Mayonnaise-like Pickering high internal phase emulsion and preparation method thereof

Abstract

This invention belongs to the field of molecular biology, specifically relating to a mayonnaise-like Pickering high internal phase emulsion and its preparation method. This invention utilizes TG modification of arginine (Arg)-induced myofibrillar protein (MP) to form a TAMP protein complex, thereby preparing a mayonnaise-like Pickering high internal phase emulsion. The MP, Arg, and TG enzymes used in this invention are all natural products, inexpensive and readily available, possessing good emulsifying properties and biodegradability, making it environmentally friendly and economical.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a mayonnaise-like Pickering high internal phase emulsion and its preparation method. Background Technology

[0002] Arginine (Arg) is a basic functional amino acid that can effectively improve the solubility of myofibrillar proteins. The alkaline environment provided by arginine induces changes in the conformation and potential of myosin, causing the acidic amino acids in myosin to interact with arginine, leading to the unfolding of myosin molecules and the exposure of functional groups. Transglutaminase (TG enzyme) can alter the spatial conformation of myofibrillar proteins or generate heteropeptide bonds through protein cross-linking or deamidation reactions, thus affecting the emulsifying properties of myofibrillar proteins. Cao et al. improved the gel properties of frozen-treated myofibrillar proteins by using a combination of arginine and TG enzyme, forming a fine and dense microstructure. However, the characteristics of the TAMP protein complex formed by arginine (Arg)-induced myofibrillar protein (MP) complex and subsequent modification with TG enzyme have not yet been reported.

[0003] The development of high internal phase emulsions (HIPEs) for mayonnaise provides an effective strategy to replace traditional mayonnaise. Zhao et al. prepared a mayonnaise-based HIPE using phosphorylated perilla protein isolate-chitosan composite nanoparticles (LZPI-CS CNPs), enabling its application in three-dimensional (3D) printing inks and as a lipid-soluble β-carotene delivery carrier. Liu (2019) used wheat gluten (WG) as a readily available and low-cost plant protein component, preparing and stabilizing an oil-in-water HIPE as a mayonnaise substitute via an emulsification-evaporation method. Li et al. prepared pea protein isolate (PPI) microgels using a gel breaking method and applied them to a mayonnaise-like HIPE. Su et al. successfully prepared HIPEs with droplet sizes less than 3.0 μm by constructing ternary composite particles using zein, cellulose gluconate (CFG), and lecithin (Lc). The development of high internal phase emulsions of mayonnaise made from plant-based ingredients has become a research focus for scholars, while research on high internal phase emulsions of mayonnaise made from animal-based ingredients is rarely reported. Summary of the Invention

[0004] To address the above problems, this invention provides a method for preparing a mayonnaise-like Picking high internal phase emulsion, comprising the following steps:

[0005] S1: Extraction of tilapia myofibril protein;

[0006] S2: Preparation of myofibrillar protein particles induced by transglutaminase-modified arginine:

[0007] Myofibrillar protein was dispersed in deionized water to obtain a myofibrillar protein solution. Arg was dissolved in the prepared myofibrillar protein solution and homogenized at high speed to obtain a mixed system. Finally, transglutaminase was added to obtain a transglutaminase-modified arginine-induced myofibrillar protein particle solution system.

[0008] S3: Preparation of Pickering high internal phase emulsion resembling mayonnaise:

[0009] Adding corn oil to a solution of myofibrillar protein particles induced by TG enzyme and arginine and homogenizing it yields a mayonnaise-like Pickering high internal phase emulsion.

[0010] Furthermore, in S2, the myofibrillar protein concentration in the transglutaminase-modified arginine-induced myofibrillar protein particle solution is 0.5-2.5 wt%; the Arg concentration is 1.0%.

[0011] Furthermore, the high-speed homogenization process described in S2 is a high-speed homogenization process under ultrasonic conditions, wherein the ultrasonic conditions are 4°C and 300W for 15 minutes.

[0012] Furthermore, the high-speed homogenization rate described in S2 is 8000 rpm / min, and the homogenization time is 2 min.

[0013] Furthermore, the amount of transglutaminase added in S2 is 10-20 u / g.

[0014] Furthermore, in the mayonnaise-like Pickering high internal phase emulsion described in S3, the Arg concentration is 0.5%-2.0%; the amount of transglutaminase added is 10-20 u / g; the amount of myofibrillar protein is 0.5-2.5 wt%; and the oil-to-protein ratio is 0.80-0.87.

[0015] Furthermore, the homogenization step described in S3 has the following conditions: 7000 rpm and a homogenization time of 2 min.

[0016] The present invention also provides a mayonnaise-like Pickering high internal phase emulsion, which is prepared by the above preparation method.

[0017] The present invention has the following beneficial effects:

[0018] (1) The MP, Arg and TG enzymes used in this invention are all natural products, the raw materials are inexpensive and readily available, and they have good emulsifying properties and biodegradability, making them environmentally friendly and economical.

[0019] (2) This invention utilizes a TAMP protein complex to prepare a mayonnaise-like Pickering high internal phase emulsion, which exhibits unique emulsifying advantages. The resulting Pickering high internal phase emulsion possesses the fine appearance, particle size, and modulus intensity G′ characteristic of mayonnaise. The bridging and flocculation effect is the primary force maintaining the stability of the HIPPEs system. Furthermore, it exhibits good thermal stability and excellent centrifugal stability. This invention is the first to utilize myofibrillar protein modification to construct O / W type HIPPEs, providing a new reference direction for the development of animal-based mayonnaises. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Appearance, microstructure, and laser confocal microscopy of HIPPEs with different MP contents;

[0022] Figure 2 The dynamic viscoelastic modulus variation of HIPPEs with different MP contents;

[0023] Figure 3 The average particle size distribution of HIPPEs with different MP contents;

[0024] Figure 4 The appearance of HIPPEs in different dispersants;

[0025] Figure 5 The particle size distribution of HIPPEs in different dispersants;

[0026] Figure 6 Strain scans of HIPPEs with different MP contents;

[0027] Figure 7 Frequency scans of HIPPEs with different MP contents;

[0028] Figure 8 Rheological properties of apparent viscosity and shear rate of HIPPEs with different MP contents;

[0029] Figure 9 Thixotropic recovery of HIPPEs with different MP contents;

[0030] Figure 10 To assess the stability of HIPPEs in solutions with different pH values;

[0031] Figure 11 To assess the stability of HIPPEs in solutions with different NaCl concentrations;

[0032] Figure 12 For the heat treatment stability of HIPPEs;

[0033] Figure 13 For the freeze-thaw stability of HIPPEs;

[0034] Figure 14 Centrifugal stability of HIPPEs. Detailed Implementation

[0035] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0036] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] The preparation methods of the materials and solutions required in this invention are as follows:

[0041] Extraction of tilapia myofibril protein (MP):

[0042] MP extraction was performed according to a laboratory study method (Pei et al., 2023). The minced muscle was centrifuged three times (16000×, 4℃, 10 min) in a refrigerated centrifuge in a low-phosphate buffer (0.05 mol / L NaCl, 3.38 mmol / L NaH2PO4·2H2O, 15.5 mmol / L Na2HPO4·12H2O, pH 7.5), with the supernatant removed each time. Then, a high-phosphate buffer (0.6 mol / L NaCl, 3.38 mmol / L NaH2PO4·2H2O, 15.5 mmol / L Na2HPO4·12H2O, pH 7.5) was added, and after centrifugation (16000×, 4℃, 10 min), the supernatant (5-fold) was collected and allowed to precipitate at 4℃ for 30 min. Centrifuge again for 15 min under the same conditions, repeat twice, to remove the supernatant from the mixture. The precipitate (MP) is collected and stored at 4°C until further use.

[0043] Preparation of TG-modified arginine-induced myofibrillar protein (AMP) particles (TAMP):

[0044] MP (0.5-2.5 wt%) was dispersed in deionized water and vortexed for 2 min to obtain an MP solution (0.5-2.5 wt%), which was stored at 4°C for further use. A certain amount of Arg was weighed and dissolved in a prepared 1.5 wt% MP solution to achieve an Arg concentration of 1.0%. The solution was then treated with ultrasound at 4°C and 300 W for 15 min, followed by homogenization at 8000 rpm for 2 min to obtain a mixed system of AMP (Arg = 1.0%, MP = (0.5-2.5 wt%)). Finally, a certain amount of transglutaminase (TG enzyme) was weighed to achieve a TG enzyme content of 20 u / g (g is protein content). The mixture was sonicated for 15 min and then homogenized at 8000 rpm for 2 min to obtain a TG enzyme-modified AMP protein particle emulsion (TAMP). This emulsion was stored at 4°C and used within 12 h.

[0045] Preparation of stable high internal phase Pickering emulsions (HIPPEs) using TAMP

[0046] A TAMP protein complex solution was obtained, and corn oil with an oil ratio of 0.87 was added. The mixture was then homogenized at 7000 rpm for 2 min using a homogenizer with a 14 mm dispersing head to prepare a mayonnaise-like Picking high internal phase emulsion.

[0047] Characterization and rheological behavior of stable high internal phase Pickering emulsions (HIPPEs) prepared by TAMP:

[0048] The appearance of HIPPEs was captured by a digital camera. The microstructure of the HIPPE system was imaged using an optical microscope. The droplet size of HIPPEs was measured using a laser particle size analyzer. Furthermore, the morphology of HIPPEs was observed using a laser confocal microscope (CLSM). Before observation, Nile blue was used for protein staining (red), and Nile red for lipid staining (green). The dye solution and emulsion gel were stained at a mass ratio of 1:25 and excited at 633 nm and 488 nm, respectively. The rheological properties of HIPPEs were determined using a rheometer equipped with a parallel plate with a diameter of 40 mm. Flow scanning tests were used to evaluate viscosity changes at shear rates of 0.1–100 s⁻¹. The linear viscoelastic region (LVR) of all samples was measured by strain scanning tests (0.01%–200%) at a fixed frequency of 1.0 Hz. Subsequently, frequency scanning tests within the LVR were performed at oscillating angular frequencies of 0.1–100 rad / s and strains of 0.1%. Thixotropic analysis was performed in three intervals by detecting the change in viscosity over time, with different shear rates (600 s for a shear rate of 0.1 s⁻¹, 600 s for a shear rate of 10.0 s⁻¹, and 600 s for a shear rate of 0.1 s⁻¹).

[0049] Environmental stability evaluation of TAMP preparation of stable high internal phase Pickering emulsions (HIPPEs):

[0050] The stability of HIPPEs under different environments was evaluated based on their visual appearance, microstructure, droplet size, and rheological properties. Centrifugal stability was determined by transferring 20g of HIPPE samples to centrifuge tubes and centrifuging at 12000 rpm for 15 min, then recording the appearance of all samples using a camera. Thermal stability was determined by heating at 100°C for 30 min, followed by rapid cooling to 25°C. Freeze-thaw stability was determined by freezing samples at -20°C for 24 h and then thawing at 25°C for 4 h, recording the appearance changes of all samples using a camera. The effect of pH on the stability of HIPPEs was observed by placing HIPPEs in solutions with pH values ​​of 3.0, 5.0, 7.0, 9.0, and 11.0 for 5 days, and photographing the appearance changes on days 0, 3, and 5. To observe the effect of ionic strength on the stability of HIPPEs: HIPPEs were placed in solution systems with ionic strengths of 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mol / L and observed for 5 days. The appearance changes were photographed on day 0, day 3 and day 5.

[0051] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a mayonnaise-like Pickering high internal phase emulsion, characterized in that, Includes the following steps: S1: Extraction of tilapia myofibril protein; S2: Preparation of myofibrillar protein particles induced by glutamine transaminase modification with arginine: Myofibrillar protein was dispersed in deionized water to obtain a myofibrillar protein solution; Arginine was dissolved in a prepared myofibrillar protein solution and homogenized at high speed to obtain a mixed system; finally, transglutaminase was added to obtain a transglutaminase-modified arginine-induced myofibrillar protein particle solution system. S3: Preparation of Pickering high internal phase emulsion similar to mayonnaise: Corn oil was added to a solution of myofibrillar protein particles modified by glutamine transaminase and homogenized to obtain a mayonnaise-like Pickering high internal phase emulsion; The myofibrillar protein particle solution modified with glutamine transaminase described in S2 has a myofibrillar protein concentration of 0.5-2.5 wt%, an arginine concentration of 1.0%, and an glutamine transaminase addition of 10-20 u / g; the oil-to-protein ratio in S3 is 0.80-0.

87.

2. The preparation method according to claim 1, characterized in that, The high-speed homogenization process described in S2 is a high-speed homogenization process under ultrasonic conditions, with the ultrasonic conditions being 4℃ and 300W for 15 minutes.

3. The preparation method according to claim 1, characterized in that, The high-speed homogenization rate described in S2 is 8000 rpm, and the homogenization time is 2 min.

4. The preparation method according to claim 1, characterized in that, The homogenization step described in S3 has the following conditions: 7000 rpm and a homogenization time of 2 min.

5. A mayonnaise-like Pickering high internal phase emulsion, characterized in that, It is prepared by any one of the preparation methods of claims 1-4.

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