Prebiotic Carbohydrates: Effect on Reconstitution, Storage, Release

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Prebiotic carbohydrates: Effect on reconstitution, storage, release, and antioxidant properties of lime essential oil microparticles Pedro Henrique Campelo-Felix, Hugo Junior Barbosa Souza, Jayne de Abreu Figueiredo, Regiane Victoria de Barros Fernandes, Diego Alvarenga Botrel, Cassiano Rodrigues de Oliveira, Maria Irene Yoshida, and Soraia Vilela Borges J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04643 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on January 4, 2017

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Journal of Agricultural and Food Chemistry

Prebiotic carbohydrates: Effect on reconstitution, storage, release, and antioxidant properties of lime essential oil microparticles

Pedro Henrique Campelo-Felixa, Hugo Júnior Barbosa Souzaa, Jayne de Abreu Figueiredoa, Regiane Victória de Barros Fernandesa*, Diego Alvarenga Botrela, Cassiano Rodrigues de Olveirab, Maria Irene Yoshidac, Soraia Vilela Borgesa

a

Department of Food Science, Federal University of Lavras, Lavras, Minas Gerais,

Brazil b

Institute of Exact Science, Campus de Rio Paranaíba, Federal University of Viçosa,

Minas Gerais, Brazil. c

Department of Chemistry, Federal University of Minas Gerais, Minas Gerais, Brazil

*Corresponding author: [email protected]

1

ACS Paragon Plus Environment


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Abstract

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The aim of this study was to include prebiotic biopolymers as wall material in

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microparticles of lime essential oil. Whey protein isolate (WPI), inulin (IN), and

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oligofructose (OL) biopolymers were used in the following combinations: WPI, WPI/IN

5

(4:1), and WPI/OL (4:1). The emulsion droplets in presence of inulin and oligofructose

6

showed larger sizes on reconstitution. There was no significant difference in solubility

7

of the particles, but the wettability was improved on addition of the polysaccharides.

8

The size of the oligofructose chains favored the adsorption of water. Prebiotic

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biopolymers reduced thermal and chemical stability of the encapsulated oil.

10

Microparticles produced with WPI showed a higher bioactive compound release rate,

11

mainly due to its structural properties that enabled rapid diffusion of oil through the

12

pores. The use of prebiotic biopolymers can be a good option to add value to

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encapsulated products, thus promoting health benefits.

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Keywords: lime essential oil; spray drying; inulin; oligofructose; controlled release;

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antioxidant activity.

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Introduction

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In recent years, growing demand for natural food ingredients has provided major

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incentive for research in the field of bioactive compounds and functional products 1.

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Natural sources of antioxidants and antimicrobial compounds are the new focus of the

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food industry. Essential oils are plant extracts that possess significant chemical and

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sensory characteristics with several benefits. The lime essential oil (Citrus aurantifolia)

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is widely used in the food industry for its antioxidant and antimicrobial properties and

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flavor, mainly applied in beverages and baked goods 2. Its chemical composition is very

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complex and can be divided into three classes of compounds: terpenes (75%), complex

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oxygenated (12%), and sesquiterpenes (3%). It consists of limonene, γ-terpene citral,

27

and β-caryophyllene, among other substances

28

food industry and the study of new encapsulating materials is necessary to promote

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widespread use as flavoring or preservatives.

2,3

. Essential oils are widely used in the

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Maintaining the physical, chemical, and biological characteristics is a major goal

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of the food encapsulation processes. The stability of essential oils can be enhanced by

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microencapsulation, entrapment, or coating of these substances within another material4.

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Spray drying is the most used technique in microencapsulation of food because of its

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well-known practical operation.

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Selection of the encapsulant material is one of the most important stages of

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microencapsulation, as its physical characteristics should maintain the chemical

37

properties of the encapsulated compounds 4. Whey protein isolates are biopolymers

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commonly used in microencapsulation processes. Due to their surface properties, these

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proteins are able to further protect the encapsulated bioactive compound 5,6.

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The growing consumer interest in food products that promote the improvement

41

of health has motivated the research and development of so-called "functional foods."

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This growing concern is leading the food industry to develop new product lines with

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particular characteristics. Among the functional foods, the use of prebiotics have been

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highlighted by the benefits 7. Inulin is a prebiotic with degree of polymerization (DP)

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ranges from 10 to 60. It is manly extracted from chicory roots and consists of chains of

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fructose units. Oligofructose is obtained by partial hydrolysis of inulin and therefore has

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a lower DP, ranging from 2 to 8 8. Inulin and oligofructose are widely used in functional

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foods throughout the world, based on their properties related to health improvement and

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technological possibilities. Prebiotics, such as inulin, are at the forefront of the

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emerging trend towards functional foods 9. Some studies have demonstrated the

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feasibility of using prebiotic biopolymers in the production of powdered foods 10,11.

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Based on the previous studies, this work aimed to study the influence of

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different prebiotic biopolymers on the physicochemical characteristics of lime essential

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oil microparticles: reconstitution properties, storage, controlled release, and stability of

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antioxidant properties.

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Materials and methods

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Materials

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Lime (Citrus aurantifolia) essential oil (Ferquima Ind. e Com. Ltda, Vargem

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Grande Paulista, Brazil) was used as the main material. The biopolymers used as wall

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materials, whey protein isolate (WPI / 90% of protein), was donated by Alibra

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(Campinas, Brazil), and inulin (IN / 93.8% purity) Frutafit DP= 2–60 and oligofructose

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(OL / 93% purity) Frutalose DP = 2–10 (Sensus Ingredientes, Netherland) were donated

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by Metachem (Itupeva, São Paulo, Brazil).

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Experimental design

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The experiments were conducted in a completely randomized design in

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triplicates, as shown in Table 1, to evaluate the effects of the three encapsulation

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formulations on the characteristics of powders of microencapsulated lime essential oil.

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Preparation of emulsion

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The ratio between the lime essential oil and each biopolymer was 1:5 (w/w).

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Each biopolymer suspension, whey protein isolate (WPI), whey protein isolate + inulin

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(4:1; w/w) (WPI/IN) and whey protein isolate + oligofructose (4:1; w/w) (WPI/OL),

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was prepared by dissolving the material at 25% (w/w) in distilled water. These

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conditions were determined through previous studies. The wall materials were dissolved

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in distilled water, and the solutions were prepared 24 h before emulsification at room

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temperature (25 °C) to ensure full saturation of the polymer molecules. Then, lime

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essential oil was added to the wall material solution and the mixture was homogenized

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(Model 450 - Branson Ultrasonic, USA). Aliquots of 60 mL were subjected to

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ultrasonication (2 min/240 W) to completely emulsify the lime essential oil. The

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emulsion was used as the feed liquid for the spray-drying process.

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Characterization of emulsion properties

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Freshly prepared emulsions were diluted to a droplet concentration of

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approximately 1:1000 with MilliQ water and placed into the measurement chamber of a

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microelectrophoresis instrument (Nano ZS Zetasizer, Malvern Instruments Ltd.,

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Worcestershire UK). The measurements were performed in triplicates at 25 °C.

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Emulsion droplet sizes were measured from dynamic light scattering

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experiments (Zetasizer Nano ZS, Malvern Instruments, Malvern, U.K.), using 1.5 mL

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emulsion samples, diluted 1000x with MilliQ water to avoid multiple light scattering

92

effects. The particle size data are reported as Z-average mean diameters and

93

polydispersity indices (PDI).

94 95

Spray-drying process

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The emulsions were dried using a spray-dryer (model MSD 1.0; Labmaq do

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Brasil, Ribeirão Preto, Brazil) equipped with a two-fluid nozzle atomizer. The following

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operational conditions were used, as described in previous studies: inlet temperature of

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170 °C, outlet temperature of 96 °C and feed rate of 0.7 L h−1. The microparticles were

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stored under refrigeration (4 °C to 7 °C) in sealed aluminum packing, protected from

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light penetration and gas permeation until further analysis 10.

102 103

Reconstitution properties

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The wettability of the powders was determined using a previously described

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method 5. One gram of powder was sprinkled over the surface of 100 mL of distilled

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water at 20 °C without agitation. The time required for the powder particles to sediment,

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sink, submerge, and disappear from the surface was recorded and used for comparing

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the extent of wettability of the samples.

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The solubility of the powders was evaluated, according to a previously reported

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method with modifications 10. The powders were weighed (3 g) and stirred into 25 mL

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of distilled water for 5 min using a blender. The solution was then centrifuged at 3300

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rpm for 15 min. An aliquot of 10 mL of the supernatant was transferred to a pre-

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weighed Petri dish and oven-dried at 105 °C overnight. The solubility (%) was 6


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calculated as the percentage of dried supernatant in relation to the amount of powder

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originally added (3 g). The bulk density and the compacted density were determined

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according to the methodology described by Fernandes et al. 5.

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Lime essential oil microparticles were reconstituted in Milli-Q water. One gram

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of powder was mixed with 4 g of Milli-Q water, and the solution was vortexed for 30 s.

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Reconstitution droplet size was measured, as described in section 2.4.

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The moisture adsorption kinetics were determined according to a previously 5

121

reported method

for hygroscopicity, by evaluating the increase in the amount of

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moisture over time. Approximately 1 g of microparticles was placed in a NaCl saturated

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solution (75% Relative Humidity) at 25 °C. Samples were weighed after 1, 2, 3, 4, 24,

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48, 72, 96, and 120 h. The hygroscopic values were expressed as percentages (g

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absorbed water/100 g of dry solid).

126 127

Scanning electron microscopy

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The morphologies of the lime essential oil microcapsules were examined using

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scanning electron microscopy (SEM). Microcapsules were distributed over a piece of

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double-sided carbon tape adhered to a metallic support (stub). Then, the samples were

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analyzed using scanning electron microscopy JSM-6360 (JEOL; Tokyo, Japan), with an

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accelerating voltage of 20 kV.

133 134

Adsorption isotherms

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Adsorption isotherms of lime essential oil microparticles were determined by the

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static method using saturated salt solutions at 25 °C. The study involved seven saturated

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salt solutions (LiCl, MgCl, K2CO3, NaNO3, Mg(NO3)2, NaCl and KCl), with water

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activity ranging from0.113 to 0.843 11. The model fit was evaluated based on percentage

139

values of the average relative deviation, E (%) (Eq. 1)

(1)

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Where n is the number of observed data, Y is the observed value and Yp is model predicted value.

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Thermal analysis

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The thermal stabilities (TGA) of the wall materials and lime essential oil

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microparticles were evaluated through thermogravimetric analysis on a TG-DTA H

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Shimadzu 60 (Shimadzu Corporation, Kyoto, Japan) under nitrogen atmosphere, at a

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rate of 10mL min-1, with heating from 25 °C to 600 °C at a rate of 10 °C min-1.

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Controlled release Controlled release was evaluated according to a previous study

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, with some

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modifications. Approximately 100 mg of microparticles was dispersed in 25 mL of

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isopropyl alcohol at Erlenmeyer flasks. Nine bottles, representing periods of 15, 30, 45,

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60, 90, 120, 150, 180, and 240 min, were placed in a shaker incubator at 30 °C with 100

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rpm rotation. At each time interval, an aliquot of the solution was centrifuged (3000

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rpm/10 min) and filtered through a nylon syringe filter (0.45 µm). The released oil

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concentration was measured using spectrophotometric analysis at a wavelength of 252

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nm. The concentration of lime essential oil was also assayed at concentrations of 6.25,

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12.5, 25.0, 50.0 and 100 µL to develop a lime essential oil standard curve.

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Concentration was given by the Equation 2 (R2 = 0.93):

(2)

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Antioxidant activity

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The antioxidant activity of lime essential oil encapsulated was evaluated by

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testing the free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) (Sigma-Aldrich, USA).

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According to methodology of Silva et al.

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microparticles were placed under a constant temperature of 60 °C for 42 days in order to

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evaluate the antioxidant activity during storage. Approximately 100 mg of

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microcapsules were collected, dissolved in methanol/water solution (1:1 w/w), and

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subjected to ultrasonic homogenization (2 min/300 W). After homogenization, the

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solution was centrifuged (10 min/3000 rpm) to decant the solid material. A stock

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solution of 0.1 mM DPPH in methanol was prepared and stored at 4 °C until analysis.

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The solutions of the microparticles (1 mL) were added to 3 mL of the stock solution of

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DPPH and stored in dark for 1 h to facilitate DPPH radical reaction with essential oil.

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The blank was prepared by substituting the microparticle solution with 1 mL of pure

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methanol. The absorbance values of the blank solution and the samples were measured

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at a wavelength of 517 nm. The DPPH scavenging capacity of lime essential oil was

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also assayed at concentrations of 6.25, 12.5, 25.0, 50.0 and 100 lM to develop a DPPH

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scavenging capacity standard curve for lime essential oil. The antioxidant activity (%

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AA) of essential oils encapsulated for 0, 7, 14, 21, 28, 35, and 42 days were determined

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using the following equation (Eq. 3):

11

with modifications. Samples of the

179

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(3)

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Where A0 is the absorbance of blank and AM is the absorbance of the sample.

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Fourier transform infrared spectroscopy

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Fourier transform infrared spectroscopy was performed on the pure essential oil,

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on the wall materials and on microcapsules produced. Measurements were carried out at

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room temperature, in the range from 400 to 4000 cm−1 (resolution of 4 cm-1 and

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accumulation of 128 scanning) using a spectrometer with attenuated total reflectance

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accessory (Jasco 4100, Tokyo, Japan).

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Statistical analysis

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Analysis of variance was also carried out using R package software to verify the

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effects of the wall materials on the characteristics of lime essential oil microparticles.

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Differences in the mean values obtained were examined by Duncan´s test of means at a

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significance level of 5% (p93%) may

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also be due to the ultrasound process changing the structure of proteins exhibiting

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hydrophilic parts of the amino acid to water 16. Inulin showed high solubility (80%) in

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water 17 but it depends on the water temperature and composition. Other authors found

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that a high solubility (>78%) for microparticles of fish oil WPI and inulin 18.

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Quick wettability is a desirable feature for instant powders

19

. It was observed

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that addition of biopolymers significantly (p

Prebiotic Carbohydrates: Effect on Reconstitution, Storage, Release

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