Self-Emulsification of Alkaline-Dissolved Clove Bud Oil by Whey

Article pubs.acs.org/JAFC

Self-Emulsification of Alkaline-Dissolved Clove Bud Oil by Whey Protein, Gum Arabic, Lecithin, and Their Combinations Yangchao Luo, Yue Zhang, Kang Pan, Faith Critzer, P. Michael Davidson, and Qixin Zhong* Department of Food Science and Technology, University of Tennessee in Knoxville, Knoxville, Tennessee 37996, United States ABSTRACT: Low-cost emulsification technologies using food ingredients are critical to various applications. In the present study, a novel self-emulsification technique was studied to prepare clove bud oil (CBO) emulsions, without specialized equipment or organic solvents. CBO was first dissolved in hot alkaline solutions, added at 1% v/v into neutral solutions with 1% w/v emulsifier composed of whey protein concentrate (WPC), gum arabic, lecithin, or their equal mass mixtures, and adjusted to pH 7.0. The self-emulsification process did not affect UV−vis absorption spectrum, reversed-phase HPLC chromatogram, or antimicrobial activity of CBO against Escherichia coli O157:H7, Listeria monocytogenes Scott A, and Salmonella Enteritidis. The entrapment efficiency after extraction by petroleum ether was determined to be about 80%. Most emulsions were stable during 7 days of storage. Emulsions prepared with WPC had smaller particles, whereas emulsions prepared with emulsifier mixtures had more stable particle dimensions. The studied self-emulsification technique may find numerous applications in the preparation of low-cost food emulsions. KEYWORDS: self-emulsification, clove bud oil, alkaline deprotonation, neutralization, whey protein concentrate, gum arabic

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INTRODUCTION Essential oils (EOs) are aromatic liquids derived from plants or plant parts, including leaves, flowers, buds, fruits, barks, and seeds.1−3 EOs are well-known for their antioxidant and antimicrobial activities and have been widely used as functional ingredients in food, pharmaceutical, and cosmetic products.1 In recent years, the application of EOs in food products has generated particular interest because of their generally recognized as safe (GRAS) regulatory status, multiple functionalities, and wide acceptance by consumers.2,3 Clove oils are the EOs obtained by distillation of the flower buds, stems, and leaves of the clove tree (Syzygium aromaticum). Among clove oils, clove bud oil (CBO) is widely used and well-known for its potent antioxidant properties and antibacterial, antifungal, and antiviral activities.4 Eugenol, 4-allyl-2-methoxyphenol, is the primary constituent (>80%) of CBO5 and is the major contributor of the above biological functions of CBO.4,6,7 The poor solubility in aqueous solutions and high volatility during processing are two major obstacles of utilizing EOs as sanitizing agents or as preservatives in food matrices. Oil-inwater emulsions carefully prepared using appropriate surfactants and emulsification processes are common choices to deliver EOs in aqueous systems. Although synthetic surfactants such as polysorbates (Tweens) have demonstrated excellent emulsifying activities, their potential toxicity and regulated use in foods limit their applications in the food industry.8,9 Therefore, there is a paradigm shift from synthetic to naturally occurring emulsifiers. Food biopolymers, that is, proteins and polysaccharides, with appropriate surface activity are promising candidates to prepare naturally occurring and potentially GRAS emulsions for food applications.10,11 For instance, the combination of whey protein concentrate (WPC) and gum arabic (GA) has been shown to effectively prepare chia (Salvia hispanica) EO microcapsules by spray-drying, and the resultant spray-dried powder can be freely © 2014 American Chemical Society

reconstituted in water, greatly improving its application in aqueous systems.12 Furthermore, nanoemulsions, with droplets smaller than approximately 200 nm in diameter, can prevent the creaming/precipitation of EO droplets and potentially enhance their antimicrobial activity.13 Nanoemulsions are commonly prepared by high-energy methods such as highpressure homogenization and microfluidization, but high capital and operating costs as well as scalability raise concerns about their practicality in the food industry.14 Therefore, the goal of the present work was to study a low-energy self-emulsifying technique to prepare EO nanoemulsions using naturally occurring emulsifiers. CBO was used as a model EO, and WPC, GA, and soy lecithin were selected as emulsifiers. The principle was based on the deprotonation of the hydroxyl group of eugenol (Scheme 1), the major Scheme 1. Chemical Structures of Native and Deprotonated Eugenol

component in CBO, under aqueous alkaline conditions and the self-emulsifying process during the subsequent neutralization in the presence of the natural emulsifiers. The first objective was to study the feasibility of the proposed self-emulsifying approach and characterize physicochemical properties of CBO emulsions. The second objective was to evaluate how the emulsification process affected the antimicrobial activity of CBO Received: Revised: Accepted: Published: 4417

February April 15, April 23, April 23,

9, 2014 2014 2014 2014

dx.doi.org/10.1021/jf500698k | J. Agric. Food Chem. 2014, 62, 4417−4424

Journal of Agricultural and Food Chemistry

Article

The mobile phase consisting of water (solvent A) and methanol (solvent B) was applied at 0.5 mL/min as follows: a linear gradient of 20−80% B from 0 to 20 min, an isocratic step with 80% B from 20 to 25 min, and a linear gradient from 80% B to 20% B from 25 to 30 min. The column chamber was controlled at 25 °C throughout the experiments. Entrapment Efficiency. The entrapment efficiency was determined by subtracting the free oil content from the total oil content. The free oil content in the prepared emulsion was measured according to the method of Rodea-González et al.12 with modifications. Briefly, 0.5 mL of a freshly prepared emulsion was mixed with 2 mL of petroleum ether. The mixture was gently stirred for 10 min, and then 0.1 mL of the upper organic solvent phase was transferred to a 20 mL vial, followed by resting in a fume hood for 5 min to evaporate petroleum ether. Then, 4 mL of ethanol was added to dissolve the extracted oil, and the absorbance was measured at 280 nm, using the above spectrophotometer. A standard curve was established using CBO standard solutions in ethanol to correlate the absorbance value with the oil concentration. The entrapment efficiency was then calculated using eq 1:

against the Gram-negative bacteria Escherichia coli O157:H7 and Salmonella Enteritidis and the Gram-positive bacterium Listeria monocytogenes.

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MATERIALS AND METHODS

Materials. Organic-certified CBO with a purity of no less than 85% was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). WPC-34, consisting of 3−4.5% fat, 34−36% protein, and 48−52% lactose, was provided by Grande Cheese Co. (Grande, WI, USA). The clear WPC dispersion was prepared by removing fat globules as described by Damodaran.15 The 4% WPC was hydrated for 2 h in deionized water, and the pH was adjusted to 4.0. After standing for 2 h at room temperature (21 °C) and centrifugation at 5000g for 20 min (SORVALL RC5B Plus centrifuge, DuPont, Wilmington, DE, USA), the transparent supernatant was collected for emulsion preparation. GA was purchased from Fisher Scientific (Pittsburgh, PA, USA). Soy lecithin (Sunlipon 50) was generously provided by Perimondo, LLC (New York, NY, USA). Other chemicals and reagents were of analytical grade and purchased from Fisher Scientific and used as received. Preparation of Emulsions. Deprotonation of CBO was accomplished by mixing 0.5 mL of CBO with 4.5 mL of 1−5 M NaOH in a glass vial and heating at 110 °C for 10 min in a glycerol bath until the mixture became transparent. The deprotonation of CBO was accelerated by heating, similarly to saponification in the edible oil industry. For emulsion preparation, 3 M NaOH was used as aqueous phase to dissolve CBO. Single or blends of emulsifiers were dissolved separately in water at an overall emulsifier concentration of 1% w/v, consisting of WPC only, GA only, lecithin only, or their equal mass mixtures (Table 1). Then 0.5 mL of alkaline-dissolved CBO was added

entrapment efficiency (%) =

WPC (%)

gum arabic (%)

lecithin (%)

A B C D E F G

1 0 0 0.5 0 0.5 0.33

0 1 0 0.5 0.5 0 0.33

0 0 1 0 0.5 0.5 0.33

(1)

Particle Size and Stability of Emulsions. The particle size of the emulsions was measured using dynamic light scattering with a Delsa Nano analyzer (Beckman Coulter, Inc., Atlanta, GA, USA) at an angle of 165°. The hydrodynamic diameter (Dh) and polydispersity index (PDI) were measured during storage at room temperature (21 °C) for 7 days. Surface Hydrophobicity (S0). The S0 of individual emulsifiers and their combinations was determined using a fluorescence probe, 8anilinonaphthalene-1-sulfonic acid (ANS), based on the method of Luo et al.,17 with minor modifications. Briefly, individual emulsifiers or their mixtures were dispersed in 0.01 M phosphate-buffered saline (PBS, pH 7.0) at 1 mg/mL as stock solutions, which were further diluted with PBS to various concentrations in the range of 5−100 μg/mL. The diluted samples (4 mL) were mixed with 20 μL of an ANS working solution that was previously prepared at 8 mM in 0.01 M PBS (pH 7.0). The mixtures were incubated for 2 h at room temperature (21 °C) in the dark. The fluorescence intensity was then recorded at an excitation wavelength of 355 nm and an emission wavelength of 507 nm using a spectrofluorometer (model RF-1501, Shimadzu Corp.,

Table 1. Compositions Used To Prepare 1% w/v Emulsifier Aqueous Solutions sample

total oil − free oil × 100% total oil

to 4.5 mL of the emulsifier aqueous solution, preadjusted to pH 7.0 at room temperature, and mixed for 5 min on a magnetic stirring plate. Then, the pH of the mixture was adjusted to pH 7.0, using 3 or 0.3 M citric acid. The emulsion spontaneously formed during the acidification process. The emulsion formed was stirred for another 10 min and then stored at 4 °C until further analyses. Structural Changes of Clove Bud Oil after Emulsification. HPLC and UV−vis absorption spectroscopy were used to study the structure of CBO before and after self-emulsification. CBO was extracted from the emulsion prepared with WPC using ethyl acetate. The solvent was then evaporated under the fume hood, and the extracted CBO was dissolved in methanol. The unprocessed CBO was directly dissolved in methanol as a control for unprocessed CBO. The absorption spectrum of each from 200 to 600 nm was determined using a UV−vis spectrophotometer (Evolution 201, Thermo Scientific, Waltham, MA, USA). For HPLC, a reversed phase system (1200 series, Agilent Technologies, Waldbronn, Germany) was used at conditions described previously.16 The HPLC system was composed of a quaternary pump module, a degasser, an autosampler, a temperaturecontrolled column chamber, and an Agilent diode array and multiple wavelength detector. Chromatograms were recorded and integrated using the 1200 LC Chromatography Data System. A ZORBAX Eclipse Plus C18 HPLC column (5 μm, 150 mm × 4.6 mm, Agilent, Palo Alto, CA, USA) equipped with a ZORBAX Eclipse Plus C-18 guard column (4.6 × 12.5 mm, 5 μm) was used in the present study. A 10 μL sample was injected, and the detector was set at a wavelength of 274 nm.

Figure 1. (a) Appearance of clove bud oil (left) after mixing at 10% v/ v with 3 M NaOH (center) at 21 °C and after heating at 110 °C for 10 min (right); (b) appearance of 10% v/v clove bud oil heated at 110 °C for 10 min in 1−5 M NaOH (as labeled on vial caps). 4418



Article

Tokyo, Japan). The initial slope of fluorescent intensity versus sample concentration plot after linear regression with R2 > 0.995 was used as an index for S0. Topographical Structures of Oil Droplets. The morphology of emulsion samples was studied using atomic force microscopy. The freshly prepared emulsion samples were diluted with distilled water to a final emulsifier concentration of 10 μg/mL. Ten microliters of the diluted sample was spread evenly onto freshly cleaved mica sheets mounted on a sample disk (Bruker Corp., Santa Barbara, CA, USA). A rectangular cantilever having an aluminum reflective coating on the backside and a quoted force constant of 2.80 N/m (FESPA, Bruker Corp.) and a Multimode VIII microscope (Bruker AXS, Billerica, MA, USA) were used to scan the sample. Images with a preset scan area of 10.0 × 10.0 μm were generated at a scanning speed of 1 Hz using the tapping mode. Antimicrobial Assays. The antimicrobial activities of emulsion samples were tested against three bacterial strains in tryptic soy broth (TSB). E. coli O157:H7 ATCC 43895, L. monocytogenes Scott A, and Salmonella Enteritidis were obtained from the culture collection of the Department of Food Science and Technology at the University of Tennessee in Knoxville. All strains were grown in TSB and stored at −20 °C in 20% glycerol as stocks. Each strain was transferred at least twice in TSB with an interval of 24 h prior to use. Experiments were

conducted at least twice for each culture, and samples were done in duplicate. The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were determined using the microbroth dilution method.18 Each culture was diluted to ca. 106 CFU/mL in TSB. Then, 120 μL of the diluted culture was added into wells of a 96-well microtiter plate and was mixed with 120 μL of TSB or an antimicrobial sample. Free CBO samples were prepared by dissolving 5% w/v CBO in 5% ethanol that was diluted in TSB to CBO concentrations of 250, 500, 750, 1000, 1250, 1500, 2000, or 2500 ppm. The emulsion samples were diluted in TSB directly to the same CBO concentrations. The microtiter plates were incubated at 32 °C for L. monocytogenes Scott A or at 37 °C for E. coli O157:H7 ATCC 43895 and Salmonella for 24 h. The absorbance at 630 nm (Abs630) of each well was acquired before and after incubation using a microtiter plate reader (Titertek Multiscan MC, Labsystems, Helsinki, Finland). The MIC was defined as the lowest concentration of CBO preventing growth of the test strain, which corresponded to an Abs630 change of 0.05, Table 2). To better understand potential synergistic effects among natural emulsifiers, the S0 values of the emulsifiers and their mixtures were measured (Figure 5). Both the WPC and lecithin exhibited high S0 values, whereas GA (a mixture of polysaccharides and glycoproteins) was very hydrophilic and hence had a low S0. Interestingly, the combination of GA with WPC or lecithin had higher S0 values than individual emulsifiers. As discussed above, this may indicate that the complex formation between these emulsifiers may have enhanced their emulsifying and stabilizing properties. The topography of oil particles prepared with different emulsifiers is shown in Figure 6. The treatments self-emulsified by WPC, GA, and their combination had spherical particles, verifying self-emulsification during neutralization. In contrast, emulsions prepared in the presence of lecithin had irregular structures. This is likely due to water-insoluble lecithin particles because soybean lecithin is a complex mixture composed of both water-soluble and -insoluble substances, such as phospholipids, glycerol, and triglycerides.23 Entrapment Efficiency. The entrapment efficiency was about 80% for all emulsifiers (Figure 7). The treatment with GA alone had significantly lower entrapment efficiency (76.8%), possibly due to its reduced ability to emulsify CBO compared to other treatments. The efficiencies of entrapping CBO using the present self-emulsification technique were comparable to, or higher than, those of many other oil-in-water emulsion systems prepared with natural emulsifiers using high-energy methods such as high-speed homogenization,24 high-pressure homogenization,25 and probe-type sonication.26 Minimum Inhibitory and Minimum Bactericidal Concentrations of Emulsions. The MICs for free CBO were 1000, 750, and 750 ppm, whereas the MBCs were 1250, 750, and 750 ppm against L. monocytogenes Scott A, E. coli O157:H7 ATCC 43895, and Salmonella Enteritidis, respectively (Table 3). The results are in general agreement with other studies.2,27 The CBO emulsions had MICs and MBCs that were similar or one to two dilutions lower than free CBO, whereas no antimicrobial activity of the emulsifiers alone was observed. Therefore, emulsification did not have a negative impact on the antimicrobial activity of CBO. Because all MICs and MBCs were below the water solubility of eugenol (1350 ppm at 21 °C and likely higher at 32 or 37 °C),16 it can be concluded that binding between the selected emulsifiers and CBO did not interfere with the antimicrobial activity of eugenol. Conversely, higher MICs or MBCs of eugenol nanoemulsions than of free eugenol were reported when Tween 8028 or whey protein−maltodextrin conjugates29 were used as emulsifiers. Additionally, the emulsions prepared with GA and its mixture with other emulsifiers exhibited lower MICs and/or MBCs against L. monocytogenes than other treatments. It has been speculated that the strong anionic properties of GA may influence surface proteins on the cell membrane of L. monocytogenes, which may enhance the antimicrobial activity of EOs.30 Growth Kinetics of Bacteria in TSB. The three transparent/translucent emulsions (WPC alone, GA alone, and WPC−GA) with 0.2% v/v CBO (Figure 2) were evaluated for

Figure 8. Growth curves of (A) Listeria monocytogenes Scott A at 32 °C, (B) Escherichia coli O157:H7 ATCC 43895 at 37 °C, and (C) Salmonella Enteritidis at 37 °C in TSB treated by no antimicrobials (negative control, black squares), 0.25% ethanol (positive control, as in free clove bud oil (CBO) treatment, green squares), 750 ppm free CBO (predissolved in 5% ethanol, red circles), and 750 ppm of CBO in the emulsion form prepared with WPC (blue triangles), GA (yellow triangles), and equal masses of WPC and GA (magenta triangles). Error bars are standard deviations from four replicates.

the influence on the growth kinetics of the test bacteria in TSB at a final CBO level of 750 ppm. The activity was compared with free CBO predissolved in 5% ethanol. An ethanol control (0.25% v/v, equivalent to final ethanol concentration in free CBO sample) in the TSB did not show any effect on the growth of the cultures (Figure 8). Free CBO exhibited significant inhibitory effects on the growth of three bacteria. For L. monocytogenes (Figure 8A), the emulsion prepared with GA exhibited a significantly better reduction (P < 0.05) at shorter times than the 4422



Article

and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytother. Res. 2007, 21, 501−506. (5) Srivastava, A. K.; Srivastava, S. K.; Syamsundar, K. V. Bud and leaf essential oil composition of Syzygium aromaticum from India and Madagascar. Flavour Fragrance J. 2005, 20, 51−53. (6) Lee, K.-G.; Shibamoto, T. Antioxidant property of aroma extract isolated from clove buds [Syzygium aromaticum (L.) Merr. et Perry]. Food Chem. 2001, 74, 443−448. (7) Ayoola, G.; Lawore, F.; Adelowotan, T.; Aibinu, I.; Adenipekun, E.; Coker, H.; Odugbemi, T. Chemical analysis and antimicrobial activity of the essential oil of Syzigium aromaticum (clove). Afr. J. Microbiol. Res. 2008, 2, 162−166. (8) Csáki, K. F. Synthetic surfactant food additives can cause intestinal barrier dysfunction. Med. Hypotheses 2011, 76, 676−681. (9) Kralova, I.; Sjöblom, J. Surfactants used in food industry: a review. J. Disper. Sci. Technol. 2009, 30, 1363−1383. (10) Dickinson, E. Hydrocolloids as emulsifiers and emulsion stabilizers. Food Hydrocolloids 2009, 23, 1473−1482. (11) Pan, K.; Chen, H.; Davidson, P. M.; Zhong, Q. Thymol nanoencapsulated by sodium caseinate: physical and antilisterial properties. J. Agric. Food Chem. 2014, 62, 1649−1657. (12) Rodea-González, D. A.; Cruz-Olivares, J.; Román-Guerrero, A.; Rodríguez-Huezo, M. E.; Vernon-Carter, E. J.; Pérez-Alonso, C. Spraydried encapsulation of chia essential oil (Salvia hispanica L.) in whey protein concentrate-polysaccharide matrices. J. Food Eng. 2012, 111, 102−109. (13) Donsì, F.; Annunziata, M.; Sessa, M.; Ferrari, G. Nanoencapsulation of essential oils to enhance their antimicrobial activity in foods. LWT−Food Sci. Technol. 2011, 44, 1908−1914. (14) McClements, D. J.; Rao, J. Food-grade nanoemulsions: formulation, fabrication, properties, performance, biological fate, and potential toxicity. Crit. Rev. Food Sci. Nutr. 2011, 51, 285−330. (15) Damodaran, S. Straightforward process for removal of milk fat globule membranes and production of fat-free whey protein concentrate from cheese whey. J. Agric. Food Chem. 2011, 59, 10271−10276. (16) Chen, H.; Davison, M.; Zhong, Q. Impacts of sample preparation methods on solubility and antilisterial characteristics of essential oil components in milk. Appl. Environ. Microbiol. 2014, 80, 907−916. (17) Luo, Y.; Pan, K.; Zhong, Q. Physical, chemical and biochemical properties of casein hydrolyzed by three proteases: partial characterizations. Food Chem. 2014, 155, 146−154. (18) Ma, Q.; Davidson, P. M.; Zhong, Q. Antimicrobial properties of lauric arginate alone or in combination with essential oils in tryptic soy broth and 2% reduced fat milk. Int. J. Food Microbiol. 2013, 166, 77− 84. ́ ch, E.; Buraczewska, L.; (19) Pastuszewska, B.; Jabłecki, G.; Swiȩ Ochtabińska, A. Nutritional value of rapeseed meal containing lecithin gums precipitated with citric acid. Anim. Feed Sci. Technol. 2000, 86, 117−123. (20) Kim, Y. D.; Morr, C. V.; Schenz, T. W. Microencapsulation properties of gum arabic and several food proteins: liquid orange oil emulsion particles. J. Agric. Food Chem. 1996, 44, 1308−1313. (21) Bylaite, E.; Nylander, T.; Venskutonis, R.; Jonsson, B. Emulsification of caraway essential oil in water by lecithin and βlactoglobulin: emulsion stability and properties of the formed oilaqueous interface. Colloid Surf. B 2001, 20, 327−340. (22) Klein, M.; Aserin, A.; Svitov, I.; Garti, N. Enhanced stabilization of cloudy emulsions with gum arabic and whey protein isolate. Colloid Surf. B 2010, 77, 75−81. (23) Rydhag, L.; Wilton, I. The function of phospholipids of soybean lecithin in emulsions. J. Am. Oil Chem. Soc. 1981, 58, 830−837. (24) Carneiro, H. C. F.; Tonon, R. V.; Grosso, C. R. F.; Hubinger, M. D. Encapsulation efficiency and oxidative stability of flaxseed oil microencapsulated by spray drying using different combinations of wall materials. J. Food Eng. 2013, 115, 443−451.

other antimicrobial treatments, especially at 8 h, which was >2.5 log CFU/mL lower than free CBO. At 24 h, all CBO emulsions demonstrated bacteriostatic inhibition, which was 2.5−3 logs lower than the control. At the end of incubation (48 h), the antilisterial activity of WPC-emulsified sample was similar to that of free CBO and was the least effective among the three emulsions, whereas the emulsion prepared with the GA−WPC mixture exhibited significant inhibitory effects compared to the control. For E. coli O157:H7 (Figure 8B), all free and emulsified CBO treatments were highly inhibitory with an initial 2−2.5 log reduction in the first 8 h. After 48 h, all treatments continued to demonstrate bacteriostatic activity against the test microorganism. For Salmonella, no initial reduction was seen, but at the end of the incubation period, all emulsions demonstrated bacteriostatic activity, whereas the free CBO had begun to show growth (Figure 8C). Overall, at the end (48 h) of incubation, all three bacteria treated by free CBO recovered to a higher population than the three emulsion samples. Therefore, the selfemulsification technique used to prepare EO emulsions did not compromise the antimicrobial activity of CBO. In summary, CBO emulsions were successfully prepared using a self-emulsification technique with naturally occurring food emulsifiers at ambient conditions without specialized equipment or organic solvents. The self-emulsification process (dissolving in a hot alkaline solution and neutralization) did not appear to have changed either the structure or antimicrobial activity of the major component in CBO (eugenol). WPC was effective in reducing emulsion droplet size, whereas the emulsifier mixtures were better emulsion stabilizers than single emulsifiers. Emulsions as prepared were stable during storage at room temperature and could be prepared as transparent or translucent samples upon dilution to CBO concentrations appropriate for application. Thus, the studied self-emulsification technique holds great potential in preparing EO emulsions for use as antimicrobial food preservatives or sanitizers in postharvest washing solutions for fresh produce production.

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AUTHOR INFORMATION

Corresponding Author

*(Q.Z.) Mail: Department of Food Science and Technology, University of Tennessee, 2510 River Drive, Knoxville, TN 37996, USA. Phone: (865) 974-6196. Fax: (865) 974-7332. Email: [email protected]. Funding

This work was supported by the University of Tennessee and the U.S. Department of Agriculture, National Institute of Food and Agriculture, under Grant TEN02012-02247. Notes

The authors declare no competing financial interest.

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REFERENCES

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Self-Emulsification of Alkaline-Dissolved Clove Bud Oil by Whey

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