Preparation method for molten-globule-state protein–fish oil gel emulsion

Abstract

Disclosed is a method for preparing a molten-globule-state protein–fish oil gel emulsion. The method comprises placing a myofibrillar protein in a pH-neutral environment, performing homogenization and sonication, and then uniformly mixing same with a fish oil gel to obtain the product, wherein the sonication is performed at 140 to 160 W for 110 to 130 s. A molten-globule-state protein is extracted from the gel emulsion prepared by the method. The gel emulsion can be applied in marine food products.

Description

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

[0001] The present application belongs to the technical field of marine food manufacturing. More specifically, it relates to a method for preparing a molten globule protein-fish oil gel emulsion. BACKGROUND

[0002] Molten globule is a transition state of protein during the folding of linear peptide chains into specific three-dimensional structures. It is characterized by having a similar secondary structure to native proteins, lacking specific tertiary structures generated by side chains, and having an increased hydrophobic surface area compared to native proteins. Molten globule proteins are widely used in food industry, biomedicine, materials science, and environmental protection due to their good emulsifying properties and stability. 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 and biosensors with specific functions and structures, and to promote the degradation and transformation of harmful substances in the environment.

[0003] However, to form molten globule proteins, existing technologies usually require extreme acidic, alkaline, high-temperature (above 100℃) or high-pressure environments. Molten globule proteins formed under extreme pH conditions may leave residues of acid or base reagents, which may have a negative impact on the stability and safety of the final product when applied in food industry, biomedicine and materials science, and may cause secondary pollution when applied in environmental protection. High-temperature and high-pressure treatments greatly increase the difficulty, cost and risk of the process, which is not conducive to large-scale production and application.

[0004] Therefore, there is an urgent need for a mild, simple, safe and environmentally friendly method for forming molten globule proteins, which is essential for the further popularization and application of molten globule proteins. SUMMARY

[0005] The present application aims to provide a method for preparing a molten globule protein-fish oil gel emulsion, which forms molten globule proteins at neutral pH, room temperature and normal pressure through ultrasonic and emulsification operations, avoiding the residues of acid or base reagents and the cumbersome high-temperature or high-pressure treatments, and providing a mild, simple, safe and environmentally friendly method for forming molten globule proteins.

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

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

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

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

[0010] The above-mentioned objects of the present application are achieved by the following technical solutions.

[0011] The present application provides a preparation method of a molten globule protein-fish oil gel emulsion, which comprises the following steps: placing myofibrillar protein in a pH-neutral environment, homogenizing to obtain a myofibrillar protein solution, and mixing with fish oil gel after ultrasonic treatment to obtain the molten globule protein-fish oil gel emulsion; wherein the ultrasonic treatment is performed at 140-160 W for 110-130 s.

[0012] The method can form the molten globule protein under the conditions of pH neutrality, normal temperature and normal pressure through simple ultrasonic treatment and emulsification, avoids the problems of residue of acid and alkali reagents and complicated high-temperature and normal-pressure treatment in the prior art, and provides a mild, simple, safe and environmentally friendly method for forming the molten globule protein, and further improves the emulsification stability and digestion characteristics of the molten globule protein.

[0013] It should be noted that, in general, the emulsification stability and the digestion characteristics of the molten globule protein 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 application has both good emulsification stability and good digestion characteristics. In addition, in the prior art high-temperature treatment method, the emulsification stability of the molten globule protein obtained under the condition of pH neutrality is not higher than that of the molten globule protein obtained under the condition of pH 9.0, while the present application does not need heating, and only ultrasonic treatment can obtain the opposite result, i.e. the emulsification stability of the molten globule protein obtained under the condition of pH neutrality is significantly higher than that of the molten globule protein obtained under the condition of pH 9.0, which is very unexpected.

[0014] The myofibrillar protein is obtained by a conventional method, and its source can be whiteleg shrimp, golden pompano, pork, etc.

[0015] The pH-neutral environment can be a PBS buffer solution with pH 7, wherein the components of the PBS buffer solution are 0.02 M Na2HPO4 / NaH2PO4 and 0.3-0.6 M NaCl, and the pH adjuster used can be a hydrochloric acid solution and / or a sodium hydroxide solution.

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

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

[0018] Preferably, the ultrasonic wave is 120 s at 150 W.

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

[0020] Preferably, the mass ratio of fish oil in the fish oil gel is 95%-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 to clarify, and cooling to form a gel. Fish oil has a lower content of saturated fatty acids than pork fat, and can be used as an excellent animal fat substitute, providing more raw material options for the development of low-fat high-nutrition marine food.

[0024] Optionally, the gelling agent is also preheated, such as at 55-65℃, preferably 60℃.

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

[0026] Optionally, the heating is also accompanied by stirring, such as at 280-320 r / min, preferably 300 r / min.

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

[0028] In the molten globule protein-fish oil gel emulsion prepared by the present application, not only molten globule protein is formed, but also the emulsion stability and digestion characteristics of the molten globule protein are significantly improved, which can be used as and / or prepared into low-fat high-nutrition marine food. Therefore, the molten globule protein-fish oil gel emulsion prepared by the above method, the application of the above molten globule protein-fish oil gel emulsion in marine food as and / or preparation, 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 application. Among them, the extraction can use a conventional method for extracting molten globule protein from emulsion.

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

[0030] The present application can form the melt globular protein under the conditions of neutral pH, normal temperature and normal pressure by simple ultrasonic and emulsification operations, avoids the problems of residue of acid and alkali reagents and complicated high-temperature and high-pressure treatment in the prior art, provides a mild, simple, safe and environmentally friendly method for forming the melt globular protein, and further improves the emulsification stability and digestion properties of the melt globular protein. BRIEF DESCRIPTION OF DRAWINGS

[0031] Fig. 1 is a result graph of the interfacial protein adsorption amount.

[0032] Fig. 2A is a result graph of the apparent viscosity, and Fig. 2B is a result graph of the viscoelastic properties.

[0033] Fig. 3 is a result graph of the emulsification activity index and the emulsification stability index.

[0034] Fig. 4A is a particle size distribution graph of the emulsion, Fig. 4B is a particle size distribution graph of the gastric digestion solution, Fig. 4C is a particle size distribution graph of the intestinal digestion solution, and Fig. 4D is a volume average diameter d 4,3 value of the emulsion, the gastric digestion solution and the intestinal digestion solution.

[0035] Fig. 5 is a result graph of the zeta potential.

[0036] Fig. 6 is a scanning electron microscope graph, in which green represents the oil phase and red represents the protein.

[0037] Fig. 7 is a result graph of the secondary structure analysis of the emulsion.

[0038] Fig. 8 is a result graph of the secondary structure analysis of the gastric digestion solution.

[0039] Fig. 9 is a result graph of the secondary structure analysis of the intestinal digestion solution.

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

[0041] Fig. 11 is a fluorescence spectrum graph of the gastric digestion solution.

[0042] Fig. 12 is a fluorescence spectrum graph of the intestinal digestion solution.

[0043] Fig. 13 is a result graph of the surface hydrophobic interaction.

[0044] Fig. 14 is a result graph of the total sulfhydryl content.

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

[0046] Among them, "control" represents the product of blank control, and "pH3.0, pH7.0, pH9.0" represent the products prepared under pH 3.0, pH 7.0, and pH 9.0 conditions, respectively. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

[0049] 1. Extraction of myofibrillar proteins

[0050] Vannamei shrimp (30-40 pieces / kg) were shocked in an ice bath. After removing the shrimp heads, shells and intestinal glands, they were washed, filtered, drained, and then mixed with PBS buffer (0.02M Na2HPO4 / NaH2PO4, 0.1M NaCl, pH 7.0) at a mass ratio of 1:4 in an ice bath at 2-5°C. The mixture was then centrifuged in a high-speed centrifuge (Hermos Sorvall LYNX 4000, Thermo Fisher Scientific Co., USA) at 4°C and 8500 r / min for 20 min. The resulting pellet was then mixed with PBS buffer (0.02M Na2HPO4 / NaH2PO4, 0.1M NaCl, pH 7.0) at a mass ratio of 1:4 in an ice bath at 2-5°C and centrifuged at 4°C and 8500 r / min for 20 min. The precipitate obtained by centrifugation was mixed with 0.1M NaCl solution at a mass ratio of 1:4 in an ice bath at 2-5°C. After centrifugation at 4°C and 8500 r / min for 20 min, it was filtered through three layers of gauze and centrifuged again (4°C, 8500 r / min) for 20 min. The resulting precipitate was myofibrillar protein (MP).

[0051] 2. Preparation of fish oil gel

[0052] 4 g of beeswax was preheated at 60°C and added to 96 g of tuna oil. The mixture was then heated at 80°C and 300 rpm on a digital magnetic stirrer (HMS-901D, Shenzhen Broadax Technology Industry Co., Ltd., China) until clear. The mixture was then rapidly cooled until a gel formed. The fish oil gel was stored in a refrigerator at 4°C until ready for use.

[0053] 3. Statistical Analysis

[0054] All experiments in the following examples were set up in triplicate, and the results were averaged. JMP Pro 16 statistical software was used for analysis of variance (ANOVA) and Tukey’s multiple comparisons with a confidence level of 95% (P < 0.05).

[0055] Example 1 Preparation of a molten globular protein-fish oil gel emulsion

[0056] Myofibrillar proteins were dissolved in PBS buffer (0.02 M Na2HPO4 / NaH2PO4, 0.6 M NaCl, pH 7.0) in a T25 digital Ultra-turrax homogenizer (IKA, Germany) and the final concentration of myofibrillar proteins was adjusted to 20 mg / mL (concentration determination method: using bovine serum albumin (BSA) as a standard, the Biuret method was used to determine the concentration). The MP solution was stored in a refrigerator at 4°C for 12 h, and then the pH of the MP solution was adjusted to 3.0, 7.0, and 9.0, respectively, using 0.5 M hydrochloric acid / sodium hydroxide solution. The MP solution was then homogenized at low speed (6000 r / min) for 5 min using a digital disperser (IKA T25, IKA Werke GmbH, Germany), and then ultrasonicated at 150 W for 120 s in an ultrasonic cell disruptor (Branson-250D, Pitney Bowes Ultrasonics, USA). Finally, the molten globular protein-fish oil gel emulsion was obtained by mixing the myofibrillar protein solution with fish oil gel (the volume fraction of fish oil gel in the mixture was 4% (v / v)) and homogenizing at 10000 r / min for 20 min.

[0057] Comparative Example 1 Preparation of a molten globular protein-pork oil gel emulsion

[0058] The method for preparing the molten globular protein-fish oil gel emulsion under the condition of pH 7.0 in Example 1 was followed, except that pork oil was used instead of fish oil. The product of this comparative example was used as a blank control.

[0059] Test Example 1

[0060] I. Interfacial adsorption capacity

[0061] The three molten globular protein-fish oil gel emulsions obtained in Example 1 and the molten globular protein-pork oil gel emulsion obtained in Comparative Example 1 (hereinafter referred to as emulsions) were each centrifuged at 10000 r / min for 20 min at 4°C, and the upper emulsion layer was mixed with 0.1% (v / v) SDS solution at a volume ratio of 1:10. After centrifugation at 10000 r / min for 10 min at 4°C, the lower clear liquid obtained by centrifugation was the interfacial protein. The content of the interfacial protein was determined using a biuret protein quantification kit T , and the interfacial adsorption capacity was calculated according to the formula: ΓS (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 is the area average particle size of the emulsion (directly measured by a laser particle size analyzer)), the adsorption amount of interfacial protein Γ s is calculated to represent the interfacial adsorption capacity of the emulsion.

[0062] The results are shown in Figure 1. It can be seen that the interfacial protein adsorption amount of the melt-spherical protein-fish oil gel emulsion prepared under pH 7.0 condition is significantly higher than those of the other three emulsions (the melt-spherical protein-fish oil gel emulsions prepared under pH 3.0 and pH 9.0 conditions, and the melt-spherical protein-pork oil gel emulsion as a blank control), i.e. the interfacial protein film thereof is the thickest, and the emulsion is the most stable. It is shown that the method of the present application can significantly change the spatial structure of the protein, so that the hydrophobic groups and sulfhydryl groups are further exposed with the unfolding of the protein, which promotes the adsorption of the protein at the O / W interface, and further significantly improves the stability of the melt-spherical protein-fish oil gel emulsion.

[0063] II. Apparent viscosity and viscoelastic properties

[0064] Each of 1 mL of the aforementioned four emulsions was uniformly dropped on the instrument plate of a modular advanced rheometer (HAAKE MARS III, Thermo Fisher Scientific, USA), a P35Ti L rotor was selected, the gap between the instrument plate and the rotor was set to 1 mm at 25°C, and the change of the apparent viscosity of each emulsion at a shear rate of 0.1-100 s -1 was recorded.

[0065] Each of 1 mL of the aforementioned four emulsions was uniformly dropped on the instrument plate of a modular advanced rheometer (HAAKE MARS III, Thermo Fisher Scientific, USA), a P35Ti L rotor was selected, the gap between the instrument plate and the rotor was set to 1 mm at 25°C, and the strain sweep was fixed at 1%, and the change of the elastic modulus (G', Pa) and the viscous modulus (G", Pa) of each emulsion at an oscillation frequency of 0.1-100 Hz was recorded to represent the viscoelastic properties of the emulsion.

[0066] The results are shown in Figure 2, wherein Figure 2A is a result graph of apparent viscosity, and Figure 2B is a result graph of viscoelasticity. It can be seen that the apparent viscosity, elastic modulus and viscous modulus of the molten globular protein-fish oil gel emulsion prepared under pH 7.0 condition are significantly higher than those of the other three emulsions (the molten globular protein-fish oil gel emulsions prepared under pH 3.0 and pH 9.0 conditions, and the molten globular protein-pork oil gel emulsion as a blank control), i.e. the emulsion stability thereof is the highest, indicating that the method of the present application can significantly improve the stability of the molten globular protein-fish oil gel emulsion.

[0067] III. Emulsifying activity index and emulsion stability index

[0068] Take 40 μL of each of the aforementioned four emulsions, dilute to 4 mL with 0.1% (w / v) SDS solution, and then measure the absorbance A0 at 500 nm with a multifunctional enzyme marker (SpectraMax M2, Meigu Molecular Instruments Company, USA), stand for 60 min, and then measure the absorbance A again. 60 According to the following formula, the emulsifying activity index (EAI) and the emulsion stability index (ESI) are calculated, respectively. (N is the dilution ratio of the emulsion (i.e. 100), C is the concentration of the myofibrillar protein solution (i.e. 20 mg / mL), and V is the volume fraction of the fish oil gel in the emulsion (i.e. 4%).

[0069] The results are shown in Figure 3. It can be seen that the emulsifying activity index and the emulsion stability index of the molten globular protein-fish oil gel emulsion prepared under pH 7.0 condition are significantly higher than those of the other three emulsions (the molten globular protein-fish oil gel emulsions prepared under pH 3.0 and pH 9.0 conditions, and the molten globular protein-pork oil gel emulsion as a blank control), i.e. the emulsifying activity and the emulsion stability (ESI as high as about 90%) thereof are the highest, indicating that the method of the present application can significantly improve the emulsifying activity and the emulsion stability of the molten globular protein-fish oil gel emulsion.

[0070] Test Example 2

[0071] I. Preparation of gastric juice

[0072] Take the aforementioned four emulsions, mix each with the gastric simulation solution containing 3.2 mg / mL pepsin at a volume ratio of 1:1, and adjust the pH of the mixed solution to 2.0 with 0.5 M hydrochloric acid solution, then incubate in a constant temperature shaker at 37°C and 90 r / min for 1 h, and then adjust the pH of the system to 7.0 to terminate the reaction, to obtain the gastric juice.

[0073] II. Preparation of intestinal juice

[0074] The gastric juice and the small intestine simulated solution containing 12.5 mg / mL pancreatin, 1.6 mg / mL lipase and 5.0 mg / mL bile salt were mixed uniformly at a volume ratio of 1:1, and then incubated in a constant temperature shaker at 37°C and 90 r / min (the pH of the system was maintained at 7.0 by using 0.5M sodium hydroxide solution) for 4h, and then removed and cooled to terminate the reaction to obtain the intestinal juice.

[0075] III. Particle size

[0076] The four emulsions, the gastric juice and the intestinal juice were added into different beakers respectively as the samples to be measured. A laser particle size analyzer (Mastersizer 3000, Malvern Instruments Ltd., UK) was used to measure the particle size of the samples to be measured. The pump speed was set to 8500 r / min, the refractive index of the dispersion medium was set to 1.330, and the refractive index of the particles to be measured 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 light blocking rate of the sample to be measured was adjusted to 5%-10%, and the measurement was started after 30s of equilibrium.

[0077] The results are shown in FIG. 4, wherein FIG. 4A is a particle size distribution diagram of the emulsion, FIG. 4B is a particle size distribution diagram of the gastric juice, FIG. 4C is a particle size distribution diagram of the intestinal juice, and FIG. 4D is a volume average diameter d 4,3 It can be seen that, no matter the pure emulsion or the gastric juice and the intestinal juice, the particle size of the product under the condition of pH 7.0 is significantly smaller than that of the products under the other three conditions (the products under the conditions of pH 3.0 and pH 9.0, and the product as a blank control), that is, the emulsion stability and dispersibility are the highest, and the oil droplet particle size formed in the small intestine digestion process is the smallest, which indicates that the method of the present application can significantly improve the stability, dispersibility and digestion characteristics of the molten globular protein-fish oil gel emulsion.

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

[0079] The four emulsions, the gastric juice and the intestinal juice were diluted to 0.5 mg / mL with PBS buffer (0.02M Na2HPO4 / NaH2PO4, 0.6M NaCl, pH 7.0), and then 1 mL of the diluted solution was placed in a Marvin zeta potential sample cell of a nanoparticle size potential instrument (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK) to measure the ζ-potential of the sample at 25°C, and the equilibrium time was set to 120s.

[0080] The results are shown in Figure 5. It can be seen that the absolute value of the zeta potential of the product under the condition of pH 7.0 is significantly greater than that of the product under the other three conditions (the product under the condition of pH 3.0 and pH 9.0, and the product as a blank control), that is, the emulsion stability is the highest, and the electrostatic repulsion generated during small intestine digestion is the highest, which can better promote the contact of digestive enzymes with bile salts, oil and fat, etc., indicating that the method of the application can significantly improve the stability and digestion characteristics of the molten globular protein-fish oil gel emulsion.

[0081] V. Microstructure

[0082] The aforementioned four emulsions, gastric juice and intestinal juice were used as test samples, and the test samples were dyed 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 emulsification on the emulsifying and rheological properties of myofibrillar protein stabilized pork fat emulsions. Foods, 10(6), 1201. 993 https: / / doi.org / 10.3390 / foods10061201], and then observed by a confocal laser scanning microscope (TCS SP8, Leica, Germany).

[0083] The results are shown in Figure 6. It can be seen that the particle size of the product under the condition of pH 7.0 is significantly smaller than that of the product under the other three conditions (the product under the condition of pH 3.0 and pH 9.0, and the product as a blank control), that is, the emulsion stability and dispersibility are the highest, and the oil droplet particle size formed during small intestine digestion is the smallest, indicating that the method of the application can significantly improve the stability, dispersibility and digestion characteristics of the molten globular protein-fish oil gel emulsion.

[0084] VI. Raman spectrum

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

[0086] The results are shown in Figures 7-9, wherein Figure 7 is a graph of the secondary structure analysis of the emulsion, Figure 8 is a graph of the secondary structure analysis of the gastric juice, and Figure 9 is a graph of the secondary structure analysis of the intestinal juice. It can be seen that, whether it is the pure emulsion, or the gastric juice and the intestinal juice, the secondary structure of the product under the condition of pH 7.0 is closer to the secondary structure of the native protein (62.03% ± 2.52% of α-helix, 14.14% ± 1.36% of β-sheet, 13.98% ± 0.79% of β-turn, and 9.85% ± 0.45% of random coil), i.e., it has the highest emulsification stability, and during the digestion in the small intestine, it tends to restore the structure before digestion and remains stable, indicating that the method of the present application can make the protein in the molten globule state protein-fish oil gel emulsion have a similar secondary structure to the native protein, and significantly improve the emulsification stability and digestion characteristics.

[0087] Seven, fluorescence spectrum

[0088] The aforementioned four emulsions, gastric juice, and intestinal juice were each diluted to 0.5 mg / mL with PBS buffer (0.02 M Na2HPO4 / NaH2PO4, 0.6 M NaCl, pH 7.0), and then the internal fluorescence spectrum of the diluted solution was scanned using a fluorescence spectrometer (RF-5301PC, Shimadzu Corporation, Japan). The excitation wavelength was 295 nm, the emission wavelength was 300-400 nm, and the slit width was 5 nm.

[0089] The results are shown in Figures 10-12, wherein Figure 10 is a graph of the fluorescence spectrum of the emulsion, Figure 11 is a graph of the fluorescence spectrum of the gastric juice, and Figure 12 is a graph of the fluorescence spectrum of the intestinal juice. It can be seen that the fluorescence intensity of the maximum absorption peak first increases 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 produced by the side chain in the native protein is lacking; and whether it is the pure emulsion, or the gastric juice and the intestinal juice, the fluorescence intensity of the maximum absorption peak of the product under the condition of pH 7.0 is significantly higher than that of the products under the other three conditions (the products under the conditions of pH 3.0 and pH 9.0, and the product as a blank control), i.e., the most obvious change in the tertiary structure occurs, indicating that the method of the present application can make the tertiary structure in the molten globule state protein-fish oil gel emulsion change significantly, and improve the digestion characteristics.

[0090] Eight, surface hydrophobic interaction

[0091] The four emulsions, gastric juice, intestinal juice were diluted to 2 mg / mL with PBS buffer (0.02M Na2HPO4 / NaH2PO4, 0.6M NaCl, pH7.0) respectively, 1 mL of the diluted solution was mixed with 40 μL of bromophenol blue solution (1 mg / mL) respectively, and then was allowed to stand for 10 min, and then was centrifuged at 4 ℃ and 4000 rpm for 15 min by using a refrigerated centrifuge, and then the supernatant obtained by centrifugation was diluted 5 times with PBS buffer (0.02M Na2HPO4 / NaH2PO4, 0.6M NaCl, pH7.0), and then the absorbance A of the solution was measured at 595 nm. In addition, 1 mL of the diluted solution was replaced by 1 mL of PBS buffer (0.02M Na2HPO4 / NaH2PO4, 0.6M NaCl, pH7.0), and then the absorbance A0 of the solution was measured at 595 nm. Finally, the bromophenol blue binding amount of the sample was calculated according to "bromophenol blue binding amount (μg) = 40 μg × (A0-A) / A0", so as to characterize the surface hydrophobicity of the sample.

[0092] The results are shown in Figure 13. It can be seen that, no matter the pure emulsion, or the gastric juice and intestinal juice, the bromophenol blue binding amount of the product under pH7.0 condition is significantly higher than that of the products under other three conditions (the products under pH3.0 and pH9.0 conditions, and the product as a blank control), i.e. the surface hydrophobicity is the largest, the hydrophobic group is exposed the most, a larger hydrophobic surface is formed, and the largest surface hydrophobicity is still maintained after the small intestine digestion. It is shown that the method of the present application can increase the protein hydrophobic surface area of the melt globular protein-fish oil gel emulsion, and significantly improve the emulsification stability and digestion characteristics.

[0093] Nine, total sulfhydryl content

[0094] 0.6 mL of the four emulsions, gastric juice, intestinal juice (as the sample to be tested) was suspended in 4 mL of Tris-glycine buffer solution (0.086M Tris, 0.09M glycine, 4 mM EDTA, pH = 8.0) containing 8M urea, and then 20 μL of Ellman reagent (Tris-Gly buffer containing 4 mg / mL of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB)) was added, and then after vigorous shaking, it was allowed to stand at 25 ℃ for 1 h, and then was centrifuged at 12000 r / min for 10 min, and then the absorbance A of the supernatant obtained by centrifugation was measured at 412 nm.

[0095] In addition, the sample to be tested was replaced by Tris-glycine buffer solution, and then the supernatant was obtained by referring to the foregoing method, and then the absorbance A0 of the supernatant was measured at 412 nm. Finally, the total sulfhydryl content of the sample was calculated according to "total sulfhydryl content (μmol / g) = 73.53 × (A-A0) / ρ" (ρ is the concentration of the myofibrillar protein solution (i.e. 20 mg / mL)).

[0096] The results are shown in Figure 14. It can be seen that the total sulfhydryl content of the product under pH 7.0 condition is significantly lower than that of the product under other three conditions (the product under pH 3.0 and pH 9.0 conditions, and the product as a blank control), that is, the most disulfide bonds are formed, the emulsion stability is the highest, and the most disulfide bonds are formed after small intestine digestion, indicating that the method of the application can significantly improve the stability and digestion characteristics of the molten globule protein-fish oil gel emulsion.

[0097] Ten, free fatty acid release rate

[0098] In the foregoing process of preparing the intestinal juice, the volume of NaOH consumed to keep the pH of the system stable at 7.0 during the incubation time of 4 h is recorded as V NaOH , the molar concentration of NaOH (0.5 M) is recorded as m NaOH , and the free fatty acid (FFA) release rate is calculated according to (M Lipid is the average molar mass of the lard / fish oil in the sample, and W Lipid is the mass of the lard / fish oil in the sample).

[0099] The results are shown in Figure 15. It can be seen that the free fatty acid release rate of the intestinal juice under pH 7.0 condition is significantly higher than that of the intestinal juice under pH 3.0 and pH 9.0 conditions, and the intestinal juice as a blank control, that is, the release amount of the emulsion prepared under pH 7.0 condition in the intestinal juice is the highest, up to 71.41%, and in combination with the result of the highest stability in the foregoing experiment, it can be determined that the emulsion prepared under pH 7.0 condition can achieve better sustained release result in the intestinal juice, and the digestion characteristics are better, that is, the phenomenon that the emulsion with higher stability has higher digestion characteristics unexpectedly appears in the test results of the application, indicating that the method of the application can significantly improve the digestion characteristics and stability of the molten globule protein-fish oil gel emulsion.

[0100] It can be known from the analysis of the results of Raman spectrum, fluorescence spectrum, surface hydrophobicity and total sulfhydryl that the molten globule protein is formed in the molten globule protein-fish oil gel emulsion prepared by the method of the application, and it can be known from the analysis of the relationship between particle size, zeta potential, microstructure, emulsion stability and free fatty acid release rate that the method of the application improves the stability and digestion characteristics of the molten globule protein-fish oil gel emulsion. In summary, it is shown that the method of the application not only can form the molten globule protein under neutral pH, normal temperature and pressure conditions, avoiding the problems such as residue of acid and alkali reagents and complicated high temperature and pressure treatment in the prior art, but also can further improve the emulsion stability and digestion characteristics of the molten globule protein.

[0101] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

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 ultrasonication to obtain the solution; wherein the ultrasonication is performed at 140 to 160W for 110 to 130 seconds.

2. The preparation method according to claim 1, characterized in that The homogenization is carried out at 5500-6500 r / min for 4-6 minutes.

3. The preparation method according to claim 1, characterized in that: In the myofibrillar protein solution, the concentration of myofibrillar protein is 10-40 mg / mL.

4. 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%.

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

6. 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.

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

8. The molten globule protein-fish oil gel emulsion prepared by the method according to any one of claims 1 to 7.

9. Use of the molten globule protein-fish oil gel emulsion according to claim 8 as and / or in the preparation of marine food.

10. The molten globule protein extracted from the molten globule protein-fish oil gel emulsion according to claim 8.

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