Ginger essential oil nanoemulsion encapsulated by zein/NaCas and antimicrobial control on chilled chicken -

Ginger essential oil nanoemulsion encapsulated by zein/NaCas and antimicrobial control on chilled chicken

An efficient antibacterial nanoemulsion was prepared using zein and NaCas to encapsulate ginger essential oil (GEO). Physical, optical, and mechanical properties as well as the antibacterial activities of GEO nanoemulsion were investigated. At 1:1 mass ratio of zein/NaCas, the GEO nanoemulsion possessed the highest solubility, entrapment efficiency and stability. The GEO/zein/NaCas complex was confirmed by ultraviolet and fluorescence spectroscopy. The addition of GEO led to more amorphous structure formation and the secondary structure changes of zein/NaCas improved the solubility and stability of GEO. GEO nanoemulsion inactivated two common foodborne bacteria, namely, Staphylococcus aureus and Pseudomonas aeruginosa, by destroying the cell membrane. Meanwhile, the GEO nanoemulsion exhibited better preservation effects on chilled chicken breasts than nonemulsified GEO and could effectively prolong the shelf life of chicken breasts for 6 days. This research provides a green and low-cost method for preparing GEO nanoemulsion to control the risk of foodborne pathogens.

Materials and methods

Zein (>92%), NaCas (>90%) and ginger essential oil (GEO) were purchased. Pseudomonas aeruginosa and Staphylococcus aureus were isolated from chicken

Emulsion preparation

2% of Zein and NaCas solutions were prepared separately first. The pH of both solutions was adjusted to 11.5 with 3 M NaOH and magnetically stirred at 600 rpm for 2 h. Next, GEO protonation [10% (v/v)] was performed in accordance with the following steps. Two mL of GEO was added to an 18 mL of 3 M NaOH in Erlenmeyer flask and heated at 120 ◦C until the solution was transparent. Then, zein and NaCas solution prepared above were mixed with zein/NaCas mass ratios of 0:1, 1:1, 1:2, and 2:1. After stirring for 30 min at 600 rpm, deprotonated GEO [10% (v/v)] was added to zein/NaCas mixture solutions at a final concentration of 1% (v/v) GEO. In addition, a series of deprotonated GEO were mixed with zein/NaCas mass ratio of 1:1 to obtain nanoemulsion with a final concentration of GEO ranging from 0.5 to 2% (v/ v). At last, the pH of zein/NaCas/GEO mixture was adjusted to 7.0 using 3 M citric acid monohydrate. The GEO nanoemulsions formed spontaneously and were freeze dried. The obtained powders were stored at − 20 ◦C for further study.

Particle size and zeta potential

Nanoparticle size and potential analyzer was used to investigate the particle size and zeta potential of prepared GEO nanoemulsion. Before measurement, the large precipitates in nanoemulsions were removed by centrifuging at 6000 rpm for 10 min. After that, the emulsions were diluted by 20 times using pure water (pH 7.0) before analyses. Each sample was determined three times and the average value was stated.

Entrapment efficiency (EE)

EE refers to the ratio of the remaining oil content in the emulsion to the total oil content after removing the free oil. Total amounts of GEO added in nanoemulsions were defined as total oil content. The determination of free oil content was mainly carried out according to previous work. Subsequently, 0.5 mL of the supernatant was added to 2 mL of petroleum ether. After standing for 10 min, 100 μL of the upper layer solution was transferred and evaporated, and 4 mL of absolute ethanol was added. Subsequently, the absorbance at 204 nm (Abs204 nm) was detected using a full-wavelength microplate reader. A standard curve is defined by Abs204 nm from standards containing known concentrations of GEO. The concentration of free oil was calculated by a standard curve made in advance.

Surface hydrophobicity (S0)

S0 value was determined using 8-anilinonaphthalene-1-sulfonic acid (ANS). Each group of protein solution (2%, w/w) was diluted to different concentrations (0.02%–0.08%, w/w) by using 10 mM PBS (pH 7.0). Then, 4 mL of the prepared proteins were mixed with 20 μL of 8 mM ANS and incubated at room temperature for 2 h in the dark. Then the fluorescence of the incubated sample was determined with the excitation wavelength of 350 nm and the emission spectrum ranging from 400 to 600 nm using the LS-55 fluorescence spectrometer. A linear graph of fluorescence intensity at 465 nm against protein concentration was drawn. The slope of the regression equation (R2 > 0.99) obtained was the S0 of the protein.

Characteristics of GEO nanoemulsion

Ten μL of nanoemulsion was pipetted onto the cover slip, spread out, and air dried naturally. All samples were then sprayed with gold. The morphology and distribution of the nanoparticles in the nanoemulsion matrix were observed using scanning electron microscopy at an accelerating voltage of 10 kV.

Fluorescence spectroscopy

Proteins could exhibit intrinsic fluorescence, which mainly came from the tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) residues. The changes in microenvironment of Tyr, Trpand Phe residue in zein and NaCas was explored using a fluorescence spectrometer. In brief, 0.02 mg/L of zein, NaCas or zein/NaCas mixture (w/w, 1:1) was prepared by PBS, respectively. Then GEO dissolved in 95% ethanol was diluted to 0–40 μg/mL using prepared protein solutions. Fluorescence spectroscopy analysis was performed with the excitation wavelength of 280 nm and the emission spectra ranging from 290 nm to 450 nm.

X-ray diffraction (XRD)

GEO nanoemulsion (zein/NaCas mass ratio was 1:1), zein, NaCas, zein/NaCas mixture (w/w, 1:1) were freeze dried into powder before analysis. The crystalline structures were measured using an X-ray diffractometer (D2 PHASER, Germany) at 20 kV and 5 mA. The scanning range (2θ) was 5◦–80◦ in 0.028 steps, and the scanning rate was 4◦/min.

Cell morphological analysis

The indicator bacteria P. aeruginosa and S. aureus were incubated in brain heart infusion (BHI) broth in a shaker (200 rpm, 37 ◦C) for 24 h. The cultures of two strains were transferred to the fresh BHI medium at the proportion of 2% and cultured to logarithmic growth phase. Five mL bacterial cells were collected by centrifuging at 6000 rpm for 5 min. Then, 5 mL of GEO nanoemulsion under minimum concentration (MIC) was added, and a sample with the 5 mL of PBS buffer added was used as the control. All the samples processed above were cultured with shaking for 3 h (200 r/min, 37 ◦C). The cells were collected by centrifuging at 6000 rpm for 5 min. One mL of 2.5% (v/v) glutaraldehyde was added to the cells for fixing the bacteria. After 12 h at 4 ◦C, the samples were centrifuged at 6000 rpm for 5 min to collect the bacterial cells. The cells were then eluted with 50%, 70%, 80%, and 90% ethanol for 15 min successively. Anhydrous ethanol was added to dehydrate the samples three times, each lasting for 30 min. Finally, the samples were fixed on glass slides, air dried, sprayed with gold, and finally observed by SEM.

Laser scanning confocal microscopic (LSCM) analysis

LSCM (Ultra View VOX, PerkinElmer, America) was performed to assess the damage to P. aeruginosa and S. aureus cell membranes following treatment with GEO nanoemulsion. The bacterial suspension was prepared as mentioned above. After GEO treatment, the bacteria were collected and resuspended in 10 mM PBS buffer. Subsequently, two fluorescent dyes were added and stained in the dark by using the LIVE/ DEAD BacLight kit in accordance with the instructions from the manufacturer. After that, cells were collected and observed via LSCM with a 100× oil lens.

Evaluation of antibacterial activity in bacterial solution

The P. aeruginosa and S. aureus activated overnight were transferred to 5 mL LB liquid medium at the proportion of 2% for further cultivation. After incubating to the logarithmic phase, the bacterial solution was diluted to 103 CFU/mL with normal saline. A two-fold dilution method was used to test the MIC of GEO nanoemulsion, with the final GEO concentrations of 0.625, 1.25, 2.5, and 5 mg/mL. The concentration of no colony growth on the plate was the MIC. In brief, 100 μL of bacteria in the logarithmic phase was mixed with 10 mL of 1% (v/v) GEO solution and GEO nanoemulsion (Final concentration of GEO is 1%). In the control group, the bacterial solution was added to the same volume of physiological saline. After treatment, serial decimal dilutions were carried out for viable counts.

Evaluation of antibacterial activity in the bacterial solution in chicken breasts

Twenty g of minced fresh chicken breast meat were sub-packaged. Then, 1 mL of PBS (control), GEO solution and GEO nanoemulsion containing 1% GEO (v/v) were added. The chicken was stored for 12 days at 4 ◦C. The samples were taken every three days. The total viable count was obtained using the serial decimal dilutions.

Results

Self-Assembly of GEO nanoemulsion

GEO nanoemulsion was prepared using NaCas and zein as the emulsifier. Except for the sample with a zein/NaCas mass ratio of 2:1, all the nanoemulsion samples were uniformly dispersed in water, and they presented a good light-yellow milky appearance. The sample with a zein/NaCas mass ratio of 2:1 showed obvious precipitation. It was probably because that no sufficient NaCas combined with zein nanoparticles to stabilize the nanoemulsions, which then tended to aggregate Particle size and zeta potential were detected to evaluate the dispersion and storage stability of the emulsion. The nanoemulsion prepared by zein/NaCas at 1:1 mass ratio showed the smallest particle size (144 nm), while the particle size obtained from the zein/ NaCas at 0:1 mass ratio was the largest (196.46 nm). The smaller the particle size of nanoemulsion was, the larger the specific surface area and the better the dispersibility. At the mass ratio of 1:1, the maximum negative charge of the emulsion was − 34.62 mV, indicating that the zein colloidal particles had the strongest electrostatic repulsion and the best stability in the water phase under this condition. Overall, the addition of NaCas decreased the content of visible precipitates in the mixture, decreased the particle size, and increased the zeta potential and stability. This finding might be due to insufficient NaCas for stabilizing the zein nanoparticles.

S0

In the present study, the S0 values of zein/NaCas mixtures were analyzed to further understand the synergy between zein and NaCas. NaCas alone had the lowest S0 value (1656.23), where only a small amount of hydrophobic surface could be used for fluorescent probes (ANS). In the zein/NaCas mixtures, S0 increased with the increase in the zein content (5824.57 of 1:2 to 7599.93 of 2:1), indicating that ANS was easily accessible to hydrophobic zein. As the size of the nanoparticle decreased, the specific surface area increased, thus exposing more hydrophobic residues. In turn, the stability of the emulsion could decrease. The mixture with a zein/NaCas mass ratio of 2:1 had the highest S0, but the EE and stability were lower than those of the sample with a zein/NaCas mass ratio of 1:1. Therefore, 1:1 was used as the optimal ratio in the experiment.

The effect of GEO concentrations on the characteristics of nanoemulsion was detected at a NaCas/zein mass ratio of 1:1. As the concentration of GEO increased, the EE decreased from 90.95% to 81.48%. It might be due to the fact that GEO escaped into the external phase through a more porous polymer matrix structure. The particle size and zeta potential increased significantly with increasing GEO concentration. In conclusion, when the concentration of GEO was 0.5%, the nanoemulsion had the optimal stability and the highest EE value. Therefore, in actual applications, the concentration of GEO in the emulsion should be reduced as much as possible under the conditions that meet the required antibacterial requirements.

During GEO nanoemulsion formation, casein micelles were dissociated and zein was dissolved first in strong alkaline. At this point, zein could diffuse in the opened NaCas lattice. When the deprotonated GEO was added, the three components could blend well in the aqueous solution. When the pH was gradually neutralized to 7.0, both zein and GEO became insoluble but GEO could be embedded into the hydrophobic interiorof the zein under the hydrophobic interaction. Steric stabilization forces and electrostatic repulsion worked together to prevent zein aggregation. Hence, GEO nanoemulsions encapsulated with NaCas/zein (m/m, 1:1) showed the highest EE and excellent stability, while the nanoemulsions encapsulated with NaCas/zein (m/m, 1:2) displayed obvious precipitation due to the shortage of NaCas.

Morphology of GEO nanoemulsion

The SEM images directly displayed the size and shape of the nanoemulsion particles. When the concentration of GEO was 1% (v/v), the morphology of GEO nanoemulsion with different mass ratios of zein/NaCas were spherical, and they had smooth surfaces. The size of the emulsion particles with zein/NaCas mass ratios of 1:1 and 1:2 was smaller than that with other ratios.

The interaction between small molecules and proteins could be analyzed through the intrinsic fluorescence characteristics of proteins. Zein had an emission peak at 310 nm which is the emission wavelength of tyr residues. As the concentration of GEO increased, the maximum emission wavelength slightly red shifted, and the fluorescence intensity gradually decreased. These results indicated that the microenvironment of Tyr residue in zein was altered due to addition of GEO. NaCas had a characteristic emission peak at 350 nm, which was the emission wavelength of Trp. The fluorescence intensity decreased with increasing GEO concentration, but the maximum emission wavelength did not change. These findings indicated that GEO changed the microenvironment of tryptophan (Trp) residues in NaCas

UV–vis spectroscopy

As the concentration of GEO increased, the maximum absorption wavelength slightly red shifted (from 195 nm to 201 nm), and the absorption peak gradually decreased. As the concentration of GEO increased, the absorption peak of the proteins at 278 nm also gradually increased, showing a hyperchromic effect. These results indicated that a π-π stacking interaction between GEO and zein/NaCas occurred. Thus, the two results indicated that the complex could be formed through the interaction of GEO and zein/NaCas.

FTIR spectroscopy

The chemical bond and functional group information contained in the molecule could be obtained from the infrared spectrum. The change in molecular structure could also be reflected by the change in the vibration frequency of the polar chemical bond. Both of the peak became more flattened after embedding the GEO, indicating the decreased stretching of O–H and C–H due to the interaction between GEO and proteins. The peaks disappeared after GEO added, further demonstrating that GEO was successfully embedded in the zein/NaCaS. Based on the changes in bands in GEO nanoemulsion, GEO might chemically react with zein/NaCas to form new substances.

XRD analysis

In this study, the four samples exhibited two intense diffraction peaks of 2θ at 8.75◦ and 19.29◦, which were associated to the crystallinity of zein and NaCas. The diffraction peak of zein was found at 28.42◦, but that of NaCaS was not, indicating that the crystal planes contained in zein was different from that in NaCas. When GEO was added to the nanoemulsion, the intensity of the characteristic peak absorption related to the protein crystal structure and the relative crystallinity of the material decreased. This decrease might be due to the interference of other components, which hindered the progress of the crystallization process and contributed to the formation of an amorphous structure in the nanoemulsion.

Damage of bacterial cell membrane

The effects of GEO nanoemulsion on the cell morphology of P. aeruginosa and S. aureus were observed via SEM. The bacterial cells in the control group were plumply dispersed uniformly with smooth cell membranes. Cells of both strains were damaged after treatment with GEO nanoemulsion. The surface of P. aeruginosa cells was severely damaged, the morphology was shriveled, and the cells overlapped one another. The S. aureus cells lost their original spherical shape. The morphology became shriveled and wrinkled, with a slight depression. P. aeruginosa and S. aureus cells were stained with two fluorescent dyes, SYTO-9 and PI. CLMS was used to intuitively observe the changes in cell-membrane permeability. Most of the untreated indicator bacterial cells showed green fluorescence and only few cells showed red light due to decay, indicating that the bacterial cell membrane was basically intact and undamaged. After treatment with GEO nanoemulsion, most of the bacterial cells showed red fluorescence, and only a small part of cells showed green fluorescence. PI entered the cell through the damaged cell membrane and combined with nucleic acid, thus making the cell show red fluorescence. The major inhibitory components of GEO are gingerol and zingiberene. The cytoplasmic membrane damage caused by GEO nanoemulsion might be due to the role of GEO.

Evaluation of antibacterial activity in bacterial solution

The MIC values on P. aeruginosa and S. aureus were 5 and 2.5 mg/mL, respectively. Thus, the GEO nanoemulsion showed better inhibitory effect on the S. aureus strain. After the GEO aqueous solution treatment for 4 h, the viable counts of P. aeruginosa and S. aureus decreased by 1.14 and 2.22 lg CFU/mL. Whereas after 4 h of GEO nanooemulsion treatment, the viable counts of P. aeruginosa and S. aureus decreased by 2.16 and 4.59 lg CFU/mL. The GEO nanoemulsion had a stronger antibacterial effect than the GEO aqueous solution. In addition, the inactivation effect of GEO on S. aureus was stronger than that on P. aeruginosa, and this finding was consistent with the results of MIC.

Evaluation of antibacterial activity in chicken breasts

Microorganisms are the main cause of meat spoilage during the storage of chilled meat and meat products. The TVC of the control group increased rapidly and reached 7.62 ± 0.19 lg CFU/g after 9 days, exceeding the TVC limit for fresh meat (range of 6–7 lg CFU/g). The TVC of GEO-treated chicken breasts reached 7.65 lg CFU/g, while that of GEO nanoemulsion-treated group was only 5.75 lg CFU/g after 12 days of storage. TVB-N content is also an important indicator of meat freshness. The TVB-N values of chicken breasts reached 25.08 ± 0.78 mg/100 g for the untreated group after 9 days. In addition, 15–25 mg/100 g of TVB-N in chicken breasts indicated poor freshness, whereas 25 mg/100 indicated rottenness. The TVB-N reached 30.9 ± 0.57 mg/100 g after 12 days for the GEO-treated group, exceeding the limit of spoilage. These results indicated that the GEO encapsulated by zein/NaCas exhibited better preservation effect on chilled chicken breasts than un emulsified GEO, and it could effectively prolong the shelf life of chicken breasts for 6 days. Chicken breast was selected as a model food containing high protein. As a consequence, the GEO nanoemulsion was environment-friendly emulsion that had promising effects on the preservation of food products.

Conclusions

In this study, GEO nanoemulsion was prepared using NaCas, zein, and naturally occurring emulsifiers. Self-emulsification did not require special equipment and organic solvents. Thus, it showed the advantages of high safety and low cost. When the mass ratio of zein/NaCas was 1:1, the emulsion showed the smallest particle size with the highest EE and the strongest stability. Spectroscopic analysis showed that GEO not only changed the secondary structure of proteins but also had a bond action with proteins, and this bond action promoted the formation of the amorphous structure of proteins. In addition, antibacterial experiments showed that nanoemulsification improved the antibacterial properties of GEO. Besides, the GEO nanoemulsion prepared by low-energy selfassembly method had a good inhibitory effect on the common spoilage bacteria, such as P. aeruginosa and S. aureus, in meat. Thus, it has a certain potential to be applied in meat preservation.

Citation:

Tang, M., Liu, F., Wang, Q., Wang, D., Wang, D., Zhu, Y., Sun, Z. and Xu, W., 2022. Physicochemical characteristics of ginger essential oil nanoemulsion encapsulated by zein/NaCas and antimicrobial control on chilled chicken. Food Chemistry, 374, p.131624.