CN115505037A - Fish skin collagen antifreeze peptide improved by glycosylation and preparation method and application thereof - Google Patents
Fish skin collagen antifreeze peptide improved by glycosylation and preparation method and application thereof Download PDFInfo
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- CN115505037A CN115505037A CN202211196420.2A CN202211196420A CN115505037A CN 115505037 A CN115505037 A CN 115505037A CN 202211196420 A CN202211196420 A CN 202211196420A CN 115505037 A CN115505037 A CN 115505037A
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- glycosylation
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- collagen peptide
- fish skin
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-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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Abstract
The invention discloses a fish skin collagen antifreeze peptide improved by glycosylation and a preparation method and application thereof, and belongs to the fields of food biochemistry and aquatic product preservation. The preparation method comprises the following steps: adding monosaccharide into the fish skin collagen peptide solution, stirring to obtain a glycopeptide mixture, then adjusting the pH of the glycopeptide mixture, stirring at the temperature of 50-80 ℃ for reaction, and obtaining the fish skin collagen antifreeze peptide improved by glycosylation after the reaction is finished. The glycosylated peptide is prepared by a tilapia collagen peptide glycosylation reaction process based on a glycosylation reaction principle, so that tilapia collagen peptide and sugar are fully crosslinked, the mass-to-charge ratio and the secondary structure of tilapia collagen peptide are changed, the thermal hysteresis activity of tilapia collagen peptide is enhanced, the anti-freezing activity of tilapia collagen peptide is improved by applying a grafting reaction, the product stability is improved, and a new thought and a new way are provided for the development of the phosphorus-free antifreeze agent in the aquatic product freezing and processing industry.
Description
Technical Field
The invention relates to the fields of food biochemistry and aquatic product preservation, in particular to a fish skin collagen antifreeze peptide improved by glycosylation and a preparation method and application thereof.
Background
Aquatic products in China are abundant in resources and are mainly transported and stored in a freezing processing mode, but the aquatic products are easy to undergo ice crystallization and recrystallization in the transportation process to cause protein freezing denaturation, so that the quality of the aquatic products is deteriorated, and functional characteristics are weakened, such as juice loss and the original flavor and properties of the aquatic products are changed. The antifreeze agent is added to prevent the freezing denaturation of aquatic products to the maximum extent, and the antifreeze agent in the food industry mainly comprises saccharides and polyphosphate. Although these antifreeze agents can effectively increase the water holding capacity of food and prevent the freezing denaturation of food, certain defects still exist, sugar antifreeze agents can increase unnecessary sweetness and calorie, and excessive intake of polyphosphate can bring health risks to consumers.
In recent years, antifreeze peptides have become a focus of research for developing green antifreeze agents. The antifreeze peptide can be combined with the surface of an ice crystal to inhibit the growth of the ice crystal, and can be adsorbed on the surface of the ice crystal through intermolecular forces such as hydrogen bonds, hydrophobic interaction, van der waals force and the like to inhibit the recrystallization of the ice crystal, so that the antifreeze peptide can reduce the freezing point of a solution to cause the difference between the freezing point and the melting point of the solution, the characteristic is called Thermal Hysteresis Activity (THA), and the Thermal hysteresis activity is an important evaluation index of the antifreeze peptide. Although antifreeze peptides have great potential to become novel food antifreeze agents, large-scale application is limited due to low active ingredients and high cost. Therefore, the development of improving the activity of antifreeze peptides is of great significance.
The glycosylation reaction is a chemical reaction between carbonyl and amino, and has high safety and simple operation. Under certain temperature and humidity conditions, the glycosylation reaction can introduce C = O in the sugar into the structure of the collagen peptide, so that the bioactivity of the anti-collagen peptide is obviously improved. In 2021, the report of the national fishery statistics yearbook, the tilapia level in the freshwater fish culture yield in China is fifth, the national tilapia yield in 2020 reaches 16.6 ten thousand tons, the total yield of the tilapia is 50 percent, and fish skin and fish scale are used as main processing byproducts and can be used for extracting collagen peptide. And tilapia collagen peptide has certain anti-freezing activity, but no method for improving the activity of tilapia collagen peptide through glycosylation reaction is reported in the prior art.
Disclosure of Invention
The invention aims to provide fish skin collagen antifreeze peptide improved by glycosylation and a preparation method and application thereof, and aims to solve the problems in the prior art. Under the conditions of specific temperature and time, the tilapia collagen peptide and sugar are fully crosslinked, so that the mass-to-charge ratio and the secondary structure of the tilapia collagen peptide are changed, the thermal hysteresis activity of the tilapia collagen peptide is enhanced, the anti-freezing activity of the tilapia collagen peptide is improved by using a grafting reaction, and the stability of the tilapia collagen peptide is improved. The glycosylated peptide prepared by the invention is white, has no peculiar smell, lower heat and sweetness, good solubility and easy absorption and utilization, and can be used as a safe and efficient green antifreeze agent to be applied to food.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for preparing fish skin collagen antifreeze peptide improved by glycosylation, which comprises the following steps:
adding monosaccharide into the fish skin collagen peptide solution, stirring to obtain a glycopeptide mixture, then adjusting the pH of the glycopeptide mixture, stirring at the temperature of 50-80 ℃ for reaction, and obtaining the fish skin collagen antifreeze peptide improved by glycosylation after the reaction is finished.
Further, the mass concentration of the fish skin collagen peptide solution is 7-9%.
Further, the mass ratio of the fish skin collagen peptide solution to glucose is 1: (1-4); the monosaccharide includes glucose, xylose or galactose.
Further, the stirring condition is that the stirring is carried out for 10-40min at normal temperature; the pH value is adjusted to 7.5-8.5, and the stirring reaction time is 60-120min.
Further, after the reaction is finished, separation and purification are carried out to obtain the fish skin collagen antifreeze peptide improved by glycosylation.
Further, the separation is purified as: adding water into the product after the reaction is finished to prepare a solution with the concentration of 10-50mg/mL, sending the solution to a sephadex chromatographic column for separation and purification, collecting an active peak, and freeze-drying; wherein, the chromatographic conditions for separation and purification are as follows: the mobile phase is ultrapure water, the loading amount is 1-5% of the volume of the column bed of the sephadex chromatographic column, the flow rate is 1mL/min, and the detection wavelength is 220nm.
Further, the fish skin collagen peptide comprises tilapia skin collagen peptide.
Furthermore, the tilapia skin collagen peptide has the molecular weight of 500-3000Da and is easy to have a grafting reaction with sugar molecules.
The invention also provides the fish skin collagen antifreeze peptide obtained by the preparation method.
The invention also provides an application of the fish skin collagen antifreeze peptide in preparation of a food antifreeze agent.
The invention also provides a food antifreeze agent which comprises the fish skin collagen antifreeze peptide.
The invention discloses the following technical effects:
1. the method adopts tilapia processing by-products as raw material sources, fully utilizes biological resources, and reduces the damage of food processing to the environment.
2. According to the invention, monosaccharide such as glucose is used for grafting in the preparation of the glycosylated peptide, so that the antifreeze activity of tilapia collagen peptide is improved, and the stability of the tilapia collagen peptide is enhanced, so that the tilapia collagen peptide serving as an antifreeze agent can better maintain the original flavor and nutritional value of food, the safety is improved, and the storage life of frozen food is prolonged.
3. The invention provides a method for preparing glycosylation-improved fish skin collagen antifreeze peptide, which has the advantages of simple operation, easy preparation, low raw material cost and easy realization of industrial production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a graph showing the effect of the extent of glycosylation on thermal hysteresis activity;
FIG. 2 shows the degree of grafting of the glycosylation product at different reaction temperatures;
FIG. 3 shows the degree of grafting of the glycosylation product at different reaction times;
FIG. 4 shows the degree of grafting of the glycosylation product at different collagen peptide concentrations;
FIG. 5 shows the degree of grafting of the glycosylation product for different glycopeptide ratios;
FIG. 6 is a DSC heat flow curve for the glycosylation product and a control sample;
FIG. 7 is a Fourier transform infrared spectrum of the glycosylation product and a control sample, wherein a: glucose; b: a collagen peptide; c: a collagen peptide-glucose mixture; d: (ii) a glycosylation product;
FIG. 8 shows the IR spectra analysis (A) and the secondary structure analysis (B) of the bands I of collagen peptide, collagen peptide-glucose mixture and glycosylation product amide;
FIG. 9 is a total ion flux profile of collagen peptides and their glycosylation products;
FIG. 10 is a 0-3min MS profile of collagen peptides and their glycosylation products;
FIG. 11 is a 5.5-9min MS spectrum of collagen peptide and its glycosylation products;
FIG. 12 is a statistical result of residual enzyme activities of hydrogen peroxide in the glycosylated peptide and collagen peptide treated group and the blank control group;
FIG. 13 is the influence of glycosylated peptides, collagen peptides and blank control groups on the thawing and water loss rate of scallop adductor muscle in the process of frozen storage;
FIG. 14 shows the effect of glycosylated peptides, collagen peptides and blank control groups on the content of salt-soluble proteins in the process of freezing and storing adductor muscles of scallops;
FIG. 15 shows the effect of glycosylated peptide, collagen peptide and blank control group on the total thiol content of scallop adductor muscle during frozen storage;
FIG. 16 shows Ga in the process of freezing scallop adductor muscle by glycosylated peptide, collagen peptide and blank control group 2+ -effect of ATPase enzyme activity.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
1 materials and methods
1.1 materials and reagents
Tilapia skin collagen peptide (model specification: CF-500-S-02) purchased from Henan Shengmeno biotechnology, inc.; anhydrous dextrose, guangdong Guanghua science and technology, inc.; ortho-phthalaldehyde (OPA), β -mercaptoethanol, shanghai mclin biochemistry science and technology ltd; bovine serum albumin, beijing solibao science and technology ltd; sodium Dodecyl Sulfate (SDS), tokyo kulai bock technologies ltd; acetonitrile, supelco, usa; acetonitrile and formic acid are mass spectra pure, and other reagents are analytical pure.
1.2 materials and reagents
Ultra-high performance liquid phase time-of-flight mass spectrometer, waters corporation, usa; fourier infrared spectrometer, BRUKER, germany; type FE28 pH meter, mettler-toledo instruments (shanghai) ltd; varioskan Flash full-automatic enzyme marking instrument, siemer heschel technologies ltd; XP205 model analytical balance, shunhua constant scientific instruments, inc., shanghai; QL-905 vortex mixer, haimen, leibel instruments manufacturing Co., ltd; DTT-A1000 electronic balance, china, inc.; SHJ-6AB magnetic stirring water bath, changzhou gold jar Liangyou Instrument Co., ltd; DSC-300C differential scanning calorimeter, nanjing Dazhan instrumentation Inc.
1.3 methods
1.3.1 preparation of the glycosylation product
Dissolving collagen peptide in pure water to prepare solutions with different concentrations (5, 6, 7, 8 and 9%), adding glucose with a certain proportion (the glycopeptide ratio is 1. Adjusting the pH value of the rest mixed solution to 8.0 by using 0.1mol/L sodium hydroxide and hydrochloric acid solution, placing the mixed solution in a magnetic stirring water bath kettle, and setting corresponding reaction time and reaction temperature. And after the reaction is finished, quickly cooling to room temperature to obtain a glycosylation product, and freeze-drying for later use. Preparing glucose solution with the same concentration as a glucose sample group, and freeze-drying for later use.
1.3.2 collagen peptide glycosylation Single factor experiment
The grafting Degree (Degree of Graft, DG) is used as a measurement index, a single-factor experiment is performed on the collagen peptide, the temperature of the glycosylation reaction is set to be 40, 50, 60, 70, 80 ℃, the reaction time is set to be 30, 60, 90, 120, 150min, the concentration of the collagen peptide is 5, 6, 7, 8, 9%, the glycopeptide ratio is 1.
1.3.3 orthogonal optimization collagen peptide glycosylation reaction process
On the basis of single factor, selecting reaction temperature, reaction time, collagen peptide concentration and glycopeptide ratio to carry out four-factor three-level L 9 (3 4 ) Orthogonal test, the design table is as follows, DG is used as an index, and the glycosylation reaction conditions are optimized through the orthogonal test to determine the optimal process conditions.
TABLE 1 glucose glycosylation reaction orthogonal test factor level coding table
1.3.4 determination of the degree of grafting
DG was measured according to the OPA method. Taking 80mg OPA sample in 2mL methanol, stirring to dissolve completely, adding 0.1mol/L borax and 20% SDS each 5mL, finally adding 200 μ L beta-mercaptoethanol, mixing well, adding pure water to constant volume of 100mL to obtain OPA reagent, and preparing at present. When determining DG, 4mL of OPA solution is taken, 200 microliter of glycosylation product is added and placed on a vortex instrument to be mixed uniformly, pure water is used as a control group, the reaction is carried out for 2min in a water bath kettle at 35 ℃ in a dark place, after the reaction is finished, the light absorption value of the glycosylation product is determined at 340nm, lysine is used as a standard curve, and DG is calculated according to the formula (1).
In the formula: DG is degree of grafting,%; a. The 0 The content of free amino groups before glycosylation reaction is mol/L; a. The 1 The content of free amino groups after glycosylation reaction is mol/L.
1.3.5 measurement of thermal hysteresis Activity
Evaluation of THA was performed by Differential Scanning Calorimetry (DSC). Dissolving a sample to be detected in pure water to prepare a solution of 10mg/mL, accurately transferring 10 mu L of the sample into a crucible by using a liquid transfer gun, and measuring the THA of the sample by taking bovine serum albumin as a control. After the instrument is stabilized, the temperature is reduced from 25 ℃ to-25 ℃ at the speed of 10 ℃/min, and then the temperature is increased to 25 ℃, so as to obtain the melting point (T) of the sample m ) And melt down break (Δ H) m ). Then, the temperature is reduced to-25 ℃ at the speed of 10 ℃/min, and the temperature is slowly increased at the speed of 3 ℃/min until the sample is in a solid-liquid mixed state, which is called as a retention temperature (T) h ) The temperature was maintained for 2min, the temperature was reduced to-25 ℃ at the same rate, and the initial crystallization temperature (T) of the sample was recorded 0 ) And crystallization break (Δ H) f ) THA and ice crystal content phi are calculated according to the formula (2) and the formula (3).
THA/℃＝T h -T 0 (2)
In the formula: THA is thermal hysteresis activity, deg.c; t is h The retention temperature, DEG C; t is 0 The initial crystallization temperature, DEG C; phi is the ice crystal content; Δ H m To melt down break; Δ H f To be a crystallization break.
1.3.4 Infrared Spectroscopy
Fourier transform-induced spectroscopy (FTIR) was performed. Weighing 100mg potassium bromide, grinding and drying, adding 1mg sample, mixing uniformly, grinding and tabletting. At 4000-400cm -1 Infrared spectral scanning is performed in the range with a resolution set at 4cm -1 。
1.3.4 Mass Spectrometry
Adopting a series quadrupole time-of-flight mass spectrum to carry out structure identification, setting a mobile phase A as ultrapure water (containing 0.1% formic acid), setting a mobile phase B as acetonitrile (containing 0.1% formic acid), and setting a chromatographic column: waters UPLC BEH C18 (2.1 mm. Times.50mm, 1.7 μm), column temperature: 30 ℃, flow rate: 0.2mL/min, the sample was prepared as a 0.1mg/mL solution and loaded with 10. Mu.L. Gradient elution conditions: 96% A → 50%;50% A → 0%;0% A → 96% A.
1.4 data processing
Each set of experiments was repeated 3 times to take the mean, and analyzed for variance using SPSS, with P <0.05 representing significant differences, plotted using Origin 2021.
2 results and analysis
2.1 Effect of extent of glycosylation on thermal hysteresis Activity
The glycosylation reaction degree of the collagen peptide and the glucose plays an important role in the biological activity of the glycosylation product, the glycosylation reaction degree of the collagen peptide can be evaluated through a DG value, and the thermal hysteresis activity of the glycosylation product can be evaluated through a THA value. Fixing the glycosylation reaction time and glycopeptide ratio, changing other reaction conditions, measuring the grafting degree and thermal hysteresis activity of glycosylation products obtained under different reaction conditions, and exploring the relationship between the glycosylation reaction degree and the thermal hysteresis activity. As shown in FIG. 1, the grafting degree of the glycosylation product is in positive correlation with the thermal hysteresis activity, and as the glycosylation product DG is increased, the THA is gradually increased, which proves that the larger the glycosylation reaction degree of the collagen peptide and the glucose is, the larger the thermal hysteresis activity of the product is.
2.2 collagen peptide glycosylation reaction Single factor test results
2.2.1 Effect of reaction temperature on glycosylated DG
Temperature is a key factor influencing the glycosylation reaction, the influence of different reaction temperatures on DG is shown in FIG. 2, and it can be seen from FIG. 2 that the DG results of glycosylation products obtained at different reaction temperatures are (27.73. + -. 0.72), (32.87. + -. 0.88), (36.37. + -. 0.07), (38.7. + -. 0.32), (32.21. + -. 1.12)%, respectively. DG increases gradually as the reaction temperature increases from 40 ℃ to 70 ℃. This is because the structure of the collagen peptide begins to become loose due to the increase of temperature, so that the free amino groups in the collagen peptide are exposed, and the peptide molecules and the sugar molecules move faster at higher temperature, so that the collision probability of the free amino groups and the carboxyl groups in the system is increased. However, DG tends to decrease at 70-80 deg.C, and it is presumed that the active site of glycosylation tends to saturate at 70 deg.C, the temperature is continuously increased, the reaction proceeds to the late stage, the sugar is degraded and the amino compound undergoes a series of reactions to form a late-stage compound, and DG begins to decrease.
2.2.2 Effect of reaction time on glycosylated DG
The collagen peptide was glycosylated at 70 ℃ for 30, 60, 90, 120, and 150min, respectively, and the results are shown in FIG. 3, where DG was (30.95. + -. 0.61), (34.43. + -. 0.61), (38.70. + -. 0.32), (35.66. + -. 0.57), and (33.45. + -. 1.07)%. The extent of glycosylation increases with increasing reaction time, peaking at 90min and then flattening out. This is because the collagen peptide undergoes high temperature hydrolysis at the early stage of the reaction to cause peptide chain breakage and expose more free amino groups, thereby promoting the progress of the glycosylation reaction, and as the reaction proceeds to 90min later, the glycosylation reaction ends and proceeds to the next stage, causing the amino groups to be consumed and the DG to begin to decrease.
2.2.3 Effect of collagen peptide concentration on glycosylated DG
The effect of different collagen peptide concentrations on DG is shown in FIG. 4, which shows that as the collagen peptide concentration increases, DG increases and then decreases, and when the concentration reaches 8%, the DG reaches the peak value (46.61 + -0.63)%. This is probably because the DG is increased because the free amino group in the system is increased by increasing the peptide concentration under the same reaction temperature and time, and the chance of condensation reaction with the free carboxyl group is increased. However, since the glucose content is fixed, that is, the carboxyl content is fixed, the glycosylation reaction tends to be saturated when the concentration of the collagen peptide reaches 8%, and the DG does not increase any more after the concentration exceeds 8%, and gradually tends to be balanced.
2.2.4 Effect of glycopeptide ratios on glycosylated DG
FIG. 5 shows the effect of glycopeptide ratio on the glycosylation product DG, which increases and then decreases as the ratio of glucose to collagen peptide increases. The glycopeptide ratio was 3. This is because when the sugar proportion in the system is low, the progress of the glycosylation reaction is promoted and the DG becomes high as the sugar content increases. When the sugar ratio is high, the viscosity of the solution in the system is increased, but the collision probability of free amino and carboxyl is hindered, and the generated glycosylation product is reduced.
2.3 glycosylation reaction orthogonal assay optimization
In order to further explore the optimal conditions of the glycosylation reaction, DG is taken as an evaluation index and L is taken as a reference on the basis of the single-factor test result 9 (3 4 ) Orthogonal table (code table is shown in table 1) optimizes the glycosylation reaction process conditions and results are shown in table 2. As can be seen from the observation of Table 2, the temperature, time, concentration and glycopeptide ratio affect the progress of the glycosylation reaction to varying degrees. According to the range (R) result, the influence of various factors on the glycosylation reaction is shown as follows: temperature of>Time>Concentration of>Glycopeptide ratios, i.e. reaction temperatures, have the greatest effect on the experiments. The optimal glycosylation process parameters obtained by orthogonal optimization are as follows: a. The 2 B 2 C 3 D 3 Namely, the reaction temperature is 70 ℃, the reaction time is 90min, the concentration is 9%, and the glycopeptide ratio is 4. This condition is not shown in the orthogonal table, and it was verified that the condition DG was (52.06. + -. 1.50)%, which is superior to each test number in the orthogonal table.
TABLE 2 orthogonal design and results of collagen peptide glycosylation reactions
2.4 Effect of glycosylation on the thermal hysteresis Activity of collagen peptides
Collagen peptides with anti-freeze activity can specifically lower the freezing point of a solution without changing its melting point, which results in a difference between the freezing point and the melting point of the solution, which is called THA. In the present study, the THA of the collagen peptide before and after the glycosylation reaction was studied by using a DSC method, and with bovine serum albumin as a reference, fig. 6 shows DSC heat flow graphs of bovine serum albumin, glucose, collagen peptide, a collagen peptide-glucose mixture, and a glycosylation product, and THA and Φ under Th were calculated based on the DSC curve, and the results are shown in table 3.
The frozen sample solution is heated to Th, i.e. kept in a solid-liquid coexisting state, and then immediately cooled to-25 ℃ to reach a complete crystallization state. At the same Th (0.1 ℃), the exothermic peak immediately appears when the temperature drops again, due to the lack of anti-freeze activity of bovine serum albumin, there is almost no THA (0.3 ℃), whereas the exothermic peaks of glucose, collagen peptides, collagen peptide-glucose mixtures and glycosylated products appear delayed, THA being 1.2, 1.1, 2.3, 0.4 ℃ respectively, THA being "moderately active" anti-freeze protein in the range of 0.2-0.5 ℃ and "active" anti-freeze protein in the range of 0.6-6 ℃, collagen peptides, collagen peptide-glucose mixtures and glycosylated products all belonging to the "extremely active" anti-freeze peptides. After glycosylation reaction, the THA of the collagen peptide is increased by 1.1 ℃, which is increased by 47.8% compared with that before reaction, and the phi is reduced by 29.94%. We find that the higher the THA of the sample, the lower its Φ, i.e. there is a linear negative correlation between THA and Φ. It is speculated that the solution viscosity increases because of the glycosylation reaction, thereby decreasing Φ of the sample and increasing THA. In addition, a large amount of hydroxyl can be introduced in glycosylation reaction, the antifreeze peptide is combined with water in ice crystals mainly through hydrogen bond interaction to achieve the effect of inhibiting the formation of the ice crystals, and the existence of the large amount of hydroxyl can increase the capability of combining the antifreeze peptide with the ice crystals in a hydrogen bond form, so that the THA of a glycosylation product is enhanced.
TABLE 3 THA and φ for the glycosylation product and control samples
2.5 Infrared Spectroscopy
FTIR spectrum is an effective technology for detecting glycosylation reaction, and the position or the intensity of an absorption peak of collagen peptide is changed after the collagen peptide is modified by glucose. FIG. 7 shows FTIR spectra of glucose (a), collagen peptide (b), collagen peptide-glucose mixture (c) and glycosylation product (d) at 1700-1600cm -1 The absorption peak (amide I) of (A) mainly represents C = O stretching vibration, 1600-1500cm -1 The absorption peak (amide II) mainly represents C-N stretching vibration and N-H angle changing vibration, and the collagen peptide is glycosylated and then positioned at 1662.6cm -1 The absorption peak of (2) is blue-shifted to 1631.7cm -1 At 1562.3cm -1 Red-shifted to 1587.3cm -1 This demonstrates that the secondary structure of the collagen peptide is changed and glucose is bound to the C = O, C-N and N-H groups of the collagen peptide by hydrophobic interactions. The glycosylation product is 3600-3100cm compared to the collagen peptide -1 (O-H stretching vibration) and 1200-950cm -1 The absorption peak positions in the range of (C-O stretching vibration and O-H angle changing vibration) are blue-shifted and the strength is increased, which is the obvious characteristic of forming glycosylation products, a great amount of C-OH is generated after the glycosylation reaction of the collagen peptide and the glucose, the successful grafting of the glucose on the collagen peptide is proved, and the glycosylation products contain more-OH groups through covalent bonds. the-CH stretching vibration frequency of the alkane is between 2960 and 2850cm -1 In the range of 1410-1260cm -1 The nearby absorption peak represents the O-H variable angle vibration, and the glycosylation product is at 2929.8, 2823.7 and 1354.0cm -1 A new peak appears, which represents that the collagen peptide may generate an R-CH-OH structure after glycosylation reaction. The specific structure in the antifreeze peptide molecule can form hydrogen bond with water molecule in ice crystal, thereby inhibiting the hydrogen bond interaction formed between ice and water. Under low temperature environment, free water is firstly frozen, and at the moment, the-OH group in the glycosylation product and the water molecule in the ice crystal form a stable hydrogen bond structure, thereby inhibiting the generation of the ice crystal and enhancing the THA.
The secondary structure composition of the collagen peptide and the glycosylation product in the amide I band was calculated by Fourier self-deconvolution and Gaussian function, and as a result, as shown in FIG. 8A and B, the secondary structure of the collagen peptide mainly consists of beta-turn and beta-sheet, which account for 40.00% and 60.00%, respectively, the secondary structure of the collagen peptide-glucose mixture and the collagen peptide is not very different, the beta-turn accounts for 59.15%, and the beta-sheet accounts for 40.85%. After the glycosylation reaction, the beta-turn is reduced to 4.82% and the beta-sheet is increased to 95.18%. This result suggests that the interaction between glucose and collagen peptide affects the secondary structure of collagen peptide. It is speculated that the increased beta-sheet may advance the binding of the glycosylation product to the ice crystal, thereby enhancing the THA of the antifreeze peptide.
2.6 Mass Spectrometry
Mass spectrometry is the core means for determining the relative molecular masses of proteins and collagen peptides, and the structural changes of the collagen peptides are analyzed by molecular weight transfer. Fig. 9 is a Base peak ion chromatogram (BPI) of tilapia skin collagen peptide and glycosylation product, wherein the chromatographic peak completely flows out within 9min, the collagen peptide ion peak is obviously weakened within 0-3min after glycosylation reaction (fig. 10), and the ion peak number is increased and the relative abundance is enhanced within 5.5-9min (fig. 11). The MS spectrum analysis of the collagen peptide and the glycosylation product was performed, and the results are shown in fig. 10 and fig. 11, which correspond to the BPI chart, the mass-to-charge ratio and peak intensity of the collagen peptide and the glycosylation product after the glycosylation reaction were changed, and the m/z value of the collagen peptide was shifted from 300-700 to around 400-800. This is probably due to the fact that glucose and tilapia skin collagen peptide have a grafting reaction, and the mass-to-charge ratio of the glucose and tilapia skin collagen peptide is shifted.
Theoretically, m/z would increase 162 when a peptide is modified with one glucose, and the mass to charge ratio would also increase exponentially if a peptide is modified with two or three glucose. The glycosylation reaction of glucose and collagen peptide is not a simple molecular weight addition, and the glucose molecule may carry or lose part of the ions in the process. Taking the two peaks with higher response values in the glycosylation product of fig. 11 as an example, we speculate that the collagen peptide with mass-to-charge ratio of 318.30 binds to one glucose and removes two O ions, resulting in the glycosylation product with mass-to-charge ratio of 448.27. Collagen peptide with mass to charge ratio of 274.28 was combined with 3 glucose molecules and two H ions to produce a glycosylation product with mass to charge ratio of 762.42.
2.7 verification of the antifreeze Properties of glycosylated peptides
2.7.1 Glycopeptide antifreeze Effect on Catalase
Adding ultrapure water into the glycosylation product powder to prepare a solution with the concentration of 10-50 mg/mL. Performing molecular sieve chromatography by using a sephadex chromatographic column according to the following molecular sieve chromatography conditions: taking ultrapure water as a mobile phase equilibrium chromatographic column, and loading the sample according to 1-5% of the volume of a column bed at a flow rate: 1mL/min, detection wavelength: 220nm; collecting active peaks, and freeze-drying to obtain glycosylated peptide powder.
Diluting the concentration of the catalase liquid (more than or equal to 20 000unit/g) to 0.1%, adding 3mg/mL glycosylated peptide into the diluted catalase liquid, comparing tilapia collagen peptide with blank treatment, measuring the initial enzyme activity of the catalase which is not frozen at 240nm, and recording the light absorption value once every 1min for 5min in total. The reduction of A240 by 0.1 in 1min is 1 enzyme activity unit (U). And (3) putting the catalase liquid into a refrigerator with the temperature of-20 ℃ for freezing for 12h, then quickly thawing, and measuring the residual enzyme activity of the hydrogen peroxide by the following formula, wherein the residual enzyme activity can express the anti-freezing activity.
In the formula: Δ 240 is the decreased enzyme activity in 5 minutes; t is 5min.
The result is shown in fig. 12, compared with the collagen peptide sample and the blank treatment group, the glycosylated peptide treatment group has the highest activity of the residual hydrogen peroxide enzyme, and the result proves that the glycosylated peptide has a remarkable anti-freezing effect on catalase.
2.7.2 protective effect of glycosylated peptide on scallop adductor muscle frozen storage
2.7.2.1, cleaning the scallop adductor muscle, adding glycosylated peptide according to 2 percent of the adductor muscle mass, comparing tilapia collagen peptide with blank treatment, putting the treated adductor muscle into a refrigerator for frozen storage, and evaluating the frozen storage protection effect of the adductor muscle after frozen storage. And (4) sucking the completely thawed sample to dry the surface water by using filter paper, weighing, and determining the thawing water loss rate according to the formula (6).
The results are shown in FIG. 13.
2.7.2.2 weighing 1 to 3g of unfrozen scallop adductor muscle, adding 10 to 30mL of phosphate buffer solution (0.05 mol/L, pH 7.0 to 8.0), grinding and extracting for 5 to 15min, centrifuging for 10 to 15min at the temperature of 4 ℃ and at the speed of 10000r/min, removing supernatant, adding phosphate buffer solution again, grinding, extracting and centrifuging, and repeating for three times. Adding 10-30 mL of NaCl solution (0.6 mol/L, pH 7.0-8.0) into the precipitate obtained after centrifugation, uniformly mixing, extracting at 4 ℃ for 10-18 h, centrifuging the obtained extracting solution at 4 ℃ and 10000r/min for 10-15 min, and determining the salt-soluble protein content of the obtained supernatant, namely the salt-soluble protein solution, by adopting a Folin kit. The results are shown in FIG. 14.
2.7.2.3 taking 0.5-1 mL of the salt-soluble protein solution extracted in the step, adding 4.0-5.0 mL of 0.2mol/L Tris-HCl buffer solution and 0.1-0.2% of dithio-2-nitrobenzoic acid solution, uniformly mixing, heating in a water bath at 40 ℃ for 20-30 min, and measuring the light absorption value at 412 nm. The total mercapto content (mol/L) was calculated according to equation (7).
In the formula: c c The total content of sulfydryl (mol/L); a is the absorbance of the sample at 412 nm; d is the dilution multiple of the sample; epsilon is a molar extinction coefficient 13600/(mol. L) -1 Cm); and c is the concentration of the sample salt soluble protein. The results are shown in FIG. 15.
2.7.2.4 according to Ga 2+ Determination of Ga by ATPase enzyme kit 2+ -ATPase enzyme activity and is calculated according to the formula (8).
In the formula: a is the light absorption value of a sample measuring tube; b is the light absorption value of the control tube; c is the light absorption value of a standard tube; d is blank tube light absorption value. The results are shown in FIG. 16.
As can be seen from the combination of FIGS. 13-16, the glycosylated collagen peptide has main anti-freezing indexes such as thawing water loss rate, salt-soluble protein content, total thiol content, and salt-soluble protein Ca for the frozen scallop column 2+ ATPase viability was either very significantly or significantly better than collagen peptide treated and placebo; the collagen peptide treated group was significantly superior to the blank control group. The experimental results show that the tilapia skin collagen peptide has certain antifreeze activity, and the antifreeze activity of the tilapia skin collagen peptide is obviously improved after glycosylation.
In conclusion, the method improves the glycosylation anti-freezing activity (thermal hysteresis activity) of tilapia collagen peptide by using glucose, and the glycosylation DG reaches (52.06 +/-1.50)%, under the optimized process parameters; the glycosylated collagen peptide THA and phi both have significant changes, and belong to the 'hyperactive' antifreeze peptide; the collagen peptide is combined with glucose molecules to enlarge the m/z of the collagen peptide; after glycosylation, most of beta-turns in the collagen peptide are converted into beta-sheets, the secondary structure of the collagen peptide is changed, and a large number of hydroxyl groups are introduced, so that the binding capacity of the collagen peptide and water molecules in ice crystals is enhanced, and the THA is increased. The antifreeze test shows that the glycosylation obviously improves the antifreeze activity of tilapia skin collagen. The research result provides important early-stage data for improving the heat retention activity of the tilapia mossambica skin collagen peptide by glycosylation and developing a green antifreeze based on the tilapia mossambica skin collagen peptide, and has important reference significance for developing other aquatic collagen antifreeze sources.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. A method for preparing fish skin collagen antifreeze peptide with improved glycosylation is characterized by comprising the following steps:
adding monosaccharide into the fish skin collagen peptide solution, stirring to obtain a glycopeptide mixture, then adjusting the pH of the glycopeptide mixture, stirring at the temperature of 50-80 ℃ for reaction, and obtaining the fish skin collagen antifreeze peptide improved by glycosylation after the reaction is finished.
2. The method according to claim 1, wherein the fish skin collagen peptide solution is contained in an amount of 7 to 9% by mass.
3. The method according to claim 1, wherein the mass ratio of the fish skin collagen peptide solution to the monosaccharide is 1: (1-4); the monosaccharide includes glucose, xylose or galactose.
4. The preparation method according to claim 1, wherein the stirring is performed at normal temperature for 10-40min; the pH is adjusted to 7.5-8.5; the stirring reaction time is 60-120min.
5. The method according to claim 1, wherein the anti-freeze peptide of fish skin collagen modified by glycosylation is obtained by separation and purification after the reaction is completed.
6. The method of claim 5, wherein the separation purification is: adding water into the product after the reaction is finished to prepare a solution with the concentration of 10-50mg/mL, sending the solution to a sephadex chromatographic column for separation and purification, collecting an active peak, and freeze-drying; wherein, the chromatographic conditions for separation and purification are as follows: the mobile phase is ultrapure water, the sample loading amount is 1-5% of the volume of the column bed of the sephadex chromatographic column, the flow rate is 1mL/min, and the detection wavelength is 220nm.
7. The method of claim 1, wherein the fish skin collagen peptide comprises tilapia skin collagen peptide.
8. A fish skin collagen antifreeze peptide obtained by the method of any one of claims 1 to 7.
9. Use of the fish skin collagen antifreeze peptide according to claim 8 in the preparation of a food antifreeze.
10. A food antifreeze comprising the fish skin collagen antifreeze peptide according to claim 8.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8604002B1 (en) * | 2010-06-23 | 2013-12-10 | University Of Notre Dame Du Lac | Saccharide antifreeze compositions |
| CN103805668A (en) * | 2014-03-05 | 2014-05-21 | 山东瀚龙生物科技有限公司 | Sugar modification-based fish skin anti-freeze protein polypeptide and preparation method thereof |
| JP2017043551A (en) * | 2015-08-25 | 2017-03-02 | 国立研究開発法人産業技術総合研究所 | Peptide for biomaterial protection |
| CN107163132A (en) * | 2017-06-23 | 2017-09-15 | 福州大学 | A kind of preparation method and application based on glucan-modified antifreeze glycopeptide |
| CN107176992A (en) * | 2017-06-23 | 2017-09-19 | 福州大学 | A kind of preparation method and application for the antifreeze glycopeptide modified based on FOS |
| CN114751958A (en) * | 2022-05-17 | 2022-07-15 | 哈尔滨腾凝科技有限公司 | Method for extracting polypeptide inhibiting DPP-4 activity from salmon skin |
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Patent Citations (6)
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|---|---|---|---|---|
| US8604002B1 (en) * | 2010-06-23 | 2013-12-10 | University Of Notre Dame Du Lac | Saccharide antifreeze compositions |
| CN103805668A (en) * | 2014-03-05 | 2014-05-21 | 山东瀚龙生物科技有限公司 | Sugar modification-based fish skin anti-freeze protein polypeptide and preparation method thereof |
| JP2017043551A (en) * | 2015-08-25 | 2017-03-02 | 国立研究開発法人産業技術総合研究所 | Peptide for biomaterial protection |
| CN107163132A (en) * | 2017-06-23 | 2017-09-15 | 福州大学 | A kind of preparation method and application based on glucan-modified antifreeze glycopeptide |
| CN107176992A (en) * | 2017-06-23 | 2017-09-19 | 福州大学 | A kind of preparation method and application for the antifreeze glycopeptide modified based on FOS |
| CN114751958A (en) * | 2022-05-17 | 2022-07-15 | 哈尔滨腾凝科技有限公司 | Method for extracting polypeptide inhibiting DPP-4 activity from salmon skin |
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| 林昊: "草鱼鱼肉蛋白酶解物及糖基化物的制备以及提高冻藏鱼糜稳定性的研究", 《中国优秀硕士学位论文全文数据库（电子期刊）工程科技辑》, no. 2022 * |
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