

3.5. Analysis of antifreeze sequences and ice crystal in simulated systems

The 3D structures of S1, S2 and S3 were modeled by AlphaFold2 and used as the ligand moleculs. As can be seen from Figure S1, pepetide S1 and S3 are both random coiled structures, while S2 has an α-helical structure. The docking and interactions of ice crystals with S1, S2 and S3 is shown in Fig. 10. As can be seen, S1 forms hydrogen bonds with ice molecules through the arginine-3, glycine-5 and glycine-10. S2 forms hydrogen bonds with ice molecules through aspartic acid-10, glutamine-13 and proline-14. S3 forms hydrogen bonds with ice molecules through alanine-2, lysine-8, and lysine-9.The docking binding energies of S1, S2 and S3 with ice crystals were −391.55, −387.30 and −370.96 kcal/mol, respectively. The higher the absolute value of the binding energy, the more stable the binding. It could be seen that the bond between S1 and ice crystals was more stable, so that it could be better adsorbed on the surface of ice crystals, hindering further growth of ice crystals [40], which was in consistent with the results of the antifreeze activity experiment. As can be seen from Fig. 10D, the binding between S1 and ice crystals was mainly through glycine and arginine residues, which was in consistent with the results reported in previous literature that glycine plays an important role in the cryoprotective activity of antifreeze peptides [41], [42].

Fig. 10.

Molecular docking (A-C) and interaction (D-F) of S1, S2, and S3 with ice crystals.

4. Conclusions

In this study, antifreeze peptides (YSHs) from yak skin were obtained by ultrasonic-assisted enzymolysis. The survival rate of Lactobacillus plantarum treated with YSHs after four freeze–thaw cycles increased from 16 % in the control group to 39–60 % in ultrasound groups, proving that YSHs had antifreeze activity. YSHs obtained by 150 W ultrasound hydrolysis had higher antifreeze activity, better hydrolysis degree and thermal stability. The peptideomics of F2 obtained by further separation of YSHs screened a total of 961 peptides, from which S1 (GERGGPGGPGPQ, 1065.29) was screened out through the antifreeze activity prediction platform and indicators such as toxicity, hydrophilicity, abundance, and molecular docking. Molecular docking showed that S1 could be adsorbed on the surface of ice crystals to inhibit ice crystal growth. DSC and cryoprotection experiments on Lactobacillus plantarum proved that YSHs, F2, and S1 had thermal hysteresis activity and cryoprotection activity, among which S1 had a more obvious effect and was better than commercial sugar antifreeze agents. In summary, this study provides a new idea for the high-value and efficient utilization of yak skin and provides a potential effective new food antifreeze agent.

CRediT authorship contribution statement

Xiaotong Ma: Writing – original draft, Investigation, Funding acquisition, Formal analysis, Data curation. Wenxing Wang: Investigation, Data curation. Hongmei Shi: Resources. Xiangying Kong: Resources. Li Zhang: Writing – review & editing, Resources, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: