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Characterization of ovalbumin-carvacrol inclusion complexes as delivery systems with antibacterial application
Food Hydrocolloids 105 (2020) 105753
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Characterization of ovalbumin-carvacrol inclusion complexes as delivery systems with antibacterial application Shengqi Rao a, b, *, 1, Guangwei Xu a, c, 1, Xiangning Lu a, c, Ruyi Zhang a, c, Lu Gao a, c, Qingyan Wang e, Zhenquan Yang a, c, Xinan Jiao b, d, ** a
School of Food Science and Engineering, Yangzhou University, Yangzhou, 225127, Jiangsu, China Postdoctoral Mobile Station of Biology, School of Bioscience and Biotechnology, Yangzhou University, Yangzhou, 225009, Jiangsu, China Jiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou University, Yangzhou, 225127, Jiangsu, China d Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture of China, Yangzhou, 225001, Jiangsu, China e State Key Laboratory of Non-Food Biomass and Enzyme Technology, Nanning, 530226, guangxi, China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Carvacrol Ovalbumin nanocarrier Antibacterial microencapsulation Ovalbumin gel
Carvacrol is an effective natural antimicrobial and antioxidant agent; however, its poor aqueous solubility and high volatility limit its application in food systems. Ovalbumin can encapsulate hydrophobic molecules, thereby improving aqueous solubility and reducing volatility. The aim of this study was to explore the potential of an ovalbumin nanocarrier to improve the application range of carvacrol. Protein structure and scanning electron microscopy showed that carvacrol increased gel hardness and tackiness at pH 5, and OVA gel formed a uniform granular shape that was beneficial for reconstitution. The dominant force in OVA gel changed from electrostatic to hydrophobic interaction, indicating that carvacrol could hydrophobically bind to the gel. The ovalbumincarvacrol (OVA-Car) complex was prepared using an oil-in-water method. The particle size was 132 � 10 nm, and the encapsulation efficiency was 51.41%. The polydispersity index was 0.355, indicating the general stability of the composite. Fluorescence spectroscopy and differential thermogram studies indicated complex formation. Antibacterial property testing against Bacillus cereus and Salmonella indicated that the OVA-Car complex inhibited bacterial growth at lower concentrations than free carvacrol. Minimum inhibitory concentration and minimum bactericidal concentration for Bacillus cereus (0.0968 and 0.3875 mg/mL) and Salmonella (0.1937 and 0.3875 mg/mL), respectively, were 2–4 times higher than those of free carvacrol. The time sterilization curve indicated that pathogenic bacterial growth did not reach the control level. The OVA-Car complex has potential application in food systems owing to its storage stability and improved antimicrobial activity.
1. Introduction Bacterial pathogen-induced food loss during processing and storage remains a persistent problem. An estimated 9.4 million illnesses are due to known foodborne pathogens every year (Scallan et al., 2011). Out breaks of Bacillus cereus and Salmonella infections are among the many safety issues linked to the food industry. In Europe, B. cereus is the pri mary pathogen in instant foods and large-scale catering operations (Wijnands, Dufrenne, Rombouts, In’t veld, & Leusden, 2006). Chinese food borne detection network data suggest that dozens of B. cereus
poisoning incidents occur in China each year, accounting for 11.4% of bacterial food poisoning incidents (Fu, Gao, & Guo, 2007). Outbreaks of Salmonella infection are often associated with a variety of foods, and 16 serious food-related Salmonella infections occurred in the United States in 2018 (Ma et al., 2019). Therefore development of improved sanitizing strategies for the food industry is urgently needed. Synthetic antibac terial agents can effectively inhibit bacterial growth. However, anti biotic resistance and the toxicity of synthetic agents should not be ignored. Owing to the adverse effects of synthetic antimicrobial agents on the environment and health, plant-based antibacterial substances are
* Corresponding author. School of Food Science and Engineering, Yangzhou University, Yangzhou, 225127, Jiangsu, China. ** Corresponding author. Postdoctoral Mobile Station of Biology, School of Bioscience and Biotechnology, Yangzhou University, Yangzhou, 225009, Jiangsu, China. E-mail addresses: [email protected] (S. Rao), [email protected] (X. Jiao). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.foodhyd.2020.105753 Received 29 September 2019; Received in revised form 6 January 2020; Accepted 7 February 2020 Available online 11 February 2020 0268-005X/© 2020 Elsevier Ltd. All rights reserved.
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gaining increasing attention (Zhang, Shu, Chen, Cao, & Jiang, 2019). Carvacrol is a major component of the essential oils derived from oregano, thyme, marjoram, and summer savory (Chalier, Arfa, Preziosi-Belloy, & Gontard, 2007). Carvacrol is Generally Recognized as Safe (GRAS) by the US Food and Drug Administration and has been included in the list of chemical flavorings that may be added to food stuffs by the Council of Europe ((De Vincenzi, Stammati, De Vincenzi, & Silano, 2004). However, carvacrol has poor stability and is oxidized, decomposed, or evaporated when exposed to air, light, or heat (Locci, Lai, Piras, Marongiu, & Lai, 2004). Carvacrol is highly insoluble in water owing to its lipophilic nature and may have limited contact with path ogens in foods with high moisture content (Kalemba & Kunicka, 2003). Therefore, carvacrol stability can be improved by nano-carrier embed ding technology. When nanocarriers are used in processing and storage, they can reduce the direct contact between active substances and external con ditions, such as heat, light, and oxygen, to improve stability. Another function of polymeric nanoparticles is to enhance the bioavailability of hydrophobic compounds such as lipophilic drugs and vitamins (Choi & Han, 2018). Different wall materials including carbohydrate polymers, proteins, and lipids have been applied for microencapsulation (Lee & Rosenberg, 2001). Protein hydrogels are used as carrier particles because of their high nutritional value and are considered safe. Hen egg white protein (EWP) is a low cost protein that is widely used as a food ingredient as it possesses several desirable functional properties including foaming, emulsification, heat setting, and binding adhesion (Somchue, Sermsri, Shiowatana, & Siripinyanond, 2009). EWP is a valued biomaterial in the nano-carrier industry owing to its excellent nutritional value, digestibility, self-assembly, and amphiphilic proper ties (Li & Nakai, 1989). Ovalbumin, the main protein component in EWP, is comprised of 385 amino acids, of which 50% are hydrophobic and 33% are charged, demonstrating the potential of EWP to be used as a high-efficiency carrier of lipophilic components (Chang et al., 2019). Complexes obtained by protein-hydrophobic compound interactions have the following advantages: (i) protection of polyunsaturated fatty acids through their entrapment and immobilization in nanoscopic par ticles which prevent contact with pro-oxidant agents (enzymes, oxygen, light, metals) (Zimet & Livney, 2009); (ii) protein improves the water solubility of hydrophobic compounds, allowing their incorporation in clear food systems such as beverages (Ilyasoglu & El, 2014). Heat treatment can form ovalbumin to nanostructures with higher surface hydrophobicity (Li et al., 2018a). However, there are few studies on the formation of ovalbumin-essential oil complexes. In order to explore the potential of ovalbumin as a nanocarrier and improve the application range of carvacrol, carvacrol was complexed with OVA and its proper ties as a protein gel, its nanoparticle state, complex characterization, and antibacterial activity were evaluated. The information on this protein-carvacrol complex provide a theoretical basis for future appli cations of ovalbumin and carvacrol.
The collected egg white protein supernatant was heated for 30 min in a 90 � C water bath and then immediately moved to an ice bath and cooled to 4 � C (Li et al., 2018b). The gels were formed after 24 h of storage at 4 � C. The gels were minced by stirring, then homogenized using an ultrasonic processor (600 W with 3 s on/off pulse for 15 min). The homogenized OVA nanoparticle dispersion was sealed and stored at 4 � C until analysis. OVA nanoparticles loaded with carvacrol were prepared following the above method, and 31 mg/mL saturated solution of carvacrol in ethanol was dropped into OVA nanoparticles and thoroughly stirred (Chang et al., 2019; Li et al., 2018b). 2.3. Texture profile analysis Texture profile analysis was carried out with a TMS-PRO texture analyzer fitted with a flat plunger. Eight EW gels (9 mm height and 20 mm diameter) were compressed twice to 50% of their original height at a crosshead speed of 2 mm/s and a 5 g trigger point load with 5 s be tween compressions. Hardness, gumminess, and springiness were calculated from force-time deformation curves using Texture Expert software version 1.22 (Stable Micro Systems). All samples from two batches were measured six times. 2.4. Turbidity measurement Protein aggregate dispersion turbidity was determined by measuring the absorbance of the solution at λ ¼ 500 nm at 25 � C. Before mea surement, the samples were diluted at a ratio of 1:10 (v/v) to remain in the linear absorbance region. In addition, the turbidity of samples diluted with 6 M urea, 0.5% SDS, 30 mM DTT were measured after reacting for 10 min to unravel the interactive forces involved in the formation and maintenance of the nanoparticle structure (Chang et al., 2016). Turbidity was expressed by light transmittance. 2.5. Estimation of carvacrol loading The nanoparticle suspension was centrifuged at 5000 � g for 10 min. The resulting nanoparticle phase (upper phase) was removed and added to 5 mL of acetonitrile. This solution was stirred at 1000 rpm for 30 min, then centrifuged at 5000 � g for 10 min. Absorption at 275 nm was measured to calculate carvacrol content by UV spectrophotometry. The entrapment efficacy for carvacrol was calculated based on the standard curve and the following entrapment efficacy (EE) (Maryan, Shakeri, & Kiani, 2015). EE(%) ¼ (loaded carvacrol (mg)) / (total added carvacrol (mg)) � 100
2.6. Size determination
2. Materials and methods
OVA–Car nanoparticle sizes were measured by dynamic light scat tering using a 5022F Goniometer System (ALV-GmbH, Germany). One milliliter of OVA–Car mixture (15%, w/v) was transferred into the measuring cell, and the temperature was set at 25 � C. For all experi ments, measurements were conducted in triplicate; each measurement consisted of 11 individual runs with a 10 s duration and 120 s equili bration before measurement.
2.1. Materials Fresh hen eggs were purchased from a local supermarket. (Yangz hou, CN). Carvacrol (99%), phosphotungstic acid, sodium 8-anilino-1naphthalenesulfonate (ANS) and C4H10O2S2 (DTT) were purchased from Sigma Aldrich. (St Louis, MO, USA). NaOH, Nacl and urea were obtained from Biological Technology Co., Ltd (Shanghai, CN).
2.7. Characterization
2.2. Protein sample preparation
Regarding the influence of carvacrol on the fluorescence spectra of OVA, the concentration of the carvacrol was held constant at 0.3 mg/mL while the OVA concentration was varied from 0 to 1 mg/mL. The resulting solution was measured on a fluorescence spectrophotometer at an excitation wavelength of 270 nm and an emission wavelength of 280
Egg white (EW) was separated from the washed hen egg and 20 mL samples were adjusted to pH 2.0, 5.0, 7.0, and 9.0, respectively, with 1 M HCl then stirred with a magnetic stirrer (Model HJ-6, CN) for 3 h. The suspension was centrifuged at 8000 � g for 15 min at room temperature. 2
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nm. A differential scanning calorimeter (DSC) model 8500 (PerkinElmer, USA) was used to study complex formation between carvacrol and OVA. Analysis was performed with a scanning rate of 90 � C/min from 25 � C to 120 � C and was maintained at 120 � C for 1 min to ensure even sample heating. The instrument was calibrated using zinc and indium metals before sample testing. OVA samples and inclusion complexes were accurately weighed (2 mg) and placed in aluminum pans (40 mL) with one hole in each lid (Mourtzinos, Kalogeropoulos, Papadakis, Kon stantinou, & Karathanos, 2008). Particle morphological features were observed by a field emission scanning electron microscope (Gemini SEM 300, Carl Zeiss, UK) with an accelerated voltage of 15 kV and photographed at 1000� magnification. Samples were lyophilized and the dried gel powder was sprinkled onto two-sided adhesive tape then coated with a thin layer of gold. TEM analysis was carried out with a Tecnai 12 (Philips, NL) at an accelerating voltage of 100 kV. The sample was stained with a 1.5% phosphotungstic acid aqueous stain, and the sample dispersions were dropped onto a copper grid for TEM observation. 2.8. Nanoparticle antimicrobial properties Salmonella CICC 21513 and Bacillus cereus CICC 21261 were obtained from China Industrial Microbial Culture Collection Management Center. Minimum inhibitory concentration (MIC) was determined by broth dilution method, as recommended by the NCCLS 2000. All bacteria were inoculated in LB broth at 37 � C for 24 h. The concentration of culture suspensions was adjusted to 106 CFU/mL by comparison with the McFarland turbidity standard no. 0.5. A series of sample concentrations (1 mL each) were prepared in tubes by twofold dilution with LB broth. Bacterial suspensions (1 mL each) were inoculated in the sample series. After incubation at 37 � C for 24 h, MIC was determined as the concentration of the sample in the tube without turbidity containing the lowest sample concentration. To evaluate the minimum bactericidal concentration (MBC), samples from all tubes without turbidity (from the MIC test) were transferred to LB agar plates and incubated at 37 � C for 24 h. The MBC was determined as the lowest concentration of the sample in the tube corresponding to the agar plate without bacterial growth. Tests were performed in triplicate for each sample.
Fig. 1. Texture profile analysis of egg white gels with different pH values. (A) Hardness; (B) Springiness; (C) Gumminess.
Fig. 2. Microstructure of OVA lyophilized gelation under various pH levels. 3
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Fig. 4. Differential scanning calorimetry thermograms of carvacrol, OVA, and OVA-Car.
In the case of a pH value far from the isoelectric point of the protein (4.8 for OVA) the gel structure appears dense, smooth, and aggregated. In contrast, the structure of gels generated at pH values near the iso electric points are average and include an aggregated spherical structure with higher hardness and tackiness. Therefore, the subsequent experi ments were carried out at pH 5. The colloidal stability of protein microparticle dispersions was investigated using turbidity measurements (Fig. 3). In addition, to further reveal the pattern of interactive forces involved in forming and maintaining the microparticle structure, the turbidity of the dispersion in the presence of various proteins denaturants (1 M NaCl, 6 M urea, 0.5% SDS, and 30 mM DTT, respectively) was investigated. Micropar ticle dispersions prepared under pH 5 were turbid with close to 20% transmission. The OVA microparticle dispersion prepared under pH 5 was gravitationally stable, with transmission increasing to 1.2% after standing for 30 min. In comparison, microparticle dispersion prepared with carvacrol showed a transmission below 20%, but was gravitation ally stable. Interestingly, the results also showed that the presence of 0.5% SDS or 6 M urea led to a significant increase in transmission, with a greater increase observed in the presence of 6 M urea (Fig. 3A). Compared to OVA-Carvacrol which was left for 30 min, the presence of 0.5% SDS resulted in a transmittance increase of 111.54%. On the other hand, the presence of 30 mM DTT did not result in decreased trans mission. SDS, urea, and DTT are used mainly to disrupt hydrophobic interactions, hydrogen bonding, and disulfide bonding, respectively (Gezimati, Creamer, & Singh, 1997; Schmitt et al., 2010). Thus, the results suggested that hydrophobic interactions and hydrogen bonds were the major intramolecular interactive forces maintaining the in ternal structure of the OVA microparticle, with hydrogen bonds acting as the dominant force. In the gel particle dispersion prepared from carvacrol, 0.5% SDS resulted in a significant increase in transmittance, and 6 M urea reduced transmittance. The experimental results showed that the surface hydrophobicity of the OVA gel after heat treatment increased by 103% compared to that of the natural OVA. Thus, hydrophobic interactions and hydrogen bonds were the major intramolecular interactive forces maintaining the internal structure of the microparticle, with hydrophobic interactions playing the dominant role.
Fig. 3. (A) Effects of various protein-perturbing solvents on the particle diameter of microparticles formed by heating at 90 � C for 45 min under pH 2.0 at protein concentrations of 2–10% with or without salt addition, followed by homogenization at 10 MPa; (B) Effect of heat treatment on ovalbumin surface hydrophobicity.
Based on the MIC testing results, the OVA–Car nanoparticles were used for further tests. The bacterial suspension was diluted to a final concentration of 5 � 105 CFU/mL in LB broth. According to the MIC of the OVA–Car nanoparticles, the experimental groups were set as blank ovalbumin, free carvacrol and 1 mic antibacterial nanoparticles, with an initial inoculum of 5 � 105 CFU/mL. Then, the tubes were incubated at 37 � C. Changes in bacterial count during exposure were monitored and tested at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h. Test tubes containing only bacteria were considered controls. 2.9. Statistical analysis All tests were conducted in triplicate. Data were expressed as the mean � standard errors. Statistical analyses were performed using SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA), and the significant differences were determined with a 95% confidence interval (p < 0.05). 3. Results and discussion 3.1. Gel characteristics The texture results of OVA gel treated with or without carvacrol are shown in Fig. 1. The hardness of the OVA gel decreased with increasing pH, and the gel hardness reached a maximum at pH 5 after adding carvacrol. For the tackiness, after the addition of carvacrol, the OVA gel showed a tendency to rise and then fall, reaching a maximum at pH 5. Egg white gels consist of polymers connected to each other in order to form a 3-dimensional network. In the case of OVA, a positive correlation between WH and stiffness (represented by Young’s modulus) was apparent (Urbonaite et al., 2016). This suggests that carvacrol can react physically or chemically with OVA gel. Carvacrol enhances the gel network structure by increasing gel hardness and tackiness, thereby facilitating encapsulation of the active substance. The microstructure of the subsequently formed OVA gel at pH 2, 5, 7, and 9, as observed using SEM, is presented in Fig. 2. The effect of pH on the gelation structure is clearly visible.
3.2. Characterization Carvacrol fluorescence intensity increased with the presence of OVA without changing the emission maximum and shape of the peaks (Fig. 4). Preliminary studies show that OVA modifies the fluorescence spectra of carvacrol, suggesting an interaction between both com pounds, and thus, a concomitant complex formation. The fluorescence of 4
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results showed that some samples were lost during the operation. This could be associated with the loss of carvacrol due to volatilization during the complexation process and drying step that are carried out in an open container at room temperature for the oil-in-water method. A dynamic light scattering technique was applied to investigate average particle size. OVA-Car particles displayed an average diameter of 132.2 nm (Fig. 7), which was larger than that observed by TEM. The particle size determined by dynamic light scattering represents a hy drodynamic diameter (Keawchaoon & Yoksan, 2011). The larger diameter might be a result of the swelling of the protein layer sur rounding the individual particles and/or aggregation of single particles while dispersed in water. Nanoparticle formulation was expected to enhance the solubility or dispersibility of the poorly water-soluble carvacrol in an aqueous system and improve its stability. Ovalbumin
Fig. 5. Fluorescence spectrum of OVA and OVA-Car.
the phosphor medium (carvacrol) is enhanced by the inclusion of the phosphor medium (Hergert & Escandar, 2003). Because the structural conformation of the medium can protect the singlet state of the encap sulated phosphor from the quencher in the cavity in water and increase its radiation rate, the molecular motion in terms of the degree of freedom is reduced, preventing collision inactivation. At the same time, phosphor can be subjected to a more suitable local micro-environment with less polarity and greater rigidity so that the quantum efficiency is improved. The formation of inclusion complexes for each encapsulation method was confirmed by DSC analysis. DSC thermograms of free OVA and freeze-dried inclusion complexes are shown in Fig. 5. The OVA DSC curve showed a sharp endothermic peak at 81.26 � C that corresponds to its boiling point. The DSC curve of OVA-Car showed a sharp endo thermic peak at 76.37 � C that corresponds to its boiling point. The transfer of this peak is indirect proof that an inclusion complex has been formed (Gomes, Moreira, & Castell-Perez, 2011) between carvacrol and OVA by comparing the thermal stability of the free carvacrol with the encapsulated form. The morphology of the particles was observed by TEM. The individual OVA particles and carvacrol-loaded OVA particles exhibited spherical shapes with an average diameter of 50–90 nm (Fig. 6A and B).
Fig. 7. Size distribution of OVA and OVA-Car nanoparticles (15% w/v). Table 1 Minimum inhibitory and bactericidal concentration (MIC, MBC) against B. cereus and Salmonella for free carvacrol and ovalbumin–carvacrol nanoparticles Strains
3.3. Encapsulation rate and size determination
Bacillus cereus CICC 21261 Salmonella CICC 21513
Entrapment efficiency was 51.37% for OVA-Car complexes. The encapsulation method used in the experiment entrapped carvacrol very effectively, suggesting that a small molecular weight compound such as carvacrol is suitable for inclusion into OVA. However, the encapsulation
a b
MICa (mg/ mL)
MBCb (mg/ mL)
Car
OVACar
Car
OVACar
0.3875
0.0968
0.775
0.3875
0.3875
0.1937
0.775
0.3875
Minimum inhibitory concentration. Minimum bactericidal concentration.
Fig. 6. Transmission electron microscope images of OVA-Car inclusion complexes. Images are representative of samples and depict free OVA (A) and OVA-Car inclusion complexes (B). 5
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of these compounds through the lipopolysaccharide covering on the outer membrane (Burt, 2004). The time-sterilization assays were performed to compare the anti microbial activities of OVA-Car nanoparticles. The results are shown in Fig. 8. Given an initial inoculum of 106 CFU/mL, it was obvious that the antibacterial effect of OVA-Car nanoparticles was better than that of the other treatments. Compared to freed carvacrol, the effect of encapsula tion was obvious. The growth curves were not obviously influenced by protein nanoparticles after 24 h. However, the encapsulation-treated OVA-Car nanoparticles showed significantly lower values than the other test groups (P < 0.05). A stronger bactericidal effect on B. cereus (Salmonella) might be exerted in OVA-Car nanoparticles (Ma, Shi, Wang, & Guo, 2017). According to a previous research (Wang, Li, Si, Lin, & Chen, 2011), since the primary action sites of essential oils are at the membrane and inside the cytoplasm of bacteria, the improved antimi crobial activity of carvacrol likely occurred because OVA enhanced the access of carvacrol to these regions by increasing carvacrol aqueous solubility. Since it is possible to use lower concentrations of carvacrol when it is administered as a complex, these results show that encapsulation with OVA improves antimicrobial delivery to active sites (pathogenic mi croorganisms) providing exciting avenues for future research. 4. Conclusions pH 5 facilitated the binding of OVA gel to carvacrol, and the binding mode changed from electrostatic to hydrophobic interaction. Inclusion complex formation with carvacrol was confirmed by DSC and fluores cence spectroscopy analysis. Moreover, this method presented a high entrapment efficiency and increased carvacrol water solubility. The type of complex inhibited B. cereus and Salmonella strains at a lower con centration than free carvacrol, indicating that encapsulation enhanced the antimicrobial action mechanism and decreased the concentration of antimicrobial compound needed for inhibition. Moreover, OVA com plexes were not degraded by light during storage, providing stable freeflowing powders. These OVA-carvacrol complexes could be useful antimicrobial delivery systems for application in a variety of food sys tems where foodborne pathogens present a risk.
Fig. 8. Time-kill curves for carvacrol, OVA, and OVA-Car nanoparticles against (A) Bacillus cereus 21,261 or (B) Salmonella 21,513.
and their complexes form water soluble aggregates in aqueous solutions, and these aggregates are able to solubilize lipophilic water-insoluble drugs through hydrophobic interactions or micelle-like structures (Gu et al., 2017). A polydispersity index is a measure of the particle size uniformity present in a suspension. Polydispersity indices higher than 0.3 could be due to the tendency of the OVA-Car inclusion complex particles to agglomerate, creating a larger particle size than anticipated. 3.4. Antimicrobial properties of the nanoparticles
Declaration of competing interest
The antibacterial properties of protein–carvacrol nanoparticles against B. cereus and Salmonella are shown in Table 1. MICs of OVA-Car nanoparticles treated with encapsulation against B. cereus decreased from 0.3875 to 0.0968 mg/mL compared to those of untreated carva crol, while MICs of those treated with encapsulation against Salmonella decreased from 0.3875 to 0.1937 mg/mL. MBCs of OVA-Car nano particles treated with encapsulation against B. cereus and Salmonella decreased from 0.775 to 0.3875 mg/mL compared to those of untreated carvacrol, proving that encapsulation had an effective antibacterial ac tion against B. cereus and Salmonella, proving that encapsulation had an effective antibacterial action against B. cereus and Salmonella. Therefore, the effective antibiotic mechanism of OVA-Car nanoparticles was investigated further. The antibacterial activity of carvacrol may be due to interactions with microbial cell membrane components. The distor tion of the structure would induce expansion, membrane destabilization, and increased membrane fluidity, which would eventually increase passive permeability (Cristani et al., 2007). The MIC for OVA-Car par ticles was lower than that of carvacrol alone (P < 0.05) for both bacteria species, probably due to the increase in the carvacrol-carrier contact surface as a consequence of the more drastic mechanical treatment (Fernandes, Vieira, & Veiga, 2002). Moreover, the MICs and MBCs differed between the two bacterial species because each bacterial strain responds differently to the essential oil (Burt, 2004). This might be due to the lower antibacterial susceptibility of hydrophobic compounds against gram-negative microorganisms, owing to the restricted diffusion
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Li, J. H., Zhang, M. Q., Chang, C. H., Gu, L. P., Peng, N., Su, Y. J., et al. (2018a). Molecular forces and gelling properties of heat-set whole chicken egg protein gel as affected by NaCl or pH. Food Chemistry, 261, 36–41. Li, J. H., Zhang, Y. F., Fan, Q., Teng, C. H., Xie, W. Y., Shi, Y., et al. (2018b). Combination effects of NaOH and NaCl on the rheology and gel characteristics of hen egg white proteins. Food Chemistry, 250, 1–6. Locci, E., Lai, S. M., Piras, A., Marongiu, B., & Lai, A. (2004). 13C-CPMAS and 1H-NMR study of the inclusion complexes of beta-cyclodextrin with carvacrol, thymol, and eugenol prepared in supercritical carbon dioxide. Chemistry and Biodiversity, 1(9), 1354–1366. Ma, Y., Ding, S. J., Fei, Y. Q., Liu, G., Jang, H. M., & Fang, J. (2019). Antimicrobial activity of anthocyanins and catechins against foodborne pathogens Escherichia coli and Salmonella. Food Control, 106. https://doi.org/10.1016/j. foodcont.2019.106712. Maryam, K., Shakeri, S., & Kiani, K. (2015). Preparation and in vitro investigation of antigastric cancer activities of carvacrol-loaded human serum albumin nanoparticles. IET Nanobiotechnology, 9(5), 294–299. Ma, S., Shi, C., Wang, C. N., & Guo, M. R. (2017). Effects of ultrasound treatment on physiochemical properties and antimicrobial activities of whey protein-totarol nanoparticles. Journal of Food Protection, 80(10), 1657–1665. Mourtzinos, I., Kalogeropoulos, N., Papadakis, S. E., Konstantinou, K., & Karathanos, V. T. (2008). Encapsulation of nutraceutical monoterpenes in β-cyclodextrin and modified starch. Journal of Food Science, 73(1), S89–S94. Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A., Roy, S. L., et al. (2011). Foodborne illness acquired in the United States-major pathogens. Emerging Infectious Diseases, 17(1), 7–15. Schmitt, C., Moitzi, C., Bovay, C., Rouvet, M., Bovetto, L., Donato, L., et al. (2010). Internal structure and colloidal behaviour of covalent whey protein microgels obtained by heat treatment. Soft Matter, 6(19), 4876–4884. Somchue, W., Sermsri, W., Shiowatana, J., & Siripinyanond, A. (2009). Encapsulation of α-tocopherol in protein-based delivery particles. Food Research International, 42(8), 909–914. Urbonaite, V., Van, D. K. S., De Jongh, H. H. J., Scholten, E., Ako, K., Van, D. L. E., et al. (2016). Relation between gel stiffness and water holding for coarse and fine-stranded protein gels. Food Hydrocolloids, 56, 334–343. Wang, T., Li, B., Si, H., Lin, L., & Chen, L. (2011). Release characteristics and antibacterial activity of solid state eugenol/β-cyclodextrin inclusion complex. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 71(1–2), 207–213. Wijnands, L. M., Dufrenne, J. B., Rombouts, F. M., In’t veld, P. H., & Leusden, F. M. van (2006). Prevalence of potentially pathogenic Bacillus cereus in food commodities in the Netherlands. Journal of Food Protection, 69(11), 2587–2594. Zhang, W. L., Shu, C., Chen, Q. Y., Cao, J. K., & Jiang, W. B. (2019). The multi-layer film system improved the release and retention properties of cinnamon essential oil and its application as coating in inhibition to penicillium expansion of apple fruit. Food Chemistry, 299. https://doi.org/10.1016/j.foodchem. 2019.125109. Zimet, P., & Livney, Y. D. (2009). Beta-lactoglobulin and its nanocomplexes with pectin as vehicles for ω-3 polyunsaturated fatty acids. Food Hydrocolloids, 23(4), 1120–1126.
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Food Hydrocolloids journal homepage: http://www.elsevier.com/locate/foodhyd
Characterization of ovalbumin-carvacrol inclusion complexes as delivery systems with antibacterial application Shengqi Rao a, b, *, 1, Guangwei Xu a, c, 1, Xiangning Lu a, c, Ruyi Zhang a, c, Lu Gao a, c, Qingyan Wang e, Zhenquan Yang a, c, Xinan Jiao b, d, ** a
School of Food Science and Engineering, Yangzhou University, Yangzhou, 225127, Jiangsu, China Postdoctoral Mobile Station of Biology, School of Bioscience and Biotechnology, Yangzhou University, Yangzhou, 225009, Jiangsu, China Jiangsu Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou University, Yangzhou, 225127, Jiangsu, China d Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture of China, Yangzhou, 225001, Jiangsu, China e State Key Laboratory of Non-Food Biomass and Enzyme Technology, Nanning, 530226, guangxi, China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Carvacrol Ovalbumin nanocarrier Antibacterial microencapsulation Ovalbumin gel
Carvacrol is an effective natural antimicrobial and antioxidant agent; however, its poor aqueous solubility and high volatility limit its application in food systems. Ovalbumin can encapsulate hydrophobic molecules, thereby improving aqueous solubility and reducing volatility. The aim of this study was to explore the potential of an ovalbumin nanocarrier to improve the application range of carvacrol. Protein structure and scanning electron microscopy showed that carvacrol increased gel hardness and tackiness at pH 5, and OVA gel formed a uniform granular shape that was beneficial for reconstitution. The dominant force in OVA gel changed from electrostatic to hydrophobic interaction, indicating that carvacrol could hydrophobically bind to the gel. The ovalbumincarvacrol (OVA-Car) complex was prepared using an oil-in-water method. The particle size was 132 � 10 nm, and the encapsulation efficiency was 51.41%. The polydispersity index was 0.355, indicating the general stability of the composite. Fluorescence spectroscopy and differential thermogram studies indicated complex formation. Antibacterial property testing against Bacillus cereus and Salmonella indicated that the OVA-Car complex inhibited bacterial growth at lower concentrations than free carvacrol. Minimum inhibitory concentration and minimum bactericidal concentration for Bacillus cereus (0.0968 and 0.3875 mg/mL) and Salmonella (0.1937 and 0.3875 mg/mL), respectively, were 2–4 times higher than those of free carvacrol. The time sterilization curve indicated that pathogenic bacterial growth did not reach the control level. The OVA-Car complex has potential application in food systems owing to its storage stability and improved antimicrobial activity.
1. Introduction Bacterial pathogen-induced food loss during processing and storage remains a persistent problem. An estimated 9.4 million illnesses are due to known foodborne pathogens every year (Scallan et al., 2011). Out breaks of Bacillus cereus and Salmonella infections are among the many safety issues linked to the food industry. In Europe, B. cereus is the pri mary pathogen in instant foods and large-scale catering operations (Wijnands, Dufrenne, Rombouts, In’t veld, & Leusden, 2006). Chinese food borne detection network data suggest that dozens of B. cereus
poisoning incidents occur in China each year, accounting for 11.4% of bacterial food poisoning incidents (Fu, Gao, & Guo, 2007). Outbreaks of Salmonella infection are often associated with a variety of foods, and 16 serious food-related Salmonella infections occurred in the United States in 2018 (Ma et al., 2019). Therefore development of improved sanitizing strategies for the food industry is urgently needed. Synthetic antibac terial agents can effectively inhibit bacterial growth. However, anti biotic resistance and the toxicity of synthetic agents should not be ignored. Owing to the adverse effects of synthetic antimicrobial agents on the environment and health, plant-based antibacterial substances are
* Corresponding author. School of Food Science and Engineering, Yangzhou University, Yangzhou, 225127, Jiangsu, China. ** Corresponding author. Postdoctoral Mobile Station of Biology, School of Bioscience and Biotechnology, Yangzhou University, Yangzhou, 225009, Jiangsu, China. E-mail addresses: [email protected] (S. Rao), [email protected] (X. Jiao). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.foodhyd.2020.105753 Received 29 September 2019; Received in revised form 6 January 2020; Accepted 7 February 2020 Available online 11 February 2020 0268-005X/© 2020 Elsevier Ltd. All rights reserved.
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gaining increasing attention (Zhang, Shu, Chen, Cao, & Jiang, 2019). Carvacrol is a major component of the essential oils derived from oregano, thyme, marjoram, and summer savory (Chalier, Arfa, Preziosi-Belloy, & Gontard, 2007). Carvacrol is Generally Recognized as Safe (GRAS) by the US Food and Drug Administration and has been included in the list of chemical flavorings that may be added to food stuffs by the Council of Europe ((De Vincenzi, Stammati, De Vincenzi, & Silano, 2004). However, carvacrol has poor stability and is oxidized, decomposed, or evaporated when exposed to air, light, or heat (Locci, Lai, Piras, Marongiu, & Lai, 2004). Carvacrol is highly insoluble in water owing to its lipophilic nature and may have limited contact with path ogens in foods with high moisture content (Kalemba & Kunicka, 2003). Therefore, carvacrol stability can be improved by nano-carrier embed ding technology. When nanocarriers are used in processing and storage, they can reduce the direct contact between active substances and external con ditions, such as heat, light, and oxygen, to improve stability. Another function of polymeric nanoparticles is to enhance the bioavailability of hydrophobic compounds such as lipophilic drugs and vitamins (Choi & Han, 2018). Different wall materials including carbohydrate polymers, proteins, and lipids have been applied for microencapsulation (Lee & Rosenberg, 2001). Protein hydrogels are used as carrier particles because of their high nutritional value and are considered safe. Hen egg white protein (EWP) is a low cost protein that is widely used as a food ingredient as it possesses several desirable functional properties including foaming, emulsification, heat setting, and binding adhesion (Somchue, Sermsri, Shiowatana, & Siripinyanond, 2009). EWP is a valued biomaterial in the nano-carrier industry owing to its excellent nutritional value, digestibility, self-assembly, and amphiphilic proper ties (Li & Nakai, 1989). Ovalbumin, the main protein component in EWP, is comprised of 385 amino acids, of which 50% are hydrophobic and 33% are charged, demonstrating the potential of EWP to be used as a high-efficiency carrier of lipophilic components (Chang et al., 2019). Complexes obtained by protein-hydrophobic compound interactions have the following advantages: (i) protection of polyunsaturated fatty acids through their entrapment and immobilization in nanoscopic par ticles which prevent contact with pro-oxidant agents (enzymes, oxygen, light, metals) (Zimet & Livney, 2009); (ii) protein improves the water solubility of hydrophobic compounds, allowing their incorporation in clear food systems such as beverages (Ilyasoglu & El, 2014). Heat treatment can form ovalbumin to nanostructures with higher surface hydrophobicity (Li et al., 2018a). However, there are few studies on the formation of ovalbumin-essential oil complexes. In order to explore the potential of ovalbumin as a nanocarrier and improve the application range of carvacrol, carvacrol was complexed with OVA and its proper ties as a protein gel, its nanoparticle state, complex characterization, and antibacterial activity were evaluated. The information on this protein-carvacrol complex provide a theoretical basis for future appli cations of ovalbumin and carvacrol.
The collected egg white protein supernatant was heated for 30 min in a 90 � C water bath and then immediately moved to an ice bath and cooled to 4 � C (Li et al., 2018b). The gels were formed after 24 h of storage at 4 � C. The gels were minced by stirring, then homogenized using an ultrasonic processor (600 W with 3 s on/off pulse for 15 min). The homogenized OVA nanoparticle dispersion was sealed and stored at 4 � C until analysis. OVA nanoparticles loaded with carvacrol were prepared following the above method, and 31 mg/mL saturated solution of carvacrol in ethanol was dropped into OVA nanoparticles and thoroughly stirred (Chang et al., 2019; Li et al., 2018b). 2.3. Texture profile analysis Texture profile analysis was carried out with a TMS-PRO texture analyzer fitted with a flat plunger. Eight EW gels (9 mm height and 20 mm diameter) were compressed twice to 50% of their original height at a crosshead speed of 2 mm/s and a 5 g trigger point load with 5 s be tween compressions. Hardness, gumminess, and springiness were calculated from force-time deformation curves using Texture Expert software version 1.22 (Stable Micro Systems). All samples from two batches were measured six times. 2.4. Turbidity measurement Protein aggregate dispersion turbidity was determined by measuring the absorbance of the solution at λ ¼ 500 nm at 25 � C. Before mea surement, the samples were diluted at a ratio of 1:10 (v/v) to remain in the linear absorbance region. In addition, the turbidity of samples diluted with 6 M urea, 0.5% SDS, 30 mM DTT were measured after reacting for 10 min to unravel the interactive forces involved in the formation and maintenance of the nanoparticle structure (Chang et al., 2016). Turbidity was expressed by light transmittance. 2.5. Estimation of carvacrol loading The nanoparticle suspension was centrifuged at 5000 � g for 10 min. The resulting nanoparticle phase (upper phase) was removed and added to 5 mL of acetonitrile. This solution was stirred at 1000 rpm for 30 min, then centrifuged at 5000 � g for 10 min. Absorption at 275 nm was measured to calculate carvacrol content by UV spectrophotometry. The entrapment efficacy for carvacrol was calculated based on the standard curve and the following entrapment efficacy (EE) (Maryan, Shakeri, & Kiani, 2015). EE(%) ¼ (loaded carvacrol (mg)) / (total added carvacrol (mg)) � 100
2.6. Size determination
2. Materials and methods
OVA–Car nanoparticle sizes were measured by dynamic light scat tering using a 5022F Goniometer System (ALV-GmbH, Germany). One milliliter of OVA–Car mixture (15%, w/v) was transferred into the measuring cell, and the temperature was set at 25 � C. For all experi ments, measurements were conducted in triplicate; each measurement consisted of 11 individual runs with a 10 s duration and 120 s equili bration before measurement.
2.1. Materials Fresh hen eggs were purchased from a local supermarket. (Yangz hou, CN). Carvacrol (99%), phosphotungstic acid, sodium 8-anilino-1naphthalenesulfonate (ANS) and C4H10O2S2 (DTT) were purchased from Sigma Aldrich. (St Louis, MO, USA). NaOH, Nacl and urea were obtained from Biological Technology Co., Ltd (Shanghai, CN).
2.7. Characterization
2.2. Protein sample preparation
Regarding the influence of carvacrol on the fluorescence spectra of OVA, the concentration of the carvacrol was held constant at 0.3 mg/mL while the OVA concentration was varied from 0 to 1 mg/mL. The resulting solution was measured on a fluorescence spectrophotometer at an excitation wavelength of 270 nm and an emission wavelength of 280
Egg white (EW) was separated from the washed hen egg and 20 mL samples were adjusted to pH 2.0, 5.0, 7.0, and 9.0, respectively, with 1 M HCl then stirred with a magnetic stirrer (Model HJ-6, CN) for 3 h. The suspension was centrifuged at 8000 � g for 15 min at room temperature. 2
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nm. A differential scanning calorimeter (DSC) model 8500 (PerkinElmer, USA) was used to study complex formation between carvacrol and OVA. Analysis was performed with a scanning rate of 90 � C/min from 25 � C to 120 � C and was maintained at 120 � C for 1 min to ensure even sample heating. The instrument was calibrated using zinc and indium metals before sample testing. OVA samples and inclusion complexes were accurately weighed (2 mg) and placed in aluminum pans (40 mL) with one hole in each lid (Mourtzinos, Kalogeropoulos, Papadakis, Kon stantinou, & Karathanos, 2008). Particle morphological features were observed by a field emission scanning electron microscope (Gemini SEM 300, Carl Zeiss, UK) with an accelerated voltage of 15 kV and photographed at 1000� magnification. Samples were lyophilized and the dried gel powder was sprinkled onto two-sided adhesive tape then coated with a thin layer of gold. TEM analysis was carried out with a Tecnai 12 (Philips, NL) at an accelerating voltage of 100 kV. The sample was stained with a 1.5% phosphotungstic acid aqueous stain, and the sample dispersions were dropped onto a copper grid for TEM observation. 2.8. Nanoparticle antimicrobial properties Salmonella CICC 21513 and Bacillus cereus CICC 21261 were obtained from China Industrial Microbial Culture Collection Management Center. Minimum inhibitory concentration (MIC) was determined by broth dilution method, as recommended by the NCCLS 2000. All bacteria were inoculated in LB broth at 37 � C for 24 h. The concentration of culture suspensions was adjusted to 106 CFU/mL by comparison with the McFarland turbidity standard no. 0.5. A series of sample concentrations (1 mL each) were prepared in tubes by twofold dilution with LB broth. Bacterial suspensions (1 mL each) were inoculated in the sample series. After incubation at 37 � C for 24 h, MIC was determined as the concentration of the sample in the tube without turbidity containing the lowest sample concentration. To evaluate the minimum bactericidal concentration (MBC), samples from all tubes without turbidity (from the MIC test) were transferred to LB agar plates and incubated at 37 � C for 24 h. The MBC was determined as the lowest concentration of the sample in the tube corresponding to the agar plate without bacterial growth. Tests were performed in triplicate for each sample.
Fig. 1. Texture profile analysis of egg white gels with different pH values. (A) Hardness; (B) Springiness; (C) Gumminess.
Fig. 2. Microstructure of OVA lyophilized gelation under various pH levels. 3
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Fig. 4. Differential scanning calorimetry thermograms of carvacrol, OVA, and OVA-Car.
In the case of a pH value far from the isoelectric point of the protein (4.8 for OVA) the gel structure appears dense, smooth, and aggregated. In contrast, the structure of gels generated at pH values near the iso electric points are average and include an aggregated spherical structure with higher hardness and tackiness. Therefore, the subsequent experi ments were carried out at pH 5. The colloidal stability of protein microparticle dispersions was investigated using turbidity measurements (Fig. 3). In addition, to further reveal the pattern of interactive forces involved in forming and maintaining the microparticle structure, the turbidity of the dispersion in the presence of various proteins denaturants (1 M NaCl, 6 M urea, 0.5% SDS, and 30 mM DTT, respectively) was investigated. Micropar ticle dispersions prepared under pH 5 were turbid with close to 20% transmission. The OVA microparticle dispersion prepared under pH 5 was gravitationally stable, with transmission increasing to 1.2% after standing for 30 min. In comparison, microparticle dispersion prepared with carvacrol showed a transmission below 20%, but was gravitation ally stable. Interestingly, the results also showed that the presence of 0.5% SDS or 6 M urea led to a significant increase in transmission, with a greater increase observed in the presence of 6 M urea (Fig. 3A). Compared to OVA-Carvacrol which was left for 30 min, the presence of 0.5% SDS resulted in a transmittance increase of 111.54%. On the other hand, the presence of 30 mM DTT did not result in decreased trans mission. SDS, urea, and DTT are used mainly to disrupt hydrophobic interactions, hydrogen bonding, and disulfide bonding, respectively (Gezimati, Creamer, & Singh, 1997; Schmitt et al., 2010). Thus, the results suggested that hydrophobic interactions and hydrogen bonds were the major intramolecular interactive forces maintaining the in ternal structure of the OVA microparticle, with hydrogen bonds acting as the dominant force. In the gel particle dispersion prepared from carvacrol, 0.5% SDS resulted in a significant increase in transmittance, and 6 M urea reduced transmittance. The experimental results showed that the surface hydrophobicity of the OVA gel after heat treatment increased by 103% compared to that of the natural OVA. Thus, hydrophobic interactions and hydrogen bonds were the major intramolecular interactive forces maintaining the internal structure of the microparticle, with hydrophobic interactions playing the dominant role.
Fig. 3. (A) Effects of various protein-perturbing solvents on the particle diameter of microparticles formed by heating at 90 � C for 45 min under pH 2.0 at protein concentrations of 2–10% with or without salt addition, followed by homogenization at 10 MPa; (B) Effect of heat treatment on ovalbumin surface hydrophobicity.
Based on the MIC testing results, the OVA–Car nanoparticles were used for further tests. The bacterial suspension was diluted to a final concentration of 5 � 105 CFU/mL in LB broth. According to the MIC of the OVA–Car nanoparticles, the experimental groups were set as blank ovalbumin, free carvacrol and 1 mic antibacterial nanoparticles, with an initial inoculum of 5 � 105 CFU/mL. Then, the tubes were incubated at 37 � C. Changes in bacterial count during exposure were monitored and tested at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h. Test tubes containing only bacteria were considered controls. 2.9. Statistical analysis All tests were conducted in triplicate. Data were expressed as the mean � standard errors. Statistical analyses were performed using SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA), and the significant differences were determined with a 95% confidence interval (p < 0.05). 3. Results and discussion 3.1. Gel characteristics The texture results of OVA gel treated with or without carvacrol are shown in Fig. 1. The hardness of the OVA gel decreased with increasing pH, and the gel hardness reached a maximum at pH 5 after adding carvacrol. For the tackiness, after the addition of carvacrol, the OVA gel showed a tendency to rise and then fall, reaching a maximum at pH 5. Egg white gels consist of polymers connected to each other in order to form a 3-dimensional network. In the case of OVA, a positive correlation between WH and stiffness (represented by Young’s modulus) was apparent (Urbonaite et al., 2016). This suggests that carvacrol can react physically or chemically with OVA gel. Carvacrol enhances the gel network structure by increasing gel hardness and tackiness, thereby facilitating encapsulation of the active substance. The microstructure of the subsequently formed OVA gel at pH 2, 5, 7, and 9, as observed using SEM, is presented in Fig. 2. The effect of pH on the gelation structure is clearly visible.
3.2. Characterization Carvacrol fluorescence intensity increased with the presence of OVA without changing the emission maximum and shape of the peaks (Fig. 4). Preliminary studies show that OVA modifies the fluorescence spectra of carvacrol, suggesting an interaction between both com pounds, and thus, a concomitant complex formation. The fluorescence of 4
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results showed that some samples were lost during the operation. This could be associated with the loss of carvacrol due to volatilization during the complexation process and drying step that are carried out in an open container at room temperature for the oil-in-water method. A dynamic light scattering technique was applied to investigate average particle size. OVA-Car particles displayed an average diameter of 132.2 nm (Fig. 7), which was larger than that observed by TEM. The particle size determined by dynamic light scattering represents a hy drodynamic diameter (Keawchaoon & Yoksan, 2011). The larger diameter might be a result of the swelling of the protein layer sur rounding the individual particles and/or aggregation of single particles while dispersed in water. Nanoparticle formulation was expected to enhance the solubility or dispersibility of the poorly water-soluble carvacrol in an aqueous system and improve its stability. Ovalbumin
Fig. 5. Fluorescence spectrum of OVA and OVA-Car.
the phosphor medium (carvacrol) is enhanced by the inclusion of the phosphor medium (Hergert & Escandar, 2003). Because the structural conformation of the medium can protect the singlet state of the encap sulated phosphor from the quencher in the cavity in water and increase its radiation rate, the molecular motion in terms of the degree of freedom is reduced, preventing collision inactivation. At the same time, phosphor can be subjected to a more suitable local micro-environment with less polarity and greater rigidity so that the quantum efficiency is improved. The formation of inclusion complexes for each encapsulation method was confirmed by DSC analysis. DSC thermograms of free OVA and freeze-dried inclusion complexes are shown in Fig. 5. The OVA DSC curve showed a sharp endothermic peak at 81.26 � C that corresponds to its boiling point. The DSC curve of OVA-Car showed a sharp endo thermic peak at 76.37 � C that corresponds to its boiling point. The transfer of this peak is indirect proof that an inclusion complex has been formed (Gomes, Moreira, & Castell-Perez, 2011) between carvacrol and OVA by comparing the thermal stability of the free carvacrol with the encapsulated form. The morphology of the particles was observed by TEM. The individual OVA particles and carvacrol-loaded OVA particles exhibited spherical shapes with an average diameter of 50–90 nm (Fig. 6A and B).
Fig. 7. Size distribution of OVA and OVA-Car nanoparticles (15% w/v). Table 1 Minimum inhibitory and bactericidal concentration (MIC, MBC) against B. cereus and Salmonella for free carvacrol and ovalbumin–carvacrol nanoparticles Strains
3.3. Encapsulation rate and size determination
Bacillus cereus CICC 21261 Salmonella CICC 21513
Entrapment efficiency was 51.37% for OVA-Car complexes. The encapsulation method used in the experiment entrapped carvacrol very effectively, suggesting that a small molecular weight compound such as carvacrol is suitable for inclusion into OVA. However, the encapsulation
a b
MICa (mg/ mL)
MBCb (mg/ mL)
Car
OVACar
Car
OVACar
0.3875
0.0968
0.775
0.3875
0.3875
0.1937
0.775
0.3875
Minimum inhibitory concentration. Minimum bactericidal concentration.
Fig. 6. Transmission electron microscope images of OVA-Car inclusion complexes. Images are representative of samples and depict free OVA (A) and OVA-Car inclusion complexes (B). 5
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of these compounds through the lipopolysaccharide covering on the outer membrane (Burt, 2004). The time-sterilization assays were performed to compare the anti microbial activities of OVA-Car nanoparticles. The results are shown in Fig. 8. Given an initial inoculum of 106 CFU/mL, it was obvious that the antibacterial effect of OVA-Car nanoparticles was better than that of the other treatments. Compared to freed carvacrol, the effect of encapsula tion was obvious. The growth curves were not obviously influenced by protein nanoparticles after 24 h. However, the encapsulation-treated OVA-Car nanoparticles showed significantly lower values than the other test groups (P < 0.05). A stronger bactericidal effect on B. cereus (Salmonella) might be exerted in OVA-Car nanoparticles (Ma, Shi, Wang, & Guo, 2017). According to a previous research (Wang, Li, Si, Lin, & Chen, 2011), since the primary action sites of essential oils are at the membrane and inside the cytoplasm of bacteria, the improved antimi crobial activity of carvacrol likely occurred because OVA enhanced the access of carvacrol to these regions by increasing carvacrol aqueous solubility. Since it is possible to use lower concentrations of carvacrol when it is administered as a complex, these results show that encapsulation with OVA improves antimicrobial delivery to active sites (pathogenic mi croorganisms) providing exciting avenues for future research. 4. Conclusions pH 5 facilitated the binding of OVA gel to carvacrol, and the binding mode changed from electrostatic to hydrophobic interaction. Inclusion complex formation with carvacrol was confirmed by DSC and fluores cence spectroscopy analysis. Moreover, this method presented a high entrapment efficiency and increased carvacrol water solubility. The type of complex inhibited B. cereus and Salmonella strains at a lower con centration than free carvacrol, indicating that encapsulation enhanced the antimicrobial action mechanism and decreased the concentration of antimicrobial compound needed for inhibition. Moreover, OVA com plexes were not degraded by light during storage, providing stable freeflowing powders. These OVA-carvacrol complexes could be useful antimicrobial delivery systems for application in a variety of food sys tems where foodborne pathogens present a risk.
Fig. 8. Time-kill curves for carvacrol, OVA, and OVA-Car nanoparticles against (A) Bacillus cereus 21,261 or (B) Salmonella 21,513.
and their complexes form water soluble aggregates in aqueous solutions, and these aggregates are able to solubilize lipophilic water-insoluble drugs through hydrophobic interactions or micelle-like structures (Gu et al., 2017). A polydispersity index is a measure of the particle size uniformity present in a suspension. Polydispersity indices higher than 0.3 could be due to the tendency of the OVA-Car inclusion complex particles to agglomerate, creating a larger particle size than anticipated. 3.4. Antimicrobial properties of the nanoparticles
Declaration of competing interest
The antibacterial properties of protein–carvacrol nanoparticles against B. cereus and Salmonella are shown in Table 1. MICs of OVA-Car nanoparticles treated with encapsulation against B. cereus decreased from 0.3875 to 0.0968 mg/mL compared to those of untreated carva crol, while MICs of those treated with encapsulation against Salmonella decreased from 0.3875 to 0.1937 mg/mL. MBCs of OVA-Car nano particles treated with encapsulation against B. cereus and Salmonella decreased from 0.775 to 0.3875 mg/mL compared to those of untreated carvacrol, proving that encapsulation had an effective antibacterial ac tion against B. cereus and Salmonella, proving that encapsulation had an effective antibacterial action against B. cereus and Salmonella. Therefore, the effective antibiotic mechanism of OVA-Car nanoparticles was investigated further. The antibacterial activity of carvacrol may be due to interactions with microbial cell membrane components. The distor tion of the structure would induce expansion, membrane destabilization, and increased membrane fluidity, which would eventually increase passive permeability (Cristani et al., 2007). The MIC for OVA-Car par ticles was lower than that of carvacrol alone (P < 0.05) for both bacteria species, probably due to the increase in the carvacrol-carrier contact surface as a consequence of the more drastic mechanical treatment (Fernandes, Vieira, & Veiga, 2002). Moreover, the MICs and MBCs differed between the two bacterial species because each bacterial strain responds differently to the essential oil (Burt, 2004). This might be due to the lower antibacterial susceptibility of hydrophobic compounds against gram-negative microorganisms, owing to the restricted diffusion
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