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NANOENCAPSULATION AND NANOEMULSION OF BIOACTIVE COM POUNDS TO

ENHANCE THEIR ANTIOXIDANT ACTIVITY IN FOOD

KHALED F. M 1, KHALED M. A. RAMADAN 2 & I. S. ASHOUSH3

1Department of Food Science and Technology, National Research Centere, Dooki, Cairo, Egypt 2Department of Agricultural Biochemistry, Faculty of Agriculture, Ain-Shams University, Cairo, Egypt

3Department of Food Science, Faculty of Agriculture, Ain- Shams University, Cairo, Egypt

ABSTRACT

This work aims at investigating the effect of the nanoemulsion and nanoencapsulation delivery systems on the

antioxidant and antimicrobial activity of different bioactive components. Thymol, carvacrol, linalool and eugenol

hydrophobic compounds were encapsulated in dextran of oil-in-water (o/w) nanoemulsions prepared by ultrasonic

homogenization method and stabilized by different types of emulsifier Tween 20, 40 or 80 and distilled water. The results

showed that the reducing power, antioxidant capacity, H2O2 scavenging and antioxidant activity of un-encapsulated

eugenol was the highest percent compared others essential oils. The highest essential oils nanoencapsulated yield (EY%)

and efficiency (EE%) were observed for eugenol 91.74 and 97.81 %, respectively by using Dex/T20 treatment.

Nanoencapsulation of eugenol can enhance the aqueous solubility of eugenol at 25, 35 and 45 °C. Thermal stability of

eugenol increased from 63.5 to 143 °C after nanoencapsulation by using Differential Scanning Coliremetry (DSC).

Antioxidant activity of nanoencapsulated eugenol 0.5 mg/ml in linolic acid system compred with BHT activity at different

periods storage were 98.97, 98.42, 98.12 and 97.89 % at 0, 60, 120 and 180 days, respectively. Sensory evaluation of jelly

prepared with different levels of nano-encapsulated eugenol compared to 1% its un-encapsulated showed the Dex/T20

(0.3 %) formation was the most accepted by testers. The antimicrobial activity of nano-encapsulated eugenol was measured

against three different microorganisms, such as Molds & yeasts, coliforms and salmonella. Significance and Impact of the

Study: nanoencapsulation affects positively essential oils main compounds, improves antioxidant activity and retains

antimicrobial activity, enhancing the quality of the oils.

KEYWORDS: Nano-Emulsion, Nano-Encapsulation, Bioactive Compounds, Thymol, Carvacrol, Eugenol, Linalool

INTRODUCTION

Plant phenolic and polyphenolic compounds are widespread components of the human diet found in vegetables,

fruits, soybean, cereals, tea, coffee, red wine and natural herb extracts. The extracts of medicinal plant and culinary herb

sources possess very strong antioxidant activity in vitro. This antioxidant activity is due mainly to phenolic compounds

present in plants (Sahaa et al, 2008). The strong antioxidant activity of the volatile extract of thyme is mostly due to its

main volatile components, thymol and carvacrol. In the case of the volatile extract of basil, eugenol, reported as a major

volatile component, probably contributes significantly to the strong antioxidant activity of the extract. Eugenol showed

high antioxidant activity in the aldehyde/carboxylic acid assay (Teissedre & Waterhouse 2000; and Lee, & Shibamoto

2001).

International Journal of Food Science and Technology (IJFST) ISSN(P): Applied; ISSN(E): Applied Vol. 4, Issue 3, Dec 2014, 1-22 © TJPRC Pvt. Ltd.

2 Khaled F. M, Khaled M. A. Ramadan & I. S. Ashhous

rc.orgeditor@tjp www.tjprc.org

Essential oils (EO) are a group of ethereal lipophilic compounds, extracted from herbs and spices such as thyme

(thymol), oregano (carvacrol) and clove (eugenol) (Burt, 2004). EO are volatile and highly aromatic, with strong

antioxidant, antibacterial and antifungal properties (Toure et al, 2007). When applying EO at a level above their solubility

limit, the visible insoluble EO, particularly in beverages, may be a quality defect unacceptable by consumers.

Encapsulation is the most commonly employed approach in the food industry to disperse lipophilic compounds.

In addition, effective encapsulation technologies protect sensitive compounds from degradation, mask unpleasant tastes of

certain compounds, reduce losses due to evaporation, prevent binding or interaction with other food matrix components,

or facilitate controlled release under desired conditions (McClements et al, 2007; de Vos et al, 2010). Proteins and

carbohydrates are frequently studied as matrices to encapsulate lipophilic compounds by spray drying (Charve and

Reineccius 2009; Gharsallaoui et al, 2007; Sliwinski et al, 2003; Zeller et al, 1998), which is a one-step, low-energy and

economical encapsulation method commonly used in the food industry (Adamiec and Kalemba 2006). Specifically relevant

to this study, mixtures of whey protein isolate (WPI) and maltodextrin (MD) have been used to encapsulate avocado

(Bae and Lee, 2008), ginger (Toure et al, 2007), oregano (Baranauskiene, et al, 2006) and caraway (Bylaite et al, 2001)

essential oils. Moreover, conjugation of WPI and MD by dry heating (the Maillard reaction) improves emulsifying

properties of whey protein (Akhtar and Dickinson 2007; Choie et al, 2010). For whey proteinbased encapsulation systems,

heat stability of capsules is an important parameter because manufacturing of many beverage products requires thermal

processing to inactivate spoilage and pathogenic microorganisms, but whey protein is known to aggregate during heating,

especially at increased ionic strength and acidity near isoelectric point when electrostatic repulsion weakens (Bryant and

McClements 1998). Further, to prepare transparent dispersions, nanoparticles with encapsulated lipophilic compounds

typically smaller than 100 nm are required to reduce the scattering of light (Farhang 2009). However, heat stable

transparent dispersions with EO-containing capsules based on whey protein or conjugates have not been reported.

Recently, studies have shown the successful approach of using nanoemulsions to improve stability in food

applications. Tan (2005) and Yuan et al, (2008) prepared EO nanodispersions using high-pressure homogenization and

studied their physicochemical properties. Other applications include the encapsulation of limonene, lutein, omega-3 fatty

acids, astaxantin, and lycopene (Chen et al, 2006), the encapsulation of a -tocopherol to reduce lipid oxidation in fish oil

(Weiss et al, 2009), and the use of nanoemulsions to incorporate essential oils, oleoresins, and oil-based natural flavors into

food products such as carbonated beverages and salad dressings (Ochomogo and Monsalve- Gonzalez 2009).

In the last two decades, nanotechnology has rapidly emerged as one of the most promising and attractive research

fields. The technology offers the potential to significantly improve the solubility and bioavailability of many functional

ingredients.

The high hydrophobicity of some bioactive substances makes them insoluble in aqueous systems, and they

therefore have a poor intake in the body. To improve antioxidants dispersibility in water, for example (Horn and Rieger

2001), and also to increase their bioavailability during gastrointestinal passage (Deming and Erdman 1999), antioxidant

crystals must be formulated, that is, to incorporate them in the fine particles of oil-in-water (O/W) emulsions.

They can be produced using two different approaches: high-energy and low energy methods. High-energy

methods use intense mechanical forces to break up macroscopic phases or droplets into smaller droplets and typically

involve the use of mechanical devices known as homogenizers, which may use high-shear mixing, high-pressure

homogenization, or ultrasonication. In contrast, low-energy methods rely on the spontaneous formation of emulsions under

Nanoencapsulation and Nanoemulsion of Bioactive Compounds to Enhance their Antioxidant Activity in Food 3


specific system compositions or environmental conditions as a result of changes in interfacial properties (McClements and

Li 2010). Spontaneous emulsification is a less expensive and energy-efficient alternative that takes advantage of the

chemical energy stored in the system (Bilbao Sáinz et al, 2010). High-energy methods are effective in reducing droplet

sizes but may not be suitable for some unstable molecules, such as proteins or peptides.

One of the most used low-energy methods is the phase inversion temperature (PIT) method, which is based on the

changes in solubility of polyoxyethylene-type nonionic surfactants with temperature. The surfactant is hydrophilic at low

temperatures but becomes lipophilic with increasing temperature due to dehydration of the polyoxyethylene chains. At low

temperatures, the surfactant monolayer has a large positive spontaneous curvature forming oil-swollen micellar solution

phases (or O/W microemulsions), which may coexist with an excess oil phase. At high temperatures, the spontaneous

curvature becomes negative and water-swollen reverse micelles (or W/O microemulsions) coexist with an excess water

phase. At a critical temperature—the hydrophilic–lipophilic balance (HLB) temperature—the spontaneous curvature is

zero and a bicontinuous microemulsion phase containing comparable amounts of water and oil phases coexists with both

excess water and oil phases.

If the cooling or heating process is not fast, coalescence predominates and polydispersed coarse emulsions are

formed (Ee et al, 2008).

Other low-energy methods that can be used to prepare nanoemulsions are the PIC (phase inversion composition)

method, in which the temperature is maintained constant and the composition is changed (the solvent quality is changed by

mixing two partially miscible phases together). By using the PIC method, different nanomaterials such as colloidosomes

(Dinsmore et al, 2002), cubosomes (Spicer 2004), and microfluidic channels (Xu et al. 2005) have been prepared. Liu et al,

(2006) used polyoxyethylene (PEO) nonionic surfactants for the preparation of paraffin oil-in water nanoemulsions also by

using the PIC method. They reported the preparation of 2.1 Methods of Formation 9 stable nanoemulsions with diameters

ranging from 100 to 200 nm. Surfactants used in this system were a combination of Span 80, a sorbitan monooleate with a

low HLB, and Tween 80, an ethoxilated sorbitan monooelate ester with a high HLB. As these two surfactants possess the

same backbone, they can mix easily, leading to a controlled change in the final HLB.

Another commonly used inversion method is transitional phase inversion (TPI) (Jahanzad et al, 2010). Before TPI

can occur, the surfactants in both phases must diffuse toward the interface, adsorb at the interface, and conform into a

mixed surfactant layer at the optimum conditions. The rate of diffusion of the surfactants depends on many parameters,

including their size, the viscosity of the phase, and the intensity of mixing, etc. For oil-in-water emulsions containing a pair

of water soluble and oil-soluble surfactants, it was found that the addition of the water phase containing the water-soluble

surfactant to the oil phase containing the oil-soluble surfactant may produce very fine emulsions if it is associated with

interfacial tension lowering in the course of addition. The rate of addition of the second phase is of great importance in

achieving an emulsion with the desired properties. This is Nano and Micro Food Emulsions because the dynamic of phase

inversion emulsification is very fast, and the emulsion properties change quickly with further addition, contrary to some

conventional emulsification methods. Therefore, it is important to find and maintain a semi equilibrated state in the course

of emulsification during which sufficient surfactant diffusion/adsorption can occur, and thus droplet rupturing is enhanced.

TPI occurs when the curvature of the oil–water interface gradually changes from positive to negative, passing through a

zero curvature at the inversion point. This is associated with a shift in the surfactant nature from water-soluble to

oil-soluble, or vice versa.



Conventional emulsions, that is, emulsions with microdroplets, are thermodynamically unstable liquid–liquid

dispersions. Emulsions with particle sizes higher than 100 nm are usually prepared by high-energy methods. As in the case

of nanoemulsions, the O/W systems consist of lipid droplets dispersed in an aqueous medium, with each lipid droplet being

surrounded by a thin emulsifier layer. The initial droplet concentration and size distribution of this type of delivery system

can be controlled, as can the nature of the emulsifier used to stabilize the droplets. A careful selection of emulsifier type

enables one to control interfacial properties such as charge, thickness (dimensions), rheology, and response to

environmental stresses (such as pH, ionic strength, temperature, and enzyme activity) (McClements and Li 2010). To form

conventional emulsions, usually a preemulsion with coarse particles, obtained by a mechanical device such as an

Ultra-Turrax, is prepared. This system is further homogenized to obtain a micro droplet emulsion. In general, a variety of

homogenizers are available to prepare emulsions, including high-shear mixers, high-pressure homogenizers, colloid mills,

ultrasonic homogenizers, and membrane homogenizers.

Encapsulation of bioactive compounds represents a feasible and efficient approach to modulate drug release,

increase the physical stability of the active substances, protect them from the interactions with the environment, decrease

their volatility, enhance their bioactivity, reduce toxicity, and improve patient compliance and convenience (Ravi 2000).

Encapsulation of Essential Oils deals with micrometric size capsules, which are used for the protection of the

active compounds against environmental factors (e.g, oxygen, light, moisture, and pH), to decrease oil volatility and to

transform the oil into a powder. Encapsulation in nanometric particles is an alternative for overcoming these problems but

additionally, due to the sub cellular size, may increase the cellular absorption mechanisms and increasing bioefficacy

(Anna et al, 2014).

Carvacrol, limonene and cinnamaldehyde were encapsulated in the sunflower oil droplets of nanoemulsions

prepared by high pressure homogenization and stabilized by different emulsifiers: lecithin, pea proteins, sugar ester and a

combination of Tween 20 and glycerol monooleate. The antimicrobial activity was measured against three different

microorganisms, such as Escherichia coli, Lactobacillus delbrueckii and Saccharomyces cerevisiae. The measured

antimicrobial activity was significantly affected by the formulation of the nanoemulsion, where the different bioactive

compounds were encapsulated. In particular, the effect of the delivery systems on the antimicrobial activity was correlated

to the concentration of the essential oil components in the aqueous phase in equilibrium with the nanoemulsion droplets,

suggesting that the ability of the active molecules to interact with cell membranes is associated to their dissolution in the

aqueous phase. (Francesco et al, 2012).

Although the antimicrobial effects of essential oils are well established and despite the several studies on their

antimicrobial activity (Bakkali et al, 2008; Ben Arfa et al, 2006; Ceylan and Fung, 2004; Helander et al, 1998; Lambert et

al, 2001; Ultee et al, 2000, 2002; Sikkema et al, 1995), it must be highlighted that their mechanisms of action are poorly

understood, even though different theories were proposed. In particular, it appears that the different essential oil

components are characterized by a peculiar mechanism of action, which depends on the nature of the specific bioactive

molecule (Di Pasqua et al, 2007).

The objective of this work was to try increase the efficiency and stability of some essential oils using

nanotechnology (nano-emulsion and nano-encapsulation) techniques on the antioxidant and antimicrobial activity for

application in the food industry.



MATERIALS AND METHODS

Materials

Pure bioactive compounds, Thymol, Euganol, Linalool and Carvacrol were delivered from Sigma – Aldrich

Chemicals.

Methods

Reducing Power

The reducing power of thymol, carvacrol, linalool, and eugenol was determined according to the method of

Oyaizu, (1986). A volume of 0.4 ml of ethanolic solutions with different concentration (0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0

mg/ml) form each tested compound were added to 1 ml potassium ferricyanide 1% and 1 ml phosphate buffer 0.2 M, pH =

6.6. Mixture was incubated at 50 oC for 20 min. Then, trichloroacetic acid 10% was added to mixture, and centrifuged at

650 g for 10 min. Two ml of the supernatant was mixed with 2 ml distilled water and 0.4 ml ferric chloride 0.1% and the

absorbance was read spectrophotometrically( Shimadzu UV-VIS 160 A) at 700 nm. Higher absorbance of the reaction

mixture indicated greater reducing power.

Total Antioxidant Capacity as Equivalents of α-Tocopheroel

The total antioxidant capacity of thymol, carvacrol, linalool and eugenol was determined according to the method

of Prieto et al. (1999). Ethanolic solution with different concentration (10, 20, 30, 40, 50, 70, 100 and 500 ppm) was added

to 3.5 ml reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4mM Ammonium molybdate). Then, the

tubes were capped and incubated in a water bath at 95 oC for 90 min. After cooling to room temperature, the absorbance

was measured at 695 nm against a blank. The antioxidant capacities expressed as equivalents of α-tocopheroel.

The Antioxidant Capacity of Thymol, Carvacrol, Eugenol and Linalool by DPPH Method

The ability of tested compounds to scavenge DPPH radical was determined according to the method of (Hatano,

et al, 1988). The reaction mixture had contained 0.1 ml of DPPH radical solution (5 mM) and different concentration of

tested compounds (from 0.0026 mM to 83 mM). Total volume of the reaction mixture was 3ml. Absorption of DPPH

radical at 515 nm was determined after 10 min against a blank solution that contained only methanol.

% DPPH radical scavenging activity = ((Ac-As)/Ac) ×100

Where: As= absorbance of sample; Ac= absorbance of control (DPPH radical solution in methanol without

sample).

Scavenging of Hydrogen Peroxide

The ability of tested compounds to scavenge hydrogen peroxide was determined according to the method of Ruch

et al, (1989). A solution (20 mM) of hydrogen peroxide was prepared in ethanol 95%. Hydrogen peroxide concentration

was determined spectrophotometrically from absorption at 230 nm by using the molar absorptive of 81M-1 cm-1. Ethanolic

solution of each sample with different concentration 10, 20, 40, 60, 80, 100, 120,140 ppm was added to a hydrogen

peroxide solution. Absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a blank.

% scavenging of hydrogen peroxide effect = (As/Ac -1) ×100



Where: As= absorbance of sample, Ac= absorbance of control (hydrogen peroxide solution in ethanol without

sample).

Antioxidant Activity in a Linoleic Acid System

An antioxidant activity assay was carried out by using the linoleic acid system Osawa and Namiki (1981). Tested

compounds in ethanolic solution with different concentration (125, 250, 375,500 ppm) were added to 2 ml ethanolic

solution of linoleic acid 0.2 M. Then, the total volume was adjusted to 4 ml with ethanol. And the reaction mixture

incubated at 40oC. the degree of oxidation was measured according to the thiocyanate method Mitsuda et al, (1996) by

adding 0.1 ml reaction mixture , 0.1 ml ammonium thiocyanate 30% , 3 ml solvent (n butanol :ethanol 1:1) and 0.1 ml

ferrous chloride (20 mM in 3.5% HCl). After mixing was stirred for 3 min, the absorbance measured at 500 nm, and the

percentage of antioxidant activity was calculated from the following equation:

Antioxidant activity %= 100 - [(absorbance of sample / absorbance of control) ×100]

All tests and analyses were run in triplicate and averaged

Preparation of Bioactive Nanoemulsions

Oil-in-water (o/w) emulsions were prepared according to Helder et al, (2011) with some modifications.

Briefly: 0.03% (w/w) bioactive compounds (higher than 97 % purity) was dissolved at 40 ºC in n-hexane (95% purity).

This organic solution was then added to an aqueous solution containing 0.5 % (w/w) Tween (20, 40 and 80) (97%, Sigma,

USA) in distilled water. The organic/aqueous phase volume ratio of 1: 9 was used. The emulsions were homogenized using

an ultrasonicator homogenizer (Homogenizer PRO 400PC). The n-hexane was then removed from the fine emulsion by

rotary evaporation. In order to simulate industrial production environment, no special care was taken to avoid contact with

oxygen.

The nanoemulsions obtained were stored at 4 ºC in the absence of light, during the evaluation period, reproducing

eventual commercial conditions.

• 2 % dextran was added to 0.5 % (w/w) Tween 80 and then 100 ml distilled water and stirring (Dex/T80).

• 2 % dextran was added to 0.5 % Tween 40 and then 100 ml distilled water (Dex/T40).

• 2 % dextran was added to 0.5 % Tween 20 and then 100 ml distilled water (Dex/T20).

Determination of Nano-Encapsulation Efficiency (NE) of the Clove Powder

The encapsulation efficiency was calculated as the amount of eugenol present in the nano-capsules compared with

the eugenol initially used in their production. The nano-capsule was completely destroyed in accordance to the procedure

reported by Robert et al, (2010), with little modifications. 5 ml of the encapsulated solution was added to 10 ml methanol.

The dispersion was agitated using a vortex (1 min). After vortexing, the samples were allowed to rest for 5 min while

protected from light and then in an Ultrasonicator (M/s Ika, Germany) for 30 min at 1000 rpm, then centrifuged at 112,000

g for 5 min and finally filtered. The supernatant was analyzed for bioactive compounds such as total eugenol content, total

phenolic content and antioxidant activity.



The nanoencapsulation efficiency (NE) was calculated according to Eq. (1) using the results from total bioactive

compounds and theoretical bioactive compounds (Ahmed et al, 2010).

(1)

Encapsulation Yield

The encapsulation yield (EY) was calculated according to Eq. (2) as the ratio of the mass of nanocapsules

obtained after ultrasonic possessing to the total mass of the initial substances added before encapsulation Duduku et al,

(2012).

(2)

Oxidation Study of Bioactive Compounds Encapsulated forms by DSC

DSC was furthermore employed to study the thermo-oxidation stability of the samples. Samples of pure terpenes

as well as their complexes with B-CD and their encapsulated forms in MS containing around 2 mg of terpenes were placed

in 40-µL aluminum pans having a hole in their lid. The specimens were heated in pure oxygen atmosphere from room

temperature to 120 ◦C at 90 ◦C/min, remained at 120 ◦C for 1 min, to ensure a uniform temperature distribution within the

sample, and then heated up to 420 ◦C at 10 ◦C /min.

Application and Preparation of Jelly

Jelly was prepared in laboratory by adding different levels of eugenol nanoencapsulated i.e. 0.10, 0.20, 0.30, 0.40

and 0.50% w/w in laboratory using the traditional procedure. The formulation of Jelly was contained the ingredients of

sucrose, gelatin, citric acid, flavoring agent, sodium benzoate and potassium citrate, ascorbic acid and antioxidant pigment

were 84.0, 15.0, 0.20, 0.10, 0.10, 0.10 and 0.1-0.5 %, respectively.

Jellies were wrapped by polyethylene and aluminum foil and packed in carton bags and were stored at room

temperature 25±5ºC. The control of jelly was prepared with 0.10% synthetic color (carmine) according to Attia et al,

(2013)

Phase Solubility Studies

Phase solubility studies were carried out as described by Higuchi and Connors (1965) and Mourtzinos et al,

(2008). Briefly, an excess amount of eugenol encapsulated was mixed with an aqueous solution of dextran of increasing

concentrations (0 to 0.015 M). A laboratory shaker was used and the samples were equilibrated for 2 days at 25, 35, and 45

ºC at gas-tight bottles. Then the samples were filtered with a syringe through 0.45-µm pore diameter PVDF filters, and the

amount of eugenol encapsulated in solution was determined spectrophotometrically by measuring the absorbance at 280.2

nm. The experiments were carried out in triplicate at each temperature.



The stability constants, KC, of the reaction of dextran toward a complex formation were calculated from the

straight-line portion of the phase solubility diagram according to the Higuchi–Connors equation:

Microbiological Analyses

For microbiological evaluation, the following three analyses were performed: yeast and mold counting, coliforms

at 45°C and Salmonella sp. research using the techniques outlined in the current legislation, RDC # 12 (BRASIL, 2001).

Sensory Evaluation

An acceptance test of the jelly samples was carried out at the Sensory Analysis Laboratory of the Food Science

and Technology Department, National Research Center (NRC) with the participation of 41 volunteer members of the NRC

community from both sexes and of all ages. Volunteers were selected based on their interest and availability to take part in

the tests. Samples were evaluated through tests of preference and intention to purchase in accordance with NBR

12806/1993 (ABNT, 1993). The testers assessed the samples using a 9-point structured hedonic scale, as follows:

1 - disliked very much; 2 - disliked quite a lot; 3 - fairly disliked; 4 - slightly disliked; 5 - neither liked nor disliked;

6 - slightly liked; 7 - fairly liked; 8 - liked quite a lot; 9 - liked very much. The samples were marked with random three

digit numbers and served to testers in individual booths under white light. Plain mineral water and biscuit means cookie

were provided for rinsing of the palate between sample evaluations.

Statistical Analyses

Experimental results were mean ± S.D. of three parallel measurements and analyzed by SPSS 10 (SPSS Inc.

Chicago, IL). Differences between means were determined using Tukey multiple comparisons and least significant

difference (LSD). Correlations were obtained by Pearson correlation coefficient in bivariate correlations. P values <0.05

were regarded significant.

RESULTS AND DISCUSSIONS

Antioxidant Activity of the Basil and Essential Oils Major Components

Reducing Power

The reducing power of thymol (T), carvacrol (C), linalool (L), and eugenol (E) was measured by the ferricyamide

method. Different concentrations of thymol, carvacrol, linalool and eugenol 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml were

investigated. Higher absorbance at 700 nm of the reaction mixture indicated greater reducing power. Table 1 represents the

reducing power of T, C, L and E measured as absorbance at 700 nm. Table 1 reveals that T, C, E exhibited a reducing

power, but to varying degrees while L has no significant reducing power. A positive correlation has been observed between

concentrations and reducing power for T, C, E especially E. There is a significant difference among the tested compounds

in their reducing power.



Table 1: Reducing Power of Thymol, Carvacrol, Linalool and Euganol as Absorbance at 700 nm

Concentration (mg/ml)

Absorbance at 700 nm

Thymol Carvacrol Linalool Eugenol Ascorbic

Acid 0.05 0.36±0.01 0.23±0.02 0.04±0.01 0.96±0.03 0.83±0.01 0.1 0.66±0.02 0.53±0.02 0.09±0.01 1.64±0.02 1.36±0.11 0.2 1.05±0.01 0.87±0.01 0.14±0.01 2.15±0.05 1.68±0.06 0.4 1.48±0.20 1.26±0.02 0.18±0.01 2.73±0.03 1.87±0.03 0.6 1.64±0.06 1.41±0.04 0.20±0.01 2.91±0.07 2.03±0.05 0.8 1.73±0.04 1.56±0.06 0.21±0.01 3.37±0.13 2.05±0.12 1.0 1.78±0.05 1.68±0.03 0.22±0.01 3.92±0.11 2.07±0.07

The reducing power of E was higher than T than C in the same order, we have notice that the reducing power of E

was about 7-fold the reducing power of T and C.

Politeo, et al. (2007) reported that the highest reducing power (FRAP) was showed samples of pure eugenol.

The reducing power of the volatile aglycones is comparable, but less than the reducing power of essential oil and BHT.

A hierarchy in the reducing capacity of samples could be observed as well: eugenol > BHT > basil essential oil > basil

volatile aglycones. Among the compounds identified in the essential oil and volatile aglycones from basil, eugenol was

considered the main contributor of the antioxidant capacity. The comparison of the antioxidant capacity of pure eugenol

and the total essential oil at corresponding to equivalent mass of eugenol in total essential oil (EC50 = 0.099 g/l) was

showed very similar antioxidant capacities for both in all concentration range.

The Antioxidant Capacity of Thymol, Carvacrol, Eugenol and Linalool by Phosphomolybdenium Method

The antioxidant capacity of T, C, E and L by phosphomolybdenium method were determined and expressed as

equivalents of α- tocopherol (nmol/g oil). Different concentrations of T, C, E and L 10, 20, 30, 40, 50, 70, 100 and 500

ppm were measured.

Table 2: The Antioxidant Capacity of Thymol, Carvacrol, Linalool and Eugenol by Phosphomolybdenium Methods as Equivalents of α-Tochopherol

Conc. (ppm)

Antioxidant Capacity % Thymol Carvacrol Linalool Eugenol

10 00.00 00.00 13.52 27.83 20 00.00 00.00 51.67 76.65 30 00.00 00.00 75.02 99.52 40 00.00 00.00 81.34 100.0 50 4.65 1.71 82.19 100.0 70 12.11 8.31 82.28 100.0 100 28.26 24.58 83.09 100.0 500 35.74 31.91 84.56 100.0

The obtained results showed that E, L, T and C have an antioxidant capacity, but with different degrees as shown

in Table 2. The antioxidant capacity of all tested compounds increased with the concentrations. There was a significant

difference among the tested compounds in their antioxidant capacity. Ranking of increasing the antioxidant capacity is E >

L > T > C in all tested concentration. We have notice that the antioxidant capacity of E and L was very high compared to

the antioxidant capacity of T and C and this result was in good agreement with the previous obtained result Islam et al,

(2006).



The antioxidant capacity of L was very high but its reducing power is very low. So, there was a very high

difference between the reducing power and the antioxidant capacity of L. linalool is a monoterpen which has an allelic

structure, its structure can be reacted with H2SO4 which present in the reaction mix in the antioxidant capacity and a

sulfunyl compound will form. So that, sulfonyl compound reacts and give a high antioxidant capacity. The difference

between the reducing power and the antioxidant capacity of L related to the reaction conditions because of the reaction

mixture of antioxidant capacity contained H2SO4 0.6 µ and the reaction mix was heated at 95°C for 90 min. so, its false

antioxidant capacity. This mechanism can explain according to March (1968). On the other hand, the reducing power and

antioxidant capacity of E were the highest activity. So, that, BO has a high reducing power and antioxidant capacity

because of its containing of E. from the obtained results, T and C have low reducing power and antioxidant capacity.

In addition, to has a low reducing power and antioxidant capacity. As previously mentioned the reducing power and the

antioxidant capacity of E is the highest activity. Mechanism of antioxidant action is reducing power and antioxidant

capacity of E could be explained according to Ou et al. (2002)

The Percent of H2O2 Scavenging Activity of Thymol, Carvacrol, Linalool and Eugenol

The percent of H2O2 scavenging activity of T, C, L and E was determined at different concentrations 10, 20, 40,

60, 80, 100, 120 and 140 ppm. As shown in Table 3, all tested compounds exerted a concentration-dependent scavenging

of H2O2 except L. The concentration of H2O2 in the systems containing T, C and E samples dropped very sharply during

the initial 10 min period of the assay especially in E samples. The percent of H2O2 scavenging activity of T, C and E

increased in the following order E > T > C at all tested concentrations. There was a significant difference among all tested

compounds in the percent of H2O2 scavenging activity.

Table 3: The Percent of H2O2 Scavenging Activity of Thymol, Carvacrol, Linalool and Eugenol

Concentration mg/ml

% H 2O2 Scavenging Activity

Thymol Carvacrol Linalool Eugenol Ascorbic

Acid 0.01 35.63±0.02 4.27±0.02 0.00±0.00 18.45±0.02 28.21±0.01 0.02 39.51±0.01 18.94±0.04 0.00±0.00 53.72±0.03 52.31±0.01 0.04 53.82±0.03 25.18±0.05 0.00±0.00 78.51±0.12 77.47±0.08 0.06 60.04±0.11 33.56±0.03 0.00±0.00 96.38±0.23 98.58±0.12 0.08 67.93±0.21 43.28±0.07 0.00±0.00 99.35±0.21 98.59±0.11 0.1 76.53±0.23 56.46±0.13 0.00±0.00 100±0.11 98.60±0.10 0.12 96.38±0.08 82.41±0.16 0.00±0.00 100±0.04 98.60±0.10 0.14 96.42±0.07 89.23±0.10 0.00±0.00 100±0.01 98.60±0.09

The obtained results showed that the percent of H2O2 scavenging activity of E was the highest percent. As shown

in Table 3, the percent of H2O2 scavenging of E was 99.35 % and 100% at concentrations 0.08 and 0.1 mg/ml respectively,

while L has no H2O2 scavenging activity related to the phenolic structure where the phenolic compounds (E, T and C) have

H2O2 scavenging activity while the non-phenolic compound (L) has no H2O2 scavenging activity. Mechanism of H2O2

scavenging activity could be explained according to Wettasinghe and Shahidi (1999) as shown in Part I. Since the phenolic

compounds (E, T and C) are good hydrogen atom donors, they may accelerate the conversion of H2O2 to H2O.

Aysun and Ayse (2011) reported that the essential oil showed a higher membrane-protective effect than thymol

and carvacrol. The three major constituents of the essential oil were linalool (50.53%), carvacrol (24.52%), and thymol

(15.66%), respectively.



The Antioxidant Activity of Thymol, Carvacrol, Lina lool and Eugenol in LInoleic Acid System

The antioxidant activity of T, C, L and E were determined in linoleic acid system and compared with BHT

activity. Different concentrations used in determination 125, 250, 375 and 500 ppm for the tested compounds and 200 ppm

for BHT. The linoleic acid and tested compounds samples mixtures were incubated at 40ºC for 3 days. Every day, samples

were taken for determination of the antioxidant activity.

Table 4: The Antioxidant Activity of Thymol, Carvacrol, Linalool and Eugenol in Linolic Acid System Compared with BHT Activity

Conc. (mg/ml)

% Antioxidant Activity Thymol Carvacrol Linalool Eugenol BHT 0.2 mg/ml

0.125 65.31 48.05 72.84 91.05 88.17 0.250 67.49 50.91 75.27 93.52 90.23 0.375 69.82 53.57 79.43 95.16 92.74 0.500 71.33 58.40 81.61 97.48 95.11

All tested compounds exhibited an antioxidant activity, but to various degrees except L as shown in Table 4.

A positive correlation has been observed between concentrations (for T, C and E) and antioxidant activity. There is a

significant difference among the tested compounds in their antioxidant activity. The antioxidant activity of the tested

compounds decreased in the following order: T > E > C > BHT > L. The best antioxidant activity of all tested compound

(T, C and E) observed from 0.5 mg/ml concentrations.

Thymol is the best antioxidant activity in all concentration then carvacrol then Eugenol then BHT then linalool.

The antioxidant activity of all tested compound, increased with concentrations except linalool. So that, the best

concentration of thymol, carvacrol and eugenol was 0.5 mg/ml in the antioxidant activity. The antioxidant activity of L was

a negative value, the negative value. The negative value of the antioxidant activity increased with concentrations.

The negative antioxidant activity of L could be explained because of the peroxide value of L samples was higher than the

peroxide value of control where that the calculation equation was: 100 – [(absorbance of sample/absorbance of control) X

100]. So that we have to notice that L has a pro-oxidant effect.

The results were hugher that that reported by Aysun and Ayse (2011) the 6 major components of the essential oil

were linalool (50.53%), carvacrol (24.52%), thymol (15.66%), cymene (2.68%), terpinen-4-ol (2.06%), and gamma-

terpinene (1.41%), respectively.

Characterization of Nanoemulsion and Nanoencapsulation of Bioactive Compounds

Nanoemulsions are not only used to change the texture, flavor, and appearance of foods, but they are increasingly

used to delivery biologically functional components that are lipophilic such as antioxidants, flavors, and nutraceuticals.

Traditionally, emulsions could only be manufactured on a commercial scale with droplets in the nano-size range 10-100

nm) (Friberg et al, 2004; McClements, 2005a; Leal-Calderon et al, 2007).

Encapsulation of essential oils at the nanoscale represents a viable and efficient approach to increasing the

physical stability of the bioactive compounds, protecting them from the interactions with the food ingredients.

Encapsulation of functional ingredients has been shown to reduce ingredient interactions and allow for better control over

mass transport phenomena and chemical reactions. Encapsulation of biologically active components such as nutraceuticals

may also improve bioactivity, e.g. by improving adsorption and uptake of components during digestion. In the case of food



antimicrobials, encapsulation may increase the effectiveness of concentration of bioactive compounds in areas of the food

system where target microorganisms are preferentially located (Jochen et al, 2009).

Nano-Encapsulation Yield (EY)

Figure 1 showed that the values EY of the essential oils nanoencapsulated. High EY values for nanocapsules

obtained by treatment Dex/T20 of eugenl was 91.74 %

Figure 1: Nano-Encapsulation Yield of of Thymol, Carvacrol, Linalool and Eugenol in Dextran

This encapsulation yield was higher than that reported by Madhu and Prabhu, (1985) and Frank and

Hans-Joachim, (2007) being 50 %.

Encapsulation Efficiency (%EE)

The results in Figure 2 showed higher values for %EE for the eugenol encapsulated by Dex/T20 was 97.81 %

Figure 2: Nano-Encapsulation Efficiency of Thymol, Carvacrol, Linolool and Eugenol in Dextran

This nano-encapsulation efficiency of eugenol and linalool and thymol were 97.81, 83.79 and 71.51 %,

respectively, but low level in carvacrol. This result was higher than that reported by Dipan and paramita (2013) of the

bioactive compounds such as eugenol, phenolic compounds and antioxidants are in the range of 60–65%.

The nano-encapsulated eugenol was choosen for subsequent studies due to its higher yield and efficiency.

Solubility of the Eugenol Nano-Encapsulated

Essential oils are slightly soluble in water and impart their odor and taste to the water. The results in Figure 3

showed that the solubility of eugenol nanoencapsulated was Increases with temperature and was higher at 45 °C as a result

of a concentration of dextran 0.01 Molar. This result was agreement with that reported by Regiane et al, (2013).

The physical properties of the nanoencapsulated eugenol related to its ease of dispersion in an aqueous solution

include moisture content and respective particle porosity, and the instantanization properties (wetting, dispersibility,

and solubility). These properties are influenced by the nature of the feed (solids content, viscosity, and temperature) as

shown in Figure 4.



Figure 3: Phase Solubility Study of Nano-Encapsulated Eugenol with Dextran in Water at 25, 35, and 45 ºC

Numerous materials, such as dextran (encapsulating agents), are available for encapsulation of essential oils and

application in foods. Dextran is used by the filer industry in applications protecting the core material from oxidation and

volatilization. Moreover, it exhibits high solubility and low viscosity in aqueous solution when compared to other

hydrocolloid dextran which facilitates the spray drying process. The objective of this study was to evaluate the influence of

operational conditions on the properties of nanoencapsulated eugenol essential oil.

Figure 4: Time Related Release of Dextran Nano-Encapsulated Eugenol in Water

Thermal Stability of Nano Eugenol Capsules DSC (Differential Scanning Coliremetry)

The thermal stability of the eugenol nanocapsulated was determined by using Differential Scanning Coliremetry

Figure 5a,b. Higher exothermal peaks still occurred at much higher temperature 143 °C in Figure 5a after nanoencapsulation

of eugenol compared to the initial eugenol before encapsulated was 63.5 °C. This result indicated that capsulation process

caused oxidation prevention and increased the thermal stability of eugenol.

Figure 5: DSC Thermogram of Eugenol before (a) and After Nanoencapsulated

(b) Transmission Electron Microscopy (TEM) of Eugenol Nanoencapsulated

a b

a b



Figure 6 showed TEM micrographs of the investigated models. The particles of the observed sample differ in size

and shape. The morphology of eugenol before encapsulation was obtained by using transmission electron microscopy with

diameter range from 4.2 to 9.8 nm in Figure 6a but was 3.9 to 4.7 nm in Figure 6b

Figure 6: Morphology of Eugenol after Nanoemulsion (a) and Nanoencapsulation (b) by Transmission Electron Microscopy

Storage Stability of Nano-Encapsulated Eugenol

The results in Table 5 showed that the antioxidant activity of eugenol nano-encapsulated was stable in long-term

180 days storage at 4 °C. However, the loading capacity of eugenol un-capsules is relatively low. To address this issue,

the loss of turmeric oil in key steps in the process was quantified and these data may facilitate improvements in the

procedure to produce an increased loading capacity.

Table 5: Antioxidant Activity of Nano-Encapsulated Eugenol (Dex/T20) in Linolic Acid System Compared with BHT Activity at Different Periods Storage

Conc. (mg/ml)

Antioxidant Activity of Nano-Encapsulated Eugenol (%) After

Storage

0 Day 60

Days 120

Days 180

Days

0.125 cap. Un-cap.

93.82 91.05

93.63 86.25

93.25 82.74

92.94 79.16

0.250 cap. Un-cap.

95.24 93.52

95.01 88.31

94.83 85.23

94.62 82.77

0.375 cap. Un-cap.

97.30 95.16

97.13 92.06

96.81 89.12

96.53 86.81

0.500 cap. Un-cap.

98.97 97.48

98.42 95.25

98.12 92.61

97.89 90.16

BHT 88.17 88.17 88.17 88.17

In order to retain their biological activity and minimize the impact on the organoleptic properties of foods where

they are incorporated, essential oil constituents, like other bioactive compounds, need to be protected from the interaction

with other food ingredients and from environmental stresses experienced during food manufacturing and storage

(McClements, 1999).

Sensory Evaluation of Jelly

Sensory properties of jelly prepared with adding different levels of eugenol nanoencapsulated compared with

control (eugenol 1 % without nano-encapsulation) are given in Table 6. Analysis of variance showed mostly significant

differences in color, taste, odor and overall acceptability for jelly as control by different levels of eugenol encapsulated in

the range 0.1 to 0.6 %. The addition of eugenol encapsulated with different levels significantly affected color, taste, odor

and overall acceptability.



In general, consumer perception has been that treatment as favorable in terms of the general acceptance of the

sample is added by a ratio of 0.3 % eugenol nanoencapsulated was 9.6 %. Addition of the particles of encapsulated

bioactive compounds at amounts up to 5.0% in bread formulation seemed reasonable in terms of general quality parameters

and sensory properties (Burce et al, 2013).

Table 6: Sensory Evaluation of Jelly Prepared with Different Levels of Nano-Encapsulated Eugenol Compared to 1 % Eugenol Untreated

Treatments Parameter

Color Taste Odor Overall

Acceptability Control * 9.80±0.83 9.80±0.79 9.70±0.79 9.70±0.82

0.1 % 7.40±0.71 7.10±0.57 7.20±0.63 7.20±0.65 0.2 % 8.40±0.32 8.20±0.62 8.30±0.71 8.30±0.74 0.3 % 9.80±0.79 9.70±0.81 9.60±0.92 9.60±0.81 0.4 % 8.50±0.63 8.30±0.75 8.30±0.74 8.30±0.77 0.5 % 7.10±0.56 7.00±0.49 7.00±0.61 7.00±0.52 0.6 % 7.00±0.53 6.50±0.47 7.00±0.60 6.50±0.46

* control (eugenol 1 % without nano-encapsulation), LSD: nd (no detected)

Sensory evaluation was applied for the development and improvement of products, quality control and studies on

process. Table 6 shows the average scores for the sensory evaluations. The Dex/T20 (0.3 %) formulation was the most

accepted by testers, receiving the largest number of indications for the responses ‘liked quite a lot’ and ‘liked very much ‘.

The Dex/T20 (0.3 %) formulation was the least accepted, receiving the largest number of responses ‘disliked very

much’, for a product to be considered acceptable in terms of its sensory properties; it needs to have a minimum 96% level

of approval.

Microbiological Analysis

The microbiological characteristics observed for the jelly without addition of nano-encapsulated antioxidant

compared to the jelly with addition of nano-encapsulated antioxidant after three months of refrigerated storage are shown

in Table 7.

Table 7: The Microbiological Characteristics of the Jelly with or without the Addition of Nano-Encapsulated Antioxidant

Control: is jelly without antioxidant; cfu g-1: colony forming units/g

The jelly maintained its microbiological quality during storage and was well within the requirements set forth by

ANVISA. The nano-encapsulated eugenol showed a remarkable inhibitory activity against the growth of jelly

microorganisms, especially Molds & yeasts and no detected salmonella in any samples. More interestingly, the extract

could inhibit strongly the growth of Coliforms, which is regarded as a major etiological concern in gastro-duodenal



disorders. Therefore, the nano-encapsulated of bioactive compounds can be used as a natural source of antimicrobial and

antioxidant agents to preserve foodstuffs against a range of food related microorganisms (Rima et al, 2013).

CONCLUSIONS

Nano-encapsulation offers great advantage for an improved stability of bioactive compounds in certain food

formulations. Jelly as a staple food is considered to be a useful model to develop new generation of functional products,

and to test the stability of nano-encapsulated bioactive compounds. In this study, bioactive compounds were encapsulated

with dextran and added to jelly formulation to test their stability during the process. The results revealed that

nano-encapsulation significantly improved the thermal stability of eugenol at elevated temperatures applied during process.

Addition of the particles of encapsulated bioactive compounds at amounts up to 0.02 % in jelly formulation seemed

reasonable in terms of general quality parameters and sensory properties. Presence of bioactive compounds in a

formulation may bring important changes in the chemical reactions occurring in food during processing. The most

bioactive compounds bear functional groups that may react with other compounds in food. These reactions may lead to the

formation of processing contaminants especially in products in which processing conditions are severe. Therefore,

prevention of the reactivity of bioactive compounds through their nano-encapsulation may also help preventing certain

food safety risks

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