Views: 0 Author: Site Editor Publish Time: 2023-06-28 Origin: Site
Abstract: Soybean lipophilic protein (SLP) and hydroxy propyl methyl cellulose (HPMC) were treated with different ultrasonic powers to form complex emulsions. Structure-activity relationships between mechanisms and structural properties of emulsion-forming complexes. The results show that after two freeze-thaw cycles, the degree of coalescence decreases with the increase of ultrasonic power, and the composite emulsion formed by 400W ultrasonically treated SLP and HPMC is the most stable; the SLP-HPMC composite emulsion formed by ultrasonically treated SLP The oil yield was significantly lower than that of SLP emulsion without ultrasonic treatment (P<0.05); different ultrasonic power treatments changed the secondary structure of SLP, and the relative content of β-sheet and β-turn in SLP treated with 400 W ultrasonic was the largest, and β -The loose structure of folding and β-turn increases the flexibility of the protein, and the structure is more likely to change and stretch, which affects the stability of the emulsion interface complex, and then affects the freeze-thaw stability of the SLP-HPMC composite emulsion.
Key words:ultrasound; soybean lipophilic protein; hydroxypropyl methylcellulose; emulsion; freeze-thaw stability
Emulsion is a mixture of two immiscible liquids, while protein emulsion is a heterogeneous heterogeneous dispersion system whose instability greatly affects the quality and sensory properties of food. For a long time, it has been considered that soy protein isolate (SPI) is composed of two main storage proteins, glycinin (11S) and β-conglycinin (7S), however, the soy storage protein fraction Soybean lipophilic protein (soybean lipoprotein, SLP) content accounts for about 30% of SPI storage protein, and is expected to be widely used in the food industry as a natural emulsifier. Hydroxypropyl methylcellulose (HPMC) is a kind of edible cellulose, which is rich in resources, healthy and non-toxic, and has good water solubility, film-forming properties and strong surface activity, which can control the surface Pressure and improve film viscoelasticity, used as a good stabilizer for emulsion preparation.
During food processing, emulsions usually undergo repeated freeze-thaw cycles. During the thawing process, a variety of physical and chemical changes will occur, including fat crystallization, water freezing, emulsion freeze concentration, interface phase transition and biomacromolecular conformation changes, etc., which affect the stability of the emulsion. At present, many researchers use physical, chemical and enzymatic methods to change the spatial structure and molecular properties of proteins, thereby improving the freeze-thaw stability of protein emulsions. Ding Jian et al. studied the characteristics of SPI and polysaccharide synergistically stabilizing the emulsion after ultrasonic treatment, and analyzed the instability mechanism of the formed emulsion during freezing-thawing; Cisneros Estevez et al. The formation mechanism and freeze-thaw stability have a certain influence; Wang Xibo et al. used trypsin to obtain SPI hydrolyzates with different degrees of hydrolysis, and then had Maillard reactions with dextran to further study its influence on the freeze-thaw stability of emulsions. In summary, there are many studies on the effect of emulsifiers on the crystallization of pure oil systems and the effects of emulsion processing methods on the freeze-thaw stability of emulsions, but the stability mechanism and structural characteristics of emulsions in the emulsification system formed by modified SLP and HPMC There are relatively few studies on structure-activity relationships.
In this experiment, SLP treated with different ultrasonic powers was used as an emulsifier, and HPMC was used as a dispersant to prepare emulsions to study the effect of ultrasonic changes on the protein structure and its interaction with cellulose on the freeze-thaw stability of the emulsion. and microstructure changes, comparative analysis of emulsion emulsification, degree of coalescence, oil yield and turbidity differences revealed that the emulsion was frozen. Instability mechanism during thawing.
1. Materials and methods
1.1 Materials and reagents
'Dongnong 42' soybean: Soybean Research Institute of Northeast Agricultural University; HPMC (Type I, viscosity 30 mPa·S): KIMA CHEMICAL CO., LTD; Sodium dihydrogen phosphate, disodium hydrogen phosphate: Tianjin Dongli District Great Chemical Reagent Factory; sunflower oil: commercially available; sodium chloride: Tianjin Guangfu Fine Chemical Research Institute; sulfuric acid: Beijing Xinguang Chemical Reagent Factory; all reagents were of analytical grade.
1.2 Instruments and equipment
KF． 250W Ultrasonic Cell Pulverizer: Ningbo Xinzhi Biotechnology Co., Ltd.; ULTRA. TURRAX UTL2000 high-pressure homogenizer: Shanghai Specimen Model Co., Ltd.; Juba SC. 15AH freeze dryer: Shanghai Zhuo Yilong Co., Ltd.; LGR20. W desktop high-speed refrigerated centrifuge: Beijing Jingli Centrifuge Co., Ltd.; Mastersizer 2000 laser particle size analyzer: Malvern Instruments Co., Ltd., UK; DELTAVISIONTMOMMX SR laser confocal microscope: Leica, Germany; UV-5100 high-performance ultraviolet-visible spectrophotometer Design: Shanghai Aoyi Scientific Instrument Factory; JASCO. J815 Circular Dichroism Spectrophotometer: Japan Sanwa Instrument Company.
1.3.1 Extraction of SLP
The separation of SLP refers to the method of Samoto et al. with modifications. Grind soybean powder, pass through a 60-mesh sieve, and degrease with n-hexane to prepare low-denatured soybean defatted powder, grind it, pass through a 60-mesh sieve, and degrease with n-hexane to prepare low-denatured soybean defatted powder, add 400 mL of distilled water to the heat-treated defatted soybean powder, and use Adjust the pH value to 8.0, stir at 20°C for 1 h, centrifuge at 3 000×g for 10 min, collect the supernatant and add 10 mmol/L Na2SO3, then adjust the pH value to 5.8 with H2SO4, and centrifuge at 3 000×g For 10 min, adjust the pH value of the supernatant to 5.0 with H2SO4, and stir at 55°C for 15 min, then add 50 mmol/L NaCl solution and adjust the pH value to 5.5 with NaOH, centrifuge at 3000×g for 10 min, Precipitation is SLP.
1.3.2 SLP ultrasonic treatment
A certain amount of SLP was dissolved in 10 mmol/L pH 7.4 phosphate buffered saline (PBS), and the SLP solution with a mass concentration of 1 g/100 mL was prepared, and the SLP solution was subjected to different ultrasonic power (0, 200, 300, 400, 500 W) for 5 min, and stirred at room temperature for 2 h after the treatment was completed.
1.3.3 Preparation of SLP-HPMC emulsion
Add the SLP solution (1g/100 mL) treated with different ultrasonic power into the same amount of HPMC solution (0.2g/100 mL), and stir magnetically while adding dropwise. After the complex is formed, stir for 30 min, then add 5g/100 mL 100 mL of sunflower oil was coarsely homogenized under the condition of 1000 r/min for 3 min, and then homogenized under high pressure at 100 MPa to prepare SLP-HPMC emulsions treated with different ultrasonic powers, denoted as SLP(0/200/300 /400/500)-HPMC, stored at 4°C for later use.
1.3.4 Cycle freeze-thaw treatment
The SLP-HPMC emulsions treated with different ultrasonic powers were immediately transferred to 50 mL plastic tubes with stoppers, stored at -20°C for 22 h, and then thawed at room temperature for 2 h, and some samples were taken for subsequent analysis. Repeat the above steps 2 times.
1.3.5 Particle size and potential analysis
Dilute each SLP-HPMC emulsion with 10 mmol/L pH 7.4 PBS to 0.1 g/100 mL, fill it into a cuvette (PCS8501), and use a laser particle size analyzer to measure the particle size distribution and potential of the emulsion. SLP (disperse phase) had a refractive index of 1.450, and 10 mmol/L pH 7.4 PBS (continuous phase) had a refractive index of 1.331. All experiments were carried out at 25°C.
1.3.6 Determination of emulsifying activity and emulsifying stability
The emulsification activity index (EAI) and emulsification stability index (emulsification stability index, ESI) were measured with reference to the method of Li Chen et al. After the emulsion was prepared, 50 μL was sampled from the bottom at 0 and 10 min respectively, diluted 200 times with sodium dodecyl sulfate (SDS), mixed thoroughly, and the absorbance was measured at a wavelength of 500 nm, using SDS as a blank control .
1.3.7 Microstructure observation of emulsion
Referring to the method of Hayashi et al., laser scanning confocal microscopy was used to observe the microstructure of the emulsion and evaluate the freeze-thaw stability of the emulsion. Take 1mL of freshly prepared emulsions and emulsions treated with different ultrasonic powers after freeze-thaw cycles, add 40μL of Nile Red and mix evenly, stain for 30min, take a drop of the stained emulsion samples on a grooved glass slide, cover with a cover glass, Glycerin seals. Laser confocal scanning was carried out at the excitation wavelength of 488nm, and oil objective observation was performed. The captured image resolution is 1024X1024. The experiment needs to avoid the influence of pollutants on the glass slide on the image.
1.3.8 Turbidity determination
The SLP-HPMC emulsion was diluted 40 times with PBS solution, and PBS was used as blank control, and UV. Absorbance was measured at a wavelength of 600 rim by a spectrophotometer.
1.3.9 Determination of degree of coalescence
Coalescence is the process by which molecules of oil droplets in an emulsion undergo contact and coalesce to form large oil droplets. Particle size was measured using a laser particle size analyzer.
1.3.10 Determination of oil yield
With reference to the method of Palanuwech et al. with slight modifications. Weigh 0.015 g of Sudan III reagent and add it to 1000 g soybean oil, stir at room temperature for 12 h to obtain Sudan III oil solution. Accurately weigh 4 g of Sudan III oil solution and 16 g of the emulsion to be tested in a 50 mL centrifuge tube, shake and mix well, centrifuge at 16 000Xg, 4°C for 20 min, collect the upper layer of oil and measure the absorbance at a wavelength of 508 nm. Soybean oil was used as blank.
1.3.11 Circular dichroism analysis
Refer to the method of Itoh et al. and make changes. The circular dichroism spectrometer was used to scan the SLP solutions treated with different ultrasonic powers in the range of 190-260 am and 298 K. The parameters were set as follows: path length 1 mm: scan rate 100 nm/min; spectral resolution 0.1 nm; response Time 1 s; step size 1 nm. The relative content of SLP secondary structure was calculated by CDPro software package.
1. 4 Data processing and analysis
The experimental data were processed with SPSS 22.0 analysis software and IOrigin 8.0 software, and the data were analyzed by variance analysis (p<0.05). All experiments were repeated three times, and the results were averaged or mean ± standard deviation.
2. Results and Analysis
2.1 Effects of freeze-thaw cycles on particle size and potential of ultrasonically modified SLP-HPMC emulsion
Generally, the smaller the average particle size of the protein emulsion droplets, the more stable the emulsion. Before the freeze-thaw cycle, the average particle size of the SLP-HPMC emulsion modified by ultrasonic power at different powers was close to 600 nm. After one freeze-thaw cycle, the average particle size of each emulsion increased by more than 1 times. The average particle size is the smallest, which is (1 250.0 ± 23.2) nm. After two freeze-thaw cycles, the average particle size of the SLP (0) -HPMC emulsion increased significantly, and the average particle size of the SLP-HPMC emulsion modified by ultrasonication at different powers also increased to varying degrees, and the increase was the smallest when the ultrasonic power was 400 W , indicating that the emulsion is relatively stable at this time.
The surface charge density of emulsion can effectively reflect the electrostatic interaction between emulsion droplets. The original zeta potential of SLP (0) -HPMC emulsion in pH 7.4 PBS was -11.29 mV. With the increase of ultrasonic power, the absolute value of zeta potential first increased and then decreased. The absolute value of zeta potential of all emulsions decreased after freeze-thaw cycles. The original zeta potential of SLP (400) -HPMC emulsion is -17mV, and the surface charge density is the largest. Combined with the particle size distribution and microstructure analysis results of the emulsion, it shows that the freeze-thaw stability of the emulsion is the best when the ultrasonic power is 400 W. After 2 freeze-thaw cycles, the charge on the surface of the emulsion was further reduced, which was due to the changes in the conformation of the flexible region and the secondary structure of the protein due to the ultrasonic treatment of different powers"), and the change of protein structure and conformation affected its combination with polysaccharide molecules, resulting in Different charges. In addition, the added polysaccharide (HPMC) can be adsorbed to the protein layer to form an interfacial complex, effectively avoiding the degradation of the interfacial complex, thereby enhancing the stability of the emulsion. Studies such as Thanasukarm believe that during the freeze-thaw process The emulsion protein molecules will be desorbed from the surface of the oil droplets, which will reduce the emulsification efficiency of the protein. At the same time, the reduction of the surface charge will lead to the weakening of the repulsion between the oil droplets after the emulsion freezing treatment and coalescence.
2.2 Effects of freeze-thaw cycles on the ESI and EAI of ultrasonically modified SLP-HPMC emulsions
Emulsifying properties are a measure of the protein's ability to adsorb to the oil-water interface, including EAI and ESI. EAI represents the unit mass occupied by the protein-stabilized oil-water interface layer, and ESI is used to measure the ability of the emulsion to resist chaos. Compared with the SLP (0) -HPMC emulsion, the EAI and ESI of the emulsion increased after ultrasonic treatment at 0-400 W, which may be due to the enhancement of the solubility of SLP by ultrasonic treatment. After ultrasonic treatment, the structure of SLP was opened and the solubility At the same time, the exposure of hydrophobic groups strengthened the interaction with HPMC. In addition, SLP adsorbed to the oil-water interface formed a protective bilayer barrier with HPMC, preventing the flocculation and coagulation of fat droplets.
After the freeze-thaw cycle, the EAI decreased significantly. After two freeze-thaw cycles, the EAI of the SLP (400) -HPMC emulsion decreased from 580.35 m²/g to 86.55m²/g. This may be due to the fact that the freeze-thaw process destroyed the SLP and The interaction force between HPMC affects the bilayer built by SLP-HPMC, leading to a sharp drop in the EAI of the emulsion. There was no obvious change in ESI of the emulsion after freezing and thawing. The ESI of SLP (400) -HPMC and SLP (500) -HPMC emulsions increased significantly after the first freeze-thaw treatment, but decreased significantly after the second freeze-thaw treatment, indicating that After one freeze-thaw cycle, part of the emulsion can maintain relatively good stability, but after two freeze-thaw cycles, the emulsion will appear different degrees of stratification.
2.3 Laser Confocal Microscopy Observation Results of Ultrasonic Modified SLP-HPMC Emulsion
The star-red fluorescent core and the green fluorescent peripheral boundary in the laser confocal scanning microscope image prove that there is a SLP-HPMC composite system in the interface layer. Without freeze-thaw cycles, the particle size of SLP (0) -HPMC emulsion was the largest, and the stability of the emulsion was poor. After SLP was ultrasonically treated, the particle size distribution of SLP (200) -HPMC emulsion was relatively uniform, but the particle size was larger, while the SLP (300) -HPMC and SLP (500) -HPMC emulsions showed obvious aggregation phenomenon, only the SLP (400) ) -HPMC emulsion is uniformly dispersed and the particle size is small, and the emulsion is the most stable. After one freeze-thaw cycle, the freeze-thaw stability of the SLP (0) -HPMC emulsion was the worst, and there was obvious water-oil separation phenomenon. Similarly, the SLP (200) -HPMC and SLP (300) -HPMC emulsions also appeared Phenomena of bulk aggregation and water-oil separation. After the second freeze-thaw, the emulsion droplets of SLP (0) -HPMC emulsion appeared to aggregate into flakes, which was due to the poor emulsification of the protein after the combination of SLP and HPMC without ultrasonic treatment, and the instability of the emulsion interfacial film, which formed during freezing. Ice crystals are very easy to pierce the interfacial film, and aggregation occurs between oil droplets when melting". With the increase of ultrasonic power, this unstable phenomenon gradually weakens, and the emulsion formed by ultrasonically treated SLP at higher power is compounded with HPMC, and the emulsion The particles are spherical and relatively small in size. After freeze-thawing of SLP (400) -HPMC, partial flocculation and aggregation of emulsion droplets also occurred, but the increase in particle size was small. Although the particle size of SLP (500) -HPMC emulsion is the smallest , but the water-oil separation phenomenon is serious, and it is no longer an O/W emulsion. Therefore, when the ultrasonic power is 400 W, the stability of the ultrasonically modified SLP-HPMC emulsion prepared is the best.
2.4 Freeze-thaw cycles on ultrasonically modified SLP. Influence of HPMC Emulsion Emulsion Turbidity, Degree of Coalescence and Oil Yield
Turbidity is related to the degree of dispersion of the emulsion and can further reflect the stability of the emulsion. The initial turbidity of the SLP-HPMC emulsion modified by different ultrasonic powers is similar, and the initial turbidity of the SLP (0) -HPMC emulsion is 12 780.43 ± 30.28, and the emulsion is uniform without stratification. After freeze-thaw cycles, part of the emulsion droplets were cracked, and the oil phase was driven by hydrophobic interaction to flocculate and settle again. The emulsion appeared to be stratified (Figure 4D), and the turbidity decreased significantly. After 2 freeze-thaw cycles, SLP (400) -HPMC The turbidity of the emulsion decreased to 2 744.61 ± 18.72. The results showed that freeze-thaw cycles had a significant effect on the turbidity of the emulsion, and it was difficult to restore the stability of the emulsion by simple mixing and stirring.
After freeze-thaw cycles, the interfacial film of the emulsion becomes unstable, and coalescence between oil droplets is easy. The more freeze-thaw cycles the emulsion undergoes, the greater its degree of coalescence. Ultrasonic treatment can change a protein with a large molecular weight into a protein with a relatively small molecular weight, thereby changing its flexibility and spatial structure, making it easier to combine with polysaccharides to form a dense interfacial film, thereby effectively inhibiting the aggregation of oil droplets. After freeze-thaw cycles, the degree of coalescence of ultrasonically modified SLP-HPMC emulsion was significantly lower than that of SLP (0) -HPMC emulsion. With the increase of ultrasonic power, the degree of coalescence showed a trend of first decreasing and then increasing. After 2 freeze-thaw cycles, the degree of coalescence of SLP (400) -HPMC emulsion remained minimum, which may be due to moderate ultrasonic treatment It can expose the hydrophobic groups of the protein, making it easier to adsorb on the surface of oil droplets to resist freezing damage to the stability of the emulsion, thereby increasing the freeze-thaw stability of the emulsion.
The oil yield of the SLP (0) -HPMC emulsion increased from (0.3 ± 0.2)% to (8.5 ± 0.07)% after 2 freeze-thaw cycles. Combined with the data of the degree of coalescence, the interfacial film of the emulsion is unstable at this time. Susceptible to rupture during freeze-thaw. Ultrasonic modified SLP-HPMC emulsion also aggregated to varying degrees, but the release of free oil was less than that of SLP (0) -HPMC emulsion, and with the increase of ultrasonic power, the release of free oil in ultrasonic modified SLP-HPMC emulsion It decreased first and then increased, which was consistent with the results of laser confocal scanning observation and particle size analysis. The generation of free oil is due to the fact that the free water content in the water phase is reduced during the freeze-thaw process of the emulsion, and the emulsion droplets are unstable and close to each other to coalesce, resulting in the release of oil.
2.5 The effect of ultrasonic power on the secondary structure of SLP
Circular dichroism is a fast, accurate and sensitive technique for determining protein secondary structure. It can directly measure and calculate the relative content of various types of protein secondary structures in water-soluble protein solutions. After ultrasonic treatment with different powers, the relative content of each secondary structure in SLP changed significantly. When treated at 400 and 500 W, the relative content of SLP α-helix decreased, and the relative content of β-sheet increased, which was consistent with the previous research results- - The reason may be that the ultrasonic effect affects the hydrophobic amino acid region in the a-helix structure, so that the protein molecule unfolds and changes its spatial conformation28-30. However, when high-intensity (500 W) ultrasound was applied to SLP, the protein aggregated insolublely, and the interaction with HPMC was weakened, so the a-helical structure increased to a certain extent. The structure of the protein is highly related to its function. The change of the secondary structure makes the SLP-HPMC complex more uniform and flexible, making it more completely adsorbed on the oil-water interface, which is consistent with the EAI analysis results in this experiment. . When the ultrasonic power is 400 W, the relative content of β-sheet in SLP is the highest, and it is easier for HPMC to bind to the hydrophilic amino acid region, thereby forming a stable complex, and the conjugate is adsorbed on the surface of the oil layer to form a stable emulsion, thereby improving the freeze-thaw of the emulsion. stability.
Treating SLP with different ultrasonic power can not only enhance the EAI and ESI of SLP-HPMC emulsion, but also significantly improve the freeze-thaw stability of the emulsion. Ultrasonic modified SLP-HPMC emulsions with different powers have different freeze-thaw stability. After 2 freeze-thaw cycles, SLP(400)-HPMC emulsion has the smallest degree of emulsion droplet coalescence and oil yield, and the stability is better, even 2 The obvious O/W emulsion structure can still be observed after several freeze-thaw cycles. Ultrasonic treatment changed the relative content and flexibility range of the secondary structure of SLP, and the relative content of β-sheet and β-turn in SLP treated with 400 W ultrasonic treatment was the largest, and the loose structure of β-sheet and β-turn increased the flexibility of the protein and the structure was more likely to occur Change and stretch, and then affect the stability of the emulsion interface complex, indicating that the freeze-thaw stability of the emulsion is related to the thickness of the emulsion interface layer and the structural properties of the protein.