Production of solid lipid microparticles loaded with lycopene by spray chilling: Structural characteristics of particles and lycopene stability

Accepted Manuscript Title: Production of solid lipid microparticles loaded with lycopene by spray chilling: Structural characteristics of particles and lycopene stability Author: Julio R. Pelissari Volnei B. Souza Ac´acio A. Pigoso Fabr´ıcio L. Tulini Marcelo Thomazini Christianne E.C. Rodrigues Alexandre Urbano Carmen S. Favaro-Trindade PII: DOI: Reference:

S0960-3085(15)00145-5 http://dx.doi.org/doi:10.1016/j.fbp.2015.12.006 FBP 664

To appear in:

Food and Bioproducts Processing

Received date: Revised date: Accepted date:

29-5-2015 17-11-2015 14-12-2015

Please cite this article as: Pelissari, J.R., Souza, V.B., Pigoso, A.A., Tulini, F.L., Thomazini, M., Rodrigues, C.E.C., Urbano, A., Favaro-Trindade, C.S.,Production of solid lipid microparticles loaded with lycopene by spray chilling: structural characteristics of particles and lycopene stability, Food and Bioproducts Processing (2015), http://dx.doi.org/10.1016/j.fbp.2015.12.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 - Lycopene was loaded into solid lipid microparticles by spray chilling. - Shortening, gum Arabic and carboxymethyl cellulose were used as carriers. - Microparticles were round shaped and in a crystalline phase, ranging from 10 to 110 µm.

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- Addition of gum Arabic into formulation delayed the degradation of lycopene.

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2 Production of solid lipid microparticles loaded with lycopene by spray chilling: structural characteristics of particles and lycopene stability

Pelissari, Julio R.1, Souza, Volnei B.1; Pigoso, Acácio A.2; Tulini, Fabrício L., Thomazini, Marcelo1;

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Rodrigues, Christianne E.C. 1, Urbano, Alexandre3; Favaro-Trindade, Carmen S.1

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1

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Departamento de Engenharia de Alimentos, Faculdade de Zootecnia e Engenharia de Alimentos (FZEA), Universidade de São Paulo (USP), Pirassununga, SP, Brazil. Centro Universitário Hermínio Ometto – UNIARARAS, Araras, SP, Brazil

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Departamento de Física, Universidade Estadual de Londrina (UEL), Londrina, PR, Brazil.

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Corresponding author: Prof. Dr. Carmen Sílvia Favaro-Trindade Faculdade de Zootecnia e Engenharia de Alimentos – Universidade de São Paulo Avenida Duque de Caxias Norte, 225 – 13635-000 - Pirassununga – São Paulo – Brazil [email protected] Phone: +55 (19) 3565 4139 Fax: +55 (19) 3565 4284

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3 Abstract Solid lipid microparticles (SLM) loaded with lycopene/sunflower oil solution have been produced using the spray chilling technique and a shortening as carrier. Six treatments were formulated and evaluated with regard to size distribution, morphology, Fourier transform infrared

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spectroscopy (FTIR) and X-ray diffraction. In addition, the stability of lycopene into SLM was

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evaluated by periodic quantification and instrumental color parameters measurements at different storage conditions. The microparticles produced in this study were spherical and no distinct bonds

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were detected by FTIR in lycopene/sunflower oil solution and microparticles. Moreover, X-ray diffraction analyses revealed the presence of polymorphic form β’ in the carrier (shortening) and in

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the microparticles. Stability studies indicated that the best conditions to delay the degradation of encapsulated lycopene was achieved with the formulation containing gum Arabic and storage under

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refrigeration and vacuum. Results obtained in the present study show that lycopene was stable after incorporation into SLM, encouraging future works to evaluate the bioavailability of encapsulated

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lycopene in both animal and human models.

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Keywords: carotenoid, spray congealing, spray cooling, X-ray diffraction, morphology.

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4 1. Introduction Lycopene is an acyclic carotenoid with 11 conjugated double bonds, which is responsible for the red color of tomatoes, guavas and watermelons. According to Wang (2012), the antioxidant capacity of lycopene may reduce the risk of several diseases. However, due to the high number of

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double bonds, lycopene is susceptible to oxidation and isomerization during processing steps and

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storage, particularly when stored in the presence of oxygen (Matioli and Rodriguez-Amaya, 2002). In this context, microencapsulation techniques such as spray drying, emulsion, molecular inclusion,

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complex coacervation and microemulsion have been used to overcome this drawback (Matioli and Rodriguez-Amaya, 2002; Matioli and Rodriguez-Amaya, 2003; Shu et al. 2006; Blanch et al. 2007;

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Chiu et al. 2007; Nunes and Mercadante, 2007; Rocha et al. 2012; Silva et al. 2012; Chen et al. 2014).

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One remarkable characteristic of carotenoids is the great solubility in apolar solvents such as edible fats and oils, and low solubility in water (Belitz and Grosch, 1999). However, this feature can

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interfere with lycopene administration, leading to extremely low bioavailability (Chen et al. 2014).

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In order to improve oral bioavailability of lycopene, Faisal et al. (2010) suggested the use of a lipid-

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based formulation. In this context, the application of spray chilling to produce solid lipid microparticles (SLM) loaded with lycopene using fat as carrier could be a good choice due to several aspects: (1) protection of the carotenoid; (2) improvement of lycopene bioavailability; (3) low interaction of lycopene with other compounds when it is applied in a food; (4) specific release in the intestine, during fat digestion.

Spray chilling is a process to produce microparticles in which a mixture of the molten carrier

and the active ingredient is sprayed into a cold chamber using an atomizing nozzle. When the droplets meet the cold environment of the chamber, microparticles are formed by fat solidification. According to Okuro et al. (2013a), spray chilling is a convenient technique for microparticles production because it is a low-cost continuous process that is easy to scale up and does not require solvents. In addition, spray chilling does not require the application of high temperatures that are

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5 found in spray drying, which should be considered when thermolabile ingredients (e.g. lycopene) need to be encapsulated. Thus, in this study, SLM loaded with lycopene were produced by spray chilling using a shortening as carrier, and the microparticles were characterized with regard to structure and

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lycopene stability.

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2. Material & Methods

2.1 Materials

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The carrier used to produce the microparticles was a shortening composed of hydrogenated and interesterified cottonseed, soy and palm oils (Tri-HS-48) supplied by Triângulo Alimentos

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(Itápolis, Brazil), with melting point at 51 °C. This carrier was chosen due to the availability in actual market, and because it is commonly used in food products. Gum Arabic (Dinâmica Química

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Contemporânea Ltda., Diadema, Brazil) and carboxymethylcellulose (CMC, Fagron do Brasil

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Farmacêutica Ltda., São Paulo, Brazil) were also used to produce the microparticles. The core or

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active material used was Redivivo®, a commercial ready to use lycopene dispersed in sunflower oil, containing 10% of lycopene (DSM, São Paulo, Brazil). The fatty acid composition of the shortening used as carrier was evaluated by FAME (fatty acid methyl esters) gas chromatography, according to the official methods AOCS Ce 2-66 and Ce 1-62 (AOCS, 1998), using a Shimadzu 2010 AF Gas Chromatograph (Kyoto, Japan) coupled with an automatic injector (AOC20i, Shimadzu, Kyoto, Japan) and a flame ionization detector. The analysis was performed in triplicate and the carrier composition is presented in Table 1.

2.2 Production of microparticles Solid lipid microparticles were produced using the spray chilling technique as described by Salvim et al. (2015), with modifications. Lycopene was incorporated into the shortening previously

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6 molten at 60 °C and atomized using an spray chiller (Model MSD 1.0, Labmaq do Brasil, Ribeirão Preto, Brazil) in a chamber kept at 13 °C by an air stream system, with an 1.2 mm nozzle, 1.0 kgf/cm2 air pressure and 40 ml/min of feed flow (controlled by peristaltic pump). Six formulations were used to produce SLM, as described in Table 2. The particles were produced three times, and all

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further analyses were performed in triplicate.

2.3 Lycopene quantification

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Lycopene was quantified by spectrophotometric methods, as described by Rodriguez-Amaya (2001). For that, lycopene was extracted from 10 mg of microparticles added to 10 ml of petroleum

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ether, and analyzed at 470 nm in a spectrophotometer (Hach, DR 2800, Loveland, USA). The

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quantification of lycopene was determined according to the following equation (1):

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In the formula: A = Absorbance

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(1)

V = final volume (ml)

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A1% = lycopene absorption coefficient in petroleum ether (3450 cm-1) m = sample mass (g)

Similarly, the degradation of the encapsulated material was determined with the following

equation (2): (2)

In the formula: I = Concentration of lycopene at the first day (mg/kg) F = Concentration of lycopene after 3, 30, 60 and 90 days (mg/kg)

2.4 Microparticles characterization

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2.4.1 Size distribution and volume weighted mean diameter (D4,3) Size distribution and volume weighted mean diameter (D4,3) of SLM produced in this study were evaluated periodically using a laser diffraction particle analyzer (Shimadzu Sald-201V, Kyoto,

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application of ultra-sound for better dispersion of the material.

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Japan). Before analyses, SLM were added to ethanol (Synth, Diadema, Brazil) with rapid

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2.4.2 Morphology

The morphology of SLM was evaluated by scanning electron microscopy (SEM) using the

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TM3000 Tabletop Microscope (Hitachi, Tokyo, Japan) along with the program TM3000. Microparticles were arranged in a double sided carbon tape (Ted Pella Inc., Redding, USA) and

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fixed in aluminum stubs. Images were acquired at 5 kV of acceleration and 1750 mA.

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2.4.3 Fourier transform infrared (FTIR) spectroscopy

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The chemical structures of samples were evaluated using the Perkin Elmer FT-IR

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Spectrometer (Massachusetts, USA) with the Spectrum One software (version 5.3.1). Sixteen scans were made and the working spectral region was from 4000-600 cm-1 (Okuro et al. 2013b).

2.4.4 X-ray diffraction

X-ray diffractograms from the crystal structure of SLM were obtained in a Bruker D8

diffractometer (Billerica - Massachusetts – USA) at room temperature and with cooper radiation kα (=1.5405 Å), tension of 40 kV, current of 30 mA, angular velocity of 0.05°/s, and under geometry of -2 with 2 varying from 3 to 35° (Medeiros et al. 2014, with modifications).

2.5 Lycopene stability in solid lipid microparticles

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8 Solid lipid microparticles were periodically evaluated with regard to lycopene concentration and instrumental color, after 0, 3, 30, 60 and 90 days of storage in desiccators kept in the dark, under four conditions: (1) 20-22 °C and relative humidity of 33%; (2) 20-22 °C under vacuum; (3) at 5-6 °C and relative humidity of 33%; (4) at 5-6 °C under vacuum. Quantification of lycopene in

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SLM was performed in triplicates, as described in section 2.3, and the amount of lycopene at the

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first day (t=0) was considered as 100%. The constant of degradation (k, equation 3) and the half-life time (t1/2, equation 4) were determined according to the first-order kinetics of degradation as

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follows:

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(3)

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(4)

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In the formula, C0 is the amount of lycopene at t=0, and Ct is the amount of lycopene

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determined at the reaction time (days).

The evaluation of SLM instrumental color was performed after 0, 3, 30, 60 and 90 days

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using the portable colorimeter MiniScan XE Plus (Hunter Lab, Reston, USA) to establish a correlation between the stability and color changes after lycopene degradation. The total color difference (ΔE) after 90 days of storage was obtained as follows (equation 5):

(5)

In the formula, ΔL* = L*time 0 – L*time 90; Δa* = a*time 0 – a*time 90; Δb* = b*time 0 – b*time 90, where “L” is a parameter for luminosity, “a” is a parameter for color variation from green to red, and “b” is a parameter for color variation from blue to yellow.

2.6 Statistical analyses Page 8 of 30

9 All data obtained in this study were analyzed using the program SAS v9.2 (Statistic Analysis Software, SAS Institute Inc., Cary, USA). Analysis of variance (ANOVA) followed by Tukey’s

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post-test (95% confidence interval) were applied to detect significant differences.

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3. Results and discussion

3.1 Carrier fatty acid composition and production of microparticles

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The shortening used in this study as carrier was analyzed with regard to fatty acid composition and the results are presented in Table 1. In addition, Supplementary Table 1 presents

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the solid fat content (SFC) in the shortening, from 10 to 45 °C, as provided by the supplier. Although the fatty acid composition of the carrier was very heterogeneous, the main fatty acids

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were the palmitic, stearic and oleic acids accounting more than 93% of the mixture. A heterogeneous fatty acid composition is interesting for the structure of lipid particles, since different

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crystals are produced and this contribute to their accommodation into the particle. This

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heterogeneous crystalline structure may avoid lipid recrystallization and consequent expulsion of

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the core from the microparticles. The production of SLM by spray chilling resulted in fine, fluid and red colored powder. At the end of each process, the powders were gathered, placed in closed vials, and stored in a dark and dry place.

3.2 Microparticles characterization

3.2.1 Morphology Photomicrographs of SLM produced in this study were obtained by SEM and represented in Figure 1. The micrographs show that there was no morphological difference among them, which indicates that it is possible to increase the core ratio without affecting particle morphology. In addition, results obtained in the present study indicate that the addition of gum Arabic and CMC

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10 into the carrier composition did not affect the morphology of the particles, as revealed by SEM analyses. The microparticles presented spherical shape, with agglomerations, variable diameters, rough surface with some pores, but no cracks. It is important to produce particles with spherical

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shape to favor powder flow properties and to facilitate powder application. Similarly, particle

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agglomeration may also positively contribute to powder application by reducing dust formation. However, particles with rough surfaces may have a reduced flow, and this may also indicate the

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occurrence of a heterogenic lipid matrix. In addition, pores in microparticles may expose lycopene to the oxygen, which is a pro-oxidant factor, thereby reducing particle functionality. Similar results

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were obtained by Chambi et al. (2008) for the production of SLM loaded with casein hydrolysates by spray chilling with lipid carriers (stearic, oleic and lauric acids). In another approach for the use

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of spray chilling technique, Pedroso et al. (2012 and 2013) encapsulated probiotic bacteria using interesterified fat and cocoa butter and obtained spherical particles with rough surfaces,

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corroborating the results of the present study. However, in 2012, Di Sabatino et al. encapsulated

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proteins by spray chilling and obtained spherical particles without agglomeration, which could be

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attributed to the high melting point of the lipid carrier used by those authors.

3.2.2 Size distribution and volume weighted mean diameter (D4,3) In this study, all formulations presented similar results (no significant difference, p>0.05)

with regard to the volume weighted mean diameter, which suggests that variations on formulations did not affect this parameter (Table 3). Overall, the volume weighted mean diameter of SLM produced in this study was in the range of 10 to 110 µm, which indicates high variability of size, as also observed by SEM analyses (Figure 1). In 2008, Albertini et al. obtained similar results for the production of SLM loaded with propafenone hydrochloride and vitamin E by spray chilling, where the volume weighted mean diameter of particles ranged from 75 to 150 µm. However, smaller particles were obtained by Chambi et al. (2008) and Leonel et al. (2010) for the production of SLM

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11 loaded with hydrophilic compounds and casein hydrolysates, respectively, by spray chilling, which may be attributed to different formulations and atomization devices. Particle size distributions of all formulations were similar throughout the storage period in each condition, and results obtained with formulation 1 after 0 and 90 days of storage in different

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conditions (33% RH, vacuum, room temperature and refrigeration temperature) are represented in

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Figure 2. When the particles were stored at room temperature, under 33% RH and vacuum (Figure 2, A and B), it was detected a slight increase in particle size distributions after 90 days of storage,

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which may be attributed to particle agglomeration, as corroborated by SEM results. This effect may occur due to the presence of melted triacylglycerols, which promotes the adherence among lipid

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particles. Otherwise, results obtained after the analyses of particles stored at refrigeration temperature showed that particle size distributions did not change until the last day of storage

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(Figure 2, C and D), which indicates that the temperature was important to keep the original size of

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SLM produced in this study.

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3.2.3 Fourier transform infrared spectroscopy (FT-IR)

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Infrared spectra provide important information that can be related to vibrations of functional groups from organic compounds (Guillén and Cabo, 1997). Thus, it is quite frequent to use FT-IR analyses to evaluate microparticles and microcapsules as it is possible to detect chemical bonds that may occur between the active compound and the carrier, in addition to changes in molecules present into the particles. Infrared absorption spectra of formulations 1 to 4, shortening and lycopene oil solution are presented in Figure 3. With regard to SLM produced in this study, the main component of the carrier and lycopene/sunflower oil solution were triacylglycerols. Thus, bands corresponding to the vibration of triacylglycerol functional groups were detected in all samples. According to Guillén and Cabo (1997), bands corresponding to the stretching vibration of ester bonds from triacylglycerols appear between 1300 and 1000 cm-1 (approximately 1175 cm-1 in the present study). In addition, according to those authors, bands corresponding to the high intensity stretching

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12 from carbonyl groups appear near 1750 cm-1, while bands between 3000-2800 cm-1 are related to the high intensity stretching of the acyl chain. In this study, infrared spectra obtained by the analyses of all formulations were very similar, with slight differences of intensity, which suggests that the process used to produce the

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microparticles and the incorporation of lycopene did not induce the formation of distinct bonds in

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atomized samples. The band located between 950-980 cm-1 in the spectra of all samples, with high intensity in the spectrum of lycopene/sunflower oil solution, correspond to the -HC=CH-trans

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bonds that are highly frequent in lycopene molecules. This band was also reported by Rubio-Diaz et al. (2010) when evaluating extracts containing lycopene. In 2010, Albertini et al. obtained similar

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results after FT-IR analyses of microparticles containing atenolol produced by spray chilling, since

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no interactions between the core material and carrier were detected.

3.2.4 X-Ray diffraction (XRD)

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Solid lipid microparticles containing lycopene (formulations 1 to 4), shortening (carrier) and

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lycopene/sunflower oil solution were stored at 4 °C for approximately 90 days and analyzed in a X-

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Ray diffractometer, and results are presented in Figure 4. It was detected that the diffraction pattern for lycopene/sunflower oil solution corresponded to an amorphous material, showing bands of width approximately 10° around of 2=19°, and low intensity peaks in 2=24.4° and 2=26.2°. However, the diffractograms of microparticles and carrier were similar and typical of crystalline materials, with highly defined peaks in 2 = 6°, 21° e 23.5°. Thus, the analyses of SLM (composed of shortening and lycopene/sunflower oil solution) resulted in diffractograms with superposed patterns of amorphous (lycopene/sunflower oil solution) and crystalline (shortening) materials. Moreover, it was also detected a gradual reduction on relative intensity of diffraction peaks when the rate of lycopene oil solution in microparticles composition was increased. The SLM and shortening presented similar diffraction patterns after X-ray diffraction analyses. This suggests that the crystalline phase of the carrier is not affected by the incorporation

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13 of lycopene/sunflower oil solution in any concentration tested, neither by the production process. Diffraction peaks present in the diffractogram of carrier correspond to the β’ polymorphic phase. Considering that triacylglycerols exhibit crystal polymorphism, which is defined as the capacity of a material to have different crystal forms, the X-ray diffraction analyses provide important

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information about the polymorphic forms that originate lipid microparticles. Usually, fat crystals

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may have the configurations α, β’ and β, which have different thermodynamic stabilities. If they undergo structural rearrangements that lead to changes in their crystal polymorphic forms, the lipid

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microparticles may expel the core and expose it to the environmental conditions, thereby compromising their functionality.

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According to Müller et al. (2002), during storage of SLM that have α-polymorph crystals, modifications in the lipid matrix may induce the transition to β-polymorph crystals, which

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contributes to core expulsion from inside the particle. In the present study, it was not possible to confirm if the fat that constituted the particles and the microparticles production process contributed

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to obtain this specific form or if there was some crystal rearrangement throughout the time, as it was

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period.

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not performed the XRD analyses immediately after the particle production neither over the storage

3.3 Lycopene stability in solid lipid microparticles Degradation of lycopene loaded into SLM over the storage period in different conditions

presented a first-order kinetics, and results are shown in Table 4. All formulations in all storage conditions presented significant first-order constants of degradation (k), which indicates an intense and accelerated degradation of lycopene in SLM, also evidenced by the half-life (t1/2) presented in Table 4. The results indicate that lycopene degradation was highly influenced by temperature and the presence of oxygen, since formulations stored at refrigeration temperatures and under vacuum presented low degradation rates and long half-life. When varying lycopene ratio in microparticles (formulations 1 to 4), the stability was reduced in formulations with elevated ratios of lycopene.

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14 This may indicates that lycopene is less protected from the environmental conditions in such formulations, since more lycopene molecules may be present on the surface of the particles. Similarly, the addition of gum Arabic into the formulation also increased lycopene stability in microparticles, despite the lycopene ratio in formulation 5 being similar to formulation 1, in which

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good results for lycopene stability were also reported.

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Until this date, only few studies on the production of SLM loaded with carotenoids have been reported in the literature. In 2013, Gomes et al. produced SLM loaded with β-carotene using

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stearic acid and sunflower oil as carriers. Those authors reported losses of less than 10% of the carotenoid, after 7 months at 7-10 °C, when α-tocopherol was added to the formulation. However,

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β-carotene was rapidly degraded when those particles were produced without α-tocopherol, which indicates that matrix composition may strongly affect carotenoid stability. On the other hand, Dos

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Santos et al. (2015) produced nanoparticles loaded with lycopene and reported 50% retention of active core after 14 days of storage at room temperature, indicating the influence of temperature on

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lycopene stability. In addition, according to Rodriguez-Amaya (2002), the main causes related to

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the degradation of carotenoids during food processing and storage are enzymatic and non-enzymatic

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oxidation. Carotenoids may also be degraded by isomerization, in which double bonds switch from trans to cis configuration. As a result, food products exhibit loss of color, especially when kept in contact with acids and high temperatures. In the present study, lycopene was not susceptible to enzymatic oxidation, as it was not incorporated into a food matrix. Color is another parameter that may be related to lycopene stability, since an increase in

average color indicates the degradation of lycopene (a red colored pigment). According to results presented in Table 5, particles stored at refrigeration temperature under vacuum presented the lowest values for ΔE because of low variation in color when compared to particles stored in other conditions. Values obtained for the parameter “L” (luminosity) corroborated with the lycopene content, since formulation 4 presented the lowest value for this parameter due to the higher content of carotenoid (data not shown). However, SLM became clearer (higher L value) throughout the

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15 storage period, indicating the degradation of lycopene, especially when stored at room temperature without vacuum. Similarly, the parameter “a” (variation from green to red) decreased throughout the time due to the loss of red color, indicating lycopene degradation (data not shown). This effect was more pronounced when SLM were stored at room temperature without vacuum. On the other

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hand, SLM stored at low temperatures and vacuum presented less variation in parameters “L” and

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“a”. Together, these results indicate that the association of low temperature and vacuum contribute to lycopene stability into SLM. According to Robert et al. (2003), the degradation process of

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carotenoids is related to oxygen permeability into the lipid matrix, which favours oxidative reactions that lead to loss of color of encapsulated lycopene. In the present study, despite being an

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indirect measure of degradation, results obtained with color analyses corroborate with results of

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lycopene stability presented in Table 4.

4. Conclusions

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In this study, lycopene dissolved in sunflower oil was loaded into SLM, resulting in round

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shaped particles, with some agglomerates and size in the expected range for the method of spray

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chilling. Moreover, it was concluded that the use of gum Arabic combined with the carrier, along with storage under low temperature and vacuum, are the best conditions to protect the carotenoid. In this condition, it was possible to reduce lycopene degradation during 90 days of storage, but even so, lycopene degradation rates were around 60%. Results obtained in this work encourage further studies on bioavailability of lycopene loaded into SLM, produced by spray chilling, in both animal and human models.

Acknowledgements Authors thank the National Council for Scientific and Technological Development (CNPq) for financial support (#470364/2012-2), the Coordination for the Improvement of Higher Education

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16 Personnel (CAPES) and the São Paulo Research Foundation (FAPESP) for the fellowships that

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were granted (#13/09090-2 and #14/14540-0).

Conlfict of interest

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J.R. Pelissari, V.B. Souza, A.A. Pigoso, F.L. Tulini, M. Thomazini, C.E.C. Rodrigues, A.

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Urbano and C.S. Favaro-Trindade declare that they have no conflict of interest.

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Matioli, G., Rodriguez-Amaya, D.B., 2003. Microencapsulation of lycopene with cyclodextrins.

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Ciênc. Tecnol. Alim., 23, 102-105.

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Medeiros, A.C.L., Thomazini, M., Correia, R.T.P., Urbano, A., Favaro-Trindade, C.S., 2014.

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Structural characterisation and cell viability of a spray dried probiotic yoghurt produced with goat milk and Bifidobacterium animalis subsp. lactis (BI-07). Int. Dairy J., 39, 71-77.

Müller, R.H., Radtke, M., Wissing S.A., 2002. Nanostructured lipid matrices for improved microencapsulation of drugs. Int. J. Pharm., 242, 121-128.

Nunes, I.L., Mercadante, A.Z., 2007. Encapsulaion of lycopene using spray-drying and molecular inclusion process. Braz Arch Biol Techn, 50, 893–900.

Okuro, P.K., Matos Jr, F.E., Favaro-Trindade, C.S., 2013a. Technological challenges for spray chilling encapsulation of functional food ingredients. Food Technol. Biotechnol., 51, 171-182.

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Okuro, P.K., Thomazini, M., Balieiro, J.C.C., Liberal, R.D.C.C, Fávaro-Trindade, C.S. 2013b. Coencapsulation of Lactobacillus acidophilus with inulin or polydextrose in solid lipid microparticles

ip t

provides portection and improves stability. Food Res. Int., 53, 96-103.

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Pedroso, D.L., Thomazini, M., Heinemann, R.J.B., Favaro-Trindade, C.S., 2012. Protection of Bifidobacterium lactis and Lactobacillus acidophilus by microencapsulation using spray-chilling.

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Pedroso, D.L., Dogenski, M., Thomazini, M., Heinemann, R.J.B., Favaro-Trindade, C.S., 2013. Microencapsulation of Bifidobacterium animalis subsp. lactis and Lactobacillus acidophilus in

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cocoa butter using spray chilling technology. Braz. J. Microbiol., 44, 777-783.

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Robert, P., Carlsson, R.M., Romero, N., Masson, L., 2003. Stability of spray-dried encapsulated

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20 Rubio-Diaz, D.E., Nardo, T., De Santos, A., Jesus, S., De Francis, D., Rodriguez-Saona, L.E., 2010. Profiling of nutritionally important carotenoids from genetically-diverse tomatoes by infrared spectroscopy. Food Chem., 120, 282-289.

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Salvim, M.A., Thomazini, M., Pelaquim, F.A., Urbano A., Moraes, I.C.F., Favaro-Trindade, C.S.,

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2015. Production and structural characterization of solid lipid microparticles loaded with soybean

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protein hydrolysate. Food Res Int, 76, 689–696.

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Silva, D.F., Favaro-Trindade, C.S., Rocha, G.A., Thomazini, M., 2012. Microencapsulation of

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Figure captions

Fig. 1 Photomicrographs of solid lipid microparticles loaded with lycopene produced by spray chilling. (A) Formulation 1 (20% of lycopene/sunflower oil solution); (B) Formulation 2 (23.1% lycopene/sunflower oil solution); (C) Formulation 3 (28.6% lycopene/sunflower oil solution); (D) Formulation

4

(33.3%

lycopene/sunflower

oil

solution);

(E)

Formulation

5

(17.9%

lycopene/sunflower oil solution and gum Arabic); (F) Formulation 6 (19.2% lycopene/sunflower oil solution and carboxymethylcellulose).

Page 20 of 30

21 Fig. 2 Size distributions of particles obtained with formulation 1 at the beginning (day 0, dotted line) and at the end of storage (90 days, continuous line). (A) particles stored at room temperature (20-22 °C) and relative humidity of 33%; (B) particles stored at room temperature (20-22 °C) under

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particles stored at refrigeration temperature (5-6 °C) under vacuum.

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vacuum; (C) particles stored at refrigeration temperature (5-6 °C) and relative humidity of 33%; (D)

Fig. 3 Infrared absorption spectra of solid lipid microparticles loaded with lycopene produced by

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spray chilling. (A) shortening (carrier); (B) active core (lycopene/sunflower oil solution); (C) formulation 1 (20% of lycopene/sunflower oil solution); (D) formulation 2 (23.1%

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lycopene/sunflower oil solution); (E) formulation 3 (28.6% lycopene/sunflower oil solution); (F)

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formulation 4 (33.3% lycopene/sunflower oil solution).

Fig. 4 Diffractograms of solid lipid microparticles containing lycopene obtained by X-Ray

d

diffraction analyses. (A) active core (lycopene/sunflower oil solution); (B) shortening (carrier); (C)

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formulation 1 (20% of lycopene/sunflower oil solution); (D) formulation 2 (23.1%

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lycopene/sunflower oil solution); (E) formulation 3 (28.6% lycopene/sunflower oil solution); (F) formulation 4 (33.3% lycopene/sunflower oil solution).

Page 21 of 30

22 Table 1. Fatty acid composition of the shortening used as carrier in this study. The analyses were performed by gas chromatography coupled with flame ionization detector, and results represent the ratio of each fatty acid in the carrier. Composition (g/100g)

0.46 ± 0.00

Myristic (C14:0)

0.83 ± 0.01

Palmitic (C16:0)

30.9 ± 0.7

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Lauric (C12:0)

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Fatty acid

Stearic (C18:0)

35.0 ± 0.7 28.0 ± 0.3

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Oleic (C18:1)

4.7 ± 0.1

0.19 ± 0.00

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Eicosatrienoic (C20:3)

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Linoleic (C18:2)

Page 22 of 30

23 Table 2. Composition of microparticles produced in this study, using shortening, lycopene, gum Arabic and carboxymethyl cellulose (CMC).

60

15

-

-

2

60

18

-

-

23.1

3

60

24

-

-

4

60

30

-

-

5

60

15

6

6

60

15

-

-

3

28.6 33.3 17.9 19.2

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d

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*Lycopene ratio in oil solution was 10%.

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CMC (g)

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Formulation

Gum Arabic (g)

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1

Percentage of lycopene/sunflower oil solution in formulation (%) 20

Lycopene/sunflower Shortening oil solution* (g) (g)

Page 23 of 30

24 Table 3. Volume weighted mean diameter (D4,3) of solid lipid microparticles loaded with lycopene, according to each formulation. No significant difference was detected among formulations (p>0.05). Volume weighted mean diameter (µm)

1

66.43 ± 4.6

2

69.25 ± 2.4

3

71.90 ± 6.9

4

70.02 ± 0.5

5

69.78 ± 6.5

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cr

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Formulation

71.28 ± 3.3

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M

6

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25 Table 3. First-order constant of degradation (k) and half-life (t1/2) of lycopene in microparticles, during 90 of days storage in different conditions (R2 – coefficient of determination) k (s-1)

t1/2 (days)

R2

1

0.025

27.506

0.970

Room temperature

2

0.027

25.961

0.992

(20-22 °C) and

3

0.049

14.117

relative humidity of

4

0.046

15.003

33%

5

0.027

25.577

6

0.058

1

0.037

2

0.049

3

0.059

vacuum

14.175

0.960

11.709

0.906

0.064

10.780

0.905

0.020

34.832

0.960

0.044

15.682

0.970

0.026

26.660

0.906

0.033

21.328

0.984

0.036

19.362

0.996

0.066

10.582

0.937

5

0.019

35.914

0.939

6

0.045

15.541

0.994

1

0.012

56.815

0.780

2

0.014

48.135

0.789

3

0.015

47.153

0.945

4

0.017

40.773

0.949

5

0.012

57.285

0.951

6

0.014

50.228

0.941

4 5

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temperature (5-6 °C)

3

and relative humidity

4

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2

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Refrigeration

temperature (5-6 °C) under vacuum

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0.873

1

Refrigeration

0.969

18.583

6

of 33%

0.980

0.858

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(20-22 °C) under

0.897

11.971

an

Room temperature

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Formulation

Storage condition

Page 25 of 30

26 Table 4. Total color difference (ΔE) in lycopene microparticles stored for 90 day in different conditions Room temperature

Refrigeration Room temperature

Refrigeration temperature (5-6 °C)

Formulation

(20-22 °C) under relative humidity of

temperature (5-6 °C)

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(20-22 °C) and

and relative humidity vacuum*

under vacuum*

of 33%*

25.47 ± 11.76 b, A

67.17 ± 0.21 a, A

23.99 ± 4.62 b, A

11.94 ± 0.68 a, B

2

65.09 ± 0.34 a, A

64.11 ± 0.50 a,b, A

30.36 ± 8.46 b, B

10.92 ± 2.42 a, C

3

67.56 ± 1.65 a, A

64.66 ± 0.73 a,b, A

57.19 ± 4.87 a, A

9.22 ± 2.58 a, B

4

67.12 ± 1.33 a, A

64.70 ± 1.30 a,b, A

62.88 ± 0.55 a, A

13.08 ± 2.28 a, B

5

65.72 ± 5.16 a, A

58.50 ± 4.82 b, A

23.62 ± 0.06 b, B

8.86 ± 2.01 a, C

6

63.57 ± 0.55 a, B

66.42 ± 0.09 a,b, A

65.16 ± 0.07 a, A

10.85 ± 3.01 a, C

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an

cr

1

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33%*

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* Mean ± standard deviation. Means in the same line with the same upper case letter, or means in the same column with the same lower case letter are not statistically different (p>0.05). All analyses were performed in triplicates.

Page 26 of 30

Figure 1

A

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1

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an

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cr

B

C

Ac

ce pt

ed

D

E

F

2

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Figure 2

1 A

B

D

Ac

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ed

M

an

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C

cr

ip t

A

Page 28 of 30

Figure 3

1 A a

B

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C

cr

D

F

an

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E

Ac

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ed

M

2

Page 29 of 30

Figure 4

F E D C B

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cr

A

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1

Ac

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ed

M

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2

Page 30 of 30