Shrimp peptide with low sensitization and high antioxidant activity as well as preparation method and application thereof

Abstract

The invention discloses shrimp peptide with low sensitization and high antioxidant activity and a preparation method and application thereof.The preparation method of the shrimp peptide with low sensitization and high antioxidant activity comprises the following steps that shrimp meat is taken and smashed into homogenate, low-temperature plasma discharge treatment is conducted, then the pH value of the shrimp paste is adjusted, protease is added for a reaction, and the shrimp peptide with low sensitization and high antioxidant activity is obtained; and carrying out enzyme deactivation on the hydrolysate, centrifuging, and freeze-drying the supernate to obtain the shrimp peptide powder. In the method, the low-temperature plasma discharge pretreatment can destroy the shrimp protein structure, so that the structure is expanded, steric hindrance is reduced, more reaction sites are provided for the enzymolysis reaction, the enzymolysis efficiency is improved, the sensitization of shrimps is reduced as much as possible, and shrimp peptide with relatively good antioxidant activity is generated.

Description

Technical Field

[0001] The present invention belongs to the field of food processing, and particularly relates to a shrimp peptide with low allergenicity and high antioxidant activity, a preparation method thereof, and an application thereof. Background Art

[0002] There are several proteins in shrimp meat that can cause allergic reactions in humans, and the most important allergenic protein is thermostable tropomyosin. In order to enable more people to consume nutritious and delicious shrimp products without burden, some low-allergenic shrimp products have emerged. However, the existing preparation methods of low-allergenic shrimp products all have certain deficiencies, and there is an urgent need to develop a new type of low-allergenic shrimp product to meet consumers' pursuit of food safety, nutrition, and deliciousness.

[0003] The enzymatic hydrolysis method is a common method for modifying proteins, and has advantages such as mild conditions and strong specificity. Through the peptide bond cleavage action catalyzed by enzymes, the structure of food protein polypeptide chains can be strongly damaged, thereby changing their physicochemical properties. Moreover, biological enzymes can destroy the integrity of the linear antigenic epitopes of allergenic proteins by cleaving peptide bonds, thereby reducing the allergenicity of allergenic proteins. However, the application of single enzyme hydrolysis cannot completely solve the allergenicity of some allergens, and even some epitopes may be exposed during the hydrolysis process, leading to an increase in allergenicity.

[0004] Since bioactive peptides have various physiological functions such as hypoglycemic, antioxidant, antihypertensive, and anti-inflammatory effects, they can play a preventive and control role in various diseases such as diabetes, hypertension, and hyperglycemia. Currently, bioactive peptides are mainly obtained by enzymatic hydrolysis of food-derived proteins. However, the enzymatic hydrolysis method has disadvantages such as high cost, low efficiency, and unpleasant odor. In addition, the long enzymatic hydrolysis process will also lead to serious losses of the nutritional components and sensory qualities of bioactive peptides, which means that their applications are limited. Therefore, the combination of various emerging processing technologies and enzymatic hydrolysis is considered a new strategy for producing bioactive peptides. Emerging physical technologies such as ultrasonic waves, high hydrostatic pressure, and microwaves mainly increase the enzymatic hydrolysis efficiency by unfolding the protein spatial conformation and increasing the enzyme cleavage sites. However, there are few studies on the production of low-allergenic and highly bioactive protein peptides assisted by low-temperature plasma technology. Summary of the Invention

[0005] The object of the present invention is to provide a shrimp peptide with low allergenicity and high antioxidant activity, and its preparation method and application. The present invention uses low-temperature plasma-assisted enzymatic hydrolysis to modify or destroy the amino acid sequence and spatial conformation of proteins to prepare shrimp peptides with low allergenicity and high antioxidant activity. Low-temperature plasma can effectively promote enzymatic hydrolysis by unfolding the protein structure and exposing the cleavage sites, thereby improving the enzymatic hydrolysis efficiency and reducing the enzyme dosage. At the same time, low-temperature plasma and biological enzymes have a synergistic modification or destruction effect on the protein structure, which can reduce the allergenicity of shrimp allergens to a greater extent, and is efficient and convenient. Moreover, shrimp peptides with higher antioxidant activity can be obtained by low-temperature plasma-assisted enzymatic hydrolysis, thus enhancing their application prospects.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] A preparation method of a shrimp peptide with low allergenicity and high antioxidant activity, comprising the following steps:

[0008] Take shrimp meat and grind it into a homogeneous slurry, perform low-temperature plasma discharge treatment, then adjust the pH value of the shrimp slurry, add protease and react, inactivate the enzyme in the obtained hydrolysis solution and then centrifuge, and freeze-dry the supernatant to obtain shrimp peptide powder.

[0009] Preferably, the low-temperature plasma discharge treatment is dielectric barrier discharge, the treatment time is 2 - 8 min, the voltage is 50 - 60 kV, and the current is 0.8 - 1.2 mA; further preferably, the voltage is 60 kV and the current is 1 mA.

[0010] Preferably, the protease is alkaline protease, trypsin or papain, more preferably alkaline protease or trypsin, and most preferably alkaline protease.

[0011] Preferably, the mass ratio of the enzyme activity of the protease to the mass of the shrimp meat is (2000 - 5000 U):1 g.

[0012] Preferably, the addition of protease reacts at 40 - 50 °C for 2 - 3 h.

[0013] Preferably, when the protease is alkaline protease, the hydrolysis conditions are as follows: the temperature is 50 °C and the pH value is 8.5.

[0014] Preferably, when the protease is trypsin, the hydrolysis conditions are as follows: the temperature is 40 °C and the pH value is 8.

[0015] Preferably, when the protease is papain, the hydrolysis conditions are as follows: the temperature is 40 °C and the pH value is 7.5.

[0016] Preferably, the method for inactivating the enzyme is a boiling water bath reaction at 100 °C for 10 min.

[0017] Preferably, the shrimp meat is obtained by removing the shrimp shell, head, tail and shrimp vein from penaeus vannamei.

[0018] Preferably, the homogenate is prepared by immersing the shrimp meat in an appropriate amount of pure water and crushing it into a homogenate with a blender.

[0019] A shrimp peptide with low allergenicity and high antioxidant activity is prepared by the method described in any one of the above.

[0020] Application of the above-mentioned shrimp peptide with low allergenicity and high antioxidant activity in food.

[0021] The present invention has the following advantages and effects compared with the prior art:

[0022] (1) In the method of the present invention, the low-temperature plasma discharge will destroy the shrimp protein structure, causing the structure to unfold, reducing the steric hindrance, providing more cleavage sites for proteases, greatly reducing the enzymatic hydrolysis reaction time and enzyme dosage, improving the efficiency and reducing the cost.

[0023] (2) The method of the present invention uses low-temperature plasma to cooperate with enzymatic hydrolysis to treat shrimp allergenic proteins, which can efficiently reduce the allergenicity of shrimp proteins and avoid the decline of food quality caused by over-treatment of a single technology.

[0024] (3) The method of the present invention uses low-temperature plasma pretreatment to assist protease hydrolysis of shrimp proteins, obtaining shrimp peptides with high antioxidant activity while reducing allergenicity, enhancing the application value of shrimp and the development prospect of shrimp products. Description of the Drawings

[0025] Figure 1 It is a diagram showing the change of the secondary structure of shrimp paste protein treated by low-temperature plasma.

[0026] Figure 2 It is an electrophoresis diagram of shrimp allergen proteins treated by low-temperature plasma assisted with different proteases.

[0027] Figure 3 It is a diagram showing the change of the allergenicity of shrimp allergen proteins, with the allergenicity of the original sample recorded as 100%.

[0028] Figure 4 It is a diagram showing the change of the remaining number of shrimp antigenic epitope peptides. Detailed Embodiments

[0029] The present invention will be further described in detail below with reference to the embodiments and the drawings, but the embodiments of the present invention are not limited thereto.

[0030] In the present invention, the protein gel electrophoresis is carried out according to the following steps:

[0031] Dilute the main allergenic protein of shrimp to 0.5 mg / mL with PBS. Take 80 μL of the sample and mix it with 20 μL of protein loading buffer. After heating at 95 °C for 10 min, perform gel electrophoresis using a precast gel (180 V, 100 mA), and the sample loading volume is 10 μL.

[0032] In the present invention, the detection of the protein secondary structure is carried out according to the following steps:

[0033] Analyze the changes in the secondary structure of shrimp paste protein after different treatments by Fourier transform infrared spectroscopy. Use a Fourier transform infrared spectrometer to obtain spectra in the wavenumber range of 400 - 4000 cm -1 Analyze the content of the secondary structure of the sample in the amide I band (1600 - 1700 cm -1 ) by using OMNIC (version 8) and Peakfit (version 4.12) software through deconvolution and curve fitting. The secondary structure is related to allergenicity. Generally, the lower the α - helix content of shrimp protein, the more disordered and extended its structure, the greater the change in allergenicity, and the easier it is to undergo enzymatic hydrolysis reactions.

[0034] In the present invention, the detection method of allergenicity is carried out according to the following steps:

[0035] (1) Coating: Dilute the shrimp paste to 20 μg / mL with carbonate coating buffer, and then add 100 μL / well to a 96 - well enzyme - linked immunosorbent assay (ELISA) plate and place it in a 4 °C refrigerator overnight.

[0036] (2) Washing: After pouring out the coating solution, wash the plate 5 times with PBST washing buffer, and the washing liquid volume is 300 μL / well. Then centrifuge to discard the washing liquid and pat dry on absorbent paper.

[0037] (3) Blocking: Add 200 μL of blocking solution to the wells, incubate at 37 °C for 2 h, take it out and wash 5 times and then pat dry.

[0038] (4) Antigen - antibody reaction: Add diluted 20 - fold human antiserum to the ELISA plate, 100 μL / well, incubate at 37 °C for 1.5 h, take it out and wash 5 times and then pat dry.

[0039] (5) Measurement: Add 100 μL of TMB substrate solution, react at 37 °C in the dark for 15 min, then add 50 μL of sulfuric acid (2 M) to terminate the reaction, and measure the absorbance at a wavelength of 450 nm. Record the absorbance value of the original sample as representing 100% allergenicity.

[0040] In the present invention, the detection method of ABTS radical scavenging rate is carried out according to the following steps:

[0041] Mix 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate in equal volumes, and react for 12 h under dark conditions at room temperature to prepare ABTS cation radical reagent. Dilute the ABTS cation radical reagent with ethanol to an absorbance of 0.70 ± 0.02 at a wavelength of 734 nm. Vortex mix 0.1 mL of the sample or deionized water (as a control group) with 1.4 mL of the ABTS cation radical reagent, and measure the absorbance at a wavelength of 734 nm after incubation at 25 °C for 30 min.

[0042] In the present invention, the detection method for the iron ion reducing ability is carried out according to the following steps:

[0043] Determine using a total antioxidant capacity detection kit (FRAP method). Add 7.5 mL of tripyridyltriazine (TPTZ) dilution solution, 750 μL of TPTZ solution, and 750 μL of detection buffer in sequence to serve as the FRAP working solution. Mix 180 μL of the FRAP working solution and 5 μL of the sample solution and place them into the sample detection well. After incubation at 37 °C for 5 min, measure the absorbance at 593 nm; in the standard curve detection well, replace the sample solution with 5 μL of FeSO4 standard solution; in the blank control well, replace the sample solution with 5 μL of distilled water. The results are expressed in μM / g.

[0044] In the present invention, the determination of the degree of hydrolysis is carried out according to the following steps:

[0045] Analyze the change in the degree of hydrolysis of TM after different treatments using the o-phthalaldehyde (OPA) method. Add 300 μL of OPA reagent to 40 μL of the sample (0.4 mg / mL), and measure the absorbance of the reaction mixture at 340 nm. Use 0.9516 mmol / L serine and PBS as the standard product and blank control respectively.

[0046] In the present invention, the identification of the antigenic epitope peptide is carried out according to the following steps:

[0047] Determine using HPLC-MS / MS. Redissolve the sample obtained by pretreatment in formic acid solution, then load it onto a C18 chromatographic column and use a gradient program for elution. The mass spectrometer operates in the DDA mode and automatically switches between MS and MS / MS acquisitions. The MS scanning range is 350 - 1500 m / z. Analyze the collected data using PEAKS Studio (version 8.5).

[0048] Example 1 Preparation of shrimp peptides assisted by low-temperature plasma and papain

[0049] After removing the head, shell, tail and shrimp vein of the prawn, the obtained shrimp meat was immersed in pure water (1:8, w / v), homogenized with a blender in a liquid environment to form shrimp paste, and then treated with dielectric barrier discharge type low-temperature plasma (discharge voltage 50 kV, discharge current 1.2 mA, discharge time 2 min), and the change of the secondary structure of shrimp paste protein was measured. As Figure 1 shown, the content of the protein α-helix structure decreased by 30%, indicating that the low-temperature plasma pretreatment unfolded the protein structure, which was beneficial to the subsequent enzymatic hydrolysis reaction. Figure 1 The original sample in

[0050] refers to the shrimp paste that has not been treated with dielectric barrier discharge type low-temperature plasma. After the treatment, the pH of the protein mixture was adjusted to 7.5, papain (5000 U / g) was added, placed in a water bath, continuously stirred, and reacted at 40 °C for 3 h. Then the mixture was placed in boiling water at 100 °C for 10 min to terminate the hydrolysis reaction. Finally, the mixture was centrifuged, and the supernatant was freeze-dried to obtain shrimp peptides. The ABTS radical scavenging rate and ferric ion reducing antioxidant power of the shrimp peptides obtained by this method were 44.10±0.53% and 221.52±4.33 μM / g, respectively.

[0051] Example 2 Preparation of shrimp peptides assisted by low-temperature plasma and trypsin

[0052] After removing the head, shell, tail and shrimp vein of the prawn, the obtained shrimp meat was immersed in pure water (1:10, w / v), homogenized with a blender in a liquid environment to form shrimp paste, and then treated with dielectric barrier discharge type low-temperature plasma (discharge voltage 60 kV, discharge current 0.8 mA, discharge time 6 min), and the change of the secondary structure of shrimp paste protein was measured. As Figure 1 shown, the content of the protein α-helix structure decreased by 35%, indicating that the low-temperature plasma pretreatment unfolded the protein structure, which was beneficial to the subsequent enzymatic hydrolysis reaction.

[0053] After the treatment, the pH of the protein mixture was adjusted to 8, trypsin (4000 U / g) was added, placed in a water bath, continuously stirred, and reacted at 40 °C for 2 h. Then the mixture was placed in boiling water at 100 °C for 10 min to terminate the hydrolysis reaction. Finally, the mixture was centrifuged, and the supernatant was freeze-dried to obtain shrimp peptide powder. The ABTS radical scavenging rate and ferric ion reducing antioxidant power of the shrimp peptides obtained by this method were 53.30±0.24% and 240.32±3.56 μM / g, respectively.

[0054] Example 3 Preparation of shrimp peptides assisted by low-temperature plasma and alkaline protease

[0055] The prawns are decapitated, shelled, de-tailed and de-veined to obtain the prawn meat, which is immersed in pure water (1:8, w / v). Under a liquid environment, it is evenly stirred into prawn paste with a blender, and then treated with dielectric barrier discharge type low-temperature plasma (discharge voltage 60 kV, discharge current 1 mA, discharge time 4 min) to measure the change in the secondary structure of the prawn paste protein. As Figure 1 shown, the content of the protein α-helix structure decreased by 15%, indicating that the low-temperature plasma pretreatment unfolds the protein structure, which is beneficial to the subsequent enzymatic hydrolysis reaction.

[0056] After the treatment, the pH of the protein mixture is adjusted to 8.5, alkaline protease (2000 U / g) is added, and it is placed in a water bath and continuously stirred. The reaction is carried out at 50 °C for 2 h. Then the mixture is placed in boiling water at 100 °C for 10 min to terminate the hydrolysis reaction. Finally, the mixture is centrifuged, and the supernatant is taken for freeze-drying to obtain shrimp peptide powder. The ABTS free radical scavenging rate and ferric ion reducing antioxidant power of the shrimp peptides obtained by this method are 62.65 ± 0.87% and 267.98 ± 3.13 μM / g, respectively.

[0057] Example 4 Preparation of Shrimp Peptides Assisted by Low-Temperature Plasma and Alkaline Protease

[0058] Same as Example 3, except that the low-temperature plasma treatment time is replaced with 8 min. The ABTS free radical scavenging rate and ferric ion reducing antioxidant power of the shrimp peptides obtained by this method are 70.56 ± 1.18% and 285.66 ± 4.98 μM / g, respectively.

[0059] Comparative Example 1 Treatment of Shrimp Protein with Low-Temperature Plasma Alone

[0060] The prawns are decapitated, shelled, de-tailed and de-veined to obtain the prawn meat, which is immersed in pure water (1:8, w / v). Under a liquid environment, it is evenly stirred into prawn paste with a blender, and then treated with dielectric barrier discharge type low-temperature plasma (discharge voltage 60 kV, discharge current 1 mA, discharge time 4 min).

[0061] Comparative Example 2 Treatment of Shrimp Protein with Low-Temperature Plasma Alone

[0062] Same as Comparative Example 1, except that the low-temperature plasma time is replaced with 8 min.

[0063] Comparative Example 3 Treatment of Shrimp Protein with Alkaline Protease Alone

[0064] The prawns are decapitated, shelled, de-tailed and de-veined to obtain the prawn meat, which is immersed in pure water (1:8, w / v). Under liquid conditions, it is evenly stirred into prawn paste with a blender. Then, the pH of the prawn paste is adjusted to 8.5, and alkaline protease (2000 U / g) is added. It is placed in a water bath and continuously stirred, and the reaction is carried out at 50 °C for 2 h. Then, the mixture is placed in boiling water at 100 °C for 10 min to terminate the hydrolysis reaction. Finally, the mixture is centrifuged, and the supernatant is taken for freeze-drying to obtain prawn peptide powder. The ABTS radical scavenging rate and ferric ion reducing antioxidant power of the prawn peptides obtained by this method are 34.63±0.37% and 192.79±6.12 μM / g, respectively.

[0065] Test Example 1 Gel Electrophoresis

[0066] Test the molecular weight change of the allergenic proteins in the prawn peptides prepared in Test Examples 1-3.

[0067] As Figure 2 , the 35 kDa band is the major allergenic protein paramyosin in prawns. After treatment with Example 1, the paramyosin in prawns did not change significantly, indicating that papain has little effect on the allergenic proteins in prawns. After treatment with Example 2, the paramyosin band in prawns became significantly lighter, indicating that this treatment has a significant effect on the allergenic proteins in prawns. Compared with Examples 1 and 2, the paramyosin in prawns treated with Example 3 has completely disappeared, indicating that alkaline protease has the strongest destructive effect on the allergenic proteins in prawns.

[0068] Test Example 2 Allergenicity

[0069] Test the allergenicity of Test Examples 3 and 4, and Comparative Examples 1-3.

[0070] As Figure 3 can be seen, the allergenicity of Comparative Examples 1 and 2 (treated with low-temperature plasma alone) decreased compared with the untreated samples, but the degree was limited. The allergenicity of Examples 3 and 4 (low-temperature plasma-assisted alkaline protease) decreased most significantly, decreasing by 88% and 87% respectively compared with the untreated samples, indicating that the combined action of low-temperature plasma and alkaline protease significantly reduces the allergenic potential of prawns.

[0071] Test Example 3 Residual Quantity of Epitope Peptides

[0072] Identify the residual quantity of epitope peptides in the prawn hydrolysates of Examples 3 and 4, and Comparative Example 3.

[0073] As Figure 4 can be seen, the residual quantity of epitope peptides in Examples 3 and 4 (low-temperature plasma-assisted enzymatic hydrolysis) is significantly lower than that in Comparative Example 3 (enzymatic hydrolysis with alkaline protease alone), indicating that low-temperature plasma-assisted enzymes reduce the allergenicity of prawns by destroying the mechanism of epitope peptides.

[0074] Hydrolysis Degree and Antioxidant Activity of Test Example 4

[0075] Hydrolysis degree and antioxidant activity (ABTS radical scavenging rate and ferric ion reducing ability) of Test Examples 1-4 and Comparative Example 3.

[0076] Table 1 Effect of Different Hydrolysis Methods on Antioxidant Activity of Shrimp Peptides

[0077]

[0078] As can be seen from Table 1, compared with Comparative Example 3, the hydrolysis degree of Examples 1-4 was significantly improved, indicating that low-temperature plasma has the effect of promoting enzymatic hydrolysis and improving the enzymatic hydrolysis efficiency. Moreover, the ABTS radical scavenging ability and ferric ion reducing ability of Examples 1-4 were significantly enhanced, indicating that the addition of low-temperature plasma pretreatment has a good effect on improving the antioxidant ability of shrimp hydrolysates.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing shrimp peptides with low allergenicity and high antioxidant activity, characterized in that: The following steps are involved: The shrimp meat is minced into a homogenate and treated with low-temperature plasma discharge. The pH value of the shrimp paste is then adjusted. Protease is added to react. The resulting hydrolyzate is inactivated and centrifuged. The supernatant is freeze-dried to obtain shrimp peptide powder.

2. The preparation method according to claim 1, characterized in that: The protease is alkaline protease, trypsin or papain.

3. The preparation method according to claim 2, characterized in that: The protease is alkaline protease or trypsin.

4. The preparation method according to any one of claims 1 to 3, characterized in that The low-temperature plasma discharge treatment is dielectric barrier discharge, the discharge time is 2-8 minutes, the discharge voltage is 50-60 kV, and the discharge current is 0.8-1.2 mA.

5. The preparation method according to any one of claims 1 to 3, characterized in that: The mass ratio of the enzyme activity of the protease to the shrimp meat is (2000-5000U):1g.

6. The preparation method according to any one of claims 1 to 3, characterized in that: Add protease and react at 40-50°C for 2-3 hours.

7. The preparation method according to any one of claims 1 to 3, characterized in that: When the protease is alkaline protease, the hydrolysis conditions are as follows: temperature is 50°C, pH value is 8.5; when the protease is trypsin, the hydrolysis conditions are as follows: temperature is 40°C, pH value is 8; when the protease is papain, the hydrolysis conditions are as follows: temperature is 40°C, pH value is 7.

5.

8. The preparation method according to any one of claims 1 to 3, characterized in that: The homogenate is prepared by immersing the shrimp meat in pure water and blending it into a homogenate using a blender.

9. A shrimp peptide with low allergenicity and high antioxidant activity, characterized in that: Prepared by the method described in any one of claims 1 to 8.

10. Use of the shrimp peptide with low allergenicity and high antioxidant activity as claimed in claim 9 in food.

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