Preparation and Stability of Soybean Lipophilic Protein-Hydroxypropyl Methyl Cellulose Emulsion

Abstract: To study the effect of 60, 100, 140 MPa high pressure treatment on 0.5% soybean lipophilic protein (lipophilic protein, LP) and 0%, 0.01%, 0.05%, 0.1%, 0.2% hydroxypropylmethylcellulose (hydroxypropylmethylcellulose, HPMC) complex emulsion formation, revealing the relationship between protein structure changes and emulsion stability, using dynamic laser scattering, contact angle measurement and infrared spectroscopy to study the hydrodynamic radius and interface of emulsions formed by LP and soluble HPMC complexes under different pressure treatments Adsorption properties and secondary structure changes of LP. The results showed that the emulsification, storage stability and interface adsorption capacity of the emulsion treated at 60 MPa were low. With the increase of pressure, the particle size of the emulsion became smaller, the shape of the emulsion droplet was regular, the distribution was uniform, and the negative charge on the surface of the emulsion increased; homogeneity When the pressure was 140 MPa and the mass fraction of cellulose was 0.1%, the emulsification activity and emulsification stability were as high as 223.05 m2/g and 290.5 min, respectively; different pressure treatments changed the secondary structure of LP, affected the combination with soluble HPMC, and then affected the The formation of emulsion has adsorption characteristics at the oil-water interface, and the results prove that the hydrophilicity is the best when the mass fraction of HPMC is 0.1%.

Key words: soybean lipophilic protein; hydroxypropyl methylcellulose; emulsion; high pressure treatment; stability

Isolated soy protein (SPI) is a typical commercial product of soybean, which is widely used in food processing. It has long been believed that SPI consists of two major storage proteins, glycinin (11S) and β-conglycinin (7S). However, the latest research found that the lipophilic protein (lipophilic protein, LP) content of soybean storage protein accounts for about 30% of SPI, so the composition and functional properties of SPI need to be further studied. It has been shown in the literature that the solubility of SPI must be regulated by the interaction between the three protein components of 11S, 7S and LP, rather than simply adding the properties of 11S and 7S together. Therefore, other functional properties of SPI should also be reconsidered based on the properties of these 3 protein components. There are many related studies on 11S and 7S in the existing literature, but there is a lack of related research on LP.

The main component of LP is oleosin-phospholipid, which is the original component of oil body and has strong lipophilicity. In the case of adding high concentrations of salt and surfactant (Tween 20), LP emulsions showed better thermal stability, while flocculation was observed for 11S and 7S emulsions. These data indicate that LP has better surface activity than 11S and 7S and can be used as an excellent emulsifier. In addition, most plant proteins lack obvious hydrophobic and hydrophilic partitions in the compact molecular structure, and are rarely used to carry hydrophobic bioactive substances. LP emulsions are expected to solve this problem.

In addition to small molecular mass surfactants, the use of polysaccharides to stabilize protein emulsions has received more and more attention. Compared with conventional emulsions stabilized by surfactants, particle-stabilized emulsions have the advantage of avoiding some of the adverse effects of small molecular weight surfactants, such as tissue irritation, metabolic syndrome, and environmental pollution, etc. For the most available biopolymer, cellulose is a good candidate because it is nutritious, low cost, non-toxic and beneficial for physical and mental health, biodegradable and biocompatible. Hydroxypropyl methylcellulose (HPMC) is another kind of edible cellulose. It is rich in resources, healthy and non-toxic, and has good water solubility and film-forming properties. It is ideal for preparing oil-in-water emulsions. raw material.

This experiment intends to explore the preparation and characterization of LP emulsions stabilized by HPMC, and to study the effects of different homogenization pressures and HPMC mass fractions on the stability of LP emulsions, in order to provide a more stable method for the preparation of LP emulsions.

1. Materials and methods

1.1 Materials and reagents

Soybean: Soybean Research Institute of Northeast Agricultural University; hydroxypropyl methylcellulose

Sodium dihydrogen phosphate and disodium hydrogen phosphate: Tianda Chemical Reagent Factory, Dongli District, Tianjin; Duoli sunflower oil: commercially available; Chlorine Sodium chloride: Tianjin Guangfu Fine Chemical Research Institute; sulfuric acid: Beijing Xinguang Chemical Reagent Factory; other reagents were of analytical grade.

1.2 Instruments and equipment

ULTRA-TURRAX UTL 2000 high-pressure homogenizer: Shanghai Specimen Model Co., Ltd.; LGR20-W desktop high-speed refrigerated centrifuge: Beijing Jingli Centrifuge Co., Ltd.; Mastersizer 2000 laser particle size analyzer: Malvern Instruments Co., Ltd.; DELTAVISIONTM OMX SR ultra- Resolution microscope: Leica, Germany; OCA20 video contact angle measuring instrument: Data Physics Instrument Co., Ltd., Germany; UV-5100 high-performance ultraviolet-visible spectrophotometer: Shanghai Aoyi Scientific Instruments; MAGNAIR560 Fourier transform infrared spectrometer: USA Nicolet Instruments Inc.

1.3 Method

1.3.1 Extraction of LP

Separation of soybean LP fractions was used with a modification of the method of Samoto et al. Soybean powder was ground, passed through a 60-mesh sieve, and degreased with n-hexane to prepare low-denaturation soybean defatted powder, which was dry-heated at 70 °C for 2 h, and the nitrogen solubility index decreased to 75%. 50 g of defatted soy flour after dry heat treatment was added to 400 mL of distilled water, and the pH value was adjusted to 8.0 with NaOH solution. After stirring for 1 h at 20 °C, centrifuge at 3000 r/min for 10 min. Separate the supernatant and add 10 mmol/L Na2SO3, then adjust the pH value to 5.8 with H2SO4, centrifuge at 3000 r/min for 10 min, then adjust the pH value of the supernatant to 5.0 with H2SO4, and treat at 55 °C for 15 min. Then add 50 mmol/L NaCl and adjust the pH value to 5.5 with NaOH solution, centrifuge at 3 000 r/min for 10 min, and the precipitate is the LP fraction.

1.3.2 Preparation of LP-HPMC emulsion

LP oil-in-water emulsion stabilized by HPMC was prepared with 10 mmol/L phosphate buffer solution at pH 7.4. LP solutions were prepared by dispersing an appropriate amount of LP in phosphate buffer, stirring at room temperature for 2 h, and then sonicating to ensure complete dissolution of the protein. Add 0.5% LP solution to 0%, 0.01%, 0.05%, 0.1%, 0.2% HPMC solution respectively, and stir magnetically while adding dropwise to form a complex. After stirring for 30 min, add 25% (V/V) sunflower Seed oil was homogenized under the condition of 1 000 r/min for 3 min, then high-pressure homogenized under the average pressure of 60, 100, and 140 MPa to prepare different types of emulsions, and stored at 4 °C.

1.3.3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Adopt Laemmli's method, in order to adapt to the experimental samples, some changes are made. The separation gel and stacking gel were 12% and 5% respectively. After the sample was mixed with the loading buffer and heated in a water bath, 10 μL of the mixed sample with a mass concentration of 1 mg/mL was drawn with a pipette gun. Start to run the gel after pressurization, and adjust the voltage in good time during the experiment. After the experiment, take out the film, stain it with Coomassie Brilliant Blue solution prepared before the experiment, and then decolorize it for 3 to 5 times to obtain the sample electrophoresis band. .

1.3.4 Dynamic Light Scattering

The size distribution and Zeta potential of protein particles were measured using Mastersizer2000 instrument. Before loading into the cuvette (PCS8501), the sample was diluted to a mass fraction of 0.1% with 10 mmol/L phosphate buffer (pH 7.4). All measurements were performed three times at 25 °C. Use a refractive index of 1.450 for the dispersion (LP) and 1.331 for the continuous phase (10 mmol/L phosphate buffer, pH 7.4).

1.3.5 EAI and ESI determination

Refer to the method of Li Chen et al. 50 μL of a (0 min) and b (10 min) samples were taken from the bottom of the emulsion sample, diluted 200 times with SDS, mixed thoroughly, and the absorbance A was measured at 500 nm, and SDS was used as a blank control.

1.3.6 Super-resolution microscopy

Sample droplets were stained with Nile Blue and Nile Red, a dye that does not fluoresce in most polar solvents but fluoresces strongly with a reddish color in lipid-rich environments, Nile blue shows green fluorescence in the protein-containing environment, which is convenient for observation. The structure of the emulsions under different pressure treatments was examined using a DELTAVISIONTM OMX SR super-resolution microscope. Place 5 µL of the stained emulsion on a microscope slide and cover gently with a coverslip. A He-Ne laser operating at an excitation wavelength of 633 nm images oil droplets and proteins. Use a 60X magnification oil immersion objective for observation.

1.3.7 Storage stability analysis

For the determination of the creaming index (CI) of the composite system, refer to the method of Zisu et al. The oil-in-water emulsion sample was left to stand at room temperature for 35 days, and the scale of the layered interface of the stoppered colorimetric tube where the emulsion was located was recorded every day to measure the degree of phase separation of the emulsion. The ratio of serum height HC to total emulsion height HE represents CI.

1.3.8 Measurement of contact angle

Measured by the sessile drop method using a contact angle analyzer at 25 °C. Briefly, emulsions were applied to glass slides to prepare films with a diameter of 13 mm and a thickness of 2 mm. Deposit a drop of water (5 µL) on the surface of the film using a high-precision syringe. An image of the droplet was recorded immediately after falling from the syringe by the camera, and the profile of the droplet was numerically solved and fitted to the Laplace-Young equation, its interfacial tension could be determined. The contact angle was measured on each of the 3 particles for each sample, and 3 measurements were taken for each particle.

1.3.9 Infrared spectral analysis

The emulsion was directly lyophilized, and the lyophilized sample was placed in a desiccator and fully dried with P2O5. 1.5 mg of the sample was ground and mixed with 200 mg of potassium bromide, and then pressed into tablets for infrared spectrum measurement. During the experiment, in order to reduce the interference of water vapor, the measurement chamber was continuously injected with dry N2. During the measurement, the wavenumber range is 4 000-400 cm-1, the number of scans is 64, and the resolution is 4 cm-1. The obtained infrared absorption curve is fitted by "peak fitting" software and Gaussian curve to analyze the condition of protein under different homogeneous pressures. Changes in secondary structure content.

1.4 Data processing

The data in this experiment are the average value of 3 parallel samples, and the results are processed by SPSS 22.0 analysis software and Origin 8.0 software, and the data are analyzed by ANOVA for significant difference (P<0.05).

2. Results and Analysis

2.1 SDS-PAGE analysis

The LP proposed in this experimental method is a complex composed of various proteins, including 11S, 7S and the residual subunit of lipoxygenase (Lx). The 34 kDa protein was originally identified as a soybean oleoprotein because it can be tightly associated with oil bodies, but was later thought to be located in the protein storage vacuole, also part of the LP. This is consistent with the findings of Gao Zhiming et al., proving that the extracted LPs are of good quality. However, some bands were clearly unclear, which may be due to the relatively high content of phospholipids in the LP fraction, resulting in relatively poor Coomassie brilliant blue staining, as reported by Samoto et al. It is precisely because of the existence of a large amount of phospholipids that the surface activity of LP is increased, which provides a theoretical basis for the formation of subsequent stable emulsions.

2.2 Dynamic light scattering analysis

The smaller and more uniform the particle size, the better the stability of the emulsion; the larger the absolute value of the same potential, the better the stability of the emulsion. The particle size of the LP-HPMC composite emulsion is related to the characteristics of the composite. The volume average particle diameter D4,3 of the LP-HPMC composite emulsion after different homogeneous pressure treatments, the D4,3 of the emulsion formed without adding HPMC, Most of the 3 values are distributed around 300 nm, and the particle size of the emulsion added with HPMC changes differently under the homogeneous conditions of different pressures with the change of the mass fraction of HPMC. As the homogenization pressure continues to rise, the particle size decreases continuously under the same HPMC mass fraction. Under different homogenization pressure conditions, when the cellulose mass fraction is 0.1%, the particle size is smaller than other additions, and the absolute value of the potential It is also the largest, which proves that the stability of the emulsion formed when the mass fraction of HPMC is 0.1% is the best, which is directly related to the combination degree of LP-HPMC complex. And when the homogeneous pressure is 100 MPa and the mass fraction of cellulose is 0.1%, the absolute value of the potential reaches the maximum value of 40.4 mV, indicating that the emulsion at this time reaches the most stable state.

2.3 Emulsification analysis

EAI represents the ability of the LP-HPMC complex to form an oil-water interface, which combines the solubility of proteins in the water phase and the ability of cellulose to strongly adsorb at the oil-water interface to form an emulsified layer; ESI refers to the ability of the emulsion to form a small Droplet stabilization capabilities [18]. It can be seen from Figure 2 that under the three conditions of 60, 100, and 140 MPa, the mass fraction of HPMC shows the maximum value at 0.1% EAI, which proves that the mass fraction of cellulose at 0.1% is the most favorable for the emulsion system, which is consistent with the previous conclusions. At 60 MPa, the ESI increases first and then decreases with the increase of the mass fraction of cellulose, and shows a sudden increase trend when the mass fraction is 0.1%. The addition of the addition of has a key effect on the stability of the emulsion, on the contrary, it is easier to form two phases that are incompatible with each other, which has a negative impact on the emulsification of the emulsion. When it reaches 0.2%, the ESI decreases obviously, indicating that too high a cellulose concentration is not conducive to the stability of the emulsion, and it is more appropriate to add 0.1% HPMC. At 100 MPa, ESI shows an upward trend with the increase of cellulose mass fraction from 0% to 0.1%. It can be speculated that 100 MPa may be the best homogeneous pressure for cellulose to act on the emulsion. At 140 MPa, the trend of ESI is similar to that at 60 MPa, and the ESI value is the highest when the mass fraction of cellulose is 0.1%.

2.4 Microstructure analysis of emulsion

Super-resolution microscopy is often used to analyze the microstructure of emulsions, which can directly reflect the particle size, dispersion and instability of emulsions. In the case of homogeneity at 60 MPa, the particle size of the emulsion droplets is large and the distribution is uneven, and the aggregation of emulsion droplets occurs. The interfacial film of the emulsion formed by soybean protein alone was not stable enough, and more obvious flocculation or aggregation between oil droplets occurred. Comparing the super-resolution images of 100 MPa and 140 MPa, it can be found that the state of the emulsion is the best under the homogeneous pressure of 100 MPa, the particle size is smaller and the most uniform, and the emulsion is the most stable when the mass fraction of HPMC is 0.1%. At 140 MPa, some emulsion droplets are larger, and there are also smaller emulsion droplet distributions. This is mainly due to the fact that LP and HPMC are separated under high pressure and are not well recombined, so that the oil droplets are partially stabilized by polysaccharides alone. The results proved that 100 MPa is the best homogeneous pressure for LP-HPMC emulsion formation, and the best HPMC mass fraction is 0.1%.

2.5 Storage stability analysis

Emulsification of an emulsion is caused by the different densities between the phases in the emulsion, usually the oil and water phases. Therefore, CI provides information on the degree of aggregation and separation of oil droplets from the aqueous phase in the emulsion. The 35-day storage stability test results of different emulsions showed that the layering phenomenon of the emulsion changed with time, and the CI of the LP-HPMC composite emulsion with a mass fraction of 0.01% HPMC was the highest, indicating that the stability of the emulsion was the worst. Consistent with the results of EAI and ESI analyzed before, it proves again that the addition of lower mass fraction HPMC cannot combine with LP well, but will form two incompatible phases, which is not conducive to the stability of the emulsion. Secondly, the CI of LP emulsion without adding HPMC is higher, which proves that LP alone is not enough to form a stable emulsion. The lowest CI is the emulsion with HPMC mass fraction of 0.1%, and it is concluded that the cellulose mass fraction of 0.1% is most conducive to the formation and stability of the emulsion.

2.6 Analysis of contact angle

The contact angle refers to the tangent of the gas-liquid interface at the intersection of gas, liquid and solid. The tangent is on the liquid side, and the angle between the tangent and the solid-liquid junction is θ, which is a measure of the degree of wetting. If θ<90°, the solid surface is hydrophilic, that is, the liquid wets the solid more easily, and the smaller the angle, the better the wettability; if θ>90°, the solid surface is hydrophobic. The experiment found that the change of contact angle is mainly affected by the mass fraction of cellulose, and has little to do with the change of homogeneous pressure. It may be because the hydrophilic cellulose is adsorbed on the surface of the emulsion, which increases the hydrophilicity of the emulsion. All emulsions are hydrophilic, but the degree of hydrophilicity varies. Without the addition of HPMC, the angle of the emulsion formed by wrapping sunflower oil with LP was 67.5°, which was significantly higher than the angle of the emulsion formed after adding HPMC. On the other hand, the stability of protein emulsion is closely related to the magnitude of interfacial tension and interfacial pressure. The smaller the interfacial tension and the greater the interfacial pressure, the more stable the emulsion. When the mass fraction of cellulose is 0.1%, the contact angle is as low as 19.6°, and the hydrophilicity is the best. At the same time, the interfacial tension value is also the smallest under this condition, indicating that the emulsion is the most stable when the mass fraction is 0.1%. It shows that the combination degree of LP and HPMC is the highest at this concentration, and the hydrophilic part of HPMC is adsorbed on the surface of the emulsion, which increases the hydrophilicity of the emulsion.

2.7 Infrared spectral analysis

The content of the protein secondary structure in the emulsion was characterized by infrared spectroscopy at different homogeneous pressures when the mass fraction of HPMC was 0.1%. The protein infrared spectrum of 1 600-1 700 cm-1 belongs to the peptide bond absorption range, including the protein main chain conformation Information. The obtained spectra were analyzed and calculated by Peak Fit fitting software, and the secondary structure content of LP in LP-HPMC emulsions treated with different homogeneous pressures was obtained. It can be seen that as the homogeneous pressure increases, the α-helix, β-turn and random coil structures all show a trend of decreasing first and then increasing, while the content of β-sheet structure is as high as 40.5% at 100 MPa. It shows that LP has certain influence on α-helix, β-sheet, β-turn and random coil structure after high-pressure homogenization treatment, the secondary structure of LP is destroyed, its flexible structure increases, and protein molecules change from ordered structure to become disordered. The α-helix and β-sheet structure of LP are mainly maintained by hydrogen bonds. After high-pressure homogenization treatment, the hydrogen bonds in the secondary structure may be destroyed, so that the protein molecules are unfolded, the secondary structure is destroyed, and the protein is changed to varying degrees and Stretching may affect the combination degree of LP and other substances, which is consistent with the previous conclusion that the emulsion is most stable when the homogeneous pressure is 100 MPa.

3. Conclusion

The particle size of the emulsion formed by the LP-HPMC composite treated by 100 MPa high-pressure homogenization is uniform and stable, the shape of the emulsion droplet is regular, and the EAI and ESI are better. At the same time, high-pressure homogenization changes the secondary structure of LP, thereby improving the emulsification ability. At 100 MPa, the content of β-sheet structure is the highest, and the conformation of LP changes. At this time, the composition of the disordered conformation and the unfolding of the flexible structure of LP affect the overall protein. The flexibility of the conformation makes it easier to form complexes with HPMC with better functional properties and increase the stability of the emulsion; more or less than 100 MPa will adversely affect the stability of the emulsion. The mass fraction of HPMC also has a certain influence on the emulsion. When the mass fraction is 0.1%, the ESI and hydrophilicity of the emulsion are the best, and the morphology of the emulsion observed by super-resolution microscopy is also the most stable. Therefore, this experiment found that the emulsion formed under the condition of HPMC mass fraction of 0.1% and homogeneous pressure of 100 MPa is the most stable.