Enzyme crosslinking fish gelatin-chitosan oligosaccharide composite microgel as well as preparation and application thereof

Abstract

The invention discloses enzyme crosslinking fish gelatin-chitosan oligosaccharide composite microgel as well as preparation and application thereof, and belongs to the technical field of food processing. After the fish gelatin and the chitosan oligosaccharide in different proportions are combined, enzymatic cross-linking is performed with the glutamine transaminase in different proportions, so that the uniformity and the stability of the obtained composite microgel under the optimal saccharification and enzyme cross-linking proportion are greatly improved, the functional characteristics of the fish gelatin are effectively improved, and the research and development of the gelatin microgel and the application of stable emulsion are promoted.

Description

Technical Field

[0001] The invention relates to preparation and application of enzyme cross-linked fish gelatin-chitosan oligosaccharide composite microgel, belonging to the technical field of food processing. Background Art

[0002] Fish gelatin (FG) is a natural polymer obtained by partial hydrolysis of fish skin, fish bones, fish scales and other raw materials. It is considered to be a potential and excellent substitute for mammalian gelatin because of its similar functional properties to mammalian gelatin. However, due to the difference in the types of raw materials, the proline and hydroxyproline content of fish gelatin is lower than that of mammalian gelatin, resulting in weaker functional and structural properties of fish gelatin than mammalian gelatin, which limits the production and application of fish gelatin. Therefore, the problem of improving the functional properties of fish gelatin needs to be solved urgently.

[0003] Numerous studies have shown that the functional properties of proteins can be significantly improved after specific chemical or physical treatments, including ultrasound, enzymatic hydrolysis, enzymatic cross-linking, saccharification, and binding with polyphenols. However, a single treatment method has a poor effect on improving the functional properties of proteins.

[0004] Chiooligosaccharide (COS) is a rare natural alkaline oligosaccharide in nature. As a hydrolysis product of chitosan, it has similar dissociation properties, as well as low hemolytic activity and low cytotoxicity. In addition, the short chain structure and free amino acids of chitosan oligosaccharide make it have better biological activity, easy absorption and utilization by the human body, and good solubility in a wider pH range.

[0005] Glutamine transferase (TGase) is a naturally occurring enzyme that exists in almost all organisms. TG enzyme can cause covalent bonding of proteins (or polypeptide chains) and has the property of cross-linking proteins. TG enzyme can catalyze the γ-carboxamide group in macromolecular proteins and various amino acids as an acyl donor to undergo amide transfer reaction with different acyl acceptors, catalyzing protein cross-linking. Cross-linked proteins form a dense and cross-linked three-dimensional network structure, thereby improving the functional properties of proteins.

[0006] Microgels are three-dimensional network structures with a size of micrometers or less formed by cross-linking between biomacromolecules to retain a large number of solvent molecules. The techniques for preparing microgels are generally divided into two types: intermolecular association and mechanical methods. Intermolecular association is divided into self-association and complex association. Self-association is the simplest method, which is generally formed by the aggregation of a single biopolymer under appropriate conditions. By adjusting the temperature, pH and ionic strength, it can form a microgel of appropriate size. Different types of polymers can be cross-linked by selecting various cross-linking substances to form microgels. Association and complexation are the formation of microgels through the interaction between mixed biopolymers.

[0007] After fish gelatin and chitosan oligosaccharide are combined, the fish gelatin-chitosan oligosaccharide microgel formed by enzymatic cross-linking with glutaminase can be used as a series of emulsion raw materials with strong stability and high added value. Different proportions of fish gelatin and chitosan oligosaccharide, as well as different concentrations of glutaminase added, make the microgel show different uniformity and stability. Under this technical research, the optimal conditions and methods for preparing uniform and stable fish gelatin-chitosan oligosaccharide microgels can be provided, promoting the improvement of the functional properties of fish gelatin, the research and development of microgels, and the application of stable emulsions. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and design an enzyme-crosslinked fish gelatin-chitosan oligosaccharide composite microgel to solve the problems of weak functional properties of fish gelatin and limitations in production and application.

[0009] In order to achieve the above object, the technical solution of the present invention is: A method for preparing enzyme-crosslinked fish gelatin-chitosan oligosaccharide composite microgel comprises the following steps: Step 1: Dissolve 3.0 g of fish gelatin.

[0010] Step 2: Mix a certain amount of fish gelatin and chitosan oligosaccharide, add glutaminase (TGase) solution for enzymatic cross-linking after mixing, and obtain composite microgel.

[0011] Step 3: Add deionized water, then shear the composite microgel, and then homogenize it through a high-pressure homogenizer to obtain a fish gelatin-chitosan oligosaccharide composite microgel dispersion.

[0012] Wherein, in step 1, the solvent for dissolving fish gelatin is 100 mL of ultrapure water.

[0013] Preferably, in step 1, the dissolution conditions are: the magnetic stirrer speed is 130 rpm, the stirring time is 60 min, and the dissolution is completed in a constant temperature shaking water bath at 37°C.

[0014] Wherein, in step 2, the pH of the fish gelatin solution and the chitosan oligosaccharide solution is adjusted to 7.0 with 0.2 mol / L NaOH.

[0015] Wherein, in step 2, the mixing ratio of fish gelatin solution and chitosan oligosaccharide is set to 5:1.

[0016] Wherein, in step 2, the amount of TGase added to the TGase solution is 20 U / g gelatin.

[0017] Preferably, in step 2, the concentration of fish gelatin in the reaction system is 30 g / L.

[0018] Preferably, in step 2, the conditions for enzymatic cross-linking are: reaction time 3 h, and enzymatic cross-linking is completed in a 37° C. constant temperature shaking water bath.

[0019] Wherein, in step three, the volume of deionized water added is twice that of the composite microgel.

[0020] Preferably, in step three, the shearing conditions are: high-speed shearing machine speed 12000 r / min, shearing time 2 min.

[0021] Preferably, in step three, the homogenization conditions are: high pressure homogenizer pressure 50 MPa, homogenization time 2 min.

[0022] 1. Beneficial effects achieved by the present invention: The present invention provides a method for preparing enzyme-crosslinked fish gelatin-chitosan oligosaccharide composite microgel. Fish gelatin is combined with chitosan oligosaccharide, and TG is used to react with fish gelatin to produce protein-like crosslinking, and amino acids are transferred from one peptide chain to another peptide chain to achieve modification of the peptide chain. Compared with unmodified whey protein, the small molecular weight peptide chain of enzyme-crosslinked whey protein is transformed into a high molecular weight peptide chain, which is enhanced and its functional properties are greatly improved.

[0023] 2. Through experiments, it was found that the fish gelatin solution and the chitosan oligosaccharide solution were mixed in a ratio of 5:1, and enzymatically cross-linked with glutaminase added at an amount of 20U / g fish gelatin. The fish gelatin-chitosan oligosaccharide composite microgel dispersion obtained after adding water, shearing and homogenization had better uniformity and stability.

[0024] 3. The composite microgel can be used for emulsion stabilization applications. The method is easy to operate, low-cost, and has mild processing conditions, and is suitable for the industrial production and application of fish gelatin raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the particle size diagram of fish gelatin-chitooligosaccharide composite microgel; Figure 2 is the PDI graph of fish gelatin-chitooligosaccharide composite microgel; Figure 3 It is the Zeta particle size diagram of fish gelatin-chitooligosaccharide composite microgel; Figure 4 It is the ferric iron reducing power diagram of fish gelatin-chitooligosaccharide composite microgel; Figure 5 This is the DHHP free radical scavenging rate graph of fish gelatin-chitooligosaccharide composite microgel; Figure 6This is the particle size diagram of the composite microgel under the conditions of different ratios of fish gelatin and chitosan oligosaccharide and 20 U / gFG of glutaminase addition. Different letters ac in the figure represent significant differences. Figure 7 This is the PDI graph of the composite microgel under the conditions of different ratios of fish gelatin and chitosan oligosaccharide and 20 U / gFG of glutaminase addition. Different letters ac in the graph represent significant differences. Figure 8 This is the Zeta particle size diagram of the composite microgel under the conditions of different ratios of fish gelatin and chitosan oligosaccharide and 20 U / gFG of glutaminase addition. Different letters in the figure represent significant differences. Fig. 9 This is the ferric iron reduction power diagram of the composite microgel under the conditions of different ratios of fish gelatin and chitosan oligosaccharide and 20 U / gFG of glutaminase addition. Different letters ac in the figure represent significant differences. Fig.10 This is the DHHP free radical scavenging rate of the composite microgel under the conditions of different ratios of fish gelatin and chitosan oligosaccharide and 20 U / gFG of glutaminase addition. Different letters in the figure represent significant differences. Fig.11 This is the particle size diagram of the composite microgel under the conditions of different amounts of glutaminase added and a fish gelatin and chitosan oligosaccharide addition ratio of 5:1. Different letters ac in the figure represent significant differences. Fig.12 This is the PDI graph of the composite microgel under the conditions of different amounts of glutaminase added and a fish gelatin and chitosan oligosaccharide addition ratio of 5:1. Different letters ac in the graph represent significant differences. Fig.13 This is the Zeta potential diagram of the composite microgel under the conditions of different amounts of glutaminase added and a fish gelatin and chitosan oligosaccharide addition ratio of 5:1. The different letters a and b in the figure represent significant differences. Fig.14 This is the ferric iron reducing power diagram of the composite microgel under the conditions of different addition amounts of glutaminase and the addition ratio of fish gelatin and chitosan oligosaccharide being 5:1. Different letters ad in the figure represent significant differences. Fig.15 This is the DHHP free radical scavenging rate of the composite microgel under the conditions of different amounts of glutaminase added and a fish gelatin and chitosan oligosaccharide addition ratio of 5:1. The different letters a and b in the figure represent significant differences. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0027] Example 1: Fish gelatin dissolution pretreatment.

[0028] 3.0 g of fish gelatin was dissolved in 100 mL of ultrapure water and fully dissolved by stirring (130 rpm) in a 37°C constant temperature shaking water bath for 60 min.

[0029] Example 2: Preparation of composite microgels by glycosylation and enzymatic cross-linking treatment.

[0030] A certain amount of fish gelatin solution (pH 7.0) and chitosan oligosaccharide solution (pH 7.0) were mixed in a ratio of 5:1. After mixing, glutaminase transfer solution (glutaminase addition amount is 20 U / g gelatin) was added to make the fish gelatin concentration in the reaction system 30 g·L-1. The reaction was shaken in a constant temperature water bath at 37℃ for 3 h.

[0031] Example 3: Preparation of composite microgel dispersion Two volumes of deionized water were added, followed by shearing at 12,000 r / min for 2 min using a high-speed shearing machine, and then homogenizing at a pressure of 50 MPa for 2 min using a high-pressure homogenizer to obtain a fish gelatin-chitooligosaccharide (FG-COS) microgel dispersion.

[0032] Depend on Figure 1-Figure 5 As shown in the figure, under the conditions of different ratios of fish gelatin and chitosan oligosaccharide and different addition amounts of glutaminase, compared with the blank group without saccharification and enzyme cross-linking treatment, saccharification and enzyme cross-linking modification can improve the uniformity, stability and antioxidant properties of fish gelatin to a certain extent. When the ratio of fish gelatin to chitosan oligosaccharide is 5:1 and the addition amount of glutaminase is 20 U / gFG, the uniformity, stability and antioxidant properties of the composite microgel are significantly improved. The composite microgel prepared under this condition has good functional properties, providing an effective method for improving the functional properties of fish gelatin.

[0033] Comparative Example 1: Different ratios of fish gelatin and chitosan oligosaccharide, and 20 U / gFG of glutaminase added 3.0 g of fish gelatin was dissolved in 100 mL of ultrapure water and fully dissolved by stirring (130 rpm) in a 37°C constant temperature shaking water bath for 60 min.

[0034] Then, a certain amount of fish gelatin solution (pH 7.0) was mixed with chitosan oligosaccharide solution (pH 7.0) at a ratio of 1:1, 2:1, 5:1, and 10:1, and then glutaminase transfer solution (glutaminase addition amount was 20 U / g fish gelatin) was added to make the fish gelatin concentration in the reaction system 30 g / L. The reaction was shaken in a constant temperature water bath at 37°C for 3 h.

[0035] Two times the volume of deionized water was added, followed by shearing for 2 min using a high-speed shearing machine at 12,000 r / min, and then homogenizing for 2 min using a high-pressure homogenizer at a pressure of 50 MPa to obtain a fish gelatin-chitooligosaccharide composite microgel dispersion.

[0036] Figure 6-8 The following are the particle size, PDI and Zeta potential diagrams of the composite microgels under different ratios of fish gelatin and chitosan oligosaccharides and 20 U / gFG of glutaminase addition. It can be seen that when the ratio of fish gelatin and chitosan oligosaccharides is 5:1, the particle size PDI of the composite microgel is lower than that under other ratios, the absolute value of Zeta potential is larger, and the stability and uniformity are better. Fig. 9 and Fig.10 It can be seen that the composite microgel under this condition has higher ferric iron reducing power and free radical scavenging rate and has better antioxidant properties.

[0037] In summary, when the ratio of fish gelatin to chitosan oligosaccharide is 5:1, the functional properties of the composite microgel are better.

[0038] Comparative Example 2: Different amounts of glutaminase added, fish gelatin and chitosan oligosaccharide added in a ratio of 5:1 3.0 g of fish gelatin was dissolved in 100 mL of ultrapure water and fully dissolved by stirring (130 rpm) in a 37°C constant temperature shaking water bath for 60 min.

[0039] Then, a certain amount of fish gelatin solution (pH 7.0) and chitosan oligosaccharide solution (pH 7.0) were mixed at a ratio of 5:1, and then glutaminase transfer solution (glutaminase addition amount was 0, 10, 20, 30 U / g fish gelatin) was added to make the fish gelatin concentration in the reaction system 30 g / L. The reaction was shaken in a constant temperature water bath at 37°C for 3 h.

[0040] Two times the volume of deionized water was added, followed by shearing for 2 min using a high-speed shearing machine at 12,000 r / min, and then homogenizing for 2 min using a high-pressure homogenizer at a pressure of 50 MPa to obtain a fish gelatin-chitooligosaccharide composite microgel dispersion.

[0041] Figure 11-13The particle size, PDI and Zeta potential of the composite microgels are shown in Figure 1. The particle size, PDI and Zeta potential of the composite microgels are shown in Figure 1. The particle size, PDI and Zeta potential of the composite microgels are shown in Figure 1. The particle size, PDI and Zeta potential of the composite microgels are shown in Figure 1. The particle size, PDI and Zeta potential of the composite microgels are shown in Figure 1. The particle size, PDI and Zeta potential of the composite microgels are shown in Figure 1. The particle size, PDI and Zeta potential of the composite microgels are shown in Figure 1. Fig.14 and Fig.15 It can be seen that the composite microgel under this condition has higher ferric iron reducing power and free radical scavenging rate and has better antioxidant properties.

[0042] In summary, when the addition amount of glutaminase was 20 U / gFG, the functional properties of the composite microgel were better.

[0043] Test methods 1. Determination of particle size and Zeta potential: The particle size and Zeta potential of the sample were analyzed using a nanoparticle size and Zeta potential analyzer. The particle mass fraction was diluted to 0.5% with ultrapure water, and 1 mL of the sample was slowly added to the sample pool to prevent the generation of bubbles.

[0044] 2. DPPH free radical scavenging rate determination The scavenging ability of different samples on DPPH free radicals was determined and improved. Briefly, 1.2×10 -4 mol / L DPPH solution, and then 2 mL of sample solution (0.5 mg / mL, dissolved in ultrapure water) was added to 2 mL of DPPH ethanol solution. The mixture was then reacted in the dark at room temperature for 1 h, and the absorbance was measured at 517 nm using a UV-visible spectrophotometer.

[0045] DPPH free radical scavenging activity (%) The calculation formula is as follows: DPPH free radical scavenging rate % = [A0-(A2-A1)] / A0 × 100% Where A2 is the absorbance value of the sample, A1 is the absorbance value of the control group (ethanol is added instead of DPPH), and A0 is the absorbance value of the blank group (DPPH is added instead of the sample).

[0046] 3. Determination of reducing power 2, 4, 6, 8, 10 mg / mL of FG, COS, and GC-MRPs were prepared respectively. 1 mL of sample solution was fully mixed with 2.5 mL of PBS buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide K3[Fe(CN)6], and then kept in a 50 ℃ water bath for 20 minutes, and then quickly placed in cold water to cool. Subsequently, 2.5 mL of 10% trichloroacetic acid (protected from light) was added and mixed. 2.5 mL was added to 2.5 mL of deionized water and 0.5 mL of 0.1% FeCl3 solution. After standing at room temperature for 10 minutes, A700 nm was measured and recorded as A. The blank group was replaced with 1 mL of deionized water and recorded as A0. The reducing power is positively correlated with the absorbance value, and the absorbance value A-A0 is finally used to represent the reducing power.

[0047] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An enzyme-crosslinked fish gelatin-chitosan oligosaccharide composite microgel, characterized in that: The preparation method thereof comprises the following steps: S1, dissolving fish gelatin; S2, mixing fish gelatin and chitosan oligosaccharide, adding glutaminase (TGase) solution to perform enzymatic cross-linking to obtain composite microgel; S3, adding deionized water, then shearing the composite microgel, and homogenizing it with a high-pressure homogenizer to obtain a fish gelatin-chitosan oligosaccharide composite microgel dispersion.

2. The enzyme-crosslinked fish gelatin-chitosan oligosaccharide composite microgel according to claim 1, characterized in that: Possess at least one of the following characteristics: In step S1, the amount of fish gelatin is 3.0 g, and the solvent for dissolving the fish gelatin is 100 mL of ultrapure water; In step S1, the dissolution conditions are: the magnetic stirrer speed is 130 rpm, the stirring time is 60 min, and the dissolution is completed in a constant temperature shaking water bath at 37°C.

3. The enzyme-crosslinked fish gelatin-chitosan oligosaccharide composite microgel according to claim 1, characterized in that: Possess at least one of the following characteristics: In step S2, the pH of the fish gelatin solution and the chitosan oligosaccharide solution is adjusted to 7.0 with 0.2 mol / L NaOH; In step S2, the mixing ratio of fish gelatin solution and chitosan oligosaccharide is set to 5:1; In step S2, the amount of TGase added to the TGase solution is 20 U / g gelatin; In step S2, the concentration of fish gelatin in the reaction system is 30 g / L; In step S2, the conditions for enzymatic cross-linking are: reaction time 3 h, and enzymatic cross-linking is completed in a 37° C. constant temperature shaking water bath.

4. The enzyme-crosslinked fish gelatin-chitosan oligosaccharide composite microgel according to claim 1, characterized in that: Possess at least one of the following characteristics: In step S3, the volume of deionized water added is twice that of the composite microgel; In step S3, the shearing conditions are: high-speed shearing machine speed 12000 r / min, shearing time 2 min; In step S3, the homogenization conditions are: high pressure homogenizer pressure 50 MPa, homogenization time 2 min.

5. Use of the enzyme-crosslinked fish gelatin-chitosan oligosaccharide composite microgel according to any one of claims 1 to 4 in preparing food.

6. The use according to claim 5, characterized in that: The food is at least one of dairy products, fresh food, air-dried food, freeze-dried food, compressed candy, solid beverage, liquid beverage, canned food or salted food.

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