A method for preparing molten globule protein-fish oil gel emulsion

Abstract

The present invention provides a method for preparing a molten globule protein-fish oil gel emulsion. The method places myofibrillar protein in a pH-neutral environment, and after homogenization and ultrasound, mixes it with fish oil gel to obtain the product; wherein, the ultrasound is ultrasound at 140 to 160 W for 110 to 130 seconds. The present invention can form a molten globule protein under pH-neutral, normal temperature and pressure conditions through simple ultrasound, emulsification and other operations, avoiding the cumbersome problems of residual acid and alkali reagents and high temperature and high pressure treatment in the existing methods, and provides a gentle, simple, safe and environmentally friendly method for forming molten globule protein, and can further improve the emulsification stability and digestion characteristics of the molten globule protein.

Description

Technical Field

[0001] The invention belongs to the technical field of marine food manufacturing, and more specifically, relates to a method for preparing a molten globule protein-fish oil gel emulsion. Background Art

[0002] The molten globule state is a transitional state in which proteins fold from linear peptide chains to specific three-dimensional structures. It is characterized by a secondary structure similar to that of natural proteins, lacks a specific tertiary structure generated by side chains, and has an increased hydrophobic surface area compared to the natural state. Molten globule proteins are widely used in many fields such as food industry, biomedicine, materials science, and environmental protection due to their good emulsification and stability. For example, they are used to improve the taste, texture, and functional properties of food, to achieve targeted delivery and controlled release of drugs, to construct tissue engineering scaffolds, biosensors, and other biomaterials with specific functions and structures, and to promote the degradation and transformation of harmful substances in the environment.

[0003] However, in order to form molten globule proteins, existing technologies usually need to be carried out in extremely acidic, extremely alkaline, high temperature (above 100°C) or high pressure environments. However, molten globule proteins formed under extreme pH conditions are prone to residual acid and alkali reagents, which may have a negative impact on the stability and safety of the final product when used in the fields of food industry, biomedicine and materials science, and may cause secondary pollution when used in the field of environmental protection; high temperature and high pressure treatments will greatly increase the difficulty, cost and danger of the process, which is not conducive to large-scale production and application.

[0004] Therefore, it is urgent to find a mild, simple, safe and environmentally friendly method to form molten globule protein, which is quite necessary for the further promotion and application of molten globule protein. Summary of the invention

[0005] The present invention aims to provide a method for preparing a molten globule protein-fish oil gel emulsion in view of the deficiencies in the prior art. Through operations such as ultrasound and emulsification, the molten globule protein can be formed under conditions of pH neutrality, normal temperature and pressure, avoiding the residue of acid and alkali reagents and the tediousness of high temperature or high pressure treatment, thereby providing a mild, simple, safe and environmentally friendly method for forming the molten globule protein.

[0006] The first object of the present invention is to provide a method for preparing a molten globule protein-fish oil gel emulsion.

[0007] The second object of the present invention is to provide the molten globule protein-fish oil gel emulsion prepared by the above method.

[0008] The third object of the present invention is to provide the use of the above molten globule protein-fish oil gel emulsion as and / or in the preparation of marine food.

[0009] The fourth object of the present invention is to provide a molten globule protein extracted from the above molten globule protein-fish oil gel emulsion.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The invention provides a method for preparing a molten globule protein-fish oil gel emulsion, which specifically comprises placing myofibrillar protein in a pH-neutral environment, homogenizing to obtain a myofibrillar protein solution, and mixing the solution with the fish oil gel after ultrasonication to obtain a molten globule protein-fish oil gel emulsion; wherein the ultrasonication is performed at 140 to 160 W for 110 to 130 s.

[0012] This method can form molten globule protein under pH neutral, normal temperature and pressure conditions through simple ultrasound, emulsification and other operations, avoiding the problems of residual acid and alkali reagents and tedious high temperature and normal pressure treatment in existing methods, providing a gentle, simple, safe and environmentally friendly method for forming molten globule protein, and can further improve the emulsification stability and digestion characteristics of molten globule protein.

[0013] It should be noted that, generally speaking, the emulsification stability and digestion characteristics of molten globule proteins are negatively correlated, and it is very difficult to improve both properties at the same time. Therefore, it is very unexpected that the molten globule protein obtained by the method of the present invention can have both good emulsification stability and good digestion characteristics. In addition, in the existing high-temperature treatment methods, the emulsification stability of the molten globule protein obtained under pH neutral conditions is not higher than that of the molten globule protein under pH 9.0 conditions, while the present invention does not require heating, and can obtain a completely opposite result only through ultrasonic treatment, that is, the emulsification stability of the molten globule protein obtained under pH neutral conditions is significantly higher than that of the molten globule protein under pH 9.0 conditions, which is a very unexpected result.

[0014] The myofibrillar protein is extracted in a conventional manner, and its source can be Penaeus vannamei, golden pomfret, pork and the like.

[0015] The pH-neutral environment can be a PBS buffer solution with a pH of 7, wherein the composition of the PBS buffer solution is 0.02M Na 2 HPO 4 / NaH 2 PO 4 , 0.3-0.6M NaCl, the pH adjuster used can be hydrochloric acid solution and / or sodium hydroxide solution.

[0016] Preferably, the homogenization is carried out at 5500-6500 r / min for 4-6 min, more preferably at 6000 r / min for 5 min.

[0017] Preferably, in the myofibrillar protein solution, the concentration of myofibrillar protein is 10-40 mg / mL, more preferably 20 mg / mL.

[0018] Preferably, the ultrasound is performed at 150 W for 120 s.

[0019] Preferably, the volume proportion of the fish oil gel in the molten globule protein-fish oil gel emulsion is 3% to 5%, more preferably 4%.

[0020] Preferably, in the fish oil gel, the mass proportion of fish oil is 95% to 97%, more preferably 96%.

[0021] Preferably, the fish oil used in the fish oil gel is tuna oil and / or anchovy oil.

[0022] Preferably, the gelling agent used in the fish oil gel is beeswax.

[0023] The fish oil gel is prepared by a conventional method that can gel the fish oil, such as mixing the fish oil with the gelling agent, heating until it is clear, and cooling until it forms a gel. Compared with lard, fish oil has a lower saturated fatty acid content and can be used as an excellent animal fat substitute, providing more raw material options for the development of low-fat and high-nutrition marine foods.

[0024] Optionally, the gel is preheated, such as at 55-65°C, preferably 60°C.

[0025] Optionally, the heating temperature is 75-85°C, preferably 80°C.

[0026] Optionally, stirring is performed while heating, such as stirring at 280 to 320 r / min, preferably 300 r / min.

[0027] Preferably, the mixing is carried out at 9500-10500 r / min for 18-22 min, more preferably at 10000 r / min for 20 min. This step is to complete emulsification.

[0028] In the molten globule protein-fish oil gel emulsion prepared by the present invention, not only molten globule protein is formed, but also the emulsification stability and digestion characteristics of the molten globule protein are significantly improved, and the molten globule protein can be used as and / or prepared as a low-fat and high-nutrition marine food. Therefore, the molten globule protein-fish oil gel emulsion prepared by the above method, the use of the above molten globule protein-fish oil gel emulsion as and / or in the preparation of marine food, and the molten globule protein extracted from the above molten globule protein-fish oil gel emulsion should also be within the protection scope of the present invention. Wherein, the extraction adopts a conventional method for extracting molten globule protein from an emulsion.

[0029] The present invention has the following beneficial effects:

[0030] The present invention uses simple ultrasound, emulsification and other operations to not only form molten globule protein under pH neutrality, normal temperature and pressure conditions, but also avoids the problems of residual acid and alkali reagents and cumbersome high temperature and high pressure treatment in existing methods, providing a mild, simple, safe and environmentally friendly method for forming molten globule protein, and can further improve the emulsification stability and digestion characteristics of molten globule protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the result diagram of the amount of interfacial protein adsorption.

[0032] Figure 2 A is the result diagram of apparent viscosity, Figure 2 B is the result diagram of viscoelastic properties.

[0033] Figure 3 This is the result graph of emulsification activity index and emulsification stability index.

[0034] Figure 4 A is the particle size distribution diagram of the emulsion, Figure 4 B is the particle size distribution diagram of gastric digestive juice, Figure 4 C is the particle size distribution diagram of intestinal digestive juice, Figure 4 D is the volume average diameter of emulsion, gastric digestive juice, and intestinal digestive juice. 4,3 value.

[0035] Figure 5 This is the result diagram of zeta potential.

[0036] Figure 6 This is a scanning electron microscope image. In the image, green represents the oil phase and red represents the protein.

[0037] Figure 7 This is the result of secondary structure analysis of the emulsion.

[0038] Figure 8 This is the result of secondary structure analysis of gastric digestive juice.

[0039] Fig. 9 This is the result of secondary structure analysis of intestinal digestive juice.

[0040] Fig.10 This is the fluorescence spectrum of the emulsion.

[0041] Fig.11 This is the fluorescence spectrum of gastric digestive juice.

[0042] Fig.12 This is the fluorescence spectrum of intestinal digestive juice.

[0043] Fig.13It is a result diagram of surface hydrophobic interaction.

[0044] Fig.14 It is a result diagram of the total thiol content.

[0045] Fig.15 It is a result diagram of the free fatty acid release rate.

[0046] Among them, "control" represents the product of the blank control, and "pH3.0, pH7.0, pH9.0" respectively represent the products prepared under the conditions of pH3.0, pH7.0, and pH9.0. Detailed implementation mode

[0047] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0048] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0049] I. Extraction of myofibrillar protein

[0050] Litopenaeus vannamei (30 - 40 shrimp / kg) was placed in an ice bath for shock. After removing the shrimp heads, shrimp shells, and intestinal glands, it was washed, filtered, and drained in sequence, and then mixed with PBS buffer (0.02M Na 2 HPO 4 / NaH 2 PO 4 , 0.1M NaCl, pH7.0) in an ice bath at 2 - 5°C, and then centrifuged at 4°C and 8500r / min for 20min in a high-speed centrifuge (hermo Sorvall LYNX 4000, ThermoFisher Scientific Co., USA). The precipitate obtained by centrifugation was then mixed with PBS buffer (0.02M Na 2 HPO 4 / NaH 2 PO 4 , 0.1M NaCl, pH 7.0) in an ice bath at 2 - 5°C, and then centrifuged at 4°C and 8500r / min for 20min. The precipitate obtained by centrifugation was mixed with 0.1M NaCl solution in an ice bath at 2 - 5°C in a mass ratio of 1:4, centrifuged at 4°C and 8500r / min for 20min, filtered through three layers of gauze, and centrifuged again (4°C, 8500r / min) for 20min. The precipitate obtained was myofibrillar protein (MP).

[0051] II. Preparation of fish oil gel

[0052] After preheating 4 g of beeswax at 60°C, add it to 96 g of tuna oil, place it on a digital heating magnetic stirrer (HMS-901D, Shenzhen Broadax Technology Industry Co., Ltd., China), heat it at 80°C and 300 r / min until it becomes clear, and then quickly cool it to form a gel to obtain fish oil gel. The fish oil gel is stored in a refrigerator at 4°C for later use.

[0053] 3. Statistical Analysis

[0054] Three parallel samples were set for all experiments in the following examples, and the results were averaged. The data were analyzed using JMP Pro 16 statistical software for analysis of variance (ANOVA) and Tukey multiple comparisons, with a confidence level of 95% (P<0.05).

[0055] Example 1 Preparation of Molten Globule Protein-Fish Oil Gel Emulsion

[0056] PBS buffer (0.02 M Na 2 HPO 4 / NaH 2 PO 4 , 0.6 M NaCl, pH 7.0) was used to dissolve myofibrillar protein in a T25 digital Ultra-turrax homogenizer (IKA, Germany), and the final concentration of myofibrillar protein was adjusted to 20 mg / mL (concentration determination method: bovine serum albumin (BSA) was used as the standard and measured by the Biuret method) to obtain an MP solution. The MP solution was placed in a refrigerator at 4°C for 12 hours, and then the pH of the MP solution was adjusted to 3.0, 7.0, and 9.0 with 0.5M hydrochloric acid / sodium hydroxide solution, and then homogenized at low speed (6000r / min) for 5 minutes using a digital disperser (IKAT25, IKA Instruments, Germany) to obtain myofibrillar protein solutions with pH values ​​of 3.0, 7.0, and 9.0. The solutions were then placed in an ultrasonic cell disruptor (Bransons-250D, Pitney Bowes Ultrasonics, USA), ultrasonicated at 150W for 120s, and finally mixed with fish oil gel (so that the volume fraction of fish oil gel in the mixture is 4% (v / v)), and homogenized at 10000r / min for 20 minutes to complete emulsification, thereby obtaining the molten globule protein-fish oil gel emulsion.

[0057] Comparative Example 1 Preparation of Molten Globule Protein-Lard Gel Emulsion

[0058] The method for preparing the molten globule protein-fish oil gel emulsion at pH 7.0 is the same as that in Example 1, except that the fish oil is replaced by lard. The product of this comparative example is used as a blank control.

[0059] Test Example 1

[0060] 1. Interface adsorption capacity

[0061] The three molten globule protein-fish oil gel emulsions obtained in Example 1 and the molten globule protein-lard gel emulsion obtained in Comparative Example 1 (hereinafter referred to as emulsions) were centrifuged at 4°C and 10000 r / min for 20 min, and the upper emulsion layer was mixed with 0.1% (v / v) SDS solution at a volume ratio of 1:10, and then centrifuged at 4°C and 10000 r / min for 10 min. The lower clear liquid obtained by centrifugation was the interface protein. The content of interface protein was determined using a biuret protein quantitative kit. T , and according to “Γ S (mg / m 2 )=Γ T S V "and (S v is the interfacial surface area of ​​the emulsion, is the volume fraction of fish oil gel in the emulsion (i.e. 4%), d 3,2 The adsorption amount of the interfacial protein is calculated by the area average particle size of the emulsion (measured directly by a laser particle size analyzer). s , to characterize the interfacial adsorption capacity of the emulsion.

[0062] The results are as follows Figure 1 As shown. It can be seen that the interfacial protein adsorption of the molten globule protein-fish oil gel emulsion prepared under pH 7.0 conditions is significantly higher than that of the other three emulsions (molten globule protein-fish oil gel emulsions prepared under pH 3.0 and pH 9.0 conditions, and the molten globule protein-lard gel emulsion as a blank control), that is, its interfacial protein film is the thickest and the emulsion is the most stable. This shows that the method of the present invention can significantly change the spatial structure of the protein, further expose the hydrophobic groups and thiol groups as the protein unfolds, promote the adsorption of the protein at the O / W interface, and thus significantly improve the stability of the molten globule protein-fish oil gel emulsion.

[0063] 2. Apparent viscosity and viscoelastic properties

[0064] Take 1 mL of each of the four emulsions and drop them evenly on the instrument plate of a modular advanced rheometer (HAAKE MARSⅢ, Thermo Fisher Scientific, USA). Select the P35Ti L rotor, set the gap between the instrument plate and the rotor to 1 mm at 25 °C, and record the shear rate of each emulsion at 0.1-100 s -1 Changes in apparent viscosity under .

[0065] 1 mL of each of the above four emulsions was taken and evenly dropped on the instrument plate of a modular advanced rheometer (HAAKE MARSⅢ, Thermo Fisher Scientific, USA). A P35Ti L rotor was selected, and the gap between the instrument plate and the rotor was set to 1 mm at 25°C. The strain scan was fixed to 1%, and the changes in the elastic modulus (G′, Pa) and viscous modulus (G″, Pa) of each emulsion at an oscillation frequency of 0.1 to 100 Hz were recorded to characterize the viscoelastic properties of the emulsion.

[0066] The results are as follows Figure 2 As shown, Figure 2 A is the result diagram of apparent viscosity, Figure 2 B is the result diagram of viscoelastic properties. It can be seen that the apparent viscosity, elastic modulus and viscous modulus of the molten globule protein-fish oil gel emulsion prepared under pH 7.0 conditions are significantly higher than those of the other three emulsions (molten globule protein-fish oil gel emulsions prepared under pH 3.0 and pH 9.0 conditions, and the molten globule protein-lard gel emulsion as a blank control), that is, its emulsification stability is the highest, indicating that the method of the present invention can significantly improve the stability of the molten globule protein-fish oil gel emulsion.

[0067] 3. Emulsification activity index and emulsification stability index

[0068] Take 40 μL of each of the four emulsions and dilute them to 4 mL with 0.1% (w / v) SDS solution. Then measure the absorbance A at 500 nm using a multifunctional enzyme labeling instrument (SpectraMax M2, Meigu Molecular Instruments, USA). 0 , let stand for 60 min, and measure the absorbance A again 60 . And according to (N is the emulsion dilution ratio (i.e. 100), C is the concentration of myofibrillar protein solution (i.e. 20 mg / mL), The emulsification activity index (EAI) and emulsion stability index (ESI) were calculated based on the volume fraction of fish oil gel in the emulsion (ie 4%).

[0069] The results are as follows Figure 3 As shown. It can be seen that the emulsification activity index and emulsification stability index of the molten globule protein-fish oil gel emulsion prepared under pH 7.0 conditions are significantly higher than those of the other three emulsions (molten globule protein-fish oil gel emulsions prepared under pH 3.0 and pH 9.0 conditions, and the molten globule protein-lard gel emulsion as a blank control), that is, its emulsification activity and emulsification stability (ESI is as high as about 90%) are the highest, indicating that the method of the present invention can significantly improve the emulsification activity and emulsification stability of the molten globule protein-fish oil gel emulsion.

[0070] Test Example 2

[0071] 1. Preparation of Gastric Digestive Juice

[0072] The above four emulsions were taken and mixed evenly with gastric simulation fluid containing 3.2 mg / mL pepsin in a volume ratio of 1:1, and the pH of the mixture was adjusted to 2.0 with 0.5 M hydrochloric acid solution. After incubation in a constant temperature shaker at 37°C and 90 r / min for 1 h, the pH of the system was adjusted to 7.0 to terminate the reaction to obtain gastric digestive fluid.

[0073] 2. Preparation of intestinal digestive juice

[0074] The gastric digestive fluid was mixed evenly with a small intestine simulation fluid containing 12.5 mg / mL pancreatic enzyme, 1.6 mg / mL lipase and 5.0 mg / mL bile salt in a volume ratio of 1:1, incubated in a constant temperature shaker at 37°C and 90 r / min for 4 h (during which the pH of the system was kept stable at 7.0 with 0.5 M sodium hydroxide solution), and then taken out and cooled to terminate the reaction to obtain intestinal digestive fluid.

[0075] 3. Particle size

[0076] The above four emulsions, gastric digestive juice, and intestinal digestive juice were added to different beakers as samples to be tested. The particle size of the samples to be tested was measured using a laser particle size analyzer (Mastersizer 3000, Malvern Instruments Ltd., UK). The pump speed was set to 8500r / min, the refractive index of the dispersion medium was set to 1.330, and the refractive index of the particles to be tested was set to 1.460; the background measurement time and the sample measurement time were both set to 5s; the light was aligned to ensure that the light energy value was less than 200; the laser shading rate of the sample to be tested was adjusted to 5% to 10%, and the measurement was started after 30s of equilibration.

[0077] The results are as follows Figure 4 As shown, Figure 4 A is the particle size distribution diagram of the emulsion, Figure 4 B is the particle size distribution diagram of gastric digestive juice, Figure 4 C is the particle size distribution diagram of intestinal digestive juice, Figure 4 D is the volume average diameter of emulsion, gastric digestive juice, and intestinal digestive juice. 4,3 It can be seen that, whether it is a pure emulsion or gastric digestive juice and intestinal digestive juice, the particle size of the product under pH 7.0 is significantly smaller than that of the products under the other three conditions (the products under pH 3.0 and pH 9.0, and the products as blank controls), that is, its emulsification stability and dispersibility are the highest, and the oil droplet particle size formed during the small intestine digestion process is the smallest, indicating that the method of the present invention can significantly improve the stability, dispersibility and digestibility of the molten globule protein-fish oil gel emulsion.

[0078] 4. Zeta potential (ζ-potential)

[0079] The four emulsions, gastric digestive juice, and intestinal digestive juice were respectively washed with PBS buffer (0.02M Na 2 HPO 4 / NaH 2 PO 4 , 0.6 M NaCl, pH 7.0) to 0.5 mg / mL, and then 1 mL of the dilution was placed in the Marvin ζ potential sample cell of a nanoparticle size potentiostat (Zetasizer NanoZS90, Malvern Instruments Ltd., UK) to measure the ζ-potential of the sample at 25°C, and the equilibrium time was set to 120 s.

[0080] The results are as follows Figure 5 As shown. It can be seen that, whether it is a pure emulsion, or a gastric digestive juice and an intestinal digestive juice, the absolute value of the ζ-potential of the product under pH 7.0 is significantly greater than that of the products under the other three conditions (the products under pH 3.0 and pH 9.0, and the products as blank controls), that is, its emulsification stability is the highest, and the electrostatic repulsion generated during the small intestinal digestion process is the highest, which can better promote the contact between the digestive enzyme and bile salts, oils, etc., indicating that the method of the present invention can significantly improve the stability and digestion characteristics of the molten globule protein-fish oil gel emulsion.

[0081] 5. Microstructure

[0082] The above-mentioned four emulsions, gastric digestive fluid and intestinal digestive fluid were used as test samples. The samples were stained according to the method of Zhou, Zhang, Yin, Zhang, & Yang (2021) [Zhou, L., Zhang, J., Yin, Y., Zhang, W., & Yang, Y. (2021). Effects of ultrasound-assisted 991 emulsification on the emulsifying and rheological properties of myofibrillar protein 992 stabilized pork fat emulsions. Foods, 10(6), 1201. 993 https: / / doi.org / 10.3390 / foods10061201] and observed under a confocal laser scanning microscope (TCS SP8, Leica, Germany).

[0083] The results are as follows Figure 6As shown. It can be seen that, whether it is a pure emulsion, or a gastric digestive juice or an intestinal digestive juice, the particle size of the product under pH 7.0 is significantly smaller than that of the products under the other three conditions (the products under pH 3.0 and pH 9.0, and the products as blank controls), that is, its emulsification stability and dispersibility are the highest, and the oil droplet particle size formed during the small intestine digestion process is the smallest, indicating that the method of the present invention can significantly improve the stability, dispersibility and digestibility of the molten globule protein-fish oil gel emulsion.

[0084] 6. Raman spectroscopy

[0085] The four emulsions, gastric digestive juice, and intestinal digestive juice were scanned by Raman spectrometer (LabRAM HR Evolution, Thermo Fisher Scientific, USA) (laser power 8 mW, scanning range 400-3600 cm -1 , exposure time 30s, each resolution 2.0cm -1 , sampling speed 120cm -1 , and each scan is performed at 1003cm -1 The percentage of secondary structure was determined by the method of Alix after normalization to the phenylalanine band at 1.

[0086] The results are as follows Figures 7 to 9 As shown, Figure 7 This is the result of secondary structure analysis of the emulsion. Figure 8 This is the result of secondary structure analysis of gastric digestive juice. Fig. 9 The secondary structure analysis result of intestinal digestive juice is shown in FIG. It can be seen that, whether it is pure emulsion, gastric digestive juice or intestinal digestive juice, the secondary structure of the product under pH 7.0 condition is closer to the secondary structure of natural protein (α-helix 62.03% ± 2.52%, β-fold 14.14% ± 1.36%, β-turn 13.98% ± 0.79%, random curl 9.85% ± 0.45%), that is, its emulsification stability is the highest, and it tends to restore the structure before digestion and remain stable during the digestion process of the small intestine, indicating that the method of the present invention can make the protein in the molten globule protein-fish oil gel emulsion have a secondary structure similar to that of natural protein, significantly improving its emulsification stability and digestion characteristics.

[0087] 7. Fluorescence spectrum

[0088] The four emulsions, gastric digestive juice, and intestinal digestive juice were respectively washed with PBS buffer (0.02M Na 2 HPO 4 / NaH 2 PO 4, 0.6 M NaCl, pH 7.0) to 0.5 mg / mL, and then the internal fluorescence spectrum of the dilution was scanned using a fluorescence spectrometer (RF-5301PC, Shimadzu Corporation, Japan) with an excitation wavelength of 295 nm, an emission wavelength of 300-400 nm, and a slit width of 5 nm.

[0089] The results are as follows Figures 10-12 As shown, Fig.10 is the fluorescence spectrum of the emulsion. Fig.11 This is the fluorescence spectrum of gastric digestive juice. Fig.12 It is a fluorescence spectrum of intestinal digestive juice. It can be seen that the fluorescence intensity of the maximum absorption peak increases first and then decreases, indicating that the tryptophan and tyrosine residues are exposed to the polar environment, the tertiary structure changes, and the specific tertiary structure generated by the side chain in the natural protein is missing; and whether it is a pure emulsion, or gastric digestive juice and intestinal digestive juice, the maximum absorption peak fluorescence intensity of the product under pH7.0 condition is significantly higher than that of the products under the other three conditions (products under pH3.0 and pH9.0 conditions, and products as blank controls), that is, its tertiary structure undergoes the most obvious changes, indicating that the method of the present invention can significantly change the tertiary structure in the molten globule protein-fish oil gel emulsion and improve its digestion characteristics.

[0090] 8. Surface hydrophobicity

[0091] The four emulsions, gastric digestive juice, and intestinal digestive juice were respectively washed with PBS buffer (0.02M Na 2 HPO 4 / NaH 2 PO 4 , 0.6M NaCl, pH7.0) to 2 mg / mL, and then 1 mL of the dilution was mixed with 40 μL of bromophenol blue solution (1 mg / mL), allowed to stand for 10 min, centrifuged at 4°C, 4000 rpm for 15 min in a refrigerated centrifuge, and then washed with PBS buffer (0.02M Na 2 HPO 4 / NaH 2 PO 4 , 0.6M NaCl, pH 7.0) was used to dilute the supernatant obtained by centrifugation 5 times, and the absorbance A of the solution was measured at 595nm. Another 1mL of the above dilution was replaced with 1mL PBS buffer (0.02M Na 2 HPO 4 / NaH 2 PO 4 , 0.6M NaCl, pH 7.0), and the absorbance of the solution was measured at 595nm. 0 Finally, according to "Bromophenol blue binding amount (μg) = 40μg × (A0 -A) / A 0 The bromophenol blue binding amount of the sample was calculated to characterize the surface hydrophobicity of the sample.

[0092] The results are as follows Fig.13 As shown. It can be seen that, whether it is a pure emulsion, or a gastric digestive juice or an intestinal digestive juice, the bromophenol blue binding amount of the product under pH 7.0 condition is significantly higher than that of the products under the other three conditions (the products under pH 3.0 and pH 9.0 conditions, and the products as blank controls), that is, its surface hydrophobic effect is the largest, the hydrophobic groups are exposed the most, a larger hydrophobic surface is formed, and the surface hydrophobic effect is still the largest after small intestinal digestion. It shows that the method of the present invention can increase the hydrophobic surface area of ​​the protein in the molten globular protein-fish oil gel emulsion, and significantly improve its emulsification stability and digestion characteristics.

[0093] IX. Total thiol content

[0094] Take 0.6 mL of the above four emulsions, gastric digestive fluid, and intestinal digestive fluid (as the sample to be tested), suspend them in 4 mL Tris-glycine buffer solution (0.086 M Tris, 0.09 M glycine, 4 mM EDTA, pH = 8.0) containing 8 M urea, and then add 20 μL Ellman's reagent (Tris-Gly buffer containing 4 mg / mL 5,5′-dithiodi-2-nitrobenzoic acid (DTNB)). After vigorous shaking, let it stand at 25°C for 1 hour, and then centrifuge it at 12000 r / min for 10 minutes. The absorbance A of the supernatant obtained by centrifugation is measured at 412 nm.

[0095] The above-mentioned sample to be tested was replaced with Tris-glycine buffer solution, and the supernatant was obtained according to the above method, and its absorbance A was measured at 412 nm. 0 Finally, according to "total thiol content (μmol / g) = 73.53 × (AA 0 ) / ρ” (ρ is the concentration of myofibrillar protein solution (i.e., 20 mg / mL)) to calculate the total thiol content of the sample.

[0096] The results are as follows Fig.14 As shown. It can be seen that, whether it is a pure emulsion, or a gastric digestive juice and an intestinal digestive juice, the total thiol content of the product under pH 7.0 is significantly lower than that of the products under the other three conditions (the products under pH 3.0 and pH 9.0, and the products as blank controls), that is, it forms the most disulfide bonds, has the highest emulsification stability, and forms the most disulfide bonds after small intestinal digestion, indicating that the method of the present invention can significantly improve the stability and digestion characteristics of the molten globule protein-fish oil gel emulsion.

[0097] 10. Free fatty acid release rate

[0098] In the process of preparing intestinal digestive juice, the volume of NaOH consumed to keep the pH of the system stable at 7.0 during the 4-hour incubation period was recorded as V. NaOH , the molar concentration of NaOH (0.5M) is denoted as m NaOH , and according to (M Lipid is the average molar mass of lard / fish oil in the sample, W Lipid The release rate of free fatty acids (FFA) was calculated by using the mass of lard / fish oil in the sample.

[0099] The results are as follows Fig.15 As shown. It can be seen that the free fatty acid release rate of the intestinal digestive juice under pH 7.0 conditions is significantly higher than that of the intestinal digestive juice under pH 3.0 and pH 9.0 conditions and the intestinal digestive juice as a blank control, that is, the emulsion prepared under pH 7.0 conditions has the highest release amount in the intestinal digestive juice, up to 71.41%, and combined with the results of the highest stability in the aforementioned experiments, it can be determined that the emulsion prepared under pH 7.0 conditions can achieve better sustained release results in the intestinal digestive juice and better digestion characteristics, that is, the test results of the present invention unexpectedly show that the emulsion with higher stability has higher digestion characteristics, indicating that the method of the present invention can significantly improve the digestion characteristics and stability of the molten globule protein-fish oil gel emulsion at the same time.

[0100] Through the analysis of Raman spectroscopy, fluorescence spectroscopy, surface hydrophobicity and total thiol results, it can be seen that molten globule protein is formed in the molten globule protein-fish oil gel emulsion prepared by the method of the present invention; and combined with the relationship analysis between particle size, zeta potential, microstructure and emulsification stability, and free fatty acid release rate, it can be seen that the method of the present invention improves the stability and digestion characteristics of the molten globule protein-fish oil gel emulsion. In summary, it is shown that the present invention can form molten globule protein under pH neutrality, normal temperature and pressure conditions through simple ultrasound, emulsification and other operations, avoiding the cumbersome problems of residual acid and alkali reagents and high temperature and high pressure treatment in the existing methods, providing a mild, simple, safe and environmentally friendly method for forming molten globule protein, and can further improve the emulsification stability and digestion characteristics of molten globule protein.

[0101] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a molten globule protein-fish oil gel emulsion, characterized in that: The myofibrillar protein is placed in a pH-neutral environment, homogenized to obtain a myofibrillar protein solution, and then mixed with fish oil gel after ultrasound. The ultrasound is performed at 140 to 160 W for 110 to 130 s. The homogenization is performed at 5500 to 6500 r / min for 4 to 6 minutes. The concentration of myofibrillar protein in the myofibrillar protein solution is 10 to 40 mg / mL.

2. The preparation method according to claim 1, characterized in that: The volume proportion of the fish oil gel in the molten globule protein-fish oil gel emulsion is 3% to 5%.

3. The preparation method according to claim 1, characterized in that: In the fish oil gel, the mass proportion of fish oil is 95% to 97%.

4. The preparation method according to claim 1, characterized in that: The fish oil used in the fish oil gel is tuna oil and / or anchovy oil.

5. The preparation method according to claim 1, characterized in that: The gelling agent used in the fish oil gel is beeswax.

6. The molten globule protein-fish oil gel emulsion prepared by the method according to any one of claims 1 to 5.

7. Use of the molten globule protein-fish oil gel emulsion according to claim 6 as or in the preparation of marine food.

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