A sarcoplasmic anti-freezing and anti-oxidation peptide, a preparation method thereof and application thereof in frozen food

Abstract

The application discloses a muscle plasma anti-freezing and anti-oxidation peptide, a preparation method thereof and application of the peptide in frozen food, and has the following characteristics: the amino acid sequence of the peptide is as follows: KELASQPDVDGFLVGGASLKPEFVDIINAK; the preparation method comprises the following steps: collecting muscle plasma juice lost in a thawing process of frozen meat, collecting permeate by using a 10 kDa filter membrane, high-pressure homogenizing, and then ultrafiltering to obtain small-molecule peptide components with a size less than 3 kDa; loading the concentrated liquid on a Sephadex G-25 column to perform gel filtration chromatography separation, collecting each elution peak component, concentrating, desalting, and then freeze-drying, and collecting a component with the highest anti-freezing activity of Streptococcus thermophilus; performing mass spectrometry on the component with the highest anti-freezing activity, and identifying the peptide segment with high anti-freezing and anti-oxidation activity by LC-MS / MS; and the peptide segment has good anti-freezing and anti-oxidation properties, and can significantly improve the texture and water holding capacity of frozen food in a frozen storage process.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioactive peptides, and particularly relates to a sarcoplasmic anti-freezing and anti-oxidation peptide, a preparation method thereof and application thereof in frozen food. BACKGROUND

[0002] Low-temperature freezing is one of the important preservation methods widely used in the storage and processing of livestock and poultry meat, aquatic products, fruits and vegetables and other food. However, during freezing or frozen storage, ice crystal structures of different sizes are easily formed inside the food tissue, which further destroys the three-dimensional structure of proteins and cell integrity, causes protein and fat oxidation, leads to juice loss, texture deterioration and nutritional value decline, and seriously affects the sensory quality of frozen food. Therefore, developing efficient, safe and edible anti-freezing and anti-oxidation quality ingredients has become a key bottleneck that needs to be solved in the frozen food industry.

[0003] In recent years, food-derived anti-freezing peptides have attracted much attention due to their characteristics of efficiently inhibiting ice crystal growth and protecting protein structure.

[0004] Note. Anti-freezing peptides mainly regulate ice crystal nucleation and growth process, adjust ice crystal morphology, slow down ice crystal recrystallization behavior, and at the same time interact with proteins and water. Anti-oxidation peptides can scavenge free radicals in the frozen food system, protect the structural stability of key biological macromolecules during frozen storage, and delay the quality deterioration induced by freezing. Existing methods mainly use whey, fish skin, gelatin, insect protein and other materials to produce single anti-freezing peptides, which have problems such as low raw material yield, unknown peptide segment structure characteristics, unstable anti-freezing activity, and no anti-oxidation ability.

[0005] Livestock and poultry meat often releases a large amount of sarcoplasm during thawing or cold chain storage, which is rich in small molecular active protein components such as sarcoplasmic proteins and myosin degradation products. According to statistical data, the total output of livestock and poultry meat in China is about 97.7 million tons, of which frozen meat products account for about 20%. Therefore, it is estimated that the annual output of frozen meat is about 19.54 million tons. According to the calculation of 8% of the sarcoplasm released during thawing, about 1.56 million tons of thawed sarcoplasm is produced, which is usually regarded as a waste by-product and discarded directly, causing resource waste and environmental burden. Therefore, there is still great development potential for developing safe, green and economic anti-freezing and anti-oxidation peptides from livestock by-product sarcoplasm. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a sarcoplasmic anti-freezing and anti-oxidation peptide with good anti-freezing and anti-oxidation properties, a preparation method thereof and application thereof in frozen food, which can significantly improve the texture and water holding capacity of frozen food during frozen storage.

[0007] The application solves the above technical problems by adopting the technical scheme of a sarcoplasm anti-freezing and anti-oxidation peptide, the amino acid sequence of which is as follows: KELASQPDVDGFLVGGASLKPEFVDIINAK.

[0008] The application further provides a preparation method of the above-mentioned sarcoplasm anti-freezing and anti-oxidation peptide, comprising the following steps:

[0009] Step 1: Collect the sarcoplasm juice lost in the thawing process of frozen meat in a temperature and humidity controlled environment, the collection time lasts for 24-36 hours, the sarcoplasm juice is preliminarily filtered through a 200-400 mesh precision stainless steel screen to remove insoluble tissue fragments and large particle precipitates, then the micro-fine particles and primary lipid impurities are further removed by low-temperature high-speed centrifugal separation, the permeate is collected by using a 10 kDa filter membrane for ultrafiltration;

[0010] Step 2: After the permeate obtained in Step 1 is subjected to high-pressure homogenization, the permeate is subjected to secondary ultrafiltration by using an ultrafiltration membrane to obtain a small-molecule peptide component with a molecular weight less than 3 kDa, and the peptide concentration is concentrated to 80-100 mg / mL by using a vacuum low-temperature concentration device to obtain a concentrated solution;

[0011] Step 3: The concentrated solution obtained in Step 2 is loaded on a Sephadex G-25 column for gel filtration chromatography separation, each elution peak component is collected, desalted and freeze-dried, and the component with the highest anti-freezing activity of Streptococcus thermophilus is collected;

[0012] Step 4: The component with the highest anti-freezing activity of Streptococcus thermophilus in Step 3 is dissolved in 40-60 mM NH4HCO3, DTT solution is added to make the final concentration 5-20 mmol / L, reduction is carried out at 45-60 ℃ in a water bath for 0.5-2 hours, iodacetamide solution is added to make the final concentration 50-60 mmol / L, desalination is carried out after dark reaction for 30-30 minutes, and the solvent is dried in a vacuum centrifugal concentrator at 40-50 ℃; after being dissolved in 0.1% formic acid aqueous solution, mass spectrometry is carried out, LC-MS / MS identification is carried out, one peptide segment with high anti-freezing and anti-oxidation activity is obtained, and the sequence of the peptide segment is KELASQPDVDGFLVGGASLKPEFVDIINAK.

[0013] Further, the parameters of the temperature and humidity controlled environment in Step 1 are 6-15°C and 85-95% relative humidity; the parameters of the low-temperature high-speed centrifugal separation are 6000-10000 x g and 4°C; and the parameters of the ultrafiltration membrane system are an operating pressure of 0.1-0.3 MPa and a temperature of 4-10 ℃.

[0014] Further, the gel filtration chromatography separation condition in step 3 is as follows: the mobile phase is 18-25 mM phosphate buffer, pH 6.8, the flow rate is 0.5-1.0 mL / min, the detection wavelength is 220 nm, the components are collected according to the elution peak shape, and after concentration and desalination, the components with the highest anti-freezing activity of Streptococcus thermophilus are collected and freeze-dried.

[0015] Further, the mass spectrometry detection condition in step 4 is as follows: 100 μm i.d. x 180 mm, packing: Reprosil-Pur 120 C18-AQ 3 μm analysis column; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid and 80% acetonitrile in water; flow rate: 550-620 nL / min; analysis time of each component: 60 min.

[0016] Further, the muscle sarcoplasm anti-freezing and antioxidant peptide is added in a mass concentration of 1-3%.

[0017] Further, the muscle sarcoplasm anti-freezing and antioxidant peptide is added in a mass concentration of 1-3%.

[0018] The application also provides the use of the above-mentioned muscle sarcoplasm anti-freezing and antioxidant peptide in the preparation of a frozen storage protective agent for pork gel products, and the muscle sarcoplasm anti-freezing and antioxidant peptide is added in a mass concentration of 1-3%.

[0019] Compared with the prior art, the application has the advantages that: the muscle sarcoplasm anti-freezing and antioxidant peptide, the preparation method thereof and the application thereof in frozen food have the following advantages: the thawed muscle sarcoplasm of frozen meat is used as the raw material for preparing the anti-freezing and antioxidant peptide, the raw material source is sufficient, and the cost is low; in addition, the step-by-step enrichment and purification of the peptide segment are realized through multi-stage ultrafiltration grading and gel chromatography, a higher-purity target anti-freezing peptide can be obtained, and the effect and stability of downstream application are improved; the prepared peptide segment KELASQPDVDGFLVGGASLKPEFVDIINAK is rich in hydrophilic polar residues (such as lysine K, glutamic acid E, aspartic acid D, glutamine Q, serine S, etc.) and hydrophobic flexible residues (such as glycine G, alanine A, proline P, valine V, etc.), and has good anti-freezing and antioxidant properties, which significantly improves the texture and water holding capacity of frozen food during frozen storage, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The Fourier infrared spectra of muscle sarcoplasm components with different molecular weights;

[0021] Figure 2 The ice crystal inhibition activity results of muscle sarcoplasm components with different molecular weights, the left side is >10 kDa, and the right side is <10 kDa. DETAILED DESCRIPTION

[0022] The application will be further described in detail below with reference to the examples of the drawings.

[0023] I. Experimental methods

[0024] Fourier infrared spectrum

[0025] The sample was characterized by 64 scans in the range of 400-4000 cm -1 , with a resolution of 4 cm -1 , and the baseline correction was achieved by subtracting the blank ATR spectrum.

[0026] Thermal hysteresis activity and ice crystal content

[0027] The thermal hysteresis activity and ice crystal content were determined using a differential scanning calorimeter. The sample (5 μL, 20 mg / mL) was sealed in a pre-weighed aluminum pan, cooled to -25 ℃ (4 ℃ / min), and held for 5 min. The temperature was increased at 1 ℃ / min until the sample was in equilibrium between ice crystals and aqueous solution, at which time the temperature was the holding temperature (T h ). The temperature was increased to the holding temperature (T h ) and held for 5 min, and then cooled again to -25 ℃ (1 ℃ / min). The onset temperature of sample crystallization (T0) was recorded. The thermal hysteresis activity (THA), ice crystal content (Φ) were calculated by the following formula:

[0028] Thermal hysteresis activity (THA, ℃) = T h -T0, Φ (%) = (1-ΔHf / ΔHm) × 100, where ΔHf represents the sample exothermic enthalpy, J / g. ΔHm represents the sample melting enthalpy, J / g.

[0029] Ice crystal inhibition activity determination

[0030] The ice crystal inhibition activity determination was observed by polarized light microscopy, the sample was dissolved in a 200 mg / mL sucrose solution to prepare a 10 mg / mL sample solution, 2 μL was taken and titrated on a glass slide, and covered with a glass cover slip, and placed in a refrigerator, quickly frozen to -50 ℃ (20 ℃ / min) and held for 1 min, then the temperature was increased to -22 ℃ (10 ℃ / min) and held for 5 min, and the ice crystal image was recorded.

[0031] Determination of total antioxidant capacity

[0032] The total antioxidant capacity of the enzyme solution was detected using a total antioxidant capacity rapid detection kit. Take 20 uL of peroxidase, dilute 10 times with detection buffer, and configure 200 uL of peroxidase working solution. Dilute the 10 mM Trolox standard solution to 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5 mM, add the configured peroxidase working solution 20 uL to the enzyme detection hole, add deionized water 10 uL to the blank control group of enzyme detection hole, and add 10 uL of various concentrations of Trolox standard solution to the standard curve detection hole, and then add the prepared enzyme extract 10 uL to the sample detection hole, then add 170 UL of A BTS working solution to each hole, and mix gently. Place at room temperature, avoid light and stand for 6 min, then place it in the enzyme marker, measure the absorbance at 414 nm. ΔA414=blank control absorbance-sample absorbance, use Δ414 and Tro10x standard solution concentration to make a standard curve graph. According to the standard curve formula, the total antioxidant capacity of the sample is calculated, and the molar concentration of Trolox standard is used to represent it.

[0033] Hydroxyl radical scavenging rate determination

[0034] In 7 colorimetric tubes, 9 mmol / L ferrous sulfate and 9 mmo / L salicylic acid-ethanol solution were added in turn, each 2.0 mL, then 1.0 mL of ASP solution with different mass concentrations was added, followed by 8.8 mmo / L hydrogen peroxide 2.0 mL, and then the volume was made up to 10 mL with ultrapure water, mixed well, reacted in a water bath at 37℃ for 30 min, and then the absorbance value at wavelength 510 nm was measured and recorded as Ax. The absorbance value of the ASP solution without hydrogen peroxide was recorded as Ax0, and the absorbance value of the blank without ASP solution was recorded as A0. The formula for calculating the ·OH scavenging rate of ASP is: ·OH scavenging rate (%) = [A0-(Ax-Ax0)] / A0x100.

[0035] Thawing loss

[0036] After thawing the meat chunks, wipe off the surface moisture and weigh again. Thawing loss (TL): TL (%) = (M0-M1) / M0x100%, where: M0-the mass of the meat before freezing (g); M1-the mass of the meat after thawing (g).

[0037] Cooking loss

[0038] The thawed meat paste was heated in a water bath at 85℃ for 30 min. Cooking loss (CL): CL (%) = (M1-M2) / M1x100%, where: M1-the mass of the meat before cooking (g), M2-the mass of the meat after cooking (g).

[0039] Gel strength, firmness, chewiness determination

[0040] The texture parameters of the gels were measured using a texture analyser equipped with a cylindrical probe (TA / 20). The TPA mode was chosen with a deformation level of 50 % of the sample height. The pre-test speed, test speed and post-test speed were set to 2.00 mm / s, 0.80 mm / s and 0.80 mm / s, respectively.

[0041] The gel strength of the samples was analysed using a TA / 0.5 probe with a pre-test speed, test speed and post-test speed of 1.5 mm / s, 1 mm / s and 1 mm / s, respectively, with a compression deformation of 50 % and a trigger force of 5 g. The gel strength was expressed as breaking strength x breaking distance.

[0042] Surface hydrophobicity

[0043] Surface hydrophobicity determination: 1 mL of protein solution (2 mg / mL) was taken and 80 μL of a Bromophenol Blue (BPB) aqueous solution (1 mg / mL) was added. After 10 min of reaction at room temperature, the supernatant was collected by centrifugation at 10 000 g for 15 min. The supernatant was taken and diluted 10-fold with 50 mmol / L phosphate buffer (pH 7.0) and the absorbance of the protein sample at 595 nm was measured. The results were expressed as μg of BPB / mg of protein.

[0044] Particle size

[0045] The myofibrillar protein concentration was adjusted to 1 mg / mL and the particle size of the protein was determined using dynamic light scattering. The detection parameters were set as follows: output wavelength 633 nm, 173° detection angle backscattering calibration, detection temperature 25 °C.

[0046] 11. Dissociation rate of myosin

[0047] Myosin extraction procedure: pork homogenate was dissolved in three volumes of solution A (0.1 M KCl, 20 mM potassium phosphate, 1 mM DTT, 2 mM EDTA Na2, pH 7.0), then homogenized at 10,000 rpm for 30 seconds, followed by centrifugation at 8,000 g for 10 minutes. The precipitate was collected, an equal volume of solution B (0.3 M KCl, 0.1 M KH2PO4, 50 mM K2HPO4, 1 mM EDTA Na2, 4 mM sodium pyrophosphate, pH 6.5) was added, stirred for 15 minutes, then centrifuged at 10,000 g for 12 minutes, the supernatant was collected and diluted with 14 volumes of 1 mM EDTA Na2, left to stand for 2.5 hours. The precipitate was then collected and centrifuged at 10,000 g for 6 minutes. The resulting precipitate was dissolved in 0.6 M phosphate buffer, and 5 mM MgCl2-6H2O, 5 mM Na4P2O7-10H2O, 2 mM ATP Na2 were added, after stirring for 30 minutes, centrifuged at 10,000 g for 20 minutes, the supernatant was diluted with 9 volumes of distilled water, left to stand for 12 hours, and the precipitated protein was treated according to the above procedure. Finally, the obtained solution was dialyzed in 0.6 M KCl buffer (pH 6.5). The protein concentration was determined using a BCA kit.

[0048] Method for determining the dissociation rate of crude myosin: the protein concentration in the protein homogenate obtained by adding buffer B and the supernatant of crude myosin obtained after centrifugation in the above myosin extraction process was determined using a BCA kit. The dissociation effect of crude myosin was evaluated by the solubility rate of crude myosin.

[0049] 12. Determination of protein carbonyl

[0050] Take 2 mg / mL myofibrillar protein solution, add an equal volume of 10 mM DNPH-hydrochloric acid solution, react in the dark for 1 h, then add trichloroacetic acid solution (10 %, w / v) to terminate the reaction, centrifuge at 10,000 g for 5 min, add ethanol / ethyl acetate (1:1, v / v) to the precipitate and repeat the washing three times, centrifuge at 10,000 g for 5 min, then dissolve the precipitate in 6 M guanidine hydrochloride-hydrochloric acid, react at 37 °C for 15 min, after the temperature drops to room temperature, measure the absorbance value (A370) of the solution at 370 nm, and the carbonyl content is expressed as nmol / mg protein.

[0051] 13. Determination of protein free sulfhydryl groups:

[0052] The protein sample was diluted to 1 mg / mL, 0.5 mL of the protein sample was accurately weighed, 4.5 mL of 8 M urea-containing phosphate buffer (0.1 mol / L K2HPO4, 0.01 mol / L EDTA, pH 6.0) was added, 100 μL of 0.01 mol / L DTNB-containing buffer (0.01 mol / L KH2PO4, pH 6.0) was added, and the mixture was shaken and mixed, then stood for 30 min, and the absorbance was measured at 412 nm. Each gradient was repeated 3 times. The concentration of sulfhydryl group was calculated according to the absorption coefficient 13600 mol -1 cm -1 .

[0053] 14. Determination of thiobarbituric acid reaction value (TBARS): according to GB 5009.181-2016 "National food safety standard for determination of malondialdehyde in food", spectrophotometry was used, and the result was expressed as mg / kg.

[0054] 15. Determination of peroxide value (POV): according to the colorimetric method in GB / T 5009.37-2003 "Analysis method for edible vegetable oil hygiene standard", the POV in the sample was determined, and the result was expressed as mmol / kg. II. Specific embodiments

[0056] A preparation of sarcoplasmic antifreeze, antioxidant peptide segment, comprising the following steps:

[0057] Step 1, a preparation method of a low molecular weight antifreeze, antioxidant sarcoplasmic component:

[0058] (1) Collect the sarcoplasm lost during the thawing process of frozen pig hind legs, and realize continuous drop filtration to collect the sarcoplasm in a temperature and humidity controlled environment (0~4°C, relative humidity 85~95%), and the collection time lasts for 36 h. The preliminary filtration is carried out through a 200 mesh precision stainless steel screen to remove insoluble tissue fragments and large particle precipitates; combined with low temperature high speed centrifugal separation technology (8000 × g, 4°C), further remove fine particles and primary lipid impurities, and ensure the liquid to be clear and stable.

[0059] (2) Ultrafiltration is carried out by using 10 kDa filter membrane, and the ultrafiltration membrane system (operating pressure 0.1 MPa, temperature control at 6°C) is used to collect the permeate (<10 kDa) and the retention liquid (>10 kDa) respectively. -1 attributed to the stretching vibration of protein N-H and O-H groups, covering amide A and amide B bands, wherein amide A (3500~3250 cm -1 ) is related to the stretching vibration of O-H bond.

[0060] As shown in Fig. 4, the main hydrogen bond absorption peaks of the thawed myoplasm after 10 kDa ultrafiltration treatment were located at 3413 cm Figure 1 and 3254 cm -1 , which belonged to the category of free hydrogen bonds, and the permeate component as a whole had greater absorption peak intensity, indicating that the strength of intramolecular / intermolecular hydrogen bonds was greater. The low molecular weight permeate component showed significant changes in the C-O-C glycosidic bond characteristic vibration region (1180-930 cm -1 ), which may be related to the formation of β-glycosidic bonds triggered by the decrease in molecular weight caused by ultrafiltration treatment, further indicating that the permeate (<10 kDa) component has higher antifreeze activity. -1

[0061] As can be seen from Fig. 5, the sample of the retained liquid (>10 kDa, left side) formed larger ice crystal structures during the low-temperature freezing process, while the sample of the permeate (<10 kDa, right side) formed significantly smaller ice crystals, indicating that there are significant differences in ice crystal recrystallization inhibition activity between the two. The low molecular weight permeate sample showed stronger inhibition of recrystallization, which may be due to the enrichment of low molecular weight peptides and other hydrophilic components in it. These substances have higher diffusivity and interfacial activity, and can be adsorbed on the surface of ice crystals during the formation of ice crystals, effectively preventing the growth and merging of ice crystals, thereby inhibiting the recrystallization phenomenon. Figure 2 Thermal hysteresis activity refers to the phenomenon that certain substances can significantly lower the freezing point of a solution below the melting point by adsorbing on the surface of ice crystals to inhibit their growth. This temperature difference (the difference between the freezing point and the melting point) can be used to quantify the antifreeze ability of the substance.

[0062]

[0063] Table 1 Thermal hysteresis activity and ice crystal content of myoplasm components of different molecular weights

[0064]

[0065] As can be seen from Table 1, compared with the retained liquid (>10 kDa) component, the thermal hysteresis activity (THA) of the permeate (<10 kDa) sample was 10.02% higher, and the ice crystal content was lower, with a significant effect on inhibiting the formation of ice crystals.

[0066] Table 2 Antioxidant activity of myoplasm components of different molecular weights

[0067]

[0068] ​​The results in Table 2 show that the permeate (<10 kDa) component has significantly better antioxidant activity than the retentate (>10 kDa) component. The total antioxidant capacity of the retentate (>10 kDa) component is 0.13 mM, while the total antioxidant capacity of the permeate (<10 kDa) sample is significantly improved to 2.20 mM, an increase of nearly 17 times, indicating that the small molecule component has a stronger free radical scavenging effect. In addition, the hydroxyl radical scavenging rate of the retentate (>10 kDa) component is 55.8%, while the hydroxyl radical scavenging rate of the permeate (<10 kDa) sample is as high as 96.9%, which is significantly better than the retentate component.

[0069] Step 2, enrichment, purification and identification of sarcoplasmic antifreeze and antioxidant peptide segments:

[0070] The permeate of the sarcoplasmic component with a molecular weight of <10 kDa was subjected to high-pressure homogenization (150 MPa, 3 cycles) to physically shear and break the proteins, obtaining a solution containing more small molecule peptides. Further secondary ultrafiltration was performed using a 3 kDa ultrafiltration membrane to obtain a <3 kDa small molecule peptide component, which was concentrated to a peptide concentration of 100 mg / mL using a vacuum low-temperature concentration device (temperature control ≤40°C) for standby. The above concentrated solution was loaded onto a Sephadex G-25 column for gel filtration chromatography separation. The separation conditions were as follows: mobile phase was 20 mM phosphate buffer, pH 6.8, flow rate was 1.0 mL / min, detection wavelength was 220 nm, and each elution peak component was collected, concentrated, desalted and freeze-dried. The component with the highest antifreeze activity of S. thermophilus was collected. The separation component with higher antifreeze activity was dissolved in 50 mM NH4HCO3, and DTT solution was added to make the final concentration 10 mmol / L. Reduction was carried out in a 45-60°C water bath for 1 h, followed by the addition of iodoacetamide solution to make the final concentration 55 mmol / L, and the reaction was carried out in the dark for 40 min. Desalting was performed using a self-filled desalting C18 column, and the solvent was dried in a 45°C vacuum centrifugal concentrator. After dissolution in 0.1% formic acid aqueous solution (volume ratio), mass spectrometry was performed for detection. The detection conditions were as follows: 100 μm i.d. x 180 mm, packing: Reprosil-Pur 120 C18-AQ 3 μm analysis column; mobile phase A: 0.1% formic acid aqueous solution (volume ratio); mobile phase B: 0.1 vt% formic acid and 80 vt% acetonitrile (ACN) in water; flow rate: 600 nL / min; analysis time of each component: 60 min.

[0071] After LC-MS / MS identification, one peptide segment with high-efficiency antifreeze and antioxidant activity was found, and the peptide segment sequence was KELASQPDVDGFLVGGASLKPEFVDIINAK.

[0072] Table 3. Identification of sarcoplasmic peptides using liquid chromatography-mass spectrometry

[0073]

[0074] The peptide KELASQPDVDGFLVGGASLKPEFVDIINAK is composed of 30 amino acids and is rich in hydrophilic polar residues (such as lysine K, glutamic acid E, aspartic acid D, glutamine Q, serine S, etc.) and hydrophobic flexible residues (such as glycine G, alanine A, proline P, valine V, etc.), possessing both good hydrophilicity and flexibility, and exhibiting significant antifreeze potential. The polar groups enriched in its sequence can form a stable hydrogen bond network with water molecules and ice crystal surfaces, effectively binding free water and inhibiting ice crystal formation and recrystallization. Simultaneously, hydrophobic regions such as GFLVGGASL have structural repetitive features similar to those of fish antifreeze proteins, facilitating adsorption and directional binding on ice crystal surfaces and preventing crystal expansion. The high proportions of Pro, Gly, and Ala in the peptide chain endow the peptide with strong flexibility and adaptive coiling ability, enabling it to form a flexible protective layer on the ice crystal surface and prevent ice crystals from puncturing the protein network. In addition, multiple lysine (K) provides protons to scavenge free radicals, while glutamic acid (E) and aspartic acid (D) chelate metal ions, reducing metal-induced oxidation.

[0075] III. Application Examples

[0076] Example 1: Application of the antifreeze and antioxidant peptides prepared in the specific example in the cryoprotection of Streptococcus thermophilus.

[0077] 1. Inoculate 50 μL of twice-activated Streptococcus thermophilus into 4 mL of M17 liquid medium and incubate at 37°C under microaerophilic conditions for 24 hours. After centrifuging the culture at 5000 g for 10 min, collect the bacterial pellet, wash twice with an equal volume of sterile water, and then resuspend in an equal volume of sterile water to prepare a bacterial suspension;

[0078] 2. Weigh out the antifreeze peptide powder with the amino acid sequence KELASQPDVDGFLVGGASLKPEFVDIINAK synthesized by biosynthesis and dissolve it in the above bacterial suspension. The dissolution ratio is set to 1.5% (mass-volume ratio).

[0079] 3. Inoculate 50 μL of the mixture into 4 mL of M17 culture medium and incubate at 37 ℃ for 12 hours. Measure the absorbance at 600 nm (A1). Freeze the remaining bacterial culture at -20 ℃ for 24 hours and perform three freeze-thaw cycles. After thawing, incubate at 37 ℃ for 10 minutes, then inoculate again with 50 μL of the mixture and incubate for 12 hours. Measure the absorbance (A2). Calculate the cell viability.

[0080] Comparative Example 1 was set to replace the antifreeze peptide powder with 4 wt% physiological saline, and the final solubility ratio was 1.5% (mass-volume ratio).

[0081] Comparative Example 2 used 8 wt% sucrose solution to replace the antifreeze peptide powder, with a final solubility ratio of 1.5% (mass-volume ratio).

[0082] Table 4 Comparison of survival rates of different groups of Streptococcus thermophilus

[0083]

[0084] As shown in Table 4, the survival rate of Streptococcus thermophilus in Example 1 was 89.30%, which was 52% higher than that of Comparative Example 1 (saline group) (58.75%) and 18.3% higher than that of Comparative Example 2 (sucrose group) (75.46%). The results indicate that the sarcoplasmic antifreeze peptides have a good protective effect on Streptococcus thermophilus under low temperature conditions, effectively improving its cryopreservation survival rate and showing good application potential.

[0085] Example 2: Application of the antifreeze and antioxidant peptides prepared in the specific example in the frozen storage protection of minced pork.

[0086] 1. Select fresh pork that has been slaughtered for 48-72 hours, remove visible fascia, fat and connective tissue, and cut the lean meat into 35 cm pieces. 3 Cut the lean meat into small pieces; place the processed lean meat pieces in a meat grinder and grind them 1-2 times using a 6 mm aperture sieve to obtain a uniform meat paste;

[0087] 2. Weigh 5 kg of minced meat and add 2.5% antifreeze peptide powder (amino acid sequence KELASQPDVDGFLVGGASLKPEFVDIINAK) by mass fraction. Place the mixture in a meat chopper, control the temperature below 10 ℃, and chop for 8 min until the peptide powder is evenly dispersed and fully combined with the protein to form a well-viscosified minced meat mixture. Place the chopped minced meat mixture into a clean container and let it stand at 4 ℃ for 8 h to promote protein structure recombination and peptide action.

[0088] 3. Using a meat patty mold, pre-form the minced meat into round patties with a diameter of approximately 810 cm and a thickness of 1.52 cm. Pack them into low-density polyethylene food packaging bags, heat-seal the bags, and then store them in a -18 ℃ cold storage for long-term freezing for a period of 6 months.

[0089] The difference between Comparative Example 3 and Example 2 is that no antifreeze agent was added before freezing the minced pork.

[0090] The difference between Comparative Example 4 and Example 2 is that instead of sarcoplasmic antifreeze peptide powder, 8% by weight of sucrose was added to the minced pork before freezing.

[0091] Table 5. Quality changes of different groups of frozen minced pork

[0092]

[0093] As shown in Table 5, compared with the additive-free group of Comparative Example 3, the peptide addition of the present invention significantly improved the quality deterioration problems of minced pork during frozen storage, such as decreased water retention, protein oxidative denaturation, and fat oxidation. After the frozen storage period, the thawing loss of Example 2 was 12.17%, which was reduced by approximately 32.95% compared with Comparative Example 3 and by approximately 11.75% compared with Comparative Example 4; at the same time, the cooking loss of Example 2 was 30.67%, which was reduced by approximately 14.28% compared with Comparative Example 3 and by approximately 4.34% compared with Comparative Example 4. This indicates that the peptide can effectively inhibit ice crystal growth and recrystallization during low-temperature frozen storage, reduce damage to myofibril structure, and significantly improve the water retention capacity and frozen storage stability of the product. After the freezing period in Example 2, the surface hydrophobicity value of the chyme protein system was only 24.67 μg BPB / mg protein, significantly lower than that of Comparative Examples 3 and 4. This indicates that the introduction of antifreeze and antioxidant peptides effectively reduced the exposed hydrophobic groups of the protein structure, enhanced the conformational stability and solubility of the protein, helped maintain its original functional conformation, and reduced protein aggregation caused by hydrophobic interactions during freezing. The protein particle size in Example 2 was 62.44 nm, which was smaller than that in Comparative Examples 3 (74.89 nm) and 4 (64.71 nm), indicating that the addition of this peptide could significantly inhibit the aggregation behavior of protein particles during freezing. Analysis of the myosin dissociation rate showed that after the freezing period, Example 2 reached 65.31%, much higher than that in Comparative Examples 3 (47.92%) and 4 (60.03%), further indicating that the addition of this peptide significantly inhibited the aggregation of myosin during freezing and improved the freezing stability of the protein system. Protein carbonyl, free thiol, and fat TBARS values ​​are commonly used indicators to characterize protein and fat oxidation. As shown in Table 5, at the end of the frozen storage period, the protein free thiol value of Example 2 was 50.35 nmol / mg, an increase of 40.9% and 13.5% compared to Comparative Examples 3 and 4, respectively; meanwhile, the protein carbonyl value of Example 2 was 6.94 nmol / mg, a decrease of 23.1% and 13.4% compared to Comparative Examples 3 and 4, respectively; furthermore, the fat TBARS value of Example 2 was 2.36 mg / kg, a decrease of 31.0% and 16.9% compared to Comparative Examples 3 and 4, respectively. These results show that the peptides provided by this invention can effectively protect cysteine ​​residues from oxidation during the frozen storage of minced pork, reduce the formation of carbon-based oxidation products and fat oxidation products, and improve the antioxidant capacity of protein and fat during the frozen storage of minced pork.

[0094] Example 3: Application of the antifreeze and antioxidant peptides prepared in the specific embodiment in the frozen storage protection of pork gel products.

[0095] 1. Select fresh pork that has been slaughtered for 48-72 hours, remove visible fascia, fat and connective tissue, and cut the lean meat into 35 cm pieces. 3 Cut the lean meat into small pieces and set aside; place the processed lean meat pieces in a meat grinder and grind them 1-2 times using a 6 mm aperture sieve to obtain a uniform meat paste.

[0096] 2. Weigh 5 kg of minced meat, add 2% sodium chloride, 2% antifreeze peptide powder (amino acid sequence KELASQPDVDGFLVGGASLKPEFVDIINAK) and 10% ice water, and chop for 4 min. Then add 10% pork back fat and chop for 4 min until the peptide powder is evenly dispersed and fully combined with the protein to form a well-viscosified minced meat mixture.

[0097] 3. Place the chopped meat mixture in a refrigerated environment at 4 ℃ and let it stand for 8 hours to promote protein structure recombination and peptide action; after filling the meat mixture, heat it at 80 ℃ for 30 minutes, cool it to 25 ℃, and then place the gel product in a cold storage at -18 ℃ for long-term frozen storage, with a storage period of 6 months.

[0098] The difference between Comparative Example 5 and Example 3 is that no antifreeze agent was added to the minced meat mixture.

[0099] The difference between Comparative Example 6 and Example 3 is that instead of sarcoplasmic antifreeze peptide powder, 8% by weight of sucrose was added to the minced meat mixture.

[0100] Table 6. Quality changes of frozen pork gel products in different groups

[0101]

[0102] As shown in Table 6, compared with the antifreeze group of Comparative Example 5, the peptide addition of the present invention significantly improved the quality deterioration problems of pork gel products during frozen storage, such as decreased water retention, protein oxidative denaturation, and fat oxidation. At the end of the frozen storage period, the thawing loss of Example 3 was 8.02%, which was reduced by approximately 43.63% compared with Comparative Example 5 and by approximately 12.63% compared with Comparative Example 6, demonstrating that the method of the present invention can significantly reduce thawing loss and reduce juice loss. Furthermore, at the end of the frozen storage period, the gel strength of Example 3 was 522.3 g / cm³. 2Compared with Comparative Example 5, the strength was increased by 25.7% and compared with Comparative Example 6 by 4.1%; the gel hardness of Example 3 was 1702 gf, which was increased by 69.7% compared with Comparative Example 5 and by about 2.1% compared with Comparative Example 6; the gel chewiness of Example 3 was 1145 gf, which was increased by about 69.2% compared with Comparative Example 5 and by about 18.3% compared with Comparative Example 6. These textural parameters indicate that Example 3 significantly improved the strength, hardness and chewiness of the pork gel system during frozen storage, demonstrating the good effect of the method of the present invention on improving the structure and textural properties of gel products. Further analysis of protein and fat oxidation indicators revealed that Example 3 exhibited low free thiol, carbonyl, and POV values ​​at freshness. Compared to Comparative Example 5, the free thiol value increased by 12.7%, while the carbonyl and POV values ​​decreased by 35.0% and 18.0%, respectively, effectively inhibiting protein and fat oxidation caused by the gelation heating process. At the end of the frozen storage period, compared to Comparative Examples 5 and 6, the free thiol value of Example 3 increased by 74.0% and 34.4%, respectively, while the carbonyl value decreased by 35.5% and 15.1%, respectively, and the POV value decreased by 21.7% and 11.5%, respectively. These results indicate that the peptides provided by this invention can effectively protect cysteine ​​residues from oxidation during the frozen storage of pork gel products, reduce the formation of carbon-based oxidation products and fat oxidation products, and improve the antioxidant capacity of proteins and fats in pork gel products during frozen storage.

[0103] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A sarcoplasmic antifreeze and antioxidant peptide, characterized in that... Its amino acid sequence is as follows: KELASQPDVDGFLVGGASLKPEFVDIINAK.

2. The application of the sarcoplasmic antifreeze and antioxidant peptide of claim 1 in the preparation of a cryoprotectant for Streptococcus thermophilus, characterized in that: The sarcoplasmic antifreeze and antioxidant peptides are added at a mass concentration of 1-3%.

3. The application of the sarcoplasmic antifreeze and antioxidant peptide of claim 1 in the preparation of a frozen preservation agent for minced pork, characterized in that: The sarcoplasmic antifreeze and antioxidant peptides are added at a mass concentration of 1-3%.

4. The application of the sarcoplasmic antifreeze and antioxidant peptide of claim 1 in the preparation of a cryopreservation protectant for pork gel products, characterized in that: The sarcoplasmic antifreeze and antioxidant peptides are added at a mass concentration of 1-3%.

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