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Effect of feed preparation on the properties and stability of ascorbic acid microparticles produced by spray chilling
LWT - Food Science and Technology 75 (2017) 251e260
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Effect of feed preparation on the properties and stability of ascorbic acid microparticles produced by spray chilling quio de Matos-Jr a, b, *, Talita Aline Comunian a, Marcelo Thomazini a, Fernando Eusta Carmen Sílvia Favaro-Trindade a ~o Paulo, College of Animal Science and Food Engineering, Av. Duque de Caxias Norte, 225, CP 23, CEP 13535 900, Pirassununga, Sa ~o Paulo, University of Sa Brazil b rio, BR 364, Km 04, Distrito industrial, CEP 69.920 900, Rio Branco, Acre, Federal University of Acre, Science Center of Health and Sport, Campus Universita Brazil a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 November 2015 Received in revised form 30 August 2016 Accepted 4 September 2016 Available online 5 September 2016
The aim of this study was to evaluate the effect of the feed form, dispersion or emulsion, on the production of solid lipid microparticles (SLM) loaded with ascorbic acid (AA) using interesterified fat as a carrier. The particles obtained were characterized according to the particle size distribution and morphology using SEM, thermal behavior using DSC and encapsulation efficiency. The stabilities of the free and encapsulated AA were evaluated over a period of 60 days. Spherical microparticles were obtained with diameters between 34 and 92 mm. The encapsulation efficiency varied according to the type of feed used and ranged from 59 to 73%. The stability of the AA after 60 days of storage exceeded 70%, even upon storage at 37 C. Microencapsulation by spray chilling was effective in maintaining the stability of AA. Microparticles produced from the dispersion promoted more protection of AA in the three storage temperature conditions. The particles produced could also allow controlled liberation of AA and masking of its acidic taste, which is not always desirable. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Vitamin C Microencapsulation Spray congealing Solid lipid microparticles Characterization
1. Introduction Ascorbic acid (AA) or vitamin C is used to fortify, as an antioxidant and as a reducing agent in many foods. However, to prepare foods enhanced with AA is not an easy task since this compound is very unstable. Microencapsulation has been successfully used to overcome the various limitations associated with the addition of bioactive compounds to food (Gharsallaoui, Roudaut, Chambin, Voilley, & Saurel, 2007). The basic process associated with this technology involves packaging solid, liquid or gaseous substances in extremely small capsules that can release their contents in a controlled manner and under specific conditions (Todd, 1970). Several techniques have been used to encapsulate AA, such as spray drying, microfluidic devices, double emulsion followed by complex coacervation, liposome preparation, O/W microemulsions and spray chilling (Comunian, Thomazini, Alves, Matos Junior,
* Corresponding author. University of S~ ao Paulo, College of Animal Science and Food Engineering, Av. Duque de Caxias Norte, 225, CP 23, CEP 13535 900, Pirassununga, S~ ao Paulo, Brazil. E-mail address: [email protected] (F.E. de Matos-Jr). http://dx.doi.org/10.1016/j.lwt.2016.09.006 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
Balieiro, & Favaro-Trindade, 2013; Comunian, Thomazini, Gambagorte, Trindade, & Favaro-Trindade, 2014; Dissanayake, Karunaratne, & Chandani Perera, 2015; Dong et al., 2016; Matos-Jr., Di Sabatino, Passerini, Favaro-Trindade, & Albertini, 2015; Trindade & Grosso, 2000). Spray chilling, which is also referred to as spray congealing or spray cooling, consists of atomization into a cold chamber of a solution dispersion or emulsion containing the active ingredient and a molten carrier for producing solid lipid microparticles (Okuro, de Matos, & Favaro-Trindade, 2013a). This encapsulation technique has the advantage of being a clean (not requiring solvents), low-cost, continuous, scalable and mild process (not requiring high temperatures). In this context, spray chilling has attracted significant interest from researchers and has been studied to encapsulate AA using dispersions as feed along with fully hydrogenated palm oil and vegetable glycerol monostearate as carriers (Matos-Jr., Di Sabatino, Passerini, Favaro-Trindade, & Albertini, 2015), dispersions as feed along with mixtures of lauric and oleic fatty acids as carriers (Sartori, Consoli, Hubinger, & Menegalli, 2015), and dispersions as feed along with mixtures of stearic fatty acid and hydrogenated vegetable fat as carriers (Alvim, Stein, Koury, Dantas, & Cruz, 2016).
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In all of these works solid lipid microparticles (SLM) were prepared using dispersions as feed, and they were effective at protecting AA. However, there are no reports describing the effectiveness of using emulsions as feed for preparing SLM loaded with AA. Hypothetically, emulsion-feed was supposed to keeps the entire content of AA inside the solid lipid matrix because in this case, the AA is surrounded by the surfactant, unlike in dispersion-feed, where the AA is dispersed all over the volume of the particles, including their surfaces. In fact, in a previous work of the group (Salvim et al., 2015), the feed preparation (by dispersion or emulsion) had a large influence on the characteristics of solid lipid microparticles loaded with soybean protein hydrolysate. Thus, the aim of this study was to evaluate the effects of the feed preparation (emulsion or dispersion) on the properties of solid lipid microparticles produced by spray chilling and on the stability of AA. 2. Materials and methods 2.1. Materials Ascorbic acid (AA) P.A. (Synth, S~ ao Paulo, Brazil) was used as the active material; interesterified fat with a melting point of 43 C, which was produced from fully hydrogenated palm oil and palm ~o Paulo, Brazil), was used as the lipid carrier; kernel oil (Vigor, Sa and soy lecithin powder (Pantec, S~ ao Paulo, Brazil) was used as the emulsifier. Chloroform, Tween 80, ethyl alcohol, sodium bicar~o Paulo, Brazil) as bonate and oxalic acid purchased from Synth (Sa well as 2,6-dichlorophenol-indophenol from Vetec (S~ ao Paulo, Brazil) were used to perform the analyses. All reagents were of analytical grade. 2.2. Treatments The established treatments are summarized in Table 1. Four treatments (T1-T4) were involved in the preparation of the emulsion with the lipid carrier, aqueous solution of AA and soya lecithin. These treatments varied in relation to the AA:carrier ratio of 1:10 and as a function of the rotational speed of the Ultra-Turrax (Ika, Staufen, Germany) during the preparation of the emulsion (523 and 1047 rad/s). The fifth treatment (T5) involved the preparation of an AA dispersion in the carrier in a ratio of 1:10. Treatments T6 and T7 consisted of the production of microparticles using pure lipid carrier and lipid carrier mixture along with soybean lecithin, respectively. The formulations of each treatment are shown in Table 2. 2.3. Methods 2.3.1. Production of solid lipid microparticles The solid lipid microparticles (SLM) were produced by a spray chilling technique that was conducted using the methodology described by Pelissari et al. (2016) with some modifications. For the
Table 1 Variables for each treatment evaluated for the production of solid lipid microparticles loaded with ascorbic acid produced by spray chilling. Treatment
Type of feed
Active material: Carrier (g:g)
Ultra-turrax (speed)
T1 T2 T3 T4 T5 T6 T7
Emulsion (E) Emulsion (E) Emulsion (E) Emulsion (E) Dispersion (D) Pure lipid Lipid þ soya lecithin
1:7 1:10 1:7 1:10 1:10 e e
5000 rpm 5000 rpm 10 000 rpm 10 000 rpm e e 5000 rpm
preparation of the emulsions, fat was weighed and melted at 58 C (15 C above the melting point); soybean lecithin in a powder form and an aqueous solution of AA (17.5 and 25 g of ascorbic acid in 100 g of water) were added to the mixture; and homogenization was performed with an Ultra-Turrax IKA®T25 (Staufen, Germany) for 3 min at a rotation that corresponded to the treatment. To prepare the dispersion, fat was weighed and melted at 58 C. Dry AA, previously ground in a porcelain pestle to a fine powder, was added and mixed using a Fisatom model 713 mechanical stirrer ~o Paulo, Brazil). To produce SLM that consisted of the carrier (Sa alone, the fat was melted at 58 C and subsequently atomized. The SLM that consisted of the mixture of the lipid carrier and soy lecithin was produced using a similar method, except the homogenization step of the process was performed on an Ultra-Turrax at 523 rad/s. To obtain the SLM, either as a dispersion or emulsion, the mixture of lipid carrier, soy lecithin and pure fat was sprayed in a cold room at 13 ± 2 C using a twin fluid atomizer (Ø ¼ 1.2 mm) with air pressure of 216 kPa in a spray chiller (Labmaq, S~ ao Paulo, Brazil). The flow rate was controlled using a peristaltic pump at 50 mL/min (Masterflex, Illinois, USA). The SLM were stored in closed glass in the presence of O2 protected from light at 7, 22 and 37 C. For characterization, the samples were stored at 7 C. 2.3.2. Microparticle characterization 2.3.2.1. Morphological analysis and particle size. The morphological analysis of the obtained SLM was performed using scanning electron microscopy (SEM). The samples were fixed on the sample holder using double-sided adhesive tape, coated with Au/Pd under an argon atmosphere using a high vacuum evaporator (Edwards, Crawley, UK) and examined using a scanning electron microscope (JEOL JSM-6500F, Welwyn Garden City, UK) with an accelerating voltage of 5 kV (Oliveira et al., 2007). The particle size was determined using a Sald-2-1V laser diffraction particle analyzer produced by Shimadzu (Tokyo, Japan). The microparticles were suspended in a Tween 80 aqueous solution (10 g/L) and stabilized for 5 min prior to analysis to prevent agglomeration (Pedroso, Thomazini, Heinemann, & Favaro-Trindade, 2012). The analysis was performed in triplicate. 2.3.2.2. Encapsulation efficiency. The encapsulation efficiency (EE) was determined as the difference between the total amount of AA and the amount of AA present on the surfaces of the SLM, as proposed by (Comunian et al., 2013). For the AA extraction from the SLM, the method proposed by (Maschke et al., 2007) using distilled water and chloroform was adopted. The washing of the SLM to determine the surface AA was performed according to the process described by (Leonel, Chambi, Barrera-Arellano, Pastore, & Grosso, 2010). The SLM were washed with distilled water so that the AA present on the particle surface was removed for quantification in the wash water. The Tillmans titration method described by the Association of Official Analytical Chemists (AOAC, 1998) was used to quantify the AA in terms of the total AA content and the content of AA on the surface of the particles. The calculation of the encapsulation efficiency was performed according to Equation (1).
Encapsulation efficiency ¼
ðtotal acid surface acidÞ 100 total acid (1)
2.3.2.3. Water activity. It was necessary to understand the correlation between the water activity and rate of chemical reactions with the AA degradation to evaluate the water activity of the microparticles after atomization. The measurement was performed
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Table 2 Formulation of each treatment evaluated for the production of solid lipid microparticles loaded with ascorbic acid produced by spray chilling. Treatment
T1 T2 T3 T4 T5 T6 T7
Ascorbic acid solution AA
Water
Concentration
5g 3.5 g 5g 3.5 g 3.5 g e e
20 20 20 20 e e e
25.0% 17.5% 25.0% 17.5% e e e
g g g g
Soybean lecithin
Lipid
0.3 0.3 0.3 0.3 e e 0.3
35 35 35 35 35 35 35
g g g g
g
g g g g g g g
T1: Emulsion with the proportion of active material: carrier of 1:7 at homogenization speed of 5000 rpm; T2: Emulsion with the proportion of active material: carrier of 1:10 at homogenization speed of 5000 rpm; T3: emulsion with the proportion of active material: carrier of 1:7 at homogenization speed of 10 000 rpm; T4: Emulsion with the proportion of active material: carrier of 1:10 at homogenization speed of 10 000 rpm; T5: Dispersion with the proportion of active material: carrier of 1:10; T6: pure lipid; T7: lipid þ soya lecithin.
using an Aqualab water activity analyzer (Decagon Devices, Pullman, USA) immediately after preparation of the SLM. 2.3.2.4. Thermal behavior. The thermal behavior of the SLM with AA was evaluated using a DSC thermal analyzer (TA Instruments, New Castle, USA). For the purposes of the analysis, approximately 10 mg of sample was placed in an aluminum capsule and heated at 10 C/min from 50 to 100 C under an inert atmosphere (45 mL/ min of N2) (Rocha, Trindade, Netto, & Favaro-Trindade, 2009). An empty capsule was used as a reference, and liquid nitrogen was used to cool the sample before each run. The lipid carrier, ascorbic acid, and soy lecithin were also subjected to thermal analysis. 2.3.2.5. Fourier-transform infrared spectroscopy (FTIR). The spectra of both ingredients and the SLM were determined using a PerkinElmer FT-IR spectrometer (Massachusetts, USA) and Spectrum One version 5.3.1 software. Sixteen scans were performed in total, and the working spectral region was from 600 to 4000 cm1 (Rocha-Selmi, Theodoro, Thomazini, Bolini, & Favaro-Trindade, 2013). 2.3.2.6. Evaluation of the stability of encapsulated ascorbic acid. The stability of the encapsulated AA was evaluated over a 60-day period, with measurements taken after 0, 7, 15, 30, 45 and 60 days of storage at 7, 22 and 37 ± 1 C. The SLM were protected from light and stored in closed glass covered with aluminum foil. Chloroform and distilled water were used to extract the AA from the microparticles; 250 mg of sample was weighed and placed in a test tube, and then, 5 mL of chloroform was added. Following homogenization, 5 mL of distilled water was added, and the homogenization was repeated. The aqueous phase was collected, and the procedure of water addition was repeated twice to ensure that the optimum extraction of ascorbic acid was achieved. The determination was carried out by titration according to the Tillmans method described by the Association of Official Analytical Chemists (AOAC, 1998). All analyses were performed in triplicate. 2.3.2.7. Evaluation of the stability of free ascorbic acid. The stability of free AA (in a 100 mL/L water solution) was evaluated over a period of 30 days, with measurements taken after 0, 1, 3, 5, 7, 15 and 30 days of storage in 7, 22 and 37 ± 1 C. The samples were stored under the same conditions as the microparticles. The determination was carried out by Tillmans titration method described by the Association of Official Analytical Chemists (AOAC, 1998). 2.4. Statistical analysis Statistical analysis was carried out in a completely randomized
design and repeated twice. The data obtained were analyzed by the analysis of variance (ANOVA) and Tukey's test using the Statistical Analysis System (SAS, 1995; Version 9.1.3). The results were considered to be statistically significant when a 0.05. The spraying process was carried out twice for each treatment. 3. Results and discussion The powders produced were white, cooled free-flowing, and different variables did not promote changes in their appearance. 3.1. Morphological analysis and particle size of the SLM The SLM loaded with AA presented a spherical shape independent of the formulation (Fig. 1). This format facilitates the flow of the powder. This feature confirms the results of two other studies where the same fat carrier was used to encapsulate probiotics and prebiotics by spray chilling (Okuro, Thomazini, Balieiro, Liberal & F avaro-Trindade, 2013b; Pedroso et al., 2012). The SLM prepared from feeds produced by emulsions (T1eT4) did not differ in their morphological characteristics. These SLM exhibited a smooth surface but with a strong presence of pores and imperfections. The presence of pores is undesirable because it can facilitate the entry of oxygen, which accelerates the degradation of AA. Furthermore, the presence of pores can facilitate the diffusion of water into the interior of the microparticles, providing the quick release of AA in an aqueous medium. Although the SLM examined in previous research have not always presented a smooth surface (Alvim, Souza, Koury, Jurt, & Dantas, 2013; Chambi, Alvim, Barrera-Arellano, & Grosso, 2008; Martins, Siqueira, & Freitas, 2012; Matos-Jr., Di Sabatino, Passerini, FavaroTrindade, & Albertini, 2015), the presence of pores is not frequently reported, except by Salvim et al. (2015), who also reported the presence of pores in the particles obtained by the atomization of emulsions when they encapsulated soy protein hydrolysate by spray chilling. In this context, the presence of pores in the SLM produced by the atomization of an emulsion can be attributed to water evaporation from the droplets during atomization. In contrast, rough and very small particle adhesion was observed on the surfaces of the SLM that were produced from the dispersion (T5). This can disturb the flow of the powder. Therefore, the evaluation of the morphology of the produced particles allows the inference that the atomization of the dispersion produces a particle structure that provides better protection to AA; however, it would offer greater resistance to the flow of powder. The values of the particle diameter are presented in Table 3. The results reveal that there were differences between the treatments due to the influence of various factors, and these deserve detailed
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Fig. 1. SEM micrographs of the following: (A, B and C) solid lipid microparticles loaded with ascorbic acid obtained from the emulsion observed at different magnifications; and (D, E and F) solid lipid microparticles loaded with ascorbic acid obtained from the dispersion observed with different magnifications.
Table 3 Encapsulation efficiency (EE), the mean volume diameter (MVD) and activity water (Aw) of solid lipid microparticles loaded with ascorbic acid produced by spray chilling. Treatment
EE (%)
T1 T2 T3 T4 T5 T6 T7
68.2 71.0 69.9 72.5 59.2 e e
± ± ± ± ±
MVD (mm) 3.4 2.6 4.0 3.1 4.2
a a a a b
73.86 74.69 76.78 72.87 91.62 44.92 33.82
± ± ± ± ± ± ±
2.1 1.9 1.1 3.8 1.5 3.1 2.4
Aw a a a a b c d
0.963 0.965 0.966 0.969 0.631 0.690 0.632
± ± ± ± ± ± ±
0.009 0.017 0.021 0.005 0.014 0.011 0.008
a a a a b c b
T1: Emulsion with the proportion of active material: carrier of 1:7 at homogenization speed of 5000 rpm; T2: Emulsion with the proportion of active material: carrier of 1:10 at homogenization speed of 5000 rpm; T3: emulsion with the proportion of active material: carrier of 1:7 at homogenization speed of 10 000 rpm; T4: Emulsion with the proportion of active material: carrier of 1:10 at homogenization speed of 10 000 rpm; T5: Dispersion with the proportion of active material: carrier of 1:10; T6: pure lipid; T7: lipid þ soya lecithin. There were no significant differences among the samples with the same letters in the same column (p < 0.05). The analyses were performed in triplicate.
analysis. The particle size distributions for all of the formulations exhibited unimodal behavior; i.e., with just one peak (Fig. 2). This allows discussion of the particle size, and all of the formulations showed poly-dispersed distributions, with values ranging from 34 ± 2 to 92 ± 2 mm. Comparing the treatments T1 with T2 and T3 with T4, which have different concentrations of AA but were emulsified using the same Ultra-Turrax rotations, it is possible to infer that the proportion of AA in the SLM produced by the atomization of the emulsion did not affect the average particle size. This may be explained by the fact that in all these treatments, the AA was always added to the water in a soluble form, and the amount of solution added did not change; only its concentration changed (17.5 g/100 g for T2 and T4; 25 g/100 g for T1 and T3). Similar results were reported by Leonel et al. (2010), who produced SLM loaded with glucose solution using mixtures of stearic and oleic fatty acids, hydrogenated vegetable fat as a carrier and soybean lecithin as a surfactant.
The variation in the speed of Ultra-Turrax (523 and 1047 rad/s) for the preparation of the emulsion also did not significantly alter the average particle sizes, possibly because the rate of 1047 rad/s (Treatments 3 and 4) was not enough to reduce the size of the emulsion droplets in comparison to the rate of 523 rad/s (T1 and T2). Most likely, this variation in the speed of the Ultra-Turrax did not induce a reduction in the viscosity of the emulsion since according to Albertini, Passerini, Pattarino, and Rodriguez (2008), the particle size produced by the spray congealing technique is heavily influenced by the viscosity of the feed. When comparing treatments involving emulsions (T1-T4) with the other treatments (T5-T7), the influence of the inlet fluid viscosity on the particle size became relevant. Particles produced only with fat (T6) were significantly lower than those produced by mixing fat with soybean lecithin (T7), which was lower than those prepared with the mixture of fat and dry AA (T5). These differences can be attributed to the fact that lecithin is a fine powder that was added in small amounts compared to AA; moreover, the lecithin acts by decreasing the viscosity of the medium. In terms of the AA added to the dispersion, although it was macerated and sieved using a 100 mm sieve, the powder was not as fine as the lecithin powder. In this context, although the viscosities of the feeds have not been studied in this work, it can be inferred that this parameter has a significant influence on the size of the particles obtained by spray chilling, as reported by other authors (Di Sabatino, Albertini, Kett, & Passerini, 2012; Maschke et al., 2007). The samples produced with emulsified AA (T1 to T4) had a significantly smaller medium particle size (p < 0.05) than those produced with AA dispersed in the carrier (T5) (Table 3). Therefore, it can be argued that the application of particles produced by an emulsion (T1 to T4) to food must have a minor impact on its texture. Process parameters, especially the atomization pressure and temperature, influence the particle size (Di Sabatino et al., 2012; Maschke et al., 2007; Matos-Jr., Di Sabatino, Passerini, FavaroTrindade, & Albertini, 2015). In addition to the process parameters, the type of active material, lipid carrier and design of the atomizer can also cause considerable variations in size. The series of
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Fig. 2. Particle size distribution of solid lipid microparticles loaded with ascorbic acid produced by spray chilling. T1: Emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 5000 rpm ( ); T2: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 5000 rpm ( ); T3: emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 10,000 rpm ( ); T4: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 10,000 rpm ( ); T5: Dispersion with the proportion of active material: carrier of 1:10 mL:mL ( ); T6: pure lipid ( ); T7: lipid þ soya lecithin ( ).
factors impacting this feature complicates comparisons between existing research studies. 3.2. Efficiency of microencapsulation The encapsulation efficiency values are shown in Table 3. There were no significant differences between the results obtained for the EE values of SLM produced by the atomization of emulsions. This implies that the ratio of active material:carrier and the rotation of the Ultra-Turrax had no influence on the amount of AA that remained on the surface of the microparticles in relation to the amount that had been effectively covered by the carrier. These results become consistent when the relationship with the particle size results was considered. As with the EE, the particle size was not affected by the variables described earlier. If there was no difference in size, the surface area of the particles was not changed, which could allow a smaller or larger presence of AA on the surface. Morselli Ribeiro, Barrera Arellano, and Ferreira Grosso (2012) encapsulated glucose solution in a mixture of stearic acid and oleic acid using a chilling spray technique and found different results. They found that an increase in the oleic acid concentration in relation to the active material had a positive influence on the EE. It is worth noting that the authors of this study used the same methodology to determine the EE as that employed in the current study. A comparison of the treatment that involved the preparation of a dispersion (T5) with the others (T1-T4) revealed there was a significant difference in terms of the EE values. The amount of AA on the surface was greater in the T5 dispersion, and this resulted in a lower EE value. In this case, the argument that the particle size influences the EE due to the surface area loses consistency because the particles of the T5 dispersion were larger than the others; as such, they had a smaller surface area. This difference can be attributed to the effect of feed preparation because for the treatments T1 to T4, the AA was emulsified so it was in the dispersed phase (water) of the W/O emulsion and covered by the emulsifier. Hence, the AA is less likely to be on the particle surface and to be entrapped in the wash water. Although the presence of pores was detected in these particles, as discussed previously, these may have contributed to the drag of AA even when emulsified. The EE values varied from 68.2 to 72.5% for these treatments. For the dispersion preparation (T5), although macerated to a
fine powder (<100 mm), the AA remained in the form of considerably large crystals, even if we take into consideration the sizes of the particles obtained. During the washing of the particles, any AA content that is not perfectly immobilized in the lipid matrix can be entrapped by the wash water. The AA is a highly hydrophilic compound so this “drag” is considerably favored. A comparison of the results obtained in the current research with those obtained from previous studies that have evaluated the EE using a similar methodology or those that have determined the active material present on the particle surface reveals that the values obtained are in the range of those reported by previous research. Leonel et al. (2010) encapsulated a glucose solution in mixtures of liquid and solid fatty acids (stearic and oleic acid) as well as hydrogenated vegetable fat, and they found values of glucose on the surface that ranged from 2 to 20%. They found that the theoretical values of glucose in the microparticles did not influence the EE or the concentration of glucose in solution. Morselli Ribeiro et al. (2012), who also encapsulated glucose solution, reported that the values of the active material on the surface ranged between 3 and 24%. Of the various analyses for the characterization of the microparticles obtained by spray chilling, the encapsulation efficiency needs to be treated more critically in the interpretation and discussion of the results. This is due to the lack of consensus on the definition of the encapsulation efficiency. In the current study, it was assumed that the encapsulation efficiency refers to the amount of active material effectively encapsulated. To determine the EE, the difference between the total content of ascorbic acid in the SLM and the content on the surfaces of the particles was calculated. In the literature, it is common to find EE values that have been determined by calculating the difference between the theoretical amount of atomized active material and the amount of active material present in the microparticles. This analysis is very useful as a means of verifying that the atomization process causes the degradation/loss of the active material; however, it does not provide insights into whether the active material is inside or on the surface of the SLM. If there is no criterion in the discussion of the results, the possibility of errors in relation to the values close to 100% reported and assigned as the EE emerges. Based on the consideration that the spray chilling technique produces microparticles and not microcapsules, where the active material is perfectly packaged within the microcapsules (Fig. 1), it is impossible to produce
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particles without active material on their surfaces. Finally, if the authors do not consider the difference between the amount of encapsulated active material and the active material present on the surfaces of the microparticles, the function of microencapsulation can be considerably impaired, especially when the purpose of the process is to protect the active material, mask taste, and/or disguise odor, for example. The results of the EE, when considered as per the methods employed in this study, provide the information required to evaluate the need for adjustments in the process, such as changes in the amount of active material in the formulation and/or manipulation of particle size, with the purpose of the latter being to reduce the surface area of the particle. 3.3. Water activity and moisture Information about the water activity in the particles is relevant because it provides insights into the extent to which the kinetics degradation of the active material can be influenced by the presence of water. The average values of water activity and moisture are shown in Table 3. Accordingly, treatments involving the emulsion preparation presented higher values of Aw and moisture in relation to the other treatments; therefore, they were subjected to more microbial and chemical changes. However, the microbial growth in the particles will be not supported due to the lack of nutrients because according to Bhat, Alias, and Paliyath (2012), relatively few types of microorganisms are capable of metabolizing lipids. Additionally, there isn't a source of nitrogen in the particles and just AA as a vitamin. However, particles produced by an emulsion-feed, as a function of their higher Aw, are subjected to more chemical reactions, which can explain the lower stability of AA in them, as discussed in the item 3.6. 3.4. Thermal behavior The DSC thermograms are presented in Fig. 3. No peak was observed for soy lecithin and AA. The absence of a peak occurs because the melting points of these ingredients are above 100 C and out of the temperature range studied. The fat used as a carrier presented a melting peak at approximately 43 C. In the analysis of the microparticles, this behavior was repeated; as such, the same melting peak was observed independent of the treatment. However, for treatments T1 to T4, a slight narrowing of the peak was observed, which can be attributed to the interaction of the fat with lecithin. Due to the similarity between the samples and the fat in relation to this peak, no fat interaction with the lipophilic material was observed. This would lead to depression of the melting point of the fat and could change the polymorphic behavior of the fat. The presence of an earlier peak at approximately 0 C for the fat was verified for the treatments that were prepared from an emulsion (T1eT4); this is related to the melting process of water. For treatment 5, with the dispersion preparation, this peak was not observed since there was no water in the formulation. 3.5. Fourier-transform infrared spectroscopy (FTIR) Fourier transform infrared spectroscopy was performed to identify possible interactions between the carrier and components of the formulations. The spectra of ingredients and the SLM are shown in Fig. 4. As the spectra of the SLM prepared with emulsions (T1eT4) were similar, the decision was made to present only one spectrum. The spectra obtained for the three pure ingredients were similar to those reported in the literature. For the AA, the characteristic
Fig. 3. Thermograms of the solid lipid microparticles loaded with ascorbic acid produced by spray chilling and pure ingredients (ascorbic acid, soya lecithin and lipid). T1: Emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 5000 rpm; T2: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 5000 rpm; T3: emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 10,000 rpm; T4: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 10,000 rpm; T5: Dispersion with the proportion of active material: carrier of 1:10 mL:mL.
peaks were obtained at 1021, 1109, 1310, 1650, 1750, 3400 and 3520 cm1. The peaks at 1109, 1310 and 1650 cm1 are assigned to the carbonyl (C]O) and hydroxyl (OH) groups present in the vitamin C molecule (Desai & Park, 2005). For soybean lecithin, the characteristic peaks were 1042, 2851 and 2919 cm1, the first of which corresponded to the links with phosphorus (PeO) (Silverstein, Webster, & Kiemle, 2005). Finally, the characteristic peaks for pure fat were 1738, 2851 and 2919 cm1, with the first band having moderate intensity and the last two bands having stronger intensities, which are related to the presence of carbonyl compounds, more specifically, carboxylic acids. Another characteristic peak in the fat was observed at 1170 cm1, which is related to the presence of esters (Silverstein et al., 2005). For the SLM, the spectra were consistent and characteristic peaks of AA, fat and soy lecithin (when in the formulation) appeared in all of the treatments. These similar characteristics confirmed that active material was present in the microparticles; thus, it was not lost in the encapsulation process. The peak that appeared in these samples near 3350 cm-1 is related to the axial strain (OeH) in intermolecular hydrogen bonding, thus indicating the presence of water (Silverstein et al., 2005). This is easily explained because there was water in the systems for treatments 1e4, which were prepared with an emulsion, and this was different than for treatment 5, which was a dispersion of anhydrous AA and fat. 3.6. Assessment of the stability of free AA and microencapsulated AA The stability of free AA in solution is shown in Fig. 5. The
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257
Fig. 4. FTIR spectra of the solid lipid microparticles loaded with ascorbic acid produced by spray chilling and pure ingredients (ascorbic acid, soya lecithin and lipid). T1: Emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 5000 rpm; T2: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 5000 rpm; T3: emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 10,000 rpm; T4: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 10,000 rpm; T5: Dispersion with the proportion of active material: carrier of 1:10 mL:mL.
influence of the storage temperature on the stability of ascorbic acid was clear. As expected, the higher the storage temperature, the greater the degradation rate of the AA. Following 15 days of storage,
the remaining contents of free AA and those in solution were 0, 20 and 40% for storage at 37, 24 and 7 C, respectively. These results are very similar to those reported by authors who studied the stability
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Fig. 5. Stability of free ascorbic acid stored for 30 days at temperatures of 7 (:), 24 (C) and 37 C (,).
of AA in aqueous solution (Comunian et al., 2013 and Comunian et al., 2014). In Fig. 6, it is possible to observe the stability of the microencapsulated AA. The storage temperature, as was the case with the free AA, considerably influenced its stability. Treatments that involved the preparation of an emulsion behaved similarly. The treatment that involved the dispersion preparation (T5) presented different results in terms of the stability of the AA across all of the storage temperatures. The maintenance of stability was much higher in T5. This difference could be readily observed due to the color changes of the samples, and this was the treatment that presented the smallest color change. The presence of water in the emulsion treatments was certainly a determining factor in the explanation of these results. In fact, according to Damodaran, Parkin, and Fennema (2008), AA is very susceptible to oxidation, and factors such as the oxygen concentration and water activity influence the rate of this reaction. Along with the water activity, values were higher in the particles produced by atomization of emulsions (Table 3); as discussed previously, these samples presented pores, which may have favored the diffusion of oxygen. If the results of the current study are compared with existing research involving the encapsulation and evaluation of the stability of AA, including those that examined spray drying (Trindade & Grosso, 2000), complex coacervation (Comunian et al., 2013), microfluidic devices (Comunian et al., 2014) and fluidized bed coating (Knezevic, Gosak, Hraste, & Jalsenjako, 1998), the stability provided by the AA microparticles produced by spray chilling with an emulsion was low; however, the particles produced with a dispersion showed satisfactory results. After 60 days of storage at 7 and 24 C, the AA present in the particles produced by the atomization of the emulsion remained stable at greater than 80%. At 37 C, the stability after 60 days was maintained above 60%. These results indicate that, compared to free AA in solution, these particles are a good alternative to confer protection to AA. Furthermore, if an improvement in their preparation can be achieved, this will improve the encapsulation efficiency and increase the stability values. The original idea behind the spray chilling technique was based on the notion of the production of microparticles through the atomization of the mixture between the carrier and active material, and there is no type of solvent involved in the process. However, some research has recently emerged in the literature (Chambi et al., 2008; Leonel et al., 2010; Morselli Ribeiro et al., 2012; Okuro, de Matos, & Favaro-Trindade, 2013a; Pedroso et al., 2012) in which, similar to this present work, atomized emulsions were used in such
Fig. 6. Stability of encapsulated ascorbic acid stored for 60 days at temperatures of 7 (A), 24 (B) and 37 C (C). T1: Emulsion with the proportion of active material: carrier of 1:7 at homogenization speed of 5000 rpm (:); T2: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 5000 rpm (d); T3: emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 10,000 rpm (C); T4: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 10,000 rpm (A); T5: Dispersion with the proportion of active material: carrier of 1:10 mL:mL (-).
a manner that the active material was mixed with the lipid carrier in aqueous solution with the aid of an emulsifier. Evaluation of the results influenced by the Aw in terms of the impact on the function of the microencapsulation revealed, in this case, that the maintenance of the stability of the AA represents an important consideration if these technical adaptations do not contradict the advantages of it. Furthermore, anticipating the conclusions of this work, it was found that the adaptation of the technique, atomization of an emulsion instead of a dispersion, did not favor it but
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prejudiced the purpose of microencapsulation, which was to confer protection to the AA. However, there is no doubt that encapsulation is a promising technology to protect ascorbic acid (Alvim et al., 2016). When AA encapsulated by spray drying and spray chilling was added into biscuits, it was reported that the AA was protected to a greater extent during baking when it was encapsulated by spray drying, followed closely by spray chilling. These authors also reported that AA encapsulation inhibited the formation of dark spots on the biscuits that were associated with the thermal degradation of this compound during baking. Comunian et al. (2014) added AA encapsulated by a double emulsion followed by complex coacervation in chicken frankfurters and observed that this compound was released from the capsules during processing and/or storage due to the oxidative stability presented in the samples. Moreover, the sausages that were produced using encapsulated AA showed good sensory acceptability. 4. Conclusion The comparison between the two forms of feed preparation proposed in this study indicates that both methods have advantages and disadvantages. However, the dispersion generated more promising results in relation to the stability of the AA. Further studies are necessary to improve the encapsulation efficiency of microparticles produced with the dispersion. These new studies may involve varying the proportion of the carrier to the active material as well as the use of other types of carriers. The lipid microparticles loaded with AA obtained by spray chilling can be applied to various types of food products, particularly those in which AA may suffer degradation during product processing, thus impeding its activity during the food storage period. Acknowledgments ~o de Amparo a Pesquisa do The authors thank the Fundaça ~o Paulo for the scholarships that were granted (ProEstado de Sa cesses 2010/13117-5 and 2012/01907-7). Favaro-Trindade C.S. thanks CNPq for the productivity grant (306708/2015-9). References Albertini, B., Passerini, N., Pattarino, F., & Rodriguez, L. (2008). New spray congealing atomizer for the microencapsulation of highly concentrated solid and liquid substances. European Journal of Pharmaceutics and Biopharmaceutics, 69, 348e357. http://doi.org/10.1016/j.ejpb.2007.09.011. Alvim, I. D., Souza, F. da S. de, Koury, I. P., Jurt, T., & Dantas, F. B. H. (2013). Use of the spray chilling method to deliver hydrophobic components: Physical characterization of microparticles. Food Science and Technology, 33, 34e39. Retrieved from http://www.scielo.br/scielo.php?script¼sci_arttext&pid¼S0101-20612013 000500006&nrm¼iso. Alvim, I. D., Stein, M. A., Koury, I. P., Dantas, F. B. H., & Cruz C. L. de C. V.. (2016). Comparison between the spray drying and spray chilling microparticles contain ascorbic acid in a baked product application. LWT - Food Science and Technology, 65, 689e694. http://doi.org/. http://dx.doi.org/10.1016/j.lwt.2015.08.049. AOAC. (1998). Official methods of analysis of AOAC international. Association of Official Analysis Chemists International, 9, CDeROM http://doi.org/10.3109/ 15563657608988149. Bhat, R., Alias, A. K., & Paliyath, G. (2012). Progress in food preservation. Oxford: Wiley Blackwell. Chambi, H. N. M., Alvim, I. D., Barrera-Arellano, D., & Grosso, C. R. F. (2008). Solid lipid microparticles containing water-soluble compounds of different molecular mass: Production, characterisation and release profiles. Food Research International, 41, 229e236. http://doi.org/10.1016/j.foodres.2007.11.012. Comunian, T. A., Thomazini, M., Alves, A. J. G., de Matos Junior, F. E., de Carvalho Balieiro, J. C., & Favaro-Trindade, C. S. (2013). Microencapsulation of ascorbic acid by complex coacervation: Protection and controlled release. Food Research International, 52(1), 373e379. Comunian, T. A., Thomazini, M., Gambagorte, V. F., Trindade, M. A., & FavaroTrindade, C. S. (2014). Effect of incorporating free or encapsulated ascorbic acid in chicken frankfurters on physicochemical and sensory stability. Journal of Food Science and Engineering, 4, 167e175.
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Food Science and Technology (Campinas), 30, 276e281. Retrieved fromhttp://www. scielo.br/scielo.php?script¼sci_arttext&pid¼S010120612010000100040&nrm¼iso. Martins, R. M., Siqueira, S., & Freitas, L. A. P. (2012). Spray congealing of Pharmaceuticals: Study on production of solid dispersions using box-behnken design. Drying Technology, 30. http://doi.org/10.1080/07373937.2011.633251. €pferich, A. (2007). Maschke, A., Becker, C., Eyrich, D., Kiermaier, J., Blunk, T., & Go Development of a spray congealing process for the preparation of insulinloaded lipid microparticles and characterization thereof. European Journal of Pharmaceutics and Biopharmaceutics, 65, 175e187. http://doi.org/10.1016/j.ejpb. 2006.08.008. Matos-, F. E., Jr., Di Sabatino, M., Passerini, N., Favaro-Trindade, C. S., & Albertini, B. (2015). Development and characterization of solid lipid microparticles loaded with ascorbic acid and produced by spray congealing. 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Effect of feed preparation on the properties and stability of ascorbic acid microparticles produced by spray chilling quio de Matos-Jr a, b, *, Talita Aline Comunian a, Marcelo Thomazini a, Fernando Eusta Carmen Sílvia Favaro-Trindade a ~o Paulo, College of Animal Science and Food Engineering, Av. Duque de Caxias Norte, 225, CP 23, CEP 13535 900, Pirassununga, Sa ~o Paulo, University of Sa Brazil b rio, BR 364, Km 04, Distrito industrial, CEP 69.920 900, Rio Branco, Acre, Federal University of Acre, Science Center of Health and Sport, Campus Universita Brazil a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 November 2015 Received in revised form 30 August 2016 Accepted 4 September 2016 Available online 5 September 2016
The aim of this study was to evaluate the effect of the feed form, dispersion or emulsion, on the production of solid lipid microparticles (SLM) loaded with ascorbic acid (AA) using interesterified fat as a carrier. The particles obtained were characterized according to the particle size distribution and morphology using SEM, thermal behavior using DSC and encapsulation efficiency. The stabilities of the free and encapsulated AA were evaluated over a period of 60 days. Spherical microparticles were obtained with diameters between 34 and 92 mm. The encapsulation efficiency varied according to the type of feed used and ranged from 59 to 73%. The stability of the AA after 60 days of storage exceeded 70%, even upon storage at 37 C. Microencapsulation by spray chilling was effective in maintaining the stability of AA. Microparticles produced from the dispersion promoted more protection of AA in the three storage temperature conditions. The particles produced could also allow controlled liberation of AA and masking of its acidic taste, which is not always desirable. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Vitamin C Microencapsulation Spray congealing Solid lipid microparticles Characterization
1. Introduction Ascorbic acid (AA) or vitamin C is used to fortify, as an antioxidant and as a reducing agent in many foods. However, to prepare foods enhanced with AA is not an easy task since this compound is very unstable. Microencapsulation has been successfully used to overcome the various limitations associated with the addition of bioactive compounds to food (Gharsallaoui, Roudaut, Chambin, Voilley, & Saurel, 2007). The basic process associated with this technology involves packaging solid, liquid or gaseous substances in extremely small capsules that can release their contents in a controlled manner and under specific conditions (Todd, 1970). Several techniques have been used to encapsulate AA, such as spray drying, microfluidic devices, double emulsion followed by complex coacervation, liposome preparation, O/W microemulsions and spray chilling (Comunian, Thomazini, Alves, Matos Junior,
* Corresponding author. University of S~ ao Paulo, College of Animal Science and Food Engineering, Av. Duque de Caxias Norte, 225, CP 23, CEP 13535 900, Pirassununga, S~ ao Paulo, Brazil. E-mail address: [email protected] (F.E. de Matos-Jr). http://dx.doi.org/10.1016/j.lwt.2016.09.006 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
Balieiro, & Favaro-Trindade, 2013; Comunian, Thomazini, Gambagorte, Trindade, & Favaro-Trindade, 2014; Dissanayake, Karunaratne, & Chandani Perera, 2015; Dong et al., 2016; Matos-Jr., Di Sabatino, Passerini, Favaro-Trindade, & Albertini, 2015; Trindade & Grosso, 2000). Spray chilling, which is also referred to as spray congealing or spray cooling, consists of atomization into a cold chamber of a solution dispersion or emulsion containing the active ingredient and a molten carrier for producing solid lipid microparticles (Okuro, de Matos, & Favaro-Trindade, 2013a). This encapsulation technique has the advantage of being a clean (not requiring solvents), low-cost, continuous, scalable and mild process (not requiring high temperatures). In this context, spray chilling has attracted significant interest from researchers and has been studied to encapsulate AA using dispersions as feed along with fully hydrogenated palm oil and vegetable glycerol monostearate as carriers (Matos-Jr., Di Sabatino, Passerini, Favaro-Trindade, & Albertini, 2015), dispersions as feed along with mixtures of lauric and oleic fatty acids as carriers (Sartori, Consoli, Hubinger, & Menegalli, 2015), and dispersions as feed along with mixtures of stearic fatty acid and hydrogenated vegetable fat as carriers (Alvim, Stein, Koury, Dantas, & Cruz, 2016).
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In all of these works solid lipid microparticles (SLM) were prepared using dispersions as feed, and they were effective at protecting AA. However, there are no reports describing the effectiveness of using emulsions as feed for preparing SLM loaded with AA. Hypothetically, emulsion-feed was supposed to keeps the entire content of AA inside the solid lipid matrix because in this case, the AA is surrounded by the surfactant, unlike in dispersion-feed, where the AA is dispersed all over the volume of the particles, including their surfaces. In fact, in a previous work of the group (Salvim et al., 2015), the feed preparation (by dispersion or emulsion) had a large influence on the characteristics of solid lipid microparticles loaded with soybean protein hydrolysate. Thus, the aim of this study was to evaluate the effects of the feed preparation (emulsion or dispersion) on the properties of solid lipid microparticles produced by spray chilling and on the stability of AA. 2. Materials and methods 2.1. Materials Ascorbic acid (AA) P.A. (Synth, S~ ao Paulo, Brazil) was used as the active material; interesterified fat with a melting point of 43 C, which was produced from fully hydrogenated palm oil and palm ~o Paulo, Brazil), was used as the lipid carrier; kernel oil (Vigor, Sa and soy lecithin powder (Pantec, S~ ao Paulo, Brazil) was used as the emulsifier. Chloroform, Tween 80, ethyl alcohol, sodium bicar~o Paulo, Brazil) as bonate and oxalic acid purchased from Synth (Sa well as 2,6-dichlorophenol-indophenol from Vetec (S~ ao Paulo, Brazil) were used to perform the analyses. All reagents were of analytical grade. 2.2. Treatments The established treatments are summarized in Table 1. Four treatments (T1-T4) were involved in the preparation of the emulsion with the lipid carrier, aqueous solution of AA and soya lecithin. These treatments varied in relation to the AA:carrier ratio of 1:10 and as a function of the rotational speed of the Ultra-Turrax (Ika, Staufen, Germany) during the preparation of the emulsion (523 and 1047 rad/s). The fifth treatment (T5) involved the preparation of an AA dispersion in the carrier in a ratio of 1:10. Treatments T6 and T7 consisted of the production of microparticles using pure lipid carrier and lipid carrier mixture along with soybean lecithin, respectively. The formulations of each treatment are shown in Table 2. 2.3. Methods 2.3.1. Production of solid lipid microparticles The solid lipid microparticles (SLM) were produced by a spray chilling technique that was conducted using the methodology described by Pelissari et al. (2016) with some modifications. For the
Table 1 Variables for each treatment evaluated for the production of solid lipid microparticles loaded with ascorbic acid produced by spray chilling. Treatment
Type of feed
Active material: Carrier (g:g)
Ultra-turrax (speed)
T1 T2 T3 T4 T5 T6 T7
Emulsion (E) Emulsion (E) Emulsion (E) Emulsion (E) Dispersion (D) Pure lipid Lipid þ soya lecithin
1:7 1:10 1:7 1:10 1:10 e e
5000 rpm 5000 rpm 10 000 rpm 10 000 rpm e e 5000 rpm
preparation of the emulsions, fat was weighed and melted at 58 C (15 C above the melting point); soybean lecithin in a powder form and an aqueous solution of AA (17.5 and 25 g of ascorbic acid in 100 g of water) were added to the mixture; and homogenization was performed with an Ultra-Turrax IKA®T25 (Staufen, Germany) for 3 min at a rotation that corresponded to the treatment. To prepare the dispersion, fat was weighed and melted at 58 C. Dry AA, previously ground in a porcelain pestle to a fine powder, was added and mixed using a Fisatom model 713 mechanical stirrer ~o Paulo, Brazil). To produce SLM that consisted of the carrier (Sa alone, the fat was melted at 58 C and subsequently atomized. The SLM that consisted of the mixture of the lipid carrier and soy lecithin was produced using a similar method, except the homogenization step of the process was performed on an Ultra-Turrax at 523 rad/s. To obtain the SLM, either as a dispersion or emulsion, the mixture of lipid carrier, soy lecithin and pure fat was sprayed in a cold room at 13 ± 2 C using a twin fluid atomizer (Ø ¼ 1.2 mm) with air pressure of 216 kPa in a spray chiller (Labmaq, S~ ao Paulo, Brazil). The flow rate was controlled using a peristaltic pump at 50 mL/min (Masterflex, Illinois, USA). The SLM were stored in closed glass in the presence of O2 protected from light at 7, 22 and 37 C. For characterization, the samples were stored at 7 C. 2.3.2. Microparticle characterization 2.3.2.1. Morphological analysis and particle size. The morphological analysis of the obtained SLM was performed using scanning electron microscopy (SEM). The samples were fixed on the sample holder using double-sided adhesive tape, coated with Au/Pd under an argon atmosphere using a high vacuum evaporator (Edwards, Crawley, UK) and examined using a scanning electron microscope (JEOL JSM-6500F, Welwyn Garden City, UK) with an accelerating voltage of 5 kV (Oliveira et al., 2007). The particle size was determined using a Sald-2-1V laser diffraction particle analyzer produced by Shimadzu (Tokyo, Japan). The microparticles were suspended in a Tween 80 aqueous solution (10 g/L) and stabilized for 5 min prior to analysis to prevent agglomeration (Pedroso, Thomazini, Heinemann, & Favaro-Trindade, 2012). The analysis was performed in triplicate. 2.3.2.2. Encapsulation efficiency. The encapsulation efficiency (EE) was determined as the difference between the total amount of AA and the amount of AA present on the surfaces of the SLM, as proposed by (Comunian et al., 2013). For the AA extraction from the SLM, the method proposed by (Maschke et al., 2007) using distilled water and chloroform was adopted. The washing of the SLM to determine the surface AA was performed according to the process described by (Leonel, Chambi, Barrera-Arellano, Pastore, & Grosso, 2010). The SLM were washed with distilled water so that the AA present on the particle surface was removed for quantification in the wash water. The Tillmans titration method described by the Association of Official Analytical Chemists (AOAC, 1998) was used to quantify the AA in terms of the total AA content and the content of AA on the surface of the particles. The calculation of the encapsulation efficiency was performed according to Equation (1).
Encapsulation efficiency ¼
ðtotal acid surface acidÞ 100 total acid (1)
2.3.2.3. Water activity. It was necessary to understand the correlation between the water activity and rate of chemical reactions with the AA degradation to evaluate the water activity of the microparticles after atomization. The measurement was performed
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253
Table 2 Formulation of each treatment evaluated for the production of solid lipid microparticles loaded with ascorbic acid produced by spray chilling. Treatment
T1 T2 T3 T4 T5 T6 T7
Ascorbic acid solution AA
Water
Concentration
5g 3.5 g 5g 3.5 g 3.5 g e e
20 20 20 20 e e e
25.0% 17.5% 25.0% 17.5% e e e
g g g g
Soybean lecithin
Lipid
0.3 0.3 0.3 0.3 e e 0.3
35 35 35 35 35 35 35
g g g g
g
g g g g g g g
T1: Emulsion with the proportion of active material: carrier of 1:7 at homogenization speed of 5000 rpm; T2: Emulsion with the proportion of active material: carrier of 1:10 at homogenization speed of 5000 rpm; T3: emulsion with the proportion of active material: carrier of 1:7 at homogenization speed of 10 000 rpm; T4: Emulsion with the proportion of active material: carrier of 1:10 at homogenization speed of 10 000 rpm; T5: Dispersion with the proportion of active material: carrier of 1:10; T6: pure lipid; T7: lipid þ soya lecithin.
using an Aqualab water activity analyzer (Decagon Devices, Pullman, USA) immediately after preparation of the SLM. 2.3.2.4. Thermal behavior. The thermal behavior of the SLM with AA was evaluated using a DSC thermal analyzer (TA Instruments, New Castle, USA). For the purposes of the analysis, approximately 10 mg of sample was placed in an aluminum capsule and heated at 10 C/min from 50 to 100 C under an inert atmosphere (45 mL/ min of N2) (Rocha, Trindade, Netto, & Favaro-Trindade, 2009). An empty capsule was used as a reference, and liquid nitrogen was used to cool the sample before each run. The lipid carrier, ascorbic acid, and soy lecithin were also subjected to thermal analysis. 2.3.2.5. Fourier-transform infrared spectroscopy (FTIR). The spectra of both ingredients and the SLM were determined using a PerkinElmer FT-IR spectrometer (Massachusetts, USA) and Spectrum One version 5.3.1 software. Sixteen scans were performed in total, and the working spectral region was from 600 to 4000 cm1 (Rocha-Selmi, Theodoro, Thomazini, Bolini, & Favaro-Trindade, 2013). 2.3.2.6. Evaluation of the stability of encapsulated ascorbic acid. The stability of the encapsulated AA was evaluated over a 60-day period, with measurements taken after 0, 7, 15, 30, 45 and 60 days of storage at 7, 22 and 37 ± 1 C. The SLM were protected from light and stored in closed glass covered with aluminum foil. Chloroform and distilled water were used to extract the AA from the microparticles; 250 mg of sample was weighed and placed in a test tube, and then, 5 mL of chloroform was added. Following homogenization, 5 mL of distilled water was added, and the homogenization was repeated. The aqueous phase was collected, and the procedure of water addition was repeated twice to ensure that the optimum extraction of ascorbic acid was achieved. The determination was carried out by titration according to the Tillmans method described by the Association of Official Analytical Chemists (AOAC, 1998). All analyses were performed in triplicate. 2.3.2.7. Evaluation of the stability of free ascorbic acid. The stability of free AA (in a 100 mL/L water solution) was evaluated over a period of 30 days, with measurements taken after 0, 1, 3, 5, 7, 15 and 30 days of storage in 7, 22 and 37 ± 1 C. The samples were stored under the same conditions as the microparticles. The determination was carried out by Tillmans titration method described by the Association of Official Analytical Chemists (AOAC, 1998). 2.4. Statistical analysis Statistical analysis was carried out in a completely randomized
design and repeated twice. The data obtained were analyzed by the analysis of variance (ANOVA) and Tukey's test using the Statistical Analysis System (SAS, 1995; Version 9.1.3). The results were considered to be statistically significant when a 0.05. The spraying process was carried out twice for each treatment. 3. Results and discussion The powders produced were white, cooled free-flowing, and different variables did not promote changes in their appearance. 3.1. Morphological analysis and particle size of the SLM The SLM loaded with AA presented a spherical shape independent of the formulation (Fig. 1). This format facilitates the flow of the powder. This feature confirms the results of two other studies where the same fat carrier was used to encapsulate probiotics and prebiotics by spray chilling (Okuro, Thomazini, Balieiro, Liberal & F avaro-Trindade, 2013b; Pedroso et al., 2012). The SLM prepared from feeds produced by emulsions (T1eT4) did not differ in their morphological characteristics. These SLM exhibited a smooth surface but with a strong presence of pores and imperfections. The presence of pores is undesirable because it can facilitate the entry of oxygen, which accelerates the degradation of AA. Furthermore, the presence of pores can facilitate the diffusion of water into the interior of the microparticles, providing the quick release of AA in an aqueous medium. Although the SLM examined in previous research have not always presented a smooth surface (Alvim, Souza, Koury, Jurt, & Dantas, 2013; Chambi, Alvim, Barrera-Arellano, & Grosso, 2008; Martins, Siqueira, & Freitas, 2012; Matos-Jr., Di Sabatino, Passerini, FavaroTrindade, & Albertini, 2015), the presence of pores is not frequently reported, except by Salvim et al. (2015), who also reported the presence of pores in the particles obtained by the atomization of emulsions when they encapsulated soy protein hydrolysate by spray chilling. In this context, the presence of pores in the SLM produced by the atomization of an emulsion can be attributed to water evaporation from the droplets during atomization. In contrast, rough and very small particle adhesion was observed on the surfaces of the SLM that were produced from the dispersion (T5). This can disturb the flow of the powder. Therefore, the evaluation of the morphology of the produced particles allows the inference that the atomization of the dispersion produces a particle structure that provides better protection to AA; however, it would offer greater resistance to the flow of powder. The values of the particle diameter are presented in Table 3. The results reveal that there were differences between the treatments due to the influence of various factors, and these deserve detailed
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Fig. 1. SEM micrographs of the following: (A, B and C) solid lipid microparticles loaded with ascorbic acid obtained from the emulsion observed at different magnifications; and (D, E and F) solid lipid microparticles loaded with ascorbic acid obtained from the dispersion observed with different magnifications.
Table 3 Encapsulation efficiency (EE), the mean volume diameter (MVD) and activity water (Aw) of solid lipid microparticles loaded with ascorbic acid produced by spray chilling. Treatment
EE (%)
T1 T2 T3 T4 T5 T6 T7
68.2 71.0 69.9 72.5 59.2 e e
± ± ± ± ±
MVD (mm) 3.4 2.6 4.0 3.1 4.2
a a a a b
73.86 74.69 76.78 72.87 91.62 44.92 33.82
± ± ± ± ± ± ±
2.1 1.9 1.1 3.8 1.5 3.1 2.4
Aw a a a a b c d
0.963 0.965 0.966 0.969 0.631 0.690 0.632
± ± ± ± ± ± ±
0.009 0.017 0.021 0.005 0.014 0.011 0.008
a a a a b c b
T1: Emulsion with the proportion of active material: carrier of 1:7 at homogenization speed of 5000 rpm; T2: Emulsion with the proportion of active material: carrier of 1:10 at homogenization speed of 5000 rpm; T3: emulsion with the proportion of active material: carrier of 1:7 at homogenization speed of 10 000 rpm; T4: Emulsion with the proportion of active material: carrier of 1:10 at homogenization speed of 10 000 rpm; T5: Dispersion with the proportion of active material: carrier of 1:10; T6: pure lipid; T7: lipid þ soya lecithin. There were no significant differences among the samples with the same letters in the same column (p < 0.05). The analyses were performed in triplicate.
analysis. The particle size distributions for all of the formulations exhibited unimodal behavior; i.e., with just one peak (Fig. 2). This allows discussion of the particle size, and all of the formulations showed poly-dispersed distributions, with values ranging from 34 ± 2 to 92 ± 2 mm. Comparing the treatments T1 with T2 and T3 with T4, which have different concentrations of AA but were emulsified using the same Ultra-Turrax rotations, it is possible to infer that the proportion of AA in the SLM produced by the atomization of the emulsion did not affect the average particle size. This may be explained by the fact that in all these treatments, the AA was always added to the water in a soluble form, and the amount of solution added did not change; only its concentration changed (17.5 g/100 g for T2 and T4; 25 g/100 g for T1 and T3). Similar results were reported by Leonel et al. (2010), who produced SLM loaded with glucose solution using mixtures of stearic and oleic fatty acids, hydrogenated vegetable fat as a carrier and soybean lecithin as a surfactant.
The variation in the speed of Ultra-Turrax (523 and 1047 rad/s) for the preparation of the emulsion also did not significantly alter the average particle sizes, possibly because the rate of 1047 rad/s (Treatments 3 and 4) was not enough to reduce the size of the emulsion droplets in comparison to the rate of 523 rad/s (T1 and T2). Most likely, this variation in the speed of the Ultra-Turrax did not induce a reduction in the viscosity of the emulsion since according to Albertini, Passerini, Pattarino, and Rodriguez (2008), the particle size produced by the spray congealing technique is heavily influenced by the viscosity of the feed. When comparing treatments involving emulsions (T1-T4) with the other treatments (T5-T7), the influence of the inlet fluid viscosity on the particle size became relevant. Particles produced only with fat (T6) were significantly lower than those produced by mixing fat with soybean lecithin (T7), which was lower than those prepared with the mixture of fat and dry AA (T5). These differences can be attributed to the fact that lecithin is a fine powder that was added in small amounts compared to AA; moreover, the lecithin acts by decreasing the viscosity of the medium. In terms of the AA added to the dispersion, although it was macerated and sieved using a 100 mm sieve, the powder was not as fine as the lecithin powder. In this context, although the viscosities of the feeds have not been studied in this work, it can be inferred that this parameter has a significant influence on the size of the particles obtained by spray chilling, as reported by other authors (Di Sabatino, Albertini, Kett, & Passerini, 2012; Maschke et al., 2007). The samples produced with emulsified AA (T1 to T4) had a significantly smaller medium particle size (p < 0.05) than those produced with AA dispersed in the carrier (T5) (Table 3). Therefore, it can be argued that the application of particles produced by an emulsion (T1 to T4) to food must have a minor impact on its texture. Process parameters, especially the atomization pressure and temperature, influence the particle size (Di Sabatino et al., 2012; Maschke et al., 2007; Matos-Jr., Di Sabatino, Passerini, FavaroTrindade, & Albertini, 2015). In addition to the process parameters, the type of active material, lipid carrier and design of the atomizer can also cause considerable variations in size. The series of
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255
Fig. 2. Particle size distribution of solid lipid microparticles loaded with ascorbic acid produced by spray chilling. T1: Emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 5000 rpm ( ); T2: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 5000 rpm ( ); T3: emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 10,000 rpm ( ); T4: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 10,000 rpm ( ); T5: Dispersion with the proportion of active material: carrier of 1:10 mL:mL ( ); T6: pure lipid ( ); T7: lipid þ soya lecithin ( ).
factors impacting this feature complicates comparisons between existing research studies. 3.2. Efficiency of microencapsulation The encapsulation efficiency values are shown in Table 3. There were no significant differences between the results obtained for the EE values of SLM produced by the atomization of emulsions. This implies that the ratio of active material:carrier and the rotation of the Ultra-Turrax had no influence on the amount of AA that remained on the surface of the microparticles in relation to the amount that had been effectively covered by the carrier. These results become consistent when the relationship with the particle size results was considered. As with the EE, the particle size was not affected by the variables described earlier. If there was no difference in size, the surface area of the particles was not changed, which could allow a smaller or larger presence of AA on the surface. Morselli Ribeiro, Barrera Arellano, and Ferreira Grosso (2012) encapsulated glucose solution in a mixture of stearic acid and oleic acid using a chilling spray technique and found different results. They found that an increase in the oleic acid concentration in relation to the active material had a positive influence on the EE. It is worth noting that the authors of this study used the same methodology to determine the EE as that employed in the current study. A comparison of the treatment that involved the preparation of a dispersion (T5) with the others (T1-T4) revealed there was a significant difference in terms of the EE values. The amount of AA on the surface was greater in the T5 dispersion, and this resulted in a lower EE value. In this case, the argument that the particle size influences the EE due to the surface area loses consistency because the particles of the T5 dispersion were larger than the others; as such, they had a smaller surface area. This difference can be attributed to the effect of feed preparation because for the treatments T1 to T4, the AA was emulsified so it was in the dispersed phase (water) of the W/O emulsion and covered by the emulsifier. Hence, the AA is less likely to be on the particle surface and to be entrapped in the wash water. Although the presence of pores was detected in these particles, as discussed previously, these may have contributed to the drag of AA even when emulsified. The EE values varied from 68.2 to 72.5% for these treatments. For the dispersion preparation (T5), although macerated to a
fine powder (<100 mm), the AA remained in the form of considerably large crystals, even if we take into consideration the sizes of the particles obtained. During the washing of the particles, any AA content that is not perfectly immobilized in the lipid matrix can be entrapped by the wash water. The AA is a highly hydrophilic compound so this “drag” is considerably favored. A comparison of the results obtained in the current research with those obtained from previous studies that have evaluated the EE using a similar methodology or those that have determined the active material present on the particle surface reveals that the values obtained are in the range of those reported by previous research. Leonel et al. (2010) encapsulated a glucose solution in mixtures of liquid and solid fatty acids (stearic and oleic acid) as well as hydrogenated vegetable fat, and they found values of glucose on the surface that ranged from 2 to 20%. They found that the theoretical values of glucose in the microparticles did not influence the EE or the concentration of glucose in solution. Morselli Ribeiro et al. (2012), who also encapsulated glucose solution, reported that the values of the active material on the surface ranged between 3 and 24%. Of the various analyses for the characterization of the microparticles obtained by spray chilling, the encapsulation efficiency needs to be treated more critically in the interpretation and discussion of the results. This is due to the lack of consensus on the definition of the encapsulation efficiency. In the current study, it was assumed that the encapsulation efficiency refers to the amount of active material effectively encapsulated. To determine the EE, the difference between the total content of ascorbic acid in the SLM and the content on the surfaces of the particles was calculated. In the literature, it is common to find EE values that have been determined by calculating the difference between the theoretical amount of atomized active material and the amount of active material present in the microparticles. This analysis is very useful as a means of verifying that the atomization process causes the degradation/loss of the active material; however, it does not provide insights into whether the active material is inside or on the surface of the SLM. If there is no criterion in the discussion of the results, the possibility of errors in relation to the values close to 100% reported and assigned as the EE emerges. Based on the consideration that the spray chilling technique produces microparticles and not microcapsules, where the active material is perfectly packaged within the microcapsules (Fig. 1), it is impossible to produce
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particles without active material on their surfaces. Finally, if the authors do not consider the difference between the amount of encapsulated active material and the active material present on the surfaces of the microparticles, the function of microencapsulation can be considerably impaired, especially when the purpose of the process is to protect the active material, mask taste, and/or disguise odor, for example. The results of the EE, when considered as per the methods employed in this study, provide the information required to evaluate the need for adjustments in the process, such as changes in the amount of active material in the formulation and/or manipulation of particle size, with the purpose of the latter being to reduce the surface area of the particle. 3.3. Water activity and moisture Information about the water activity in the particles is relevant because it provides insights into the extent to which the kinetics degradation of the active material can be influenced by the presence of water. The average values of water activity and moisture are shown in Table 3. Accordingly, treatments involving the emulsion preparation presented higher values of Aw and moisture in relation to the other treatments; therefore, they were subjected to more microbial and chemical changes. However, the microbial growth in the particles will be not supported due to the lack of nutrients because according to Bhat, Alias, and Paliyath (2012), relatively few types of microorganisms are capable of metabolizing lipids. Additionally, there isn't a source of nitrogen in the particles and just AA as a vitamin. However, particles produced by an emulsion-feed, as a function of their higher Aw, are subjected to more chemical reactions, which can explain the lower stability of AA in them, as discussed in the item 3.6. 3.4. Thermal behavior The DSC thermograms are presented in Fig. 3. No peak was observed for soy lecithin and AA. The absence of a peak occurs because the melting points of these ingredients are above 100 C and out of the temperature range studied. The fat used as a carrier presented a melting peak at approximately 43 C. In the analysis of the microparticles, this behavior was repeated; as such, the same melting peak was observed independent of the treatment. However, for treatments T1 to T4, a slight narrowing of the peak was observed, which can be attributed to the interaction of the fat with lecithin. Due to the similarity between the samples and the fat in relation to this peak, no fat interaction with the lipophilic material was observed. This would lead to depression of the melting point of the fat and could change the polymorphic behavior of the fat. The presence of an earlier peak at approximately 0 C for the fat was verified for the treatments that were prepared from an emulsion (T1eT4); this is related to the melting process of water. For treatment 5, with the dispersion preparation, this peak was not observed since there was no water in the formulation. 3.5. Fourier-transform infrared spectroscopy (FTIR) Fourier transform infrared spectroscopy was performed to identify possible interactions between the carrier and components of the formulations. The spectra of ingredients and the SLM are shown in Fig. 4. As the spectra of the SLM prepared with emulsions (T1eT4) were similar, the decision was made to present only one spectrum. The spectra obtained for the three pure ingredients were similar to those reported in the literature. For the AA, the characteristic
Fig. 3. Thermograms of the solid lipid microparticles loaded with ascorbic acid produced by spray chilling and pure ingredients (ascorbic acid, soya lecithin and lipid). T1: Emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 5000 rpm; T2: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 5000 rpm; T3: emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 10,000 rpm; T4: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 10,000 rpm; T5: Dispersion with the proportion of active material: carrier of 1:10 mL:mL.
peaks were obtained at 1021, 1109, 1310, 1650, 1750, 3400 and 3520 cm1. The peaks at 1109, 1310 and 1650 cm1 are assigned to the carbonyl (C]O) and hydroxyl (OH) groups present in the vitamin C molecule (Desai & Park, 2005). For soybean lecithin, the characteristic peaks were 1042, 2851 and 2919 cm1, the first of which corresponded to the links with phosphorus (PeO) (Silverstein, Webster, & Kiemle, 2005). Finally, the characteristic peaks for pure fat were 1738, 2851 and 2919 cm1, with the first band having moderate intensity and the last two bands having stronger intensities, which are related to the presence of carbonyl compounds, more specifically, carboxylic acids. Another characteristic peak in the fat was observed at 1170 cm1, which is related to the presence of esters (Silverstein et al., 2005). For the SLM, the spectra were consistent and characteristic peaks of AA, fat and soy lecithin (when in the formulation) appeared in all of the treatments. These similar characteristics confirmed that active material was present in the microparticles; thus, it was not lost in the encapsulation process. The peak that appeared in these samples near 3350 cm-1 is related to the axial strain (OeH) in intermolecular hydrogen bonding, thus indicating the presence of water (Silverstein et al., 2005). This is easily explained because there was water in the systems for treatments 1e4, which were prepared with an emulsion, and this was different than for treatment 5, which was a dispersion of anhydrous AA and fat. 3.6. Assessment of the stability of free AA and microencapsulated AA The stability of free AA in solution is shown in Fig. 5. The
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Fig. 4. FTIR spectra of the solid lipid microparticles loaded with ascorbic acid produced by spray chilling and pure ingredients (ascorbic acid, soya lecithin and lipid). T1: Emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 5000 rpm; T2: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 5000 rpm; T3: emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 10,000 rpm; T4: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 10,000 rpm; T5: Dispersion with the proportion of active material: carrier of 1:10 mL:mL.
influence of the storage temperature on the stability of ascorbic acid was clear. As expected, the higher the storage temperature, the greater the degradation rate of the AA. Following 15 days of storage,
the remaining contents of free AA and those in solution were 0, 20 and 40% for storage at 37, 24 and 7 C, respectively. These results are very similar to those reported by authors who studied the stability
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Fig. 5. Stability of free ascorbic acid stored for 30 days at temperatures of 7 (:), 24 (C) and 37 C (,).
of AA in aqueous solution (Comunian et al., 2013 and Comunian et al., 2014). In Fig. 6, it is possible to observe the stability of the microencapsulated AA. The storage temperature, as was the case with the free AA, considerably influenced its stability. Treatments that involved the preparation of an emulsion behaved similarly. The treatment that involved the dispersion preparation (T5) presented different results in terms of the stability of the AA across all of the storage temperatures. The maintenance of stability was much higher in T5. This difference could be readily observed due to the color changes of the samples, and this was the treatment that presented the smallest color change. The presence of water in the emulsion treatments was certainly a determining factor in the explanation of these results. In fact, according to Damodaran, Parkin, and Fennema (2008), AA is very susceptible to oxidation, and factors such as the oxygen concentration and water activity influence the rate of this reaction. Along with the water activity, values were higher in the particles produced by atomization of emulsions (Table 3); as discussed previously, these samples presented pores, which may have favored the diffusion of oxygen. If the results of the current study are compared with existing research involving the encapsulation and evaluation of the stability of AA, including those that examined spray drying (Trindade & Grosso, 2000), complex coacervation (Comunian et al., 2013), microfluidic devices (Comunian et al., 2014) and fluidized bed coating (Knezevic, Gosak, Hraste, & Jalsenjako, 1998), the stability provided by the AA microparticles produced by spray chilling with an emulsion was low; however, the particles produced with a dispersion showed satisfactory results. After 60 days of storage at 7 and 24 C, the AA present in the particles produced by the atomization of the emulsion remained stable at greater than 80%. At 37 C, the stability after 60 days was maintained above 60%. These results indicate that, compared to free AA in solution, these particles are a good alternative to confer protection to AA. Furthermore, if an improvement in their preparation can be achieved, this will improve the encapsulation efficiency and increase the stability values. The original idea behind the spray chilling technique was based on the notion of the production of microparticles through the atomization of the mixture between the carrier and active material, and there is no type of solvent involved in the process. However, some research has recently emerged in the literature (Chambi et al., 2008; Leonel et al., 2010; Morselli Ribeiro et al., 2012; Okuro, de Matos, & Favaro-Trindade, 2013a; Pedroso et al., 2012) in which, similar to this present work, atomized emulsions were used in such
Fig. 6. Stability of encapsulated ascorbic acid stored for 60 days at temperatures of 7 (A), 24 (B) and 37 C (C). T1: Emulsion with the proportion of active material: carrier of 1:7 at homogenization speed of 5000 rpm (:); T2: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 5000 rpm (d); T3: emulsion with the proportion of active material: carrier of 1:7 mL:mL at homogenization speed of 10,000 rpm (C); T4: Emulsion with the proportion of active material: carrier of 1:10 mL:mL at homogenization speed of 10,000 rpm (A); T5: Dispersion with the proportion of active material: carrier of 1:10 mL:mL (-).
a manner that the active material was mixed with the lipid carrier in aqueous solution with the aid of an emulsifier. Evaluation of the results influenced by the Aw in terms of the impact on the function of the microencapsulation revealed, in this case, that the maintenance of the stability of the AA represents an important consideration if these technical adaptations do not contradict the advantages of it. Furthermore, anticipating the conclusions of this work, it was found that the adaptation of the technique, atomization of an emulsion instead of a dispersion, did not favor it but
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prejudiced the purpose of microencapsulation, which was to confer protection to the AA. However, there is no doubt that encapsulation is a promising technology to protect ascorbic acid (Alvim et al., 2016). When AA encapsulated by spray drying and spray chilling was added into biscuits, it was reported that the AA was protected to a greater extent during baking when it was encapsulated by spray drying, followed closely by spray chilling. These authors also reported that AA encapsulation inhibited the formation of dark spots on the biscuits that were associated with the thermal degradation of this compound during baking. Comunian et al. (2014) added AA encapsulated by a double emulsion followed by complex coacervation in chicken frankfurters and observed that this compound was released from the capsules during processing and/or storage due to the oxidative stability presented in the samples. Moreover, the sausages that were produced using encapsulated AA showed good sensory acceptability. 4. Conclusion The comparison between the two forms of feed preparation proposed in this study indicates that both methods have advantages and disadvantages. However, the dispersion generated more promising results in relation to the stability of the AA. Further studies are necessary to improve the encapsulation efficiency of microparticles produced with the dispersion. These new studies may involve varying the proportion of the carrier to the active material as well as the use of other types of carriers. The lipid microparticles loaded with AA obtained by spray chilling can be applied to various types of food products, particularly those in which AA may suffer degradation during product processing, thus impeding its activity during the food storage period. Acknowledgments ~o de Amparo a Pesquisa do The authors thank the Fundaça ~o Paulo for the scholarships that were granted (ProEstado de Sa cesses 2010/13117-5 and 2012/01907-7). Favaro-Trindade C.S. thanks CNPq for the productivity grant (306708/2015-9). References Albertini, B., Passerini, N., Pattarino, F., & Rodriguez, L. (2008). New spray congealing atomizer for the microencapsulation of highly concentrated solid and liquid substances. European Journal of Pharmaceutics and Biopharmaceutics, 69, 348e357. http://doi.org/10.1016/j.ejpb.2007.09.011. Alvim, I. D., Souza, F. da S. de, Koury, I. P., Jurt, T., & Dantas, F. B. H. (2013). Use of the spray chilling method to deliver hydrophobic components: Physical characterization of microparticles. Food Science and Technology, 33, 34e39. Retrieved from http://www.scielo.br/scielo.php?script¼sci_arttext&pid¼S0101-20612013 000500006&nrm¼iso. Alvim, I. D., Stein, M. A., Koury, I. P., Dantas, F. B. H., & Cruz C. L. de C. V.. (2016). Comparison between the spray drying and spray chilling microparticles contain ascorbic acid in a baked product application. LWT - Food Science and Technology, 65, 689e694. http://doi.org/. http://dx.doi.org/10.1016/j.lwt.2015.08.049. AOAC. (1998). Official methods of analysis of AOAC international. Association of Official Analysis Chemists International, 9, CDeROM http://doi.org/10.3109/ 15563657608988149. Bhat, R., Alias, A. K., & Paliyath, G. (2012). Progress in food preservation. Oxford: Wiley Blackwell. Chambi, H. N. M., Alvim, I. D., Barrera-Arellano, D., & Grosso, C. R. F. (2008). Solid lipid microparticles containing water-soluble compounds of different molecular mass: Production, characterisation and release profiles. Food Research International, 41, 229e236. http://doi.org/10.1016/j.foodres.2007.11.012. Comunian, T. A., Thomazini, M., Alves, A. J. G., de Matos Junior, F. E., de Carvalho Balieiro, J. C., & Favaro-Trindade, C. S. (2013). Microencapsulation of ascorbic acid by complex coacervation: Protection and controlled release. Food Research International, 52(1), 373e379. Comunian, T. A., Thomazini, M., Gambagorte, V. F., Trindade, M. A., & FavaroTrindade, C. S. (2014). Effect of incorporating free or encapsulated ascorbic acid in chicken frankfurters on physicochemical and sensory stability. Journal of Food Science and Engineering, 4, 167e175.
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