Ice crystal recrystallization inhibiting peptide and application thereof

Abstract

The invention discloses an ice crystal recrystallization inhibitory peptide, the structure of which is as follows: X1-X2-Val-Thr-X3-X4-Met-X5, in the formula, X1 is Leu or Ile; x2 is Ile, Ser, Val or Thr; x3 is Gln or Asn; x4 is Thr, Val or Ser; and X5 is Lys or Arg. The invention also provides an application of the ice crystal recrystallization inhibiting peptide as an antifreeze peptide. The ice crystal recrystallization inhibitory peptide provided by the invention can be used as a food antifreeze additive or cryoprotectant, and the addition amount is only 2.0-5.0 mg / mL. The ice crystal recrystallization inhibitory peptide provided by the invention has strong ice crystal recrystallization inhibitory activity, the addition amount is about 10% of that of similar peptides reported in current literatures, and the product preparation method is simple, natural, safe and suitable for industrial application.

Description

Technical Field

[0001] The present invention relates to an ice crystal recrystallization inhibition peptide and its application. Background Art

[0002] The free energy of water molecules at the ice-water interface is relatively high. Therefore, the ice-water system spontaneously reduces the total free energy by decreasing the area of the ice-water interface. The macroscopic manifestation of this process is the ice crystal recrystallization phenomenon. Ice crystal recrystallization destroys the microstructure of frozen foods, induces protein denaturation and lipid oxidation, and causes frost damage and inactivation of yeasts and probiotics. It is an important reason for the deterioration of the quality of frozen foods during storage. For example, during the transportation and storage of frozen minced meat, the ice crystals inside it continuously grow through recrystallization. Ice crystal recrystallization will squeeze and damage the cell membrane structure, resulting in juice loss and texture degradation after thawing of frozen minced meat. In addition, the exuded intracellular substances contain free radicals and enzymes, which can induce lipid oxidation and protein denaturation, further reducing the sensory quality and shelf life of frozen minced meat. Larger ice crystals in ice cream will destroy the delicate texture of ice cream, resulting in a decline in the sensory quality of ice cream. Ice crystal recrystallization will exert mechanical stress on the yeasts in frozen dough, destroying the cell integrity of the yeasts, causing yeast death and reducing the wake-up property of the dough after thawing.

[0003] There is a class of antifreeze proteins in polar animals and plants that can efficiently inhibit ice crystal recrystallization. Although antifreeze proteins have extremely strong ice crystal recrystallization inhibition activity, they are expensive and still cannot be widely applied in the food industry. Therefore, finding safe, efficient, and inexpensive antifreeze protein mimics is a current research hotspot. In the chemical field, some artificially synthesized small molecules, polymers, and nanomaterials have been successively found to have ice crystal recrystallization inhibition activity. However, the safety of these materials remains to be verified and they cannot be directly used in foods.

[0004] The natural ice crystal recrystallization inhibition active materials that have been studied more in the food field are κ-carrageenan and antifreeze peptides. κ-carrageenan has strong IRI activity and can efficiently inhibit ice crystal recrystallization at a concentration of 1 mg / mL. However, κ-carrageenan forms a gel and loses its activity under the induction of trace amounts of Na+, K+, and Ca2+, and has poor compatibility with food components. The ice crystal recrystallization inhibition activity of antifreeze peptides is weaker than that of antifreeze proteins and carrageenan. Usually, a dosage of 10 - 40 mg / mL is required, which may affect the original physical and chemical properties of foods. In addition, the prior art has also used animal or plant-derived proteins for enzymatic hydrolysis and purification to prepare antifreeze polypeptides, but the activity of the proteins obtained by enzymatic hydrolysis is low and the composition is unclear. The purification steps of the enzymatic hydrolysis products are cumbersome, with high costs and low yields.

[0005] In summary, there is still a need to develop food-grade IRI active materials that are inexpensive, have strong IRI activity, and good compatibility with food components. Summary of the Invention

[0006] The object of the present invention is to provide an ice crystal recrystallization inhibiting peptide Leu-Ile-Val-Thr-Gln-Thr-Met-Lys derived from whey protein isolate hydrolysate and other derivatives with strong ice crystal recrystallization activity. The amino acid groups of the following peptides fit the two crystal planes of ice crystals {0001} and {1010} in terms of spatial arrangement, so ice crystal recrystallization can be inhibited through the "adsorption-inhibition" mechanism. In the standard drop splashing method experiment, the following peptides can completely inhibit ice crystal recrystallization at the milligram / milliliter (mM) concentration level.

[0007] The technical solution adopted by the present invention is as follows: An ice crystal recrystallization inhibiting peptide, the basic structure of which is: X1-X2-Val-Thr-X3-X4-Met-X5 Wherein: X1 is Leu or Ile; X2 is Ile, Ser, Val or Thr, preferably Ile; X3 is Gln or Asn, preferably Gln X4 is Thr, Val or Ser, preferably Thr; X5 is Lys or Arg, preferably Lys; The N-terminus of X1 can be acetylated or without special treatment; The C-terminus of X5 can be amidated or without special treatment.

[0008] Furthermore, preferably, the sequence of the ice crystal recrystallization inhibiting peptide is Leu-Ile-Val-Thr-Gln-Thr-Met-Lys or Ile-Ile-Val-Thr-Gln-Thr-Met-Lys.

[0009] The ice crystal recrystallization inhibiting peptide provided by the present invention can completely inhibit ice crystal recrystallization in sucrose solution and physiological saline at an addition concentration as low as 2.0 - 5.0 mg / mL.

[0010] The ice crystal recrystallization inhibiting peptide provided by the present invention can be prepared by solid-phase polypeptide synthesis method.

[0011] The present invention also provides the application of the ice crystal recrystallization inhibiting peptide as an antifreeze peptide.

[0012] The present invention also provides the application of the ice crystal recrystallization inhibiting peptide as a food antifreeze additive or cryoprotectant. When the ice crystal recrystallization inhibiting peptide is used as a food antifreeze additive or cryoprotectant, the addition amount is 1.0 - 5.0 mg / mL, preferably 2.5 - 5.0 mg / L, more preferably 3.0 - 5.0 mg / mL.

[0013] The present invention also provides a food anti-freezing additive, which comprises the ice crystal recrystallization inhibition peptide of the present invention.

[0014] The present invention also provides a cryoprotectant for cells and microorganisms, which comprises the ice crystal recrystallization inhibition peptide of the present invention.

[0015] The beneficial effects of the present invention are as follows: (1) The ice crystal recrystallization inhibition peptide provided by the present invention has strong ice crystal recrystallization inhibition activity, and the addition amount is about 10% of the similar peptides reported in the current literature.

[0016] (2) The above-mentioned ice crystal recrystallization inhibition peptide has a small addition amount, is colorless and odorless within the active concentration range, and the solution viscosity is almost similar to that of pure water, which is suitable for being used as a food additive and does not affect the original physical and chemical qualities of the food.

[0017] (3) After the above-mentioned polypeptide is added to the cryoprotectant for cells and microorganisms, the peptide in the supernatant can be removed by simple centrifugation after thawing, without affecting the subsequent resuscitation and culture of cells and microorganisms.

[0018] (4) Among them, the two peptides Leu-Ile-Val-Thr-Gln-Thr-Met-Lys and Ile-Ile-Val-Thr-Gln-Thr-Met-Lys are derived from the trypsin hydrolysates of milk and sheep milk proteins, are natural and safe, and the pure products can be mass-produced by solid-phase synthesis method, which is suitable for industrial application. Description of the Drawings

[0019] Figure 1 It is an optical microscope photograph of ice crystal recrystallization phenomenon.

[0020] Figure 2 Artificially synthesized ice crystal recrystallization inhibition active materials, which are small molecules, polymers and nanomaterials from left to right.

[0021] Figure 3 Chromatogram of crude peptide purified by HPLC.

[0022] Figure 4 Mass spectrum of pure peptide.

[0023] Figure 5 Photographs of ice crystal sizes of sucrose solution without ice crystal recrystallization inhibition peptide and sucrose solution added with 3.0 mg / mL ice crystal recrystallization inhibition peptide after storage at -8°C for a certain period of time.

[0024] Figure 6 Quantitative analysis results of the inhibition of ice crystal sizes by peptides with different concentrations.

[0025] Figure 7Comparison chart of ice crystal sizes of ice cream with and without added peptides after storage at -8°C for 30 min and 60 min.

[0026] Figure 8 Photos of the inhibition of ice crystal size in sucrose solution by ice crystal recrystallization inhibition peptides with different amino acid sequences. Detailed implementation manners

[0027] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto.

[0028] Optical microscope photos of ice crystal recrystallization phenomenon are as Figure 1 shown, and it can be seen that the internal ice crystals continuously grow by recrystallization.

[0029] Synthetic ice crystal recrystallization inhibitory active materials are as Figure 2 shown, from left to right are small molecules, polymers and nanomaterials respectively. However, the safety of such materials needs to be further verified and cannot be directly used in frozen foods.

[0030] Example 1 The preparation method and activity determination of ice crystal recrystallization inhibition peptides are as follows: Taking LIVTQTMK-NH2 as an example, the solid-phase synthesis method can be divided into three main steps: resin treatment, amino acid coupling reaction and trifluoroacetic acid cleavage, which are detailed as follows: (1) Resin treatment 1.1 Resin swelling Select Rink Amide-AM Resin (molar substitution coefficient is 0.65 mmol / g) as the starting resin and add it to a 1000 mL reaction column, add DCM to soak, drain, and complete the swelling of the resin.

[0031] 1.2 Resin deprotection Add a DMF solution containing 20% piperidine, stir for 30 minutes while passing N2, filter dry the solvent; then wash the resin with DMF 6 times and drain to complete the deprotection of the resin.

[0032] (2) Amino acid coupling reaction Reaction monitoring: The reaction process is monitored by the ninhydrin method.

[0033] The raw materials and reagents used are shown in Table 1 below: The specific operation process is as follows: Weigh the corresponding amounts of TBTU and the protected amino acid into a beaker, add DMF to dissolve; then add the reaction solution to the resin, add DIEA, and introduce N2 to blow for about 90 minutes, and detect by ninhydrin reaction. After the reaction is completed, remove the solvent and wash the resin with DMF 3 times. Then add a DMF solution of 20% piperidine to the resin, introduce N2 and continue to blow for 30 minutes, then remove the solvent and wash the resin with DMF 6 times to complete the coupling of this amino acid.

[0034] Repeat the above reaction procedure until the condensation reaction of all protected amino acids is completed.

[0035] Condensing agent (10 mmol feeding amount): TBTU: 12.85 g, DIEA: 25 mL The protected amino acids used in this step are shown in Table 2 below: After coupling to the last amino acid is completed, contract the polypeptide, wash it 3 times in sequence with DMF / DCM / methanol, weigh it after drying by suction. (3)Trifluoroacetic acid cleavage (dissociate the polypeptide from the resin and remove the protecting groups on the amino acid side chains) Add the resin to the pre-prepared cleavage solution (86% trifluoroacetic acid, 5% ethylenediaminetetraacetic acid, 5% benzyl methyl sulfide, 3% phenol / 2% pure water), and stir for 150 min. Then separate the resin and the cleavage solution, add ether to fully precipitate the polypeptide. Filter the polypeptide with a Buchner funnel and wash it thoroughly with ether 6 times to obtain the crude peptide. Dissolve the crude peptide in 50% aqueous acetonitrile solution, and purify the crude product by high performance liquid chromatography. Put the collected pure product solution in an eggplant-shaped flask, remove acetonitrile with a rotary evaporator, and freeze-dry for 2 days to obtain the powder pure product.

[0036] The HPLC chromatogram is as Figure 3 shown, from Figure 3 it can be seen that for the purified peptide, its purity is greater than 95%, and from Figure 4 the mass spectrum, the relative molecular weight of the peptide can be obtained as 931 Da.

[0037] Measure the ice crystal recrystallization inhibition activity of the peptide prepared in step (3). The specific steps are as follows: Dissolve the peptide at 3.0 mg / mL in a sucrose solution (5 - 40%) or a phosphate buffer at pH 7.4. Place a glass slide in a dry ice bath and pre-cool it for 30 minutes. Using a medical syringe, drop a sample (about 10 µL) from a height of 1.4 m onto the glass slide pre-cooled with dry ice. At the moment of hitting the glass slide, the sample will form a thin ice sheet with a diameter of about 1 cm and a thickness of about 10 µm. Then place the glass slide in a freezing stage pre-set at -8°C and incubate for a certain period of time. Take pictures every 2 minutes using a polarized light microscope to record the process of ice crystal recrystallization growth. Use Image J software to count the area of ice crystals in the obtained pictures.

[0038] Carry out experiments on peptides with different concentrations according to the above method for quantitative analysis. Detect the inhibitory degree of peptides with different concentrations on ice crystals.

[0039] % Ice crystal size = sample ice crystal size / ice crystal size in the sucrose solution of the same concentration without polypeptide.

[0040] Take the example of the polypeptide dispersed in a 7.2% sucrose solution. The obtained results are as Figure 5 、 6 shown, Figure 5 are the pictures of ice crystal size after storing for a certain period of time at -8°C for the sucrose solution without adding ice crystal recrystallization inhibitory peptide and the sucrose solution adding 3.0 mg / mL ice crystal recrystallization inhibitory peptide. Among them, Figure A is the picture of ice crystal size after storing for 0.5 hour at -8°C for the sucrose solution without adding ice crystal recrystallization inhibitory peptide, Figure B is the picture of ice crystal size after storing for 0.5 hour at -8°C for the sucrose solution adding 3.0 mg / mL ice crystal recrystallization inhibitory peptide; Figure C is the picture of ice crystal size after storing for 24 hours at -8°C for the sucrose solution without adding ice crystal recrystallization inhibitory peptide; Figure D is the picture of ice crystal size after storing for 24 hours at -8°C for the sucrose solution adding 3.0 mg / mL ice crystal recrystallization inhibitory peptide. Figure 5 It can be seen that the ice crystal area in the sucrose solution without adding inhibitor increases significantly after freezing storage for 24 hours, while the ice crystals adding 3.0 mg / mL ice crystal recrystallization inhibitory peptide are very tiny and no obvious increase is seen after long-term storage.

[0041] Add 3.0 mg / mL ice crystal recrystallization inhibitory peptide into sucrose solutions with different concentrations of 5 - 40%. The results show that there is ice crystal recrystallization inhibitory activity in all cases. The sucrose content of 5 - 40% covers all frozen desserts on the market. Therefore, the ice crystal recrystallization inhibitory peptide of the present invention can be applied to various frozen desserts.

[0042] The results of the quantitative analysis of the inhibitory situation of peptides with different concentrations on ice crystal size (incubated at -8°C for 30 minutes) are as Figure 6 shown, Figure 6It can be seen that when the addition amount of the peptide is 2.5 mg / mL, the ice crystal size can be reduced to 30% of that of the control group's pure sucrose solution, and when the addition amount is 3.0 mg / mL, the ice crystal size can be reduced to 5% of that of the control group's pure sucrose solution.

[0043] Example 2 The application of the ice crystal recrystallization inhibition peptide in ice cream is as follows: (1) Disperse the following ice cream raw materials into water and shear and stir evenly: 12% milk fat; 10% non-fat milk powder; 16% sucrose; 0.1% emulsifier; 0.3% polysaccharide, 0.5% ice crystal recrystallization inhibition peptide, and the balance is water.

[0044] (2) Sterilize the raw materials in (1) and then use an ice cream maker to freeze them into ice cream, and store them at -8°C.

[0045] After keeping the ice cream with the added ice crystal recrystallization inhibition peptide and the ice cream without the added ice crystal recrystallization inhibition peptide at -8°C for 30 min and 60 min, the photos of the ice crystal size are as Figure 7 shown Figure 7 The upper row is the comparison chart of the ice crystal size of the ice cream with and without the added peptide after storage at -8°C for 30 min, Figure 7 and the lower row is the comparison chart of the ice crystal size of the ice cream with and without the added peptide after storage at -8°C for 60 min.

[0046] Figure 7 It can be seen that the ice crystals in the pure ice cream increase significantly after long-term refrigeration, while the ice crystal size of the ice cream added with the ice crystal recrystallization inhibition peptide is only 7-8% of that of the pure ice cream. Therefore, the ice crystal recrystallization inhibition peptide (5.0 mg / mL) can significantly slow down the growth of ice crystal recrystallization.

[0047] Example 3 The application of the ice crystal recrystallization inhibition peptide in frozen meat paste is as follows: (1) Mix 87.7% fresh raw meat, 12% ice water, and 0.3% ice crystal recrystallization inhibition peptide, and chop and stir evenly to make meat paste.

[0048] (2) Store the meat paste prepared in (1) at -18°C.

[0049] Example 4 The application of the ice crystal recrystallization inhibition peptide in cell cryopreservation is as follows: Collect the cells or microorganisms after expansion culture, and inoculate them into the corresponding culture medium at a density of 10 3-5 / mL.

[0050] Add 0.3-1.0% ice crystal recrystallization inhibition peptide to (1), and then dispense it into cryopreservation tubes.

[0051] Place the cryopreservation tube in a programmable freezing container and place the programmable freezing container in an -80 °C refrigerator. After the temperature drops to -80 °C, transfer the cryopreservation tube to liquid nitrogen for long-term storage.

[0052] Example 5 Polypeptides with the following sequences were synthesized according to the method of Example 1 (-CONH2 in the sequence represents C-terminal amidation treatment, and the absence of -CONH2 represents no special treatment at the C-terminus): IIVTQTMK, LSVTQTMK-CONH2, LTVTQTMK-CONH2, LVVTQTMK-CONH2, LTVTQSMK-CONH2, LIVTQVMK-CONH2, LIVTQSMK-CONH2, LIVTNTMK-CONH2, LSVTQTMK-CONH2, with a purity greater than 95%.

[0053] In a 7.2% sucrose solution, 4.0 mg / mL of the above ice crystal recrystallization inhibitory peptide was added respectively, and incubated at -8 °C for 30 minutes. The size of % ice crystals could be maintained below 20% of the control 7.2% sucrose solution, as Figure 8 shown.

Claims

1. An ice crystal recrystallization inhibiting peptide, characterized in that The structure is as follows: X1-X2-Val-Thr-X3-X4-Met-X5 Wherein: X1 is Leu or Ile; X2 is Ile, Ser, Val or Thr; X3 is Gln or Asn X4 is Thr, Val or Ser X5 is Lys or Arg.

2. The ice crystal recrystallization inhibiting peptide according to claim 1, wherein X1 is Leu.

3. The ice crystal recrystallization inhibition peptide according to claim 1, characterized in that X2 is Ile.

4. The ice crystal recrystallization inhibiting peptide according to claim 1, characterized in that X3 is Gln; X4 is Thr; X5 is Lys.

5. The ice crystal recrystallization inhibiting peptide according to claim 1, wherein The sequence of the ice crystal recrystallization inhibition peptide is Leu-Ile-Val-Thr-Gln-Thr-Met-Lys or Ile-Ile-Val-Thr-Gln-Thr-Met-Lys.

6. Use of the ice crystal recrystallization inhibition peptide according to any one of claims 1 to 5 as an antifreeze peptide.

7. Use of the ice crystal recrystallization inhibition peptide according to any one of claims 1 to 5 as a food antifreeze additive or cryoprotectant.

8. The application according to claim 7, wherein When the ice crystal recrystallization inhibition peptide is used as a food antifreeze additive or cryoprotectant, the addition amount is 2 to 5 mg / mL.

9. A food antifreeze additive, comprising the ice crystal recrystallization inhibition peptide according to any one of claims 1 to 5.

10. A cryoprotectant for cells and microorganisms, comprising the ice crystal recrystallization inhibition peptide according to any one of claims 1 to 5.

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