Antifreeze peptide, antifreeze agent and application thereof in food freezing protection

Abstract

The present invention discloses an antifreeze peptide, an antifreeze agent and their application in food freezing protection, and belongs to the technical field of bioactive peptides. The amino acid sequence of the antifreeze peptide of the present invention is at least one of DINFRY and LGDGMSLST. The present invention uses defatted Antarctic krill protein as raw material, and obtains two Antarctic krill antifreeze peptides by enzymatic hydrolysis, ultrafiltration, gel filtration chromatography, LC-MS / MS and other methods. Experiments have shown that these polypeptides have good antifreeze effects and can be used as food antifreeze agents. Compared with traditional antifreeze agents, they are green and safe, and have broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides, and in particular relates to an antifreeze peptide, an antifreeze agent and applications thereof in food freezing protection. Background Art

[0002] Substances that can stabilize the quality of frozen food during the freeze-thaw cycle are called antifreeze agents. Traditional commercial antifreeze agents are generally sugars, alcohols, salts and their complexes.

[0003] Sugar antifreeze agents are mostly a mixture of sucrose and sorbitol. Although this mixture has obvious antifreeze effects, sucrose is high in calories and sweetness, which may affect the flavor of the product and easily cause symptoms such as hyperglycemia and hypertension, severely limiting its use by people with diabetes and obesity.

[0004] Salt antifreeze agents such as phosphates and complex phosphate antifreeze agents have also been proven to be good antifreeze agents, but excessive intake of phosphates will affect the body's absorption of calcium and even lead to symptoms such as osteoporosis, hypertension, and renal failure. Moreover, when the amount of phosphate added is too high, the food will have a bitter taste and will also reduce the taste and flavor of the product. In addition, although high concentrations of sugar alcohols and salt antifreeze agents can reduce the ice crystal particles formed during refrigeration, they cannot effectively prevent the recrystallization of small ice crystals caused by possible temperature fluctuations during frozen storage, thereby having a negative impact on the texture characteristics of the product.

[0005] In summary, although artificially synthesized antifreeze agents are effective and low in cost, tripolyphosphate and pyrophosphate are very susceptible to degradation when phosphate antifreeze agents are added, indicating that the use of chemical reagents during the freezing process poses certain toxic and carcinogenic risks.

[0006] Antifreeze peptides, natural antifreeze agents that improve the freezing properties of food, can effectively reduce the formation and recrystallization of ice crystals in food during the cold chain process, thereby improving the quality of low-temperature cold chain food. At the same time, antifreeze peptides have multiple properties such as thermal hysteresis activity, recrystallization inhibition activity, and cell membrane protection. Only a very small amount of them needs to be added to achieve a high antifreeze effect. In addition, natural antifreeze peptides are usually derived from natural protein hydrolysates and do not contain additional chemical additives or synthetic ingredients. This is in line with consumers' preference for concise food lists and simple raw materials, so they have broad application prospects in the food industry. Summary of the invention

[0007] In view of the problems existing in the prior art, the object of the present invention is to provide an antifreeze peptide, an antifreeze agent and their application in food freezing protection.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] An antifreeze peptide, wherein the amino acid sequence of the antifreeze peptide is at least one of DINFRY and LGDGMSLST.

[0010] An antifreeze agent, comprising at least one of the above-mentioned antifreeze peptides; preferably, comprising DINFRY and LGDGMSLST.

[0011] Based on the above scheme, the mass ratio of DINFRY and LGDGMSLST is 1:1.

[0012] On the basis of the above scheme, it also contains food acceptable auxiliary materials.

[0013] The application of the above antifreeze agent in food freezing protection.

[0014] On the basis of the above scheme, the above antifreeze agent is added to the food before freezing.

[0015] On the basis of the above scheme, the freezing protection of food is achieved by improving the residual activity of catalase.

[0016] Based on the above scheme, the food is at least one of dough, yogurt, fish paste, and probiotics.

[0017] Based on the above scheme, the probiotics are at least one of yeast and Lactobacillus rhamnosus.

[0018] The preparation method of the above-mentioned natural antifreeze peptide comprises the following steps:

[0019] (1) dissolving defatted Antarctic krill protein in deionized water to prepare an Antarctic krill protein solution;

[0020] (2) adding protease to the Antarctic krill protein solution for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, centrifuging to obtain a supernatant;

[0021] (3) ultrafiltration of the supernatant of step (2) to obtain a protease hydrolysate with a molecular weight range of <3 kDa, 3-10 kDa, and >10 kDa;

[0022] (4) taking the protease hydrolysate with a molecular weight of <3 kDa and further purifying it by gel filtration chromatography to obtain multiple polypeptide components;

[0023] (5) Determine the residual catalase activity of the polypeptide components obtained in step (4), and select the component with the highest residual catalase activity for LC-MS / MS analysis.

[0024] Based on the above scheme, the protease in step (2) is alkaline protease.

[0025] Based on the above scheme, the amount of alkaline protease added is 3000U alkaline protease per gram of Antarctic krill protein.

[0026] Based on the above scheme, the enzymatic hydrolysis conditions are 55°C, pH 9.5, and enzymatic hydrolysis for 4 h.

[0027] Advantages of the technical solution of the present invention

[0028] The present invention uses defatted Antarctic krill protein as raw material, and obtains two Antarctic krill antifreeze peptides by enzymatic hydrolysis, ultrafiltration, gel filtration chromatography, LC-MS / MS and other methods. At present, although there have been studies on other biological activities of Antarctic krill peptides such as regulating blood sugar and blood pressure, improving osteoporosis, and relieving fatigue, there have been no reports on its antifreeze activity, especially the separation, purification and identification of Antarctic krill antifreeze peptides.

[0029] The preparation of the Antarctic krill antifreeze peptide of the present invention uses defatted Antarctic krill, a food-derived protein source, as a raw material and is obtained by hydrolysis at specific enzyme cleavage sites. It has the characteristics of controllable and efficient preparation and can solve the problems of traditional antifreeze agents that are difficult to control in concentration during application and difficult to ensure uniform distribution in food after treatment.

[0030] The Antarctic krill antifreeze peptide prepared by the invention provides a certain theoretical basis for the high-value utilization, added value improvement and industrialized production of Antarctic krill, and promotes the resource utilization of Antarctic krill.

[0031] The present invention provides certain theoretical support for the development of new antifreeze agents or functional products in food cold chain logistics. Compared with traditional commercial antifreeze agents, such as sugars, alcohols, salts and their compounds, they may cause bad flavors, high fat content and high calories, and organic agents, such as DMSO, may cause irreversible cytotoxic damage to probiotic cells. The raw material of the present invention is natural and non-toxic Antarctic krill protein, which is easy to operate, green and safe. No organic reagents are introduced in the preparation process, and the equipment requirements are low. It is easy to promote and can be potentially used as an innovative antifreeze agent in food cold chain logistics.

[0032] Compared with other natural antifreeze peptides, the Antarctic krill antifreeze peptide of the present invention has abundant raw material sources, a higher protein peptide yield, and is easy to extract. In summary, the Antarctic krill antifreeze peptide has broad application prospects and great potential in frozen food systems, such as dough, yogurt, and fish paste, and can also effectively improve the survival rate of common probiotics such as yeast and Lactobacillus rhamnosus after freezing.

[0033] The present invention prepares an antifreeze agent by compounding the two Antarctic krill antifreeze peptide segments in a certain ratio. After testing, the residual activity of the catalase of the antifreeze agent is significantly higher than that of the antifreeze agent containing a single peptide segment. The antifreeze agent is used for the freezing test of Lactobacillus rhamnosus. The results show that the survival rate of Lactobacillus rhamnosus added with DY+LT after freezing is the highest, which is significantly higher than the positive control of adding 1 mg / mL sucrose, 1 mg / mL skim milk and 20% glycerol, and is better than the antifreeze agent added with a single peptide segment. It can be seen that the antifreeze agent prepared by compounding the two Antarctic krill antifreeze peptide segments of the present invention has a high antifreeze activity and has great application prospects in food freezing protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Effects of different proteases on the hydrolysis degree of Antarctic krill protein hydrolysates;

[0035] Figure 2 Effects of different proteases on the antifreeze activity of Antarctic krill protein hydrolysates;

[0036] Figure 3 The residual catalase activity of each fraction after ultrafiltration and centrifugation;

[0037] Figure 4 Gel filtration chromatography was used to separate and purify the components;

[0038] Figure 5 Residual catalase activity of each fraction after gel filtration chromatography;

[0039] Figure 6 The mass spectrum of the peptide sequence is Asp-Ile-Asn-Phe-Arg-Tyr;

[0040] Figure 7 The mass spectrum of the peptide sequence is Leu-Gly-Asp-Gly-Met-Ser-Leu-Ser-Thr;

[0041] Figure 8 The mass spectrum of the peptide sequence obtained by biosynthesis is Asp-Ile-Asn-Phe-Arg-Tyr;

[0042] Fig. 9 The mass spectrum of the peptide sequence obtained by biosynthesis is Leu-Gly-Asp-Gly-Met-Ser-Leu-Ser-Thr;

[0043] Fig.10 The residual catalase activity of single peptides and complex peptides after biosynthesis;

[0044] Fig.11 The coating morphology of the activated Lactobacillus rhamnosus strain;

[0045] Fig.12 Plate streaking morphology of the second generation strain of Lactobacillus rhamnosus after activation;

[0046] Fig.13 Growth curve of Lactobacillus rhamnosus;

[0047] Fig.14 The morphology of the bacterial suspension of Lactobacillus rhamnosus after adding different antifreeze agents;

[0048] Fig.15 Effects of different antifreeze agents on the relative survival rate of Lactobacillus rhamnosus. DETAILED DESCRIPTION

[0049] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0050] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0051] In the following embodiments,

[0052] The hydrolysis degree of Antarctic krill protein was determined by the ophthaldialdehyde (OPA) method. The specific method is as follows:

[0053] 80 mg of OPA was dissolved in 2 mL of anhydrous ethanol, 200 μL of β-mercaptoethanol, 5 mL of 10% SDS (w / v) and 92.8 mL of 0.1 mol / L sodium tetraborate to prepare 100 mL of OPA reagent solution. 40 μL of Antarctic krill enzymatic hydrolysate was mixed with 4 mL of OPA reagent and incubated at room temperature for 2 min, and the absorbance at 340 nm was measured.

[0054] Antarctic krill protein was placed in 6 mol / L HCl and reacted at 115 ℃ for 24 h. The number of free amino groups was determined using a serine standard curve as the number of free amino acids produced by complete hydrolysis of Antarctic krill protein.

[0055] The degree of hydrolysis of Antarctic krill protein is calculated according to the following formula:

[0056] Degree of hydrolysis (%) = [(NH2) t -(NH2) t0 )] / (NH2)T

[0057] Where: (NH2) t Indicates the amount of free amino acids in the enzymatic hydrolysis product at time t; (NH2) t0 Indicates the amount of free amino acids that have not been hydrolyzed by enzymes; (NH2) T Represents the amount of free amino acids after complete hydrolysis.

[0058] The antifreeze activity of Antarctic krill enzymatic hydrolysate was evaluated by the catalase residual activity method, as follows:

[0059] A certain amount of catalase was weighed and dissolved in 0.05 mol / L NaOH buffer (pH 7.0) to a final concentration of 0.5 mg / mL. The catalase solution and 2 mg / mL Antarctic krill antifreeze peptide solution were mixed in equal volumes to determine the initial enzyme activity. The remaining mixed solution was frozen in a -20 ℃ refrigerator for 24 h, taken out and thawed at 25 ℃, and then placed in a -33 ℃ refrigerator for 6 h. The freeze-thaw cycle was repeated 4 times, and the enzyme activity after freeze-thaw was determined. The ratio of the enzyme activity after freeze-thaw to the initial enzyme activity (residual activity of catalase) was used to indicate the antifreeze activity. The greater the residual enzyme activity, the greater the antifreeze activity of the sample to be tested.

[0060] Determination of catalase activity: Add 1.9 mL of distilled water, 0.1 mL of catalase and Antarctic krill antifreeze peptide mixture and 1 mL of 0.1 mol / mL hydrogen peroxide solution to a quartz cuvette, shake well immediately, put into the sample cell of the spectrophotometer, record the initial absorbance at a wavelength of 240 nm, and record the absorbance every 1 min for a total of 5 min. The absorbance value reduced by 0.1 enzyme amount within 1 min is 1 enzyme activity unit (U).

[0061] The calculation formulas of catalase activity and catalase residual activity are as follows:

[0062] Catalase activity = △A240 × [(dilution factor × total reaction volume) / (time × sample volume)] × 10;

[0063] Residual activity of catalase = (enzyme activity after freeze-thaw / initial enzyme activity) × 100%.

[0064] Different lowercase letters in the figures of the present invention indicate significant differences between different treatments.

[0065] Example 1

[0066] An antifreeze peptide, wherein the amino acid sequence of the antifreeze peptide is shown in at least one of SEQ ID NO: 1 to SEQ ID NO: 2.

[0067] SEQ ID NO:1 DINFRY

[0068] SEQ ID NO:2 LGDGMSLST

[0069] Example 2

[0070] A method for preparing an antifreeze peptide comprises the following steps:

[0071] (1) Weigh 100 g of washed, headless and shelled Antarctic krill meat, place it in a beaker, and add deionized water (4 °C) with a liquid-to-solid ratio of 3.17 mL / g to homogenize. Subsequently, adjust the pH to 11.38 with 2 mol / L sodium hydroxide, let it stand for 0.5 h, and centrifuge it at 10,000 g for 10 min at 4 °C to obtain the supernatant. The entire extraction process was repeated 3 times. Finally, adjust the pH of the collected supernatant to 4.5 with 2 mol / L phosphoric acid, let it stand for 1.0 h, and centrifuge it at 10,000 g for 10 min at 4 °C. Collect the precipitate and freeze-dry it at -80 °C.

[0072] (2) Weigh 5 g of extracted Antarctic krill protein, add acetone solution at a ratio of 1:4 (w:v), stir with a constant temperature magnetic stirrer for 3 h, and let it stand for 0.5 h until the liquid becomes clear. The upper red liquid is Antarctic krill oil. Pour it out and continue to add acetone. Stir for 2 h and let it stand for 0.5 h. Pour out the upper red liquid and repeat the above steps until the upper liquid is colorless. Recover the defatted Antarctic krill protein in a fume hood.

[0073] (3) Weigh the defatted Antarctic krill protein and dissolve it in deionized water at a ratio of 2% (w / v). Add alkaline protease to the solution at a rate of 3000 U of alkaline protease per gram of defatted Antarctic krill protein. Stir at a low speed until evenly distributed, adjust the pH value to 9.5, and place the solution at a temperature of 55 °C in a constant temperature incubator for enzymatic reaction for 4 h.

[0074] (4) After enzymatic hydrolysis, the reaction solution was heated in boiling water for 15 min to inactivate the protease and terminate the reaction. The solution was quickly cooled to 4.0 °C with ice water and centrifuged at 12,000 g for 15 min to retain the supernatant (called AKPH), which was then freeze-dried.

[0075] Effects of different proteases on the hydrolysis degree and antifreeze activity of Antarctic krill protein hydrolysates

[0076] Acidic protease, neutral protease, trypsin, pepsin and papain were used to enzymolyze Antarctic krill protein, and the enzymolysis temperature and pH were carried out at the optimal temperature and pH of each protease (Table 1), and the other conditions were the same as those of alkaline protease. After the enzymolysis was completed, the effects of different proteases on the hydrolysis degree and antifreeze activity of Antarctic krill protein hydrolysates were determined.

[0077] Table 1 Optimal temperature and pH of different proteases

[0078]

[0079] Proteases are very important for the enzymatic hydrolysis of Antarctic krill protein. Different enzymes have different cleavage sites and will produce different peptides, which will affect the enzymatic hydrolysis effect and functional characteristics of the enzymatic hydrolysis products, including the degree of hydrolysis of the protein after enzymatic cleavage and the biological activity of the produced peptides. The results of the determination of the degree of hydrolysis of Antarctic krill protein and the antifreeze activity of the enzymatic hydrolysis products by 6 proteases are shown in Figure 1 and Figure 2 .

[0080] The degree of protein hydrolysis is one of the important indicators for evaluating the high biological activity of enzymatic hydrolysis products. Good hydrolysis effect can release more active groups.

[0081] Depend on Figure 1 It can be seen that compared with the other four proteases, alkaline protease and trypsin have a higher degree of hydrolysis of Antarctic krill protein, which are 37.77% and 34.10% respectively. This may be because under the condition of sufficient substrate, these two enzymes have more cleavage sites and the protein is more easily hydrolyzed into small molecular peptides.

[0082] Depend on Figure 2 It can be seen that compared with the other four proteases, the residual catalase activity of the hydrolyzed products obtained by alkaline protease and trypsin was stronger, which was 70.00% and 64.29% respectively. Among them, alkaline protease had the best effect.

[0083] The lyophilized sample of the Antarctic krill protein product hydrolyzed by alkaline protease was treated with 6 mol / L HCl at 110°C for 24 h under a nitrogen atmosphere. The digested mixture was transferred to a centrifuge tube and dried under reduced pressure at 50°C. The dried sample was dissolved in distilled water twice. Finally, the dried sample was dissolved in sodium citrate buffer (pH 2.2) and filtered through a 0.22 µm filter. The amino acid composition of the Antarctic krill protein product hydrolyzed by alkaline protease was determined using an amino acid analyzer. The results are shown in Table 2.

[0084] As shown in Table 2, the amino acid composition of the Antarctic krill protein hydrolyzed by alkaline protease is rich in proline and glycine, which are closely related to the antifreeze activity of polypeptides, accounting for 20.67% and 9.24% respectively; followed by a high content of glutamic acid, 8.11g / 100g. Glutamic acid contains strong polar hydroxyl groups, which are related to the ice affinity and cryoprotective activity of the hydrolyzed product and can increase the surface force of the polypeptide adsorbed on ice crystals. At the same time, the hydrolyzed product is also rich in aliphatic amino acids such as alanine (9.53%), arginine (7.91%), and aspartic acid (1.54%). The presence of aliphatic amino acids can enhance the antifreeze activity because the alkyl side chain of the residue provides a non-polar environment. They maintain the hydrogen bonds formed by hydrophobic interactions with water molecules, thereby inhibiting the growth of ice crystals.

[0085] Table 2 Amino acid composition of the product of Antarctic krill protein hydrolyzed by alkaline protease

[0086]

[0087] (5) The alkaline protease-hydrolyzed Antarctic krill protein product obtained in step (4) above was separated into different molecular weights by ultrafiltration centrifuge tube filtration, and passed through a 10 KDa ultrafiltration centrifuge tube and a 3 KDa ultrafiltration centrifuge tube in sequence. The ultrafiltration parameters were set to 4000×g and 25 min until all the liquid passed through the membrane. The components: peptides >10 KDa, 3~10 KDa, and <3 KDa were collected, and the obtained components were lyophilized. The residual catalase activity of different components was measured, and then their antifreeze activity was analyzed. The results are as follows: Figure 3 shown.

[0088] Ultrafiltration centrifuge tube filtration was used to separate and purify antifreeze peptides from alkaline protease hydrolysates, and peptides with molecular weights >10 KDa, 3-10 KDa, and <3 KDa were collected. Ultrafiltration separation was used to separate and enrich small molecule peptides. Figure 3 As shown, the antifreeze activity of <3 KDa was the most obvious, and the residual activity of its catalase was 75.19%. The content of short peptides with smaller molecular weight retained by the proteinase hydrolysate was higher, and short peptides were easily adsorbed on the surface of ice crystals, inhibiting the growth of ice crystals and playing a vital role in cryoprotective activity.

[0089] (6) Take the enzymatic fraction <3 KDa and further filter and purify it on a Sephadex G-25 gel filtration column (2.0*40 cm). First balance it with ultrapure water, and then elute it with ultrapure water at a flow rate of 0.3 mL / min. Each part of the eluate is monitored at 280 nm, and the resulting peaks are collected. The antifreeze activity of different eluted fractions after gel filtration chromatography is also tested.

[0090] like Figure 4As shown, after separation by Sephadex G-25 gel filtration column, three peaks were collected, named as components F1, F2 and F3. The eluted components were collected by peak collection method. Component F2 was the second to be separated in the chromatographic column, and its molecular weight was in the middle. F1 had the largest molecular weight, and F3 had the smallest molecular weight.

[0091] Depend on Figure 5 It can be seen that the residual activity of catalase in the three components separated by Sephadex G-25 gel filtration column was significantly higher than that of the enzymatic hydrolysis product before separation and the F2 and F1 components. When the F3 component was mixed with catalase, the residual activity of catalase after freezing and thawing was 80.12%. This may be because the molecular weight of the F3 fragment hydrolyzed by protease is the smallest, and the molecular weight is an important factor affecting antifreeze activity.

[0092] (7) LC-MS / MS analysis was performed on the F3 fraction of the above gel filtration chromatography. After centrifugation and drying, the cleaved peptide sample was redissolved in Nano-LC mobile phase A (0.1% formic acid / water) and loaded into a bottle for online LC-MS / MS analysis. The dissolved sample was loaded onto a nanoViper C18 pre-column (3 μm, 100 Å) and desalted by washing. The liquid phase was an Easy nLC 1200 nanoliter liquid phase system (ThermoFisher, USA). The sample was desalted and retained on the pre-column and then separated by an analytical column. The analytical column specification was a C18 reverse phase chromatography column (Acclaim PepMap RSLC, 75 μm×25 cm C18-2 μm 100 Å). The gradient used in the experiment increased the mobile phase B (80% acetonitrile, 0.1% formic acid) from 5% to 38% within 30 min. The mass spectrometer used a ThermoFisher QExactive system (ThermoFisher, USA) combined with a nanospray Nano Flex ion source (ThermoFisher, USA), with a spray voltage of 1.9 kV and an ion transfer tube heating temperature of 275 °C. The mass spectrometer scan mode was in the information-dependent acquisition mode (DDA, Data Dependent Analysis), with a primary mass spectrometer scan resolution of 70000, a scan range of 350-2000 m / z, and a maximum injection time of 100 ms. A maximum of 20 secondary spectra with charges of 2+ to 5+ were collected in each DDA cycle, and the maximum injection time of the secondary mass spectrometer ions was 50 ms. The collision cell energy (high-energy collision-induced dissociation, HCD) was set to 28 eV, applicable to all precursor ions, and the dynamic exclusion was set to 25 seconds.

[0093] LC-MS / MS analysis of the F3 component yielded two peptides, as shown in Table 3; the typical peaks corresponding to the peptides are as follows: Figure 6 , Figure 7 Then the two peptides obtained by LC-MS / MS analysis were biosynthesized, and the corresponding mass spectra were shown as follows: Figure 8 , Fig. 9 shown.

[0094] Table 3 Identification of antifreeze peptides from Antarctic krill by HPLC-MS

[0095]

[0096] Example 3

[0097] An antifreeze agent, comprising a peptide segment with amino acid sequences such as DINFRY and LGDGMSLST; wherein the mass ratio of DINFRY to LGDGMSLST is 1:1.

[0098] Determination of the antifreeze effect of antifreeze peptides

[0099] Accurately weigh 2 mg of DINFRY (abbreviated as DY) and 2 mg of LGDGMSLST (abbreviated as LT) and dissolve them in 4 mL of deionized water, and dissolve them in each other to prepare the composite peptide DINFRY+LGDGMSLST (abbreviated as DY+LT). The antifreeze activity was further verified by measuring the residual activity of catalase. The results are as follows: Fig.10 As shown. The three components of DY, LT, and DY+LT all showed high antifreeze activity, and the residual activity of catalase was 83.12%, 85.94%, and 93.33%, respectively. The DY+LT composite peptide has more polar amino acids and acidic amino acids. With the help of these amino acids, the polypeptide is more hydrophilic and forms more hydrogen bonds with water, which enhances the stability of water during freezing and reduces the formation of large ice crystals, thereby avoiding open chain denaturation caused by changes in the spatial conformation of the protein, improving its antifreeze performance, and further verifying the high antifreeze activity of the two peptides DY and LT. According to the mass spectrometry results, the two peptides obtained by biosynthesis are rich in amino acids that can play an antifreeze effect and have a suitable peptide length. Therefore, the amino acid sequences of the peptides with high antifreeze activity in the Antarctic krill antifreeze polypeptide are Asp-Ile-Asn-Phe-Arg-Tyr and Leu-Gly-Asp-Gly-Met-Ser-Leu-Ser-Thr.

[0100] Effect of antifreeze peptides on the survival rate of probiotics

[0101] Activation and subculture of Lactobacillus rhamnosus (BNCC275495): inject 0.3-0.5mL sterile water or liquid culture medium into the freeze-dried tube, and put it back into the liquid test tube (5mL MRS broth medium) after it is fully dissolved, and mix it; evenly apply the bacterial suspension to the surface of 2 agar plates (about 200μL / plate), spread it evenly, and place the liquid test tube and the plate in a constant temperature incubator at 37℃ and 5% CO2 for cultivation. Fig.11 Take the second generation bacterial suspension and streak it on the plate, as shown. Fig.12 shown.

[0102] Determine the growth curve of Lactobacillus rhamnosus: inoculate the activated second-generation bacterial suspension of Lactobacillus rhamnosus into MRS broth medium at a volume fraction of 1%, 2%, 5%, and 10%, draw 200 μL of the fermentation liquid into a 96-well plate, and measure its OD with an enzyme-labeled instrument 600 The culture medium without inoculation of strains was used as control, and OD 600 At a suitable concentration gradient between 0.1 and 0.2, 1 mL of fermentation liquid was placed in a growth curve plate and placed on a microbial growth measuring instrument. The culture was incubated at 37°C for 48 h and the OD of the culture medium was measured every 30 min. 600 And record, with OD 600 Characterize the maximum biomass of the strain. Each sample is set up 3 times in parallel. The horizontal axis is time, and the OD 600 As the ordinate, the growth curve of Lactobacillus rhamnosus at 37°C was obtained, as shown in Fig.13 As shown. From 0h to 4h, Lactobacillus rhamnosus grows slowly, which is the hysteresis period. From 4h to 15h, it grows rapidly, which is the logarithmic period. From 15h to 20h, the growth rate slows down and reaches the stable period at about 20h. After 20h of culture, Lactobacillus rhamnosus enters the stable period, and the growth state and physiological characteristics of the bacteria are relatively stable. Therefore, the bacteria are treated with different cryoprotectants at -20℃ for 24h, and the OD is measured after 20h of recovery culture. 600 Values, OD before and after freezing treatment 600 The ratio is the relative survival rate of Lactobacillus rhamnosus.

[0103] Cryoprotective effect of antifreeze peptides on probiotics: The bacterial suspension of Lactobacillus rhamnosus was subcultured at a ratio of 1:50. The bacteria were collected during the logarithmic growth period, centrifuged (8000 rpm, 10 min), washed twice with sterile saline, and then diluted with sterile saline to an OD of 0. 600The concentration of antifreeze peptides DY, LT and DY+LT was 1.0, and the diluted bacteria (0.1 mL) were transferred to a 2 mL centrifuge tube. 0.9 mL of antifreeze peptides DY, LT and DY+LT were added respectively, all at a concentration of 1 mg / mL. The same volume of normal saline was added as the negative control group. The positive control group included 1 mg / mL sucrose, 1 mg / mL skim milk and 20% glycerol (v / v). The centrifuge tubes with different antifreeze protectants were placed in a -20℃ refrigerator for 24 h. The frozen bacterial solution was thawed at 37℃ for 15 min, 0.2 mL of the bacterial solution was transferred and inoculated into a 3.8 mL MRS broth culture tube and cultured at 37℃ for 20 h. Fig.14 OD of Lactobacillus rhamnosus after 20 h of culture with and without freezing treatment 600 The ratio is the relative survival rate. Fig.15 It can be seen that the relative survival rates of Lactobacillus rhamnosus in the 20% glycerol group, DY group, LT group and DY+LT group were significantly higher than those in other groups, among which the DY+LT composite peptide had the strongest antifreeze protection ability, with a relative survival rate of 94.11%. This may be because the composite peptide increased the accumulation of amino acids in the culture medium, and by inhibiting the growth of ice crystals, reduced the osmotic pressure after freezing, protected the cell membrane, reduced protein leakage, and maintained the activity of Lactobacillus rhamnosus. This shows that the antifreeze peptide against Antarctic krill can alleviate low temperature stress during cold storage and prevent the decline of probiotic cell viability. And the low-temperature protection activity of the composite peptide is significantly better than that of DINFRY and LGDGMSLST. The above results show that the two peptides DINFRY and LGDGMSLST have the appropriate number of amino acids, structure, and peptide length, which meet the characteristics of antifreeze peptides, especially the higher proportion of hydrophobic amino acids and acidic amino acids, which gives the composite peptide DINFRY+LGDGMSLST the strongest ability to inhibit the growth of single ice crystals and recrystallization inhibition activity, which is conducive to the development and utilization of new antifreeze agents and functional products, and has great potential as a probiotic antifreeze protectant.

[0104] In summary, the Antarctic krill antifreeze peptide obtained by the method of the present invention has high antifreeze activity.

[0105] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. An antifreeze peptide, characterized in that: The amino acid sequences of the antifreeze peptides are DINFRY and LGDGMSLST.

2. An antifreeze agent, characterized in that: The invention comprises the antifreeze peptide according to claim 1.

3. The antifreeze agent according to claim 2, characterized in that: The mass ratio of DINFRY to LGDGMSLST is 1:

1.

4. The antifreeze agent according to any one of claims 2-3, characterized in that: Also contains food acceptable excipients.

5. Use of the antifreeze agent according to claim 4 in food freezing protection.

6. The use of the antifreeze agent in food freezing protection according to claim 5, characterized in that: Before freezing, the antifreeze agent according to claim 4 is added to the food.

7. The use of the antifreeze agent in food freezing protection according to claim 6, characterized in that: The food is at least one of dough, yogurt, minced fish and probiotics.

8. The use of the antifreeze agent in food freezing protection according to claim 7, characterized in that: The probiotics are at least one of yeast and Lactobacillus rhamnosus.

Images