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LWT - Food Science and Technology 60 (2015) 207e212

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LWT - Food Science and Technology

journal homepage: www.elsevier .com/locate/ lwt

Influence of emulsion droplet size on antimicrobial activityof interesterified Amazonian oils

Paula Speranza a, *, Ana Paula Badan Ribeiro b, Rosiane Lopes Cunha c,Juliana Alves Macedo d, Gabriela Alves Macedo a

a Department of Food Science, Faculty of Food Engineering, University of Campinas, Rua Monteiro Lobato 80, Caixa Postal 6121, CEP 13083-970,Campinas, SP, Brazilb Department of Food Technology, Faculty of Food Engineering, University of Campinas, Brazilc Department of Food Engineering, Faculty of Food Engineering, University of Campinas, Brazild Department of Food and Nutrition, Faculty of Food Engineering, University of Campinas, Brazil

a r t i c l e i n f o

Article history:Received 11 April 2014Received in revised form10 July 2014Accepted 14 July 2014Available online 23 July 2014

Keywords:AntimicrobialAmazonian oilEmulsionDroplet sizeInteresterification

* Corresponding author. Tel.: þ55 19 3521 2175; faE-mail addresses: paulasperanza09@gmail.com (

unicamp.br (G.A. Macedo).

http://dx.doi.org/10.1016/j.lwt.2014.07.0220023-6438/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

This study evaluated the antimicrobial potential of emulsions formulated with interesterified Amazonianoils. The results indicated that the antimicrobial activity of these emulsions are directly related to thecharacteristics of the emulsion, and these characteristics are influenced by the interesterification processof these oils. When the antimicrobial emulsion was produced with interesterified Amazonian oils (2 mL/100 mL), the oil droplet presented smaller size, and resulted in the complete inhibition of pathogensgrowth, Bacillus cereus and Escherichia coli, after 24 h (bactericidal effect). When the antimicrobialemulsion was produced with non-interesterified oils (only physical mixing), the oil droplets were largerand the emulsions did not completely inhibit the growth of the pathogens after 24 h (bacteriostaticeffect). The results suggest that the interesterification of these oils may be responsible for changes in thephysicochemical characteristics of the emulsions, producing droplets with smaller size and greaterantimicrobial activity.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The enzymatic interesterification is one of the techniquesavailable to improve the properties of oils and fats. The changes inthe original tryaclyglycerol composition can modify the physical,nutritional and biological properties of the lipid produced,increasing its potential application (Iwasaki & Yamane, 2000;Nunes, Paula, Castro, & Santos, 2011). Furthermore, in these re-actions it is possible to produce lipids that do not exhibit phaseseparation due to incompatibility between the fat and oil fractions;an essential feature for the production of emulsions (Meten &Hartel, 2005).

Thus, this technique may be an alternative for the production oflipids with a higher commercial value. In this sense, Amazonian oilsstand out. These oils are little explored, and use them in these re-actions, can enlarge the interest in these materials due to the newcharacteristics of the lipids produced.

x: þ55 193521 2153.P. Speranza), gmacedo@fea.

Among the oils and fats of the Amazon, some due to thecomposition of fatty acids and minor compounds, are highlighted:buriti (Mauritia flexuosa L.f. e Arecaceae) and patau�a (Oenocarpusbataua Mart. e Arecaceae) oils have higher concentration ofmonounsaturated fatty acids, and some minor compounds such astocols, carotenoids and phenolics (Montúfar et al., 2010; Zanatta,Ugartondo, Mitjans, Rocha-Filho, & Vinardell, 2010); murumuru(Astrocaryum murumuru Mart. e Arecaceae) fat is rich in lauric andmyristic fatty acids (Mambrim & Barrera-Arellano, 1997) and palmstearin (Elaeis guineensis Jacq. e Arecaceae) is rich in palmitic andoleic acids, and contains significant amounts of stearic and linoleicacids (Adhikari et al., 2010). Several of the compounds present inthese oils and fats have antimicrobial activity (Batista et al., 2012;Desbois, 2012; García-Ruiz et al., 2013). Therefore produce blendswith these oils and fats and use them in interesterification reactionscan produce lipids that individual characteristics of these materialscan be modificated, and the antimicrobial potential can beincreased.

Among pathogenic bacteria capable of causing human illnessand food spoilage, some stand out: bacteria of the Bacillus genus aregram-positive and form stable spores that are resistant to harsh

P. Speranza et al. / LWT - Food Science and Technology 60 (2015) 207e212208

conditions and extreme temperatures. Bacillus cereus is a commonpathogen involved in foodborne diseases, local sepsis and woundand systemic infections (Hamouda et al., 1999). Bacteria of theEscherichia genus are gram-negative and resistant to many antibi-otics. Escherichia coli is a common pathogen responsible for variousextra intestinal or intestinal infections (Croxen et al., 2013; Ratheret al., 2013).

Thus, this study aimed to evaluate the antimicrobial effectagainst B. cereus and E. coli of emulsions formulated with inter-esterified Amazonian oils produced by different enzymes. In addi-tion, this study evaluated whether the physical characteristics ofthe emulsion influenced in their antimicrobial potential. Theinteresterified lipids tested were composed of the buriti oil andmurumuru fat (first blend) and patau�a oil and palm stearin fat(second blend). The lipids were produced using two lipases in threedifferent enzyme systems: first, a commercial lipase; second, acrude lipase from the fungus Rhizopus sp.; and third, a mixture ofboth lipases (commercial and Rhizopus sp.).

2. Materials

2.1. Chemicals

Buriti, patau�a oils and murumuru fat were bought in a localmarket in the city of Bel�em, State of Par�a, in the Brazilian Amazon.Palm stearin was kindly supplied by Agropalma (Par�a, Brazil).Commercial purified and immobilized lipase (Lipozyzme TL-IM)was kindly supplied by Novozymes. Crude lipase from Rhizopussp. was produced in a solid medium in our laboratory (Macedo,Pastore, & Rodrigues, 2004). All other reagents and solvents wereof analytical grade.

2.2. Microorganism

The bacterial strains Bacillus cereus (B. cereus) and Escherichiacoli (E. coli) were provided by Pluridisciplinary Center for Chemical,Biological and Agricultural Research (Cpqba e Unicamp, Brazil).

3. Methods

3.1. Lipases activities

Lipase activities in both enzymes were quantified using olive oilas substrate. One unit of lipase activity (U) is defined as 1 mmol ofoleic acid released per minute (Macedo et al., 2004).

3.2. Enzymatic interesterification

The enzymatic interesterification between buriti oil and mur-umuru was performed in an orbital-shaking water bath at 150 rpmfor 24 h at 40 �C under vacuum. The enzymatic interesterificationbetween patau�a oil and palm stearin was performed in an orbital-shaking water bath at 150 rpm for 24 h at 50 �C under vacuum.The weight ratio of oil to fat was 70:30, with a total weight of 10 g.The reactions were performed in three different enzyme systems:commercial lipase, lipase from Rhizopus sp. and a mixture of bothenzymes. In all systems, the final enzyme concentration was 2.5 g/100 g. Before the reaction, the enzymes were dried in a vacuumoven at 40 �C for 30 min. After completion of the reaction, theinteresterified blend was immediately filtered using a 0.45 mmmembrane filter and frozen (Rodrigues & Ayub, 2011; Sim~oes,Valero, Tecel~ao, & Ferreira-Dias, 2013). The non-interesterifiedblend (physical blend) was also subjected to the same reactionconditions.

3.3. Preparation of emulsions and inoculation

The emulsions were prepared in tubes with screw using33.5 mL/100 mL of culture medium (Nutrient broth e Merck Mil-lipore), different concentrations of water (31, 29, 25, 21, 17, 13 and9 mL/100 mL), and interesterified and non-interesterified oils (0, 2,6, 10, 14, 18 and 22 mL/100 mL), followed by 2 mL/100 mL of TritonX-100 (Buthelezi, Southway, Govinden, Bodenstein, & Toit, 2012).The emulsions were shaken (vortex) for 70 s. Finally it was added33.5 mL/100 mL of culture medium containing the microorganismwith the optical density of 0.4e0.6 at 490 nm. The final volume ofthe emulsions was kept constant at 1500 mL. After inoculation of themicroorganism, the emulsion was kept under stirring at 150 rpmfor 24 h at 37 �C. This stage was performed under asepticconditions.

3.4. Antimicrobial activity assay

After the incubation period, aliquots of 150 mL of the emulsionswere transferred to 96-well sterile microplates. Adding 20 mL of thedye of p-iodonitrotetrazoliumviolet, and after 15min, themicrobialgrowth was read by colorimetry at 490 nm in a microplate reader(BMG Labtech e Fluostar Optima) (Buthelezi et al., 2012). Thegrowth of microorganisms was compared to plates preparedwithout the presence of oil (control).

3.5. Droplet size distribution

Emulsions droplet size distribution (PsD) were measured usinga laser light scattering analyzer after 1 h of preparation (MalvernMastersizer 2000; Malvern Instruments Ltd., Worcestershire, UK)(Sato, Moraes,& Cunha, 2014). Aliquots of emulsions were added todistilled water under stirring (1750 rpm) until an obscuration rateof 10% was gained. The average droplet size was determined usingthe Sauter Mean Diameter (D32), which is defined as the diameterof the sphere having the same volume/surface area of the particle ofinterest (Equation (1)). The Specific Surface Area (SSA) was calcu-lated and this parameter is defined as the total area of the dropletsdivided by their total weight. It was calculated based on theassumption that the particles are both spherical and nonporous(Equation (2)).

D32 ¼P

ni$d3i

Pni$d

2i

(1)

Where ni is the number of particles with diameter di

SSA ¼6P V i

di

rP

V i¼ 6

rD½3;2� (2)

Where Vi is the relative volume of particles diameter of di and r isthe particle density.

4. Results and discussion

4.1. Lipases

In this study, two different lipases were used in interester-ification reactions. The commercial lipase, purified and immobi-lized presented lipolytic activity of 12.7 U/g. The semi-purified andnon-immobilized lipase from Rhizopus sp. presented lipolytic ac-tivity of 8.0 U/g. These enzymes were able to catalyze the reactionof interesterification of oils and fats from the Amazon, and effectson lipids formed can be observed below.

Table 1Mean diameter and specific surface area of emulsions droplets produced with non-interesterified and interesterified buriti oil and murumuru fat blends.

Buriti:Murumuru Enzyme system Oil in theemulsion(mL/100 mL)

D3,2 (mm) Specificsurfacearea (m2/g)

Non-interesterified blend 2 2.8 ± 0.01 2.1 ± 0.0114 5.0 ± 0.01 1.2 ± 0.02

Interesterified blend(commercial lipase)

2 2.3 ± 0.01 2.6 ± 0.014 3.0 ± 0.01 2.0 ± 0.0

Interesterified blend(lipase from Rhizopus sp.)

2 2.0 ± 0.02 3.1 ± 0.016 5.0 ± 0.01 1.2 ± 0.01

Interesterified blend(commercial þ Rhizopussp. lipases)

2 1.8 ± 0.01 3.3 ± 0 .0122 9.8 ± 0.02 0.6 ± 0.0

P. Speranza et al. / LWT - Food Science and Technology 60 (2015) 207e212 209

4.2. Antimicrobial activity of Amazonian oils in emulsions

The pathogens used to evaluate the antimicrobial activity ofthese emulsions were B. cereus and E. coli, bacteria that cause foodpoisoning, chronic skin infections, contamination of pharmaceu-tical products, food and water (Bottone, 2010; Croxen et al., 2013;Rather et al., 2013).

In this study, it was observed that the Amazonian interester-ifieds oils produced using different lipases were able to inhibit thegrowth of microorganisms B. cereus and E. coli when emulsifiedwith Triton X-100. Bacteria were tested with different concentra-tions of non-interesterified and interesterified oils in the emulsion(between 2 and 22 mL/100 mL), in order to observe the effect onbacterial growth. All oils were evaluated at a concentration of 2 mL/100 mL in the emulsion. The other concentrations (between 6 and22 mL/100 mL), varied according to the oil.

A different effect was observed for each type of oil used,although in all of them the lowest concentration of oil in theemulsion (2 mL/100 mL) had greater antimicrobial effect, regard-less of whether the oil was interesterified or not (Fig. 1). However, itwas observed that the emulsions formulated with oils inter-esterified at this concentration (2 mL/100 mL) presented bacteri-cidal effects after 24 h, while the emulsions formulated with non-interesterified oils shared bacteriostatic effects. Furthermore,emulsions prepared with interesterified oils had higher antimi-crobial activity at most of the concentrations tested.

Thus, it can be concluded that the composition of the oils wasnot primarily responsible for antimicrobial activity, since the bestresults were observed at lower concentrations of oil, that means itdoes not present a doseeresponse effect. However, the emulsionsprepared with interesterified oils showed higher antimicrobial ac-tivity, suggesting that the interesterification of these oils may beresponsible for changes in the characteristics of the emulsion, andthis factor influenced the results of antimicrobial activity.

4.3. Characteristics of the emulsions

In order to the verify whether the interesterification of oilschanged the characteristics of the emulsions the diameter (D32) and

Fig. 1. Effect of non-interesterified and interesterified blends at different concentrations (

specific surface area of the droplets in the emulsionwere evaluated(Tables 1 and 2).

The results showed that there are differences in these parame-ters when using the same concentration of the tested oils in theemulsions (2 mL/100 mL). The emulsions prepared with the inter-esterified blends showed a smaller droplet diameter and higherspecific surface area. These characteristics can be observed in bothblends, with all three enzymatic systems used forinteresterification.

Thus, the results indicate that indeed chemical modificationcaused by enzymatic interesterification was responsible for thechange in the characteristics of the emulsions.

4.4. Emulsion, Droplet size and Antimicrobial activity

In order to verify whether the characteristics of the emulsionwere related to antimicrobial activity, a table with of these pa-rameters is shown below (Tables 3 and 4). The results confirm thatthe antimicrobial activity of the emulsions is more closely related tothe droplet size than to the chemical characteristics of the oils. Inemulsions in which the specific surface area was larger, the anti-microbial feature was more effective to both microorganismstested.

2, 6, 10, 14, 18 or 22 mL/100 mL) on the antimicrobial activity of B. cereus and E. coli.

Table 2Mean diameter and specific surface area of emulsions droplets produced with non-interesterified and interesterified patau�a oil and palm stearin fat blends.

Patau�a:Palm stearinenzyme system

Oil in theemulsion(mL/100 mL)

D3,2 (mm) Specificsurfacearea (m2/g)

Non-interesterified blend 2 3.5 ± 0.0 1.7 ± 0.0218 4.6 ± 0.01 1.3 ± 0.01

Interesterified blend(commercial lipase)

2 2.1 ± 0.0 2.8 ± 0.026 2.2 ± 0.01 2.7 ± 0.01

Interesterified blend(lipase from Rhizopus sp.)

2 1.6 ± 0.01 3.7 ± 0.010 3.2 ± 0.02 1.8 ± 0.01

Interesterified blend(commercial þ Rhizopussp. lipases)

2 2.2 ± 0.0 2.7 ± 0.0222 3.6 ± 0.0 1.7 ± 0.01

Table 3Concentration of the non-interesterified and interesterified buriti oil andmurumurufat blends in the emulsion, specific surface area of the droplets in the emulsions,microbial inhibition of B. cereus and E. coli.

Buriti: MurumuruEnzyme system

Oil in theemulsion(mL/100 mL)

Specificsurfacearea (m2/g)

Inhibitionof B. cereuscompared tocontrol (%)

Inhibition ofE. colicompared tocontrol (%)

Non-interesterifiedblend

2 2.1 ± 0.01 85.1 ± 0.11 80.0 ± 0.1314 1.2 ± 0.02 43.0 ± 0.09 20.0 ± 0.06

Interesterifiedblend (commerciallipase)

2 2.6 ± 0.0 100.0 ± 0.0 100.0 ± 0.014 2.0 ± 0.0 77.5 ± 0.05 28.0 ± 0.04

Table 5Specific surface area of the droplets in the emulsions produced with 2 mL/100 mL ofthe non-interesterified and interesterified blends and microbial inhibition of B. ce-reus and E. coli.

Oil blends enzyme system Specificsurfacearea (m2/g)

Inhibition ofB. cereuscomparedto control (%)

Inhibition ofE. colicomparedto control (%)

Buriti: MurumuruNon-interesterified blend 2.1 ± 0.01 85.1 ± 0.09 80.0 ± 0.07

P. Speranza et al. / LWT - Food Science and Technology 60 (2015) 207e212210

Interesterified oils showed greater antimicrobial activity prob-ably because it allows for the formation of an emulsion withreduced droplets size. The interesterification reaction may producelipids that do not have or have reduced eutectic behavior that is, nophase separation occurs due to the incompatibility between thefractions of fats and oils. After interesterification, the lipid producedpresents a single phase completely homogeneous, whereas, in thenon-interesterified blend, separation of the triglycerides may bedue to differences in the melting points (Grimaldi, Gonçalves,Gioielli, & Sim~oes, 2001; Meten & Hartel, 2005). This phase sepa-ration that occurs in the non-interesterified blend can influence thedroplet size of the emulsion.

This paper only shows results of the interesterified oils bycommercial enzyme, nonetheless the same behavior has beenobserved for the other interesterified blends (data not shown).

Enzymatic interesterification induces exchange of fatty acids inthe structure of glycerol, and this change is related to the specificityof the enzyme (Kapoor & Gupta, 2012). The interesterified blendswith different enzymes show different behavior in emulsion,thereby modifying the drop size, hydrophobicity and their effec-tiveness as an antimicrobial agent. Interesterified blends used inthis study showed a significant change in the relationship betweensaturated and unsaturated fatty acids. There was a significant in-crease of unsaturated fatty acids, especially in the sn-1and sn-3

Table 4Concentration of the non-interesterified and interesterified patau�a oil and palmstearin fat blends in the emulsion, specific surface area of the droplets in theemulsions, microbial inhibition of B. cereus and E. coli.

Patau�a: Palm stearinEnzyme system

Oil in theemulsion(mL/100 mL)

Specificsurfacearea (m2/g)

Inhibition ofB. cereuscompared tocontrol (%)

Inhibition ofE. colicompared tocontrol (%)

Non-interesterifiedblend

2 1.7 ± 0.02 79.0 ± 0.09 68.0 ± 0.1018 1.3 ± 0.01 0.0 ± 0.0 0.0 ± 0.0

Interesterified blend(commercial lipase)

2 2.8 ± 0.02 100.0 ± 0.0 100.0 ± 0.06 2.7 ± 0.01 100.0 ± 0.0 0.0 ± 0.0

positions of the triacylglycerol, and the predominance of tri-acylglycerols mono and di-unsaturated (unpublished results). Theproduction of triacylglycerols with intermediate melting pointsexhibit characteristics of lubricity and structure, important char-acteristics for emulsion stability (O'Brien, 2004). This profile oftriacylglycerols obtained may have influenced the physicochemicalproperties of the emulsions formed.

The characteristic of the interesterified Amazonian oils used inthis study is of great interest since recent studies have indicatedthat the interesterification may influence the biological activity(Berry, 2009; Farf�an, Villal�on, Ortíz, Nieto, & Bouchon, 2013;Michalski et al., 2013; Speranza & Macedo, 2012). The intra-molecular triacylglycerol structure can influence the digestion andabsorption of lipids, affecting some metabolic responses (Michalskiet al., 2013). Thus, the use of these interesterified oils for the pro-duction of emulsions for biological applications appears to be moreadvantageous than the use of simple blending of these oils and fats.By presenting droplets with higher specific surface area, the per-formance of this emulsified oil becomes more effective in biologicalsystems.

Wang et al. (2008) evaluated the effects of curcumin (poly-phenol) encapsulated in oil in water emulsion regarding anti-inflammatory activity in rats. The authors observed increased in-hibition of edema in the ears of mice with decreasing diameter ofthe emulsion droplets. This effect is due to the increase in thesurface area-to-volume ratio as the droplet size decreases. As theinterfacial area increases, the number of dispersed droplets be-comes higher, and the number of interactions is also higher (Nunezet al., 2000).

In order to facilitate visualization, Table 5 shows only the resultsof the emulsions that produced higher microbial inhibition(formulated with a 2 mL/100 mL oil concentration). It is clearlyobserved that the emulsions produced with interesterified blendsshow larger specific surface area and higher inhibition ofpathogens.

Another study showed that there is a close relation between thephysicochemical properties of emulsions and the potential effectsas antimicrobial agents (Al-Adham, Al-Nawajeh, Khalil, & Collier,2012). The results suggested that the high levels of antimicrobialactivity are due to the unique characteristic of the structure of oil-in-water emulsion system, rather than the chemical activity of theirindividual components. Our results confirmed that the

Interesterified blend(comm. lipase)

2.6 ± 0.0 100.0 ± 0.0 100.0 ± 0.0

Interesterified blend(lipase from Rhizopus sp.)

3.1 ± 0.01 98.6 ± 0.01 100.0 ± 0.0

Interesterified blend(comm. þ Rhizopus sp. lipases)

3.3 ± 0.01 100.0 ± 0.0 100.0 ± 0.0

Patau�a: Palm stearinNon-interesterified blend 1.7 ± 0.02 79.0 ± 0.07 68.0 ± 0.05Interesterified blend

(comm. lipase)2.8 ± 0.02 100.0 ± 0.0 100.0 ± 0.0

Interesterified blend(lipase from Rhizopus sp.)

3.7 ± 0.0 100.0 ± 0.0 100.0 ± 0.0

Interesterified blend(comm. þ Rhizopus sp. lipases)

2.7 ± 0.02 98.7 ± 0.01 100.0 ± 0.0

P. Speranza et al. / LWT - Food Science and Technology 60 (2015) 207e212 211

physicochemical characteristics of the emulsion are related to theantimicrobial activity. In the emulsions in which the droplet sizewas smaller the antimicrobial effect was more relevant. This cor-relation was observed for all oils, interesterified or not (Table 5).Droplet with smaller sizes has significantly larger specific surfacearea, ensuring that the components of the emulsion have greatercontact with the bacterial membrane. The interaction between thedroplets and the bacterial membrane decreases the hydrophobicityof the bacterial cell, causing rapid loss of cell viability (Zhang et al.,2009).

Two studies have evaluated the antimicrobial activity of emul-sions and related it to the droplet size (Buranasuksombat, Kwon,Turner, & Bhandari, 2011; Terjung, Loffler, Gibis, Hinrichs, &Weiss, 2012). In these studies, the authors find no correlation be-tween droplet size and antimicrobial activity. However, many otherstudies correlate biological activity with droplet size (Acosta, 2009;McClements & Xiao, 2012; Salvia-Trujillo, Qian, Martín-Belloso, &McClements, 2013; Wang et al., 2008). These latter studies indicatethat the reduction in droplet size produces larger absorption ofactive ingredients and increased intake of these droplets in cellsand tissues.

Therefore, the results obtained in this study are in accordancewith those that relate the increase in biological activity with areduction in droplets size. The interesterification of these oilsproduces emulsions with smaller droplet size, and this featureenhances the biological potential of the emulsion. New applicationsfor interesterified oils can be investigated from this finding.

5. Conclusion

This study verified that the antimicrobial activity of emulsi-fied Amazonian oils is influenced by the oil droplet sizes in theemulsions, and these sizes are determined by the proprieties ofthe oils used. The emulsions produced with the non-interesterified blends of Amazonian oils showed a higher oildroplet size and presented a bacteriostatic effect against B. cereusand Escherichia coli pathogens, while the emulsions producedwith interesterified blends showed smaller oil droplets size andpresented bactericidal effect. The use of these interesterified oilsfor biological applications seems to be more advantageous thanthe use of simple blending of these oils.

Acknowledgments

The authors wish to thank Dr. Luiza H. Meller da Silva and Dr.Antonio M. da Cruz Rodrigues of the University of Par�a forproviding the oils. The authors are also grateful to Dr. MartaCristina T. Duarte of the CPQBA- Unicamp for providing themicroorganisms.

Financial support was provided by Coordenaç~ao de Aperfei-çoamento de Pessoal de Nível Superior (Capes) and Fundaç~ao deAmparo �a Pesquisa do Estado de S~ao Paulo (Fapesp) 2012/22774-5.

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