Effects of Emulsifier on Emulsification, Physical and Chemical Properties of Soybean Protein

Emulsification is an important functional characteristic of soybean protein, but the emulsifying ability and stability of natural soybean protein are not ideal, which limits its application in food. This paper mainly studies the application of other emulsifiers in the production of soybean protein, and studies the properties of the final product, in order to optimize the preparation technology of soybean protein, to provide new ideas for the development of high emulsifying soybean protein products and expand the scope of soybean protein application. The paper points out that soybean protein products with high emulsification can be obtained by the complex reaction of sucrose ester with the modified soybean protein as the main raw material.


Materials and reagents
The frozen defatted soybean meal was purchased from Shandong Yuwang Co., Ltd; BSA, SDS, edta-2na, Na2HPO4 ꞏ 12H2O, NaH2PO4 ꞏ 2H2O, CuSO4 ꞏ 5H2O, potassium sodium tartrate, sodium hydroxide, 95% ethanol, acetic acid, bromophenol blue (BPB, 5,5 '-disulfide bis (2-nitrobenzoic acid) (DNTB), copper sulfate (CuSO4 ꞏ 5H2O), potassium sodium tartrate, potassium sodium hydroxide, sodium bisphenol blue (BPB, sodium bisphenol blue (2-nitrobenzoic acid) (dntb), sodium dodecahydrate (BSA), sodium dodecyl sulfate (SDS), sodium edta-2na, sodium dodecyl phosphate (Na2HPO4 ꞏ 12H2O) Urea, Coomassie brilliant blue R250, glycine, glycerol, Tris, dialysis bag (molecular weight 14000) ± 8-anilino-1-naphthalenesulfonic acid and other reagents were purchased from Guangzhou Congyuan science and Technology Instrument Co., Ltd. the reagents were analytical pure; Electrophoretic preformed gel (4% concentrated gel, 12% separated gel), molecular weight standard (PM2510, 10 kDa -180 kDa) and sample buffer (reduced) were purchased from biyuntian Biotechnology Co., Ltd. [8]  Defatted soybean meal was dissolved in 8 times water, then adjusted to pH 7.80 with 3mol / L sodium hydroxide, and stirred at 50 ℃ for 45min. The solution was centrifuged at 3300g centrifugal force for 10min, and the supernatant was taken. The bottom precipitate was redissolved with 5 times water, stirred at 50 ℃ for 10 min, centrifuged at 3300 g for 10 min, and the supernatant was taken. The supernatant obtained from two centrifugations was mixed at 40 ℃ and adjusted to pH 4.50 with 3mol / L hydrochloric acid. Hydrochloric acid was added while stirring. After reaching the isoelectric point, the stirring was stopped immediately, standing for 10min, centrifuging at 3300g for 10min, and the bottom precipitated. Add a certain amount of water to dissolve the precipitate, adjust the pH to 7.20 with sodium hydroxide, and prepare 10% soy protein isolate solution. The solution was homogenized by highpressure homogenizer 20MPa and spray dried. The inlet air temperature was 170 C and the outlet temperature was 80 C. Soybean protein concentrate was extracted by dilute acid extraction. Defatted soybean powder was dissolved in 10 times water, then adjusted to pH 4.50 with 3mol / L hydrochloric acid, stirred at 50 ℃ for 20min, and centrifuged at 3300g for 10min. After repeating the above steps for 2 times, the precipitate was redissolved with water and adjusted to pH 7.20 with 3mol / L sodium hydroxide to prepare 10% soybean protein concentrate solution. The solution was homogenized by highpressure homogenizer 20MPa and spray dried. The inlet air temperature was 170 C and the outlet temperature was 80 C. The modification process of soybean protein is based on the traditional extraction process of alkali soluble acid precipitation and dilute acid extraction, adding acid-base modification, heat treatment and enzyme modification.

Effect of emulsifier on Emulsification of soybean protein
Emulsifying property refers to the property that oil and water are mixed together to form emulsion. Emulsification is the main functional property of soybean protein. The protein has the characteristic structure of emulsifier, the two affinity structure, which contains hydrophilic groups and affinity groups in protein molecules. It can aggregate on the oil water interface, reduce interfacial tension and promote emulsion formation. Soy protein isolate can not only reduce the surface tension of water and oil, but also reduce the surface tension of water and air, so it is easy to form a stable emulsion. The evaluation indexes of emulsifying properties include emulsifying capacity (EC), emulsifying stability (ES) and emulsifying activity (EA). Emulsifying ability refers to the ability of protein to dissolve or emulsify oil. The emulsifying activity refers to the ability of protein to participate in the formation of emulsion and the ability of emulsion to stabilize. Emulsion stability refers to the ability of emulsion to remain stable, and there is no ability to float, flocculate or aggregate within a certain period of time.
On the basis of previous experiments, we selected the unmodified SPI prepared by traditional alkali soluble acid precipitation, alkali modified SPI treated at ph11 for 6h, heat modified SPI treated at 80 ℃ for 5min and enzyme modified SPI hydrolyzed by trypsin for 30min to study the addition of different kinds of emulsifiers (lecithin, monoglyceride, sucrose ester The effect of Tween on the emulsification of protein was studied. The addition amount was 3G emulsifier / 100g protein [9].   The emulsifying activity of the products hydrolyzed by trypsin for 30 min and then added with Tween was lower than that of unmodified SPI; When tween was added to SPI, lecithin was added to alkali modified protein, lecithin or monoglyceride was added to heat-treated protein, and lecithin or sucrose ester was added to enzyme modified protein, the emulsifying activity of each protein was significantly  Figure 3 shows the effect of different emulsifiers on the stability of protein emulsification [10]. It can be seen from the figure that the emulsification stability of alkali modified protein and enzymatic hydrolyzed protein decreased significantly after adding tween, which was lower than that of unmodified SPI; The stability of SPI and protein hydrolysate emulsification was significantly improved by adding monoglyceride; The emulsifying stability of alkali modified protein and heat-treated protein was significantly improved by adding sucrose ester. After heat treatment at 80 ℃ for 5 min and adding sucrose ester, the product has the highest emulsifying stability. Through the previous analysis, we can find that different kinds of emulsifiers have different effects in improving the emulsifying activity and emulsifying stability of different kinds of soybean protein. After adding tween, the emulsification of alkali modified protein and enzyme hydrolyzed protein decreased; After adding emulsifier to SPI, the improvement of emulsification effect is not obvious; Lecithin played a good role in improving the emulsification of alkali modified protein; Monoglyceride has a good effect on improving the emulsification of protein hydrolysate; After heat treatment at 80 ℃ for 5 min and adding sucrose ester, the product has good emulsifying effect.

Effects of emulsifier on protein solubility
Solubility is the main index that affects the functional properties of protein, and it is the key physical and chemical properties that determine the application of protein. The solubility of protein is affected by some factors, such as pH, ionic strength, temperature and solvent type [11]. When the pH value is higher or lower than the isoelectric point, the net charge of protein is negative or positive, and its solubility increases. Although the solubility of protein is the lowest at pH, there are some differences for different proteins. Some proteins, such as casein and soybean protein, are almost insoluble at isoelectric point, while whey protein is still very soluble at isoelectric point. It is very convenient to extract and separate proteins whose solubility changes greatly with pH value by changing the pH value of the medium; For the protein whose solubility changes little with pH value, other methods are needed to achieve the purpose of separation and purification. Figure 3 shows the effect of different emulsifiers on protein solubility. It can be seen from the figure that after adding lecithin, the solubility of each protein is significantly improved; After adding tween, the solubility of alkali modified protein and enzyme hydrolyzed protein decreased; In addition, other kinds of emulsifiers can improve the solubility of protein, but the effect is not very obvious.

Effects of emulsifier on protein particle size distribution
The particle size distribution of SPI was determined by PALS laser particle size analyzer. SPI sample was prepared into 0.2% protein solution with 50 mmol / L phosphate buffer (pH 7.0) and 0.45% protein solution μ The measurement was carried out at room temperature. The average particle size is the equivalent diameter of the largest particle with 50% cumulative distribution in the particle size distribution curve. For an actual particle swarm composed of particles of different sizes and shapes, compared with a hypothetical particle swarm composed of uniform spherical particles, if the full length of the particles is the same, the diameter of the spherical particles is called the average diameter of the actual particle swarm. Micro particulate matter is widely used in daily life and industrial production. Its size and distribution are directly related to the industrial process, product quality, energy consumption and safety of production process [12]. Therefore, it is very meaningful to measure the size and diameter of micro particles accurately and conveniently and get the particle size distribution function. After adding emulsifier, the shape of particle size distribution of each product was similar, although the peak and average particle size increased slightly, and the dispersion index decreased slightly, but the changes were not significant. The reason may be due to the interaction between the hydrophilic and hydrophobic groups of emulsifier and protein, the emulsifier attached to the protein and changed the protein particle size, but the addition of small molecular emulsifier had no significant effect on the existing state of soybean protein, so the protein particle size changed little.

Effects of emulsifier on protein surface tension
Surface tension refers to the increment of surface potential energy when the unit surface area is increased. When the protein denatured at the interface, hydrophobic group would be inserted into oil phase, and the hydrophobic amino acids in oil phase had lower activation energy and spontaneous. As emulsifier protein, it must be able to adsorb to new oil water interface quickly and then reduce the interfacial tension to a low level. The decrease of surface tension is the primary condition for emulsification and foaming, and the degree of surface tension decrease can reflect the ability of protein to expand rapidly, rearrange and expose hydrophobic groups on the interface, which is an important factor of emulsifying force and foaming force. Generally speaking, the stronger the hydrophobicity of proteins, the higher the concentration of proteins adsorbed at the interface; The lower the interfacial tension, the more stable the emulsion [13]. The decrease of surface tension is the first condition of foaming, and the degree of surface tension decrease is an important factor of foaming capacity. Surface tension refers to the work needed to increase the unit surface area of the material on the interface. The surface tension can reflect the ability of the material to expand and rearrange rapidly on the interface, and it is an important factor affecting the emulsification of protein. Figure 4 shows the change of surface tension of the modified protein and the complex of the modified protein and emulsifier. It can be seen from the figure that the surface tension of the modified products has different degrees of decrease compared with SPI, among which the surface tension of the heat modified protein decreases the largest; The surface tension of the complex decreased after adding emulsifier to the modified protein, and the surface tension decreased significantly after SPI was added with Tween and other modified proteins were added with lecithin; The surface tension of the product was the lowest after heat treatment at 80 ℃ for 5min and then lecithin was added.

Effects of emulsifier on protein surface hydrophobicity
Surface hydrophobicity is a key indicator of the number of hydrophobic groups on the surface of proteins in polar water environment. Hydrophobic interaction is the main force to maintain the tertiary structure of proteins, which plays an important role in the stable conformation and functional properties of proteins. The surface hydrophobicity has more influence on the structural and functional properties of proteins than the overall hydrophobicity because of its intermolecular interaction. ANS fluorescence probe is a classical method to explore the hydrophobicity of protein surface, which can reflect the changes of protein tertiary structure in aqueous solution. The principle is that ANS, as a fluorescent probe, does not emit light in the water environment, but can emit relatively strong fluorescence after binding to protein [14]. Therefore, it can be used as a fluorescent probe to reflect the change of the polarity of the environment where the binding site is located, so as to know the change of protein tertiary structure. Surface hydrophobicity is one of the surface structural properties of protein molecules, which reflects the changes of hydrophobic groups on the surface of protein molecules. It is of great significance to the stability, conformation and functionality of proteins. The dissociation of protein subunits and the unfolding of polypeptide chains expose the hydrophobic groups hidden in protein molecules, resulting in the increase of hydrophobicity of protein surface, while the aggregation of protein molecules may cause the decrease of hydrophobicity. Figure 5 shows the change of surface hydrophobicity of different protein molecules. It can be seen from the figure that after alkali modification, heat treatment and enzyme modification, the surface hydrophobicity index of protein is significantly improved, which is consistent with previous studies. The reason for the increase of hydrophobicity is probably due to the change of conformation, the expansion of protein structure and the exposure of hydrophobic groups embedded in the molecule. Small molecular emulsifiers can combine with protein molecules through hydrophobic interaction [15], thus changing the surface hydrophobicity of protein. It can be seen from the figure that the surface hydrophobicity of each modified protein increased after adding tween; After adding lecithin, monoglyceride and sucrose ester, the surface hydrophobicity of SPI, alkali modified protein and enzymatic hydrolyzed protein decreased in varying degrees, but the surface hydrophobicity of heat-treated protein did not change significantly. Figure 5 The influence of different emulsifiers on surface hydrophobicity of soy proteins

Conclusions
Different kinds of emulsifiers have different effects on the emulsification of soybean protein. Among them, lecithin, monoglyceride and sucrose ester can improve the emulsifying activity and stability of soy protein in varying degrees [16], while tween only plays a limited role in promoting the emulsifying activity of SPI and the emulsifying stability of heat modified protein. After adding emulsifier, the surface tension of protein molecules decreased in varying degrees; except for tween, the surface hydrophobicity index of the complexes formed by other kinds of emulsifiers with SPI, alkali modified protein and enzymatic hydrolyzed protein decreased, but the thermal modified protein had no significant change; the addition of emulsifier had no significant effect on the protein particle size distribution.