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Development of W/O emulsion for encapsulation of “Pitanga” (Eugenia uniflora L.) leaf hydroethanolic extract: droplet size, physical stability and rheology

Abstract

The low chemical stability under environmental conditions of “Pitanga” leaf hydroethanolic extract (PLHE) can limit its application in industrial scale. It is known that this extract has high antioxidant and antimicrobial activities and that it must be stored under special conditions or encapsulated into W/O emulsions. The objective of this research was to encapsulate PLHE in a W/O emulsion, analyzing the effect of the concentration of the emulsifier and phase ratios on its droplet size, physical stability and viscosity. In general, the droplet size and stability of the W/O emulsions were affected by the concentrations of emulsifiers and phase ratios. The emulsion with a 20/80 W/O ratio and 3 g PGPR/100 g oil was chosen as the most stable formulation because they presented well distributed droplet sizes (unimodal distribution), the lowest D3,2 (0.25 ± 0.02 μm), the highest physical stability at 60 °C and presented Newtonian behavior. In conclusion, the W/O emulsion is able to encapsulate PHLE and can be applied to thermal processed foods.

Keywords:
plant extract; hydroethanolic solvent; microencapsulation; colloidal system; viscosity

1 Introduction

The “Pitangueira” tree (Eugenia uniflora L.) can be found in South America, Southern Asia and Africa (Arai et al., 1999Arai, I., Amagaya, S., Komatsu, Y., Okada, M., Hayashi, T., Kasai, M., Arisawa, M., & Momose, Y. (1999). Improving effects of the extracts from Eugenia uniflora on hyperglycemia and hypertriglyceridemia in mice. Journal of Ethnopharmacology, 68(1–3), 307-314. http://dx.doi.org/10.1016/S0378-8741(99)00066-5. PMid:10624893.
http://dx.doi.org/10.1016/S0378-8741(99)...
). Its fruits, called “Pitanga” or the Brazilian cherry, are edible and eaten fresh or in marmalade (Rattmann et al., 2012Rattmann, Y. D., de Souza, L. M., Malquevicz-Paiva, S. M., Dartora, N., Sassaki, G. L., Gorin, P. A. J., & Iacomini, M. (2012). Analysis of flavonoids from eugenia uniflora leaves and its protective effect against murine sepsis. Evidence-Based Complementary and Alternative Medicine, 2012, 623940. http://dx.doi.org/10.1155/2012/623940. PMid:23320032.
http://dx.doi.org/10.1155/2012/623940...
). The leaves of these trees are also well known and used in folk medicine to assist in the treatment of several diseases (Arai et al., 1999Arai, I., Amagaya, S., Komatsu, Y., Okada, M., Hayashi, T., Kasai, M., Arisawa, M., & Momose, Y. (1999). Improving effects of the extracts from Eugenia uniflora on hyperglycemia and hypertriglyceridemia in mice. Journal of Ethnopharmacology, 68(1–3), 307-314. http://dx.doi.org/10.1016/S0378-8741(99)00066-5. PMid:10624893.
http://dx.doi.org/10.1016/S0378-8741(99)...
; Consolini & Sarubbio, 2002Consolini, A. E., & Sarubbio, M. G. (2002). Pharmacological effects of Eugenia uniflora (Myrtaceae) aqueous crude extract on rat’s heart. Journal of Ethnopharmacology, 81(1), 57-63. http://dx.doi.org/10.1016/S0378-8741(02)00039-9. PMid:12020928.
http://dx.doi.org/10.1016/S0378-8741(02)...
; Rattmann et al., 2012Rattmann, Y. D., de Souza, L. M., Malquevicz-Paiva, S. M., Dartora, N., Sassaki, G. L., Gorin, P. A. J., & Iacomini, M. (2012). Analysis of flavonoids from eugenia uniflora leaves and its protective effect against murine sepsis. Evidence-Based Complementary and Alternative Medicine, 2012, 623940. http://dx.doi.org/10.1155/2012/623940. PMid:23320032.
http://dx.doi.org/10.1155/2012/623940...
).

Recently, several researchers have demonstrated the high antioxidant activity of the “Pitanga” leaf’s extracts (Garmus et al., 2014Garmus, T. T., Paviani, L. C., Queiroga, C. L., Magalhães, P. M., & Cabral, F. A. (2014). Extraction of phenolic compounds from pitanga (Eugenia uniflora L.) leaves by sequential extraction in fixed bed extractor using supercritical CO2, ethanol and water as solvents. The Journal of Supercritical Fluids, 86, 4-14. http://dx.doi.org/10.1016/j.supflu.2013.11.014.
http://dx.doi.org/10.1016/j.supflu.2013....
; Lorenzo et al., 2018Lorenzo, J. M., Vargas, F. C., Strozzi, I., Pateiro, M., Furtado, M. M., Sant’Ana, A. S., Rocchetti, G., Barba, F. J., Dominguez, R., Lucini, L., & Sobral, P. J. A. (2018). Influence of pitanga leaf extracts on lipid and protein oxidation of pork burger during shelf-life. Food Research International, 114(May), 47-54. http://dx.doi.org/10.1016/j.foodres.2018.07.046. PMid:30361026.
http://dx.doi.org/10.1016/j.foodres.2018...
; Schumacher et al., 2015Schumacher, N., Colomeu, T., de Figueiredo, D., Carvalho, V., Cazarin, C., Prado, M., Meletti, L., & Zollner, R. (2015). Identification and Antioxidant activity of the extracts of eugenia uniflora leaves. Characterization of the anti-inflammatory properties of aqueous extract on diabetes expression in an experimental model of spontaneous Type 1 diabetes (NOD Mice). Antioxidants, 4(4), 662-680. http://dx.doi.org/10.3390/antiox4040662. PMid:26783951.
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; Vargas et al., 2016Vargas, F. C., Arantes-Pereira, L., Costa, P. A., Melo, M. P., & Sobral, P. J. A. (2016). Rosemary and pitanga aqueous leaf extracts on beef patties stability under cold storage. Brazilian Archives of Biology and Technology, 59(0). http://dx.doi.org/10.1590/1678-4324-2016160139.
http://dx.doi.org/10.1590/1678-4324-2016...
, 2019Vargas, F. C., Gómez, B., Mousavi Khaneghah, A., Strozzi, I., Gavahian, M., Barba, F. J., Sobral, P. J. A., & Lorenzo, J. M. (2019). Assessment of the suitability of pitanga leaf extract as a natural antioxidant for enhancing canola oil stability: monitoring lipid oxidation parameters. European Journal of Lipid Science and Technology, 121(5), 1800447. http://dx.doi.org/10.1002/ejlt.201800447.
http://dx.doi.org/10.1002/ejlt.201800447...
). These bioactivities are due to the presence of some phenolic acids and flavonoids (e.g. gallic acid, ellagic acid and myricitrin) (Bezerra et al., 2018Bezerra, I. C. F., Ramos, R. T. D. D. M., Ferreira, M. R. A., & Soares, L. A. L. (2018). Chromatographic profiles of extractives from leaves of Eugenia uniflora. Revista Brasileira de Farmacognosia, 28(1), 92-101. http://dx.doi.org/10.1016/j.bjp.2017.11.002.
http://dx.doi.org/10.1016/j.bjp.2017.11....
; de Oliveira et al., 2018Oliveira, F. M. G., Romão, W., & Kuster, R. M. (2018). Identification of phenolic compounds in Eugenia uniflora leaves by FTICR MS in association with different ionization sources. Analytical Methods, 10(14), 1647-1655. http://dx.doi.org/10.1039/C8AY00129D.
http://dx.doi.org/10.1039/C8AY00129D...
) and terpenoids (i.e. germacrene D and trans-caryophyllene) (Garmus et al., 2014Garmus, T. T., Paviani, L. C., Queiroga, C. L., Magalhães, P. M., & Cabral, F. A. (2014). Extraction of phenolic compounds from pitanga (Eugenia uniflora L.) leaves by sequential extraction in fixed bed extractor using supercritical CO2, ethanol and water as solvents. The Journal of Supercritical Fluids, 86, 4-14. http://dx.doi.org/10.1016/j.supflu.2013.11.014.
http://dx.doi.org/10.1016/j.supflu.2013....
). Nevertheless, these active compounds are very sensitive to environmental conditions, such as light and oxygen, and their storage can be an important challenge.

The encapsulation of PLHE in a W/O emulsion by adding this product in the water phase can be an approach to guarantee its stability (Ali & Akhtar, 2014Ali, A., & Akhtar, N. (2014). Changes in the characteristics of water-in-oil-based high internal phase emulsion containing moringa leaves extract at various storage conditions. Tropical Journal of Pharmaceutical Research, 13(5), 677. http://dx.doi.org/10.4314/tjpr.v13i5.4.
http://dx.doi.org/10.4314/tjpr.v13i5.4...
, Kaimainen et al., 2015Kaimainen, M., Marze, S., Järvenpää, E., Anton, M., & Huopalahti, R. (2015). Encapsulation of betalain into w/o/w double emulsion and release during in vitro intestinal lipid digestion. Lebensmittel-Wissenschaft + Technologie, 60(2), 899-904. http://dx.doi.org/10.1016/j.lwt.2014.10.016.
http://dx.doi.org/10.1016/j.lwt.2014.10....
, Tepsongkroh et al., 2015Tepsongkroh, B., Harnsilawat, T., Maisuthisakul, P., & Chantrapornchai, W. (2015). Influence of polyglycerol polyricinoleate and biopolymers on physical properties and encapsulation efficiency of water-in-oil-in-water emulsions containing mango seed kernel extract. Journal of Dispersion Science and Technology, 36(8), 1126-1133. http://dx.doi.org/10.1080/01932691.2014.956116.
http://dx.doi.org/10.1080/01932691.2014....
, Liu et al., 2018Liu, L., Jin, Z., Wang, M., Shen, W., Zhu, Z., Wang, Z., & Liu, L. (2018). W/O Nano-Emulsions with Olive Leaf Phenolics Improved Oxidative Stability of Sacha Inchi Oil. European Journal of Lipid Science and Technology, 120(5), 1-8. http://dx.doi.org/10.1002/ejlt.201700471.
http://dx.doi.org/10.1002/ejlt.201700471...
, Rahpeyma & Sekhavatizadeh, 2020Rahpeyma, E., & Sekhavatizadeh, S. S. (2020). Effects of encapsulated green coffee extract and canola oil on liquid kashk quality. Foods and Raw Materials, 8(1), 40-51. http://dx.doi.org/10.21603/2308-4057-2020-1-40-51.
http://dx.doi.org/10.21603/2308-4057-202...
). A predominantly hydrophobic emulsifier, such as Polyglycerol polyricinoleate (PGPR), is necessary to produce a W/O emulsion (Fraj et al., 2017Fraj, J., Petrovic, L., Milinkovic, J., Katona, J., Bucko, S., & Spasojevic, L. (2017). Properties of water in oil emulsions (W/O) stabilized with mixtures of PGPR and polyglycerol fatty acid esters. Acta Periodica Technologica, 48(48), 95-107. http://dx.doi.org/10.2298/APT1748095F.
http://dx.doi.org/10.2298/APT1748095F...
). The PGPR has been widely used in researches on W/O emulsions because it is an effective hydrophobic emulsifier, and has been used in several similar studies (Márquez et al., 2010Márquez, A. L., Medrano, A., Panizzolo, L. A., & Wagner, J. R. (2010). Effect of calcium salts and surfactant concentration on the stability of water-in-oil (W/O) emulsions prepared with polyglycerol polyricinoleate. Journal of Colloid and Interface Science, 341(1), 101-108. http://dx.doi.org/10.1016/j.jcis.2009.09.020. PMid:19822323.
http://dx.doi.org/10.1016/j.jcis.2009.09...
, Ushikubo & Cunha, 2014Ushikubo, F. Y., & Cunha, R. L. (2014). Stability mechanisms of liquid water-in-oil emulsions. Food Hydrocolloids, 34, 145-153. http://dx.doi.org/10.1016/j.foodhyd.2012.11.016.
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, Tepsongkroh et al., 2015Tepsongkroh, B., Harnsilawat, T., Maisuthisakul, P., & Chantrapornchai, W. (2015). Influence of polyglycerol polyricinoleate and biopolymers on physical properties and encapsulation efficiency of water-in-oil-in-water emulsions containing mango seed kernel extract. Journal of Dispersion Science and Technology, 36(8), 1126-1133. http://dx.doi.org/10.1080/01932691.2014.956116.
http://dx.doi.org/10.1080/01932691.2014....
, Dridi et al., 2016Dridi, W., Essafi, W., Gargouri, M., Leal-Calderon, F., & Cansell, M. (2016). Influence of formulation on the oxidative stability of water-in-oil emulsions. Food Chemistry, 202, 205-211. http://dx.doi.org/10.1016/j.foodchem.2016.01.145. PMid:26920286.
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, Matos et al., 2018Matos, M., Gutiérrez, G., Martínez-Rey, L., Iglesias, O., & Pazos, C. (2018). Encapsulation of resveratrol using food-grade concentrated double emulsions: Emulsion characterization and rheological behaviour. Journal of Food Engineering, 226, 73-81. http://dx.doi.org/10.1016/j.jfoodeng.2018.01.007.
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, Velderrain-Rodríguez et al., 2019Velderrain-Rodríguez, G. R., Acevedo-Fani, A., González-Aguilar, G. A., & Martín-Belloso, O. (2019). Encapsulation and stability of a phenolic-rich extract from mango peel within water-in-oil-in-water emulsions. Journal of Functional Foods, 56, 65-73. http://dx.doi.org/10.1016/j.jff.2019.02.045.
http://dx.doi.org/10.1016/j.jff.2019.02....
). It has been also widely used in food industries to stabilize W/O emulsions such as margarines, butter, salad dressing and chocolat (Okuro et al., 2019Okuro, P. K., Gomes, A., Costa, A. L. R., Adame, M. A., & Cunha, R. L. (2019). Formation and stability of W/O-high internal phase emulsions (HIPEs) and derived O/W emulsions stabilized by PGPR and lecithin. Food Research International, 122, 252-262. http://dx.doi.org/10.1016/j.foodres.2019.04.028. PMid:31229079.
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).

W/O emulsions containing plant extracts in the W inner phase can be applied in different industries, such as foods, pharmaceuticals and cosmetics. In the food industry, for example, W/O emulsions containing plant extracts are widely used to produce functional oily foods with a lower total fat content and desirable texture (i.e. dairy products) (Rahpeyma & Sekhavatizadeh, 2020Rahpeyma, E., & Sekhavatizadeh, S. S. (2020). Effects of encapsulated green coffee extract and canola oil on liquid kashk quality. Foods and Raw Materials, 8(1), 40-51. http://dx.doi.org/10.21603/2308-4057-2020-1-40-51.
http://dx.doi.org/10.21603/2308-4057-202...
), to increase the oxidative stability of the dispersing oil phase (Liu et al., 2018Liu, L., Jin, Z., Wang, M., Shen, W., Zhu, Z., Wang, Z., & Liu, L. (2018). W/O Nano-Emulsions with Olive Leaf Phenolics Improved Oxidative Stability of Sacha Inchi Oil. European Journal of Lipid Science and Technology, 120(5), 1-8. http://dx.doi.org/10.1002/ejlt.201700471.
http://dx.doi.org/10.1002/ejlt.201700471...
), and to protect and control the release of active aqueous compounds (Mohammadi et al., 2016Mohammadi, A., Jafari, S. M., Assadpour, E., & Faridi Esfanjani, A. (2016). Nano-encapsulation of olive leaf phenolic compounds through WPC–pectin complexes and evaluating their release rate. International Journal of Biological Macromolecules, 82, 816-822. http://dx.doi.org/10.1016/j.ijbiomac.2015.10.025. PMid:26459167.
http://dx.doi.org/10.1016/j.ijbiomac.201...
; Rabelo et al., 2018Rabelo, C. A. S., Taarji, N., Khalid, N., Kobayashi, I., Nakajima, M., & Neves, M. A. (2018). Formulation and characterization of water-in-oil nanoemulsions loaded with açaí berry anthocyanins: insights of degradation kinetics and stability evaluation of anthocyanins and nanoemulsions. Food Research International, 106, 542-548. https://doi.org/10.1016/j.foodres.2018.01.017.
https://doi.org/10.1016/j.foodres.2018.0...
). In the pharmaceutical and cosmetic industries, W/O emulsions containing plant extracts can be used to produce topical products for the treatment of diseases (Rasul et al., 2011Rasul, A., Akhtar, N., Khan, B. A., Mahmood, T., Zaman, S. U., Ali, A., & Parveen, R. (2011). Evaluation for antierythmic and depigmenting effects of a newly formulated emulsion containing basil extract. Journal of Medicinal Plants Research, 5(26), 6249-6253. http://dx.doi.org/10.5897/JMPR11.1200.
http://dx.doi.org/10.5897/JMPR11.1200...
) or pro-age (Huma et al., 2020Huma, S., Khan, H. M. S., Sohail, M., Akhtar, N., Rasool, F., Majeed, F., & Daniyal, M. (2020). Development, in-vitro characterization and assessment of cosmetic potential of Beta vulgaris extract emulsion. Journal of Herbal Medicine, 23, 100372. http://dx.doi.org/10.1016/j.hermed.2020.100372.
http://dx.doi.org/10.1016/j.hermed.2020....
), respectively.

The aim of this study was to encapsulate PLHE in a W/O emulsion and analyze the effects of emulsifier concentrations and phase ratios on its droplet size and physical stability. Some physical properties, including viscosity, of W/O emulsion with the more stable formulation, were also studied. To the best of our knowledge, no works on PHLE encapsulation in an W/O emulsion has been previously published.

2 Materials and methods

2.1 Material

“Pitanga” (Eugenia uniflora L) leaves were collected in Pirassunuga (SP), Brazil. Grinsted Polyglycerol Polyricinoleate (PGPR®) was donated by DuPont (São Paulo, Brazil).

2.2 Production of “pitanga” (Eugenia uniflora L.) leaf hydroethanolic extract (PLHE)

“Pitanga” leaves were washed and dried in an air circulating oven (MA035/5, Marconi, Brazil) at 42 °C/72 h, then they were ground and sifted in a 48-mesh sieve. The leaf powder was dispersed (1g/10 mL) in a 60% ethanol solution and treated by ultrasound at 40 kHz (Odontobras, 1440 DA, Brazil) at 25 °C/40 min. Subsequently, this dispersion was treated in a magnetic heater (Gehaka, AA-2050, Brazil) at 80 °C for 30 min and then filtered using filter paper (Whatman n°1). This extract was evaporated under vacuum (Tecnal, TE-211, Brazil) at 40 °C/4 h and then water was removed by freeze-drying (Heto, model FD 1.0-60, Germany) for 5 days and stored in the dark at 4 °C (Vargas et al., 2019Vargas, F. C., Gómez, B., Mousavi Khaneghah, A., Strozzi, I., Gavahian, M., Barba, F. J., Sobral, P. J. A., & Lorenzo, J. M. (2019). Assessment of the suitability of pitanga leaf extract as a natural antioxidant for enhancing canola oil stability: monitoring lipid oxidation parameters. European Journal of Lipid Science and Technology, 121(5), 1800447. http://dx.doi.org/10.1002/ejlt.201800447.
http://dx.doi.org/10.1002/ejlt.201800447...
). The freeze-dried powder was re-suspended in distilled water (1 g/10 mL water) prior to the production of W/O emulsions.

2.3 Production of water-in-oil (W/O) emulsions

The W/O emulsion was prepared using PLHE in the aqueous phase (10, 20 and 30% w/w) and soybean oil containing hydrophobic emulsifier PGPR (3 and 5 g/100 g oil) as the oil phase (90, 80 and 70% w/w). Briefly, the W/O emulsion was prepared by dropwise addition of PLHE in the oil phase under homogenization at 15,000 rpm for 5 minutes (Labotechnik, Ultraturrax® IKA T25, Germany). Then, this coarse W/O emulsion was homogenized through power ultrasound (Branson Ultrasonics, Sonifier® SFX550, USA) operating at 20 kHz with an amplitude of 30% during 3 cycles/30 s (Leong et al., 2018Leong, T. S. H., Zhou, M., Zhou, D., Ashokkumar, M., & Martin, G. J. O. (2018). The formation of double emulsions in skim milk using minimal food-grade emulsifiers – A comparison between ultrasonic and high pressure homogenisation efficiencies. Journal of Food Engineering, 219, 81-92. http://dx.doi.org/10.1016/j.jfoodeng.2017.09.018.
http://dx.doi.org/10.1016/j.jfoodeng.201...
), both preparations were in ice bath. These emulsions were produced in triplicate and characterized just after their production.

2.4 Characterization of the emulsions

All emulsions characterizations were done in triplicate, at least.

Droplet size distributions and zeta-potential measurements

Prior to these analyses, the W/O emulsion samples were diluted 100x in chloroform, according to Souilem et al. (2014)Souilem, S., Kobayashi, I., Neves, M. A., Sayadi, S., Ichikawa, S., & Nakajima, M. (2014). Preparation of monodisperse food-grade oleuropein-loaded W/O/W emulsions using microchannel emulsification and evaluation of their storage stability. Food and Bioprocess Technology, 7(7), 2014-2027. http://dx.doi.org/10.1007/s11947-013-1182-9.
http://dx.doi.org/10.1007/s11947-013-118...
. Droplet size distributions and Zeta-potential of the W/O emulsions were determined by dynamic light scattering (Malvern Instruments, Zeta Sizer Nano ZS, UK) at 25 °C.

The droplet size distributions and the surface-based average droplet size of the W/O emulsions were calculated using ZetaSizer Nano software (Dammak & Sobral, 2018Dammak, I., & Sobral, P. J. A. (2018). Effect of different biopolymers on the stability of hesperidin-encapsulating O/W emulsions. Journal of Food Engineering, 237, 33-43. http://dx.doi.org/10.1016/j.jfoodeng.2018.05.004.
http://dx.doi.org/10.1016/j.jfoodeng.201...
; Matos et al., 2014Matos, M., Gutiérrez, G., Coca, J., & Pazos, C. (2014). Preparation of water-in-oil-in-water (W1/O/W2) double emulsions containing trans-resveratrol. Colloids and Surfaces. A, Physicochemical and Engineering Aspects, 442, 69-79. http://dx.doi.org/10.1016/j.colsurfa.2013.05.065.
http://dx.doi.org/10.1016/j.colsurfa.201...
). The Sauter size (D3,2) and span were calculated using the Equations 1 and 2, respectively, where di is the droplet size and ni the surface frequency of droplets with size di.

D 3,2 = n i d 1 3 n i d 1 2 (1)
S p a n = D v ,90 D v ,10 D v ,50 (2)

When bimodal droplet size distribution occurred, D3,2 and span values were calculated for each peak using Equations 1 and 2, respectively, where Dv,10, Dv,50 and Dv,90 corresponded to the surface-based size at 10, 50 and 90% of cumulative surface for each peak (Dammak & Sobral, 2017Dammak, I., & Sobral, P. J. A. (2017). Formulation and Stability Characterization of Rutin-Loaded Oil-in-Water Emulsions. Food and Bioprocess Technology, 10(5), 926-939. http://dx.doi.org/10.1007/s11947-017-1876-5.
http://dx.doi.org/10.1007/s11947-017-187...
, 2018Dammak, I., & Sobral, P. J. A. (2018). Effect of different biopolymers on the stability of hesperidin-encapsulating O/W emulsions. Journal of Food Engineering, 237, 33-43. http://dx.doi.org/10.1016/j.jfoodeng.2018.05.004.
http://dx.doi.org/10.1016/j.jfoodeng.201...
).

2.5 Analysis of the physical stability

The physical stability of W/O emulsions was studied using a multi-sample analytical photocentrifuge (L.U.M. GmbH, LUMiSizer, Germany) with the following parameters: 1.8 mL of sample, 2,32 5x g, time 3,600 s, time interval 10 s, at 20, 40 and 60 °C (Dammak & Sobral, 2017Dammak, I., & Sobral, P. J. A. (2017). Formulation and Stability Characterization of Rutin-Loaded Oil-in-Water Emulsions. Food and Bioprocess Technology, 10(5), 926-939. http://dx.doi.org/10.1007/s11947-017-1876-5.
http://dx.doi.org/10.1007/s11947-017-187...
, 2018Dammak, I., & Sobral, P. J. A. (2018). Effect of different biopolymers on the stability of hesperidin-encapsulating O/W emulsions. Journal of Food Engineering, 237, 33-43. http://dx.doi.org/10.1016/j.jfoodeng.2018.05.004.
http://dx.doi.org/10.1016/j.jfoodeng.201...
). The instability Index (iI) was calculated as the ratio of the height of the creamy layer in relation of the height of the initial emulsion.

2.6 Confocal laser scanning microscopy (CLSM)

The morphology of the droplets of the W/O emulsion with the more stable formulation was qualitatively analyzed using a confocal laser scanning microscope (Leica Microsystems GmbH, SP5, Germany), with objective of 63x (1.4 aperture and oil immersion). The W/O emulsion was prepared with rhodamine B solution (0.1% m/v) added in the PLHE. Then, the W/O was diluted 200x in chloroform, and the rhodamine B was excited with HeNe laser at 543 nm and the emitted light was recorded between 570 to 640 nm. These analyses were made in the Multi-User Laboratory for Confocal Microscopy – LMMC of FCMRP-USP.

2.7 Physical properties

The W/O emulsion that had the more stable formulation was analyzed for determination of its density, pH, refractive index and rheological behavior. The density was assessed with the help of a digital densimeter (Anton Paar GmbH, DMA 4500, Austria); the pH was measured using an electronic pHmeter (Ind. e Com. Eletrônica Gehaka Ltda., PG1400, Brazil), and the refractive index was measured with an Abbe type refractometer, always at 25 ºC. For these three characterizations, the W/O emulsions were analyzed without any prior preparation.

The viscosity of the W/O emulsions was also studied. Steady-state flow measurements were carried out with shear rates ranging from 0 to 125 s-1 at 25 °C, after the sample remained unperturbed for 5 min (Dammak & Sobral, 2017Dammak, I., & Sobral, P. J. A. (2017). Formulation and Stability Characterization of Rutin-Loaded Oil-in-Water Emulsions. Food and Bioprocess Technology, 10(5), 926-939. http://dx.doi.org/10.1007/s11947-017-1876-5.
http://dx.doi.org/10.1007/s11947-017-187...
). These analyses were performed using a rheometer (TA Instruments, AR-2000, UK), using a cone and plate (cone diameter = 60 mm, angle = 2° and gap = 2 mm) geometry.

2.8 Statistical analysis

All the experiments were carried out in triplicate, with three measurements for each replicate. All the experiments were submitted to variance analysis for determining significant differences between the averages using one-way or two-way (ANOVA) analyzes and Tukey test comparison (p < 0.05) with Statistica 7 software.

3 Results and discussion

3.1 Characteristics of water-in-oil (W/O) emulsions

All W/O emulsions were opaque and presented a white-yellowish color resembling milk due to the presence of PLHE (Figure 1). This appearance is usual due to the effect of droplet on light diffraction.

Figure 1
Visual aspects of produced W/O emulsions. PHLE concentration and W/O ratios are indicated in the figure. Source: own authorship.

Droplet size distribution and average droplet size

The droplet size distributions of the W/O emulsions were mono- or bimodal (Figure 2), according to the concentration of the emulsifier and the dispersed phase. The emulsions containing both 10/90 and 20/80 W/O ratios and 5 g PGPR/100 g oil presented monomodal droplet size distributions, and emulsions with 10/90 W/O ratio and 3 g PGPR/100 g oil and both emulsions with 30/70 W/O ratio presented bimodal droplet size distributions (Figure 2).

Figure 2
Droplet size distributions of W/O emulsions.

Bimodal distribution can occur, for example, when there is insufficient emulsifier present in an emulsion to stabilize all of the droplets formed during homogenization (McClements, 2004McClements, D. J. (2004). Food emulsions : principles, practices, and techniques (2nd ed.). Boca Raton: CRC Press. http://dx.doi.org/10.1201/9781420039436. ). Thus, formed droplets were well stabilized only in emulsions having the higher amount of PGPR and low amount of water (10 and 20%). Nevertheless, these emulsions were produced under the same processing conditions than those described by Tepsongkroh et al. (2015)Tepsongkroh, B., Harnsilawat, T., Maisuthisakul, P., & Chantrapornchai, W. (2015). Influence of polyglycerol polyricinoleate and biopolymers on physical properties and encapsulation efficiency of water-in-oil-in-water emulsions containing mango seed kernel extract. Journal of Dispersion Science and Technology, 36(8), 1126-1133. http://dx.doi.org/10.1080/01932691.2014.956116.
http://dx.doi.org/10.1080/01932691.2014....
, which observed only monomodal distributions, probably because they added biopolymers into the W inner phase, which contributed to stabilize droplets. Nevertheless, for a precise explanation of these behaviors, the knowledge of the critical micellar concentration would be necessary.

For the purpose of comparison, the D3,2 of W/O emulsions was calculated, and it ranged from 0.3 to 4.5 µm (Table 1), which is in the range of those found in the literature (Table 2). The increase in PGPR concentration from 3 to 5 g/100 g of oil resulted in a decrease (p < 0.05) in the D3,2 for the W/O emulsions with 10/90 (4.5 to 0.4 µm) and 30/70 (1.5 to 0.9 µm) W/O ratios. Similarly, Márquez et al. (2010)Márquez, A. L., Medrano, A., Panizzolo, L. A., & Wagner, J. R. (2010). Effect of calcium salts and surfactant concentration on the stability of water-in-oil (W/O) emulsions prepared with polyglycerol polyricinoleate. Journal of Colloid and Interface Science, 341(1), 101-108. http://dx.doi.org/10.1016/j.jcis.2009.09.020. PMid:19822323.
http://dx.doi.org/10.1016/j.jcis.2009.09...
observed that the increase of the PGPR concentration from 0.2 to 1% (in whole emulsion) in 20:80 W/O emulsions provoked a reduction in D3,2 from 2.9 to 1.3 μm (Table 2), as in this work (Table 1). These behaviors were due to the effectiveness of PGPR for W/O emulsions, as observed by Tepsongkroh et al. (2015)Tepsongkroh, B., Harnsilawat, T., Maisuthisakul, P., & Chantrapornchai, W. (2015). Influence of polyglycerol polyricinoleate and biopolymers on physical properties and encapsulation efficiency of water-in-oil-in-water emulsions containing mango seed kernel extract. Journal of Dispersion Science and Technology, 36(8), 1126-1133. http://dx.doi.org/10.1080/01932691.2014.956116.
http://dx.doi.org/10.1080/01932691.2014....
, who reported similar behavior for W/O produced with different concentrations of PGPR emulsifier. Furthermore, Matos et al. (2018)Matos, M., Gutiérrez, G., Martínez-Rey, L., Iglesias, O., & Pazos, C. (2018). Encapsulation of resveratrol using food-grade concentrated double emulsions: Emulsion characterization and rheological behaviour. Journal of Food Engineering, 226, 73-81. http://dx.doi.org/10.1016/j.jfoodeng.2018.01.007.
http://dx.doi.org/10.1016/j.jfoodeng.201...
compared the D3,2 values of W/O emulsions with different emulsifiers (PGPR, Span 80, Plurol oleique and Peceol) and observed that the W/O emulsion containing PGPR had the smallest D3,2 value (Table 2), confirming that PGPR was a more effective emulsifier in the production and stabilization of W/O emulsions. Nevertheless, for the W/O emulsion with 20/80 ratio, the PGPR concentration did not affect (p>0.05) the D3,2 values (Table 1). A possible explanation is that the presence some bioactive compound present in PLHE extracts could act as W/O stabilizer considering that W/O ratio (Tepsongkroh et al., 2015Tepsongkroh, B., Harnsilawat, T., Maisuthisakul, P., & Chantrapornchai, W. (2015). Influence of polyglycerol polyricinoleate and biopolymers on physical properties and encapsulation efficiency of water-in-oil-in-water emulsions containing mango seed kernel extract. Journal of Dispersion Science and Technology, 36(8), 1126-1133. http://dx.doi.org/10.1080/01932691.2014.956116.
http://dx.doi.org/10.1080/01932691.2014....
). However, further experiments are needed to better understand and explain these results.

Table 1
Surface-based average droplet size (D3,2) of W/O emulsions.* * Different lowercase letters in the same column and different uppercase letters on the same line indicate significant differences between averages according to Tukey test (p < 0.05).
Table 2
Examples of average droplet size values determined by others authors.

Further, the W/O ratios also affected (p < 0.05) the D3,2 values (Table 1). Considering that the expected behavior must be similar to that observed by Katsouli et al. (2017)Katsouli, M., Polychniatou, V., & Tzia, C. (2017). Influence of surface-active phenolic acids and aqueous phase ratio on w/o nano-emulsions properties; model fitting and prediction of nano-emulsions oxidation stability. Journal of Food Engineering, 214, 40-46. http://dx.doi.org/10.1016/j.jfoodeng.2017.06.017.
http://dx.doi.org/10.1016/j.jfoodeng.201...
, even if working with nanoemulsions (NE): increasing W phase content in W/O NE led to higher droplet sizes; it can be considered that for emulsions containing 3 g PGPR/100 g oil, the D3,2 increased from 0.25 to 1.53 μm when the W phase content increased from 20 to 30% (Table 1). The higher value (4.52 μm) for 10/90 emulsion containing 3 g PGPR/100 g oil was due to the insufficient stabilization of droplets, as explained above. For emulsions containing 5 g PGPR/100 g oil, overall, the D3,2 increased with the W phase content and the higher value (0.85 μm) was observed in the 30/70 W/O emulsion (Table 1). But, Ushikubo & Cunha (2014)Ushikubo, F. Y., & Cunha, R. L. (2014). Stability mechanisms of liquid water-in-oil emulsions. Food Hydrocolloids, 34, 145-153. http://dx.doi.org/10.1016/j.foodhyd.2012.11.016.
http://dx.doi.org/10.1016/j.foodhyd.2012...
, studying the effect of two W/O ratios on emulsions produced with PGPR as emulsifier, didn’t find such effect {D4,3(30:70) = 1.15 μm ≈ D4,3(60:40) = 1.19 μm}. And, curiously, Okuro et al. (2019)Okuro, P. K., Gomes, A., Costa, A. L. R., Adame, M. A., & Cunha, R. L. (2019). Formation and stability of W/O-high internal phase emulsions (HIPEs) and derived O/W emulsions stabilized by PGPR and lecithin. Food Research International, 122, 252-262. http://dx.doi.org/10.1016/j.foodres.2019.04.028. PMid:31229079.
http://dx.doi.org/10.1016/j.foodres.2019...
produced a W/O emulsion with very high-water phase ratio (75:25) and emulsified by PGPR, and determined D3,2 (2.1 μm, Table 2) in the same range of this work (Table 1), which means that the W/O ratio could not have so important effect on droplet size. Similar results were observed by Gomes et al. (2016)Gomes, A., Costa, A. L. R., de Assis Perrechil, F., & da Cunha, R. L. (2016). Role of the phases composition on the incorporation of gallic acid in O/Wand W/O emulsions. Journal of Food Engineering, 168, 205-214. http://dx.doi.org/10.1016/j.jfoodeng.2015.07.041.
http://dx.doi.org/10.1016/j.jfoodeng.201...
, also working with 75:25 W/O emulsion and with Gallic acid solution as W phase. By the way, these authors determined higher D3,2 values when using pure water as W phase.

Matos et al. (2018)Matos, M., Gutiérrez, G., Martínez-Rey, L., Iglesias, O., & Pazos, C. (2018). Encapsulation of resveratrol using food-grade concentrated double emulsions: Emulsion characterization and rheological behaviour. Journal of Food Engineering, 226, 73-81. http://dx.doi.org/10.1016/j.jfoodeng.2018.01.007.
http://dx.doi.org/10.1016/j.jfoodeng.201...
determined Dv,0.5 = 0.66 µm for W/O emulsions produced with 20% (v/v) W phase (water/ethanol) containing trans-Resveratrol, and 80% of miglyol 812 containing 5% of PGPR, as O phase (Table 2). This value was similar to those determined in this study (Table 1).

Physical stability of W/O emulsions

The physical stability of W/O emulsions was studied at 20, 40 and 60 °C. This temperature range was interesting because these emulsions can be applied to thermal processed foods. For instance, it can be used to produce active films loaded with encapsulated PLHE in such emulsions, and film processing imply the preparation of film-forming solution in temperature above the room temperature (Tessaro et al. 2021Tessaro, L., Luciano, C. G., Bittante, A. M. Q. B, Lourenço, R. V., Martelli-Tosi, M., & Sobral, P. J. A. (2021). Gelatin and/or chitosan-based films activated with “Pitanga” (Eugenia uniflora L.) leaf hydroethanolic extract encapsulated in double emulsion. Food Hydrocolloids, 113, 106523. https://doi.org/10.1016/j.foodhyd.2020.106523.
https://doi.org/10.1016/j.foodhyd.2020.1...
). The good stability of these emulsions will guarantee, thus, that the PLHE will be well dispersed into biopolymer matrix.

The curves of the W/O emulsion’s light transmission profiles, as determined by the change in laser transmission in a layer (33-35 mm) of W/O emulsion, were basically straight horizontal lines (Figure 3) and always demonstrated a low transmission (<10%) for all studied temperatures. This behavior was a consequence of the opacity in the whole sample, which was due to a uniform droplet distribution throughout the sample layer inside the cuvette (Figure 3), not destroyed by the high gravity force during analysis, confirming the high stability of these emulsions (Dammak & Sobral, 2017Dammak, I., & Sobral, P. J. A. (2017). Formulation and Stability Characterization of Rutin-Loaded Oil-in-Water Emulsions. Food and Bioprocess Technology, 10(5), 926-939. http://dx.doi.org/10.1007/s11947-017-1876-5.
http://dx.doi.org/10.1007/s11947-017-187...
).

Figure 3
Profiles transmission of W/O emulsions determined using an analytical centrifuge (2.325 x g, time 3600 s) at 20, 40 and 60 °C (photo of cuvettes with samples after analysis were attached in figures). Red-green lines are the light transmition profiles measured during analysis for each time.

The instability Index (iI), calculated from the spectra presented in the Figure 3, has an arbitrary scale ranging from 0 (more stable) to 1 (less stable) (Figure 4). Regardless of temperature and the emulsion’s formulation, the iI ranged from 0.009 to 0.063 (iI<<0.1), meaning that these systems were highly stable. Overall, iIs were correlated with D3,2, but it was significant only for iI determined at 60 ºC: iI= 7.6 x 103D3,2+ 0.026, R2= 0.81. Moreover, the high stability of W/O emulsions can be also associated to two factors: the viscosity of the lipid phase (and consequently, the high viscosity of the whole emulsion, as can be seen in 3.1.4) and the use of PGPR as emulsifier (Dickinson, 2011Dickinson, E. (2011). Double emulsions stabilized by food biopolymers. Food Biophysics, 6(1), 1-11. http://dx.doi.org/10.1007/s11483-010-9188-6.
http://dx.doi.org/10.1007/s11483-010-918...
; Ilić et al., 2017Ilić, J. D., Nikolovski, B. G., Lončarević, I. S., Petrović, J. S., Bajac, B. M., & Vučinić-Vasić, M. (2017). Release Properties and Stability of Double W1/O/W2 Emulsions Containing Pumpkin Seed Oil. Journal of Food Process Engineering, 40(2), e12349. http://dx.doi.org/10.1111/jfpe.12349.
http://dx.doi.org/10.1111/jfpe.12349...
; Tepsongkroh et al., 2015Tepsongkroh, B., Harnsilawat, T., Maisuthisakul, P., & Chantrapornchai, W. (2015). Influence of polyglycerol polyricinoleate and biopolymers on physical properties and encapsulation efficiency of water-in-oil-in-water emulsions containing mango seed kernel extract. Journal of Dispersion Science and Technology, 36(8), 1126-1133. http://dx.doi.org/10.1080/01932691.2014.956116.
http://dx.doi.org/10.1080/01932691.2014....
). According to Gomes et al. (2016)Gomes, A., Costa, A. L. R., de Assis Perrechil, F., & da Cunha, R. L. (2016). Role of the phases composition on the incorporation of gallic acid in O/Wand W/O emulsions. Journal of Food Engineering, 168, 205-214. http://dx.doi.org/10.1016/j.jfoodeng.2015.07.041.
http://dx.doi.org/10.1016/j.jfoodeng.201...
, the good stability of the emulsions with PGPR could be attributed to the attachment of PGPR hydrophobic chains in the branched structure of the oil.

Figure 4
Instability index* of the W/O emulsions with different W/O ratios, at 20, 40 and 60 °C. *Different lowercase letters at the same concentration of emulsifiers and different uppercase letters in same ratio indicate significant differences between averages according to Tukey test (p < 0.05).

Ushikubo & Cunha (2014)Ushikubo, F. Y., & Cunha, R. L. (2014). Stability mechanisms of liquid water-in-oil emulsions. Food Hydrocolloids, 34, 145-153. http://dx.doi.org/10.1016/j.foodhyd.2012.11.016.
http://dx.doi.org/10.1016/j.foodhyd.2012...
studied the stability of W/O emulsions produced with different oils and emulsifiers and observed that emulsions produced with PGPR and soybean oil were the most stable. On the other side, Rabelo et al. (2018)Rabelo, C. A. S., Taarji, N., Khalid, N., Kobayashi, I., Nakajima, M., & Neves, M. A. (2018). Formulation and characterization of water-in-oil nanoemulsions loaded with açaí berry anthocyanins: insights of degradation kinetics and stability evaluation of anthocyanins and nanoemulsions. Food Research International, 106, 542-548. https://doi.org/10.1016/j.foodres.2018.01.017.
https://doi.org/10.1016/j.foodres.2018.0...
studied the stability of nanoemulsions with different W/O ratio and Açaí extract (AE) concentrations by measuring its creaming index, and observed that samples with higher AE concentrations were able to stabilize the nanoemulsions systems, especially for 30:70 W/O. They explained these results considering that AE is rich in amino acids, such as methionine, threonine, and lysine; hence, they might play a role in stabilizing the nanoemulsions.

Although no significant differences were observed between emulsions containing 3 and 5 g PGPR/100 g oil for 20/80 W/0 condition at 20 and 40 °C, a significant difference (p<0.05) was observed at 60 ºC: 3 g PGPR/100 g oil produced a more stable 20/80 W/O emulsion (Figure 4). And, this can be important for eventual application of this emulsion on thermal processed food, for instance. This W/O emulsion showed iI of 0.009 (20 ºC), 0.016 (40 ºC) and 0.024 (60 °C) (Figure 4). Therefore, the emulsion with a 20/80 W/O ratio and 3 g PGPR/100 g oil was chosen as the most stable formulation because they presented the well distributed droplet size (unimodal), the lowest D3,2 (0.25 ± 0.02 mm), and the highest physical stability at 60 °C.

Confocal laser scanning microscopy (CLSM)

The micrographs obtained using the CLSM allowed observation of a drop of PLHE dispersed in the oil phase (Figure 5). Due to the limited sensitivity of the microscope, it was not possible to observe the smallest drops of PLHE. The red color at the PLHE-oil interface corresponds to the excitation of rhodamine B present in the PLHE. This showed that the active compounds present in PLHE, predominantly polyphenols, tended to remain at the PLHE-oil interface after the process of emulsification.

Figure 5
Confocal laser scanning micrographs of the W/O emulsion with the more stable formulation and stained with Rhodamine B. Source: own authorship.

In fact, it is known that some antioxidant compounds, such as polyphenols, can remain at the water-oil interface of emulsions, interacting with both phases contributing to the reduction of the interfacial tension between them (Katsouli et al., 2017Katsouli, M., Polychniatou, V., & Tzia, C. (2017). Influence of surface-active phenolic acids and aqueous phase ratio on w/o nano-emulsions properties; model fitting and prediction of nano-emulsions oxidation stability. Journal of Food Engineering, 214, 40-46. http://dx.doi.org/10.1016/j.jfoodeng.2017.06.017.
http://dx.doi.org/10.1016/j.jfoodeng.201...
, Velderrain-Rodríguez et al., 2019Velderrain-Rodríguez, G. R., Acevedo-Fani, A., González-Aguilar, G. A., & Martín-Belloso, O. (2019). Encapsulation and stability of a phenolic-rich extract from mango peel within water-in-oil-in-water emulsions. Journal of Functional Foods, 56, 65-73. http://dx.doi.org/10.1016/j.jff.2019.02.045.
http://dx.doi.org/10.1016/j.jff.2019.02....
). That is: the PLHE may have some contribution to the high stability of the W/O emulsions produced. Gomes et al. (2016)Gomes, A., Costa, A. L. R., de Assis Perrechil, F., & da Cunha, R. L. (2016). Role of the phases composition on the incorporation of gallic acid in O/Wand W/O emulsions. Journal of Food Engineering, 168, 205-214. http://dx.doi.org/10.1016/j.jfoodeng.2015.07.041.
http://dx.doi.org/10.1016/j.jfoodeng.201...
determined lower sedimentation index (higher stability) for emulsions prepared with Gallic acid solution as W phase compared with this result for emulsion with pure water, as W phase. According to these authors, the Gallic acid could increase emulsions stability in two ways: a) changing the polarity of the phases leading to increased solubility between them or b) penetrating the interfacial film as a surfactant causing increased flexibility of the interface. The result presented in the Figure 5 corroborates with this last explanation (b).

Some physical properties of W/O emulsion with the more stable formulation

The best W/O emulsion presented a density equal to 0.937 ± 0.001 g/cm3, lower than that of water due to the presence of the oil phase. Considering the soybean oil density (ρO = ~ 0.92 g/cm3) (De Almeida et al., 2013De Almeida, K. M., De Medeiros, E. P., Gomes, J. P., & De, E. P. (2013). Caracterização físico-química de misturas de óleos vegetais para fins alimentares Physico-chemical characterization of mixtures of vegetable oils for food. Revista Verde de Agroecologia e Desenvolvimento Sustentável, 8, 218-222.), and the water density (ρW = 0.999 g/cm3), the emulsion density (ρ) can be calculated using the principle of volume additivity (1/ρ = WOO + WWW) as 0.935 g/cm3, very close of the experimental value. The contribution of PGPR and PLHE can be neglected because its concentrations in the whole emulsion were very low.

The pH of this emulsion was 5.12 ± 0.01, due to the presence of some acids in the PLHE whose pH is 4.12 ± 0.02. An acidic pH (4.2-5.5) in W/O emulsion containing Moringa leafs extract in W phase at various storage conditions was also observed by Ali & Akhtar (2014)Ali, A., & Akhtar, N. (2014). Changes in the characteristics of water-in-oil-based high internal phase emulsion containing moringa leaves extract at various storage conditions. Tropical Journal of Pharmaceutical Research, 13(5), 677. http://dx.doi.org/10.4314/tjpr.v13i5.4.
http://dx.doi.org/10.4314/tjpr.v13i5.4...
. Similar values were determined by Huma et al. (2020)Huma, S., Khan, H. M. S., Sohail, M., Akhtar, N., Rasool, F., Majeed, F., & Daniyal, M. (2020). Development, in-vitro characterization and assessment of cosmetic potential of Beta vulgaris extract emulsion. Journal of Herbal Medicine, 23, 100372. http://dx.doi.org/10.1016/j.hermed.2020.100372.
http://dx.doi.org/10.1016/j.hermed.2020....
. The pH value determined in this work is acceptable for its use as a food additive or even in the pharmaceutical and cosmetic industries, since human skin has a pH between 4.5 and 6.0, and cosmetics must have a pH in this range (Ali & Akhtar, 2014Ali, A., & Akhtar, N. (2014). Changes in the characteristics of water-in-oil-based high internal phase emulsion containing moringa leaves extract at various storage conditions. Tropical Journal of Pharmaceutical Research, 13(5), 677. http://dx.doi.org/10.4314/tjpr.v13i5.4.
http://dx.doi.org/10.4314/tjpr.v13i5.4...
).

The refractive index of this emulsion was 1.640 ± 0.004, which is typical of a mixture with phases with different refractive indices values (water = 1.333; soybean oil = 1.467) (Chantrapornchai et al., 2001Chantrapornchai, W., Clydesdale, F. M., & McClements, D. J. (2001). Influence of flocculation on optical properties of emulsions. Journal of Food Science, 66(3), 464-469. http://dx.doi.org/10.1111/j.1365-2621.2001.tb16130.x.
http://dx.doi.org/10.1111/j.1365-2621.20...
), as in the case of emulsions, which consequently appear to be opaque (Figure 1). This result is in accordance with the low light transmittance through the sample, as can be observed in Figure 3.

Regarding the emulsion viscosity, it was observed that best W/O emulsion behaves as a Newtonian fluid (Figure 6). Its viscosity was calculated (R2 > 0.99) as 104.6 ± 0.6 mPa.s. This value was higher than that of pure water (~1 mPa.s) and even that of soybean oil (~60 mPa.s) at 25 °C (Ilić et al., 2017Ilić, J. D., Nikolovski, B. G., Lončarević, I. S., Petrović, J. S., Bajac, B. M., & Vučinić-Vasić, M. (2017). Release Properties and Stability of Double W1/O/W2 Emulsions Containing Pumpkin Seed Oil. Journal of Food Process Engineering, 40(2), e12349. http://dx.doi.org/10.1111/jfpe.12349.
http://dx.doi.org/10.1111/jfpe.12349...
), because the droplets contributed to increasing the viscosity of the system, according to the Krieger Dougherty relation (Shrestha et al., 2011Shrestha, L. K., Shrestha, R. G., Abe, M., & Ariga, K. (2011). Reverse micelle microstructural transformations induced by oil and water. Soft Matter, 7(21), 10017-10024. http://dx.doi.org/10.1039/c1sm06047c.
http://dx.doi.org/10.1039/c1sm06047c...
). Tepsongkroh et al. (2015)Tepsongkroh, B., Harnsilawat, T., Maisuthisakul, P., & Chantrapornchai, W. (2015). Influence of polyglycerol polyricinoleate and biopolymers on physical properties and encapsulation efficiency of water-in-oil-in-water emulsions containing mango seed kernel extract. Journal of Dispersion Science and Technology, 36(8), 1126-1133. http://dx.doi.org/10.1080/01932691.2014.956116.
http://dx.doi.org/10.1080/01932691.2014....
observed that the increase of PGPR concentration, from 2 to 8%, increased the viscosity of 20:80 W/O emulsions from ~90 to ~116 mPa.s, that is, in the range of that determined in this work.

Figure 6
Flow curve of the W/O emulsions, at 25 °C.

Nevertheless, a non-Newtonian behavior has been observed by Ushikubo & Cunha (2014)Ushikubo, F. Y., & Cunha, R. L. (2014). Stability mechanisms of liquid water-in-oil emulsions. Food Hydrocolloids, 34, 145-153. http://dx.doi.org/10.1016/j.foodhyd.2012.11.016.
http://dx.doi.org/10.1016/j.foodhyd.2012...
, working with 30:70 W/O emulsions prepared with soybean oil and PGPR, and water, and by Rahpeyma & Sekhavatizadeh (2020)Rahpeyma, E., & Sekhavatizadeh, S. S. (2020). Effects of encapsulated green coffee extract and canola oil on liquid kashk quality. Foods and Raw Materials, 8(1), 40-51. http://dx.doi.org/10.21603/2308-4057-2020-1-40-51.
http://dx.doi.org/10.21603/2308-4057-202...
, working with 10:90 W/O emulsions prepared with canola oil with GMS, and water containing green coffee extract. Okuro et al. (2019)Okuro, P. K., Gomes, A., Costa, A. L. R., Adame, M. A., & Cunha, R. L. (2019). Formation and stability of W/O-high internal phase emulsions (HIPEs) and derived O/W emulsions stabilized by PGPR and lecithin. Food Research International, 122, 252-262. http://dx.doi.org/10.1016/j.foodres.2019.04.028. PMid:31229079.
http://dx.doi.org/10.1016/j.foodres.2019...
also observed a non-Newtonian behavior working on 75:25 W/O emulsions emulsified by PGPR.

The high viscosity found in this work also could have contributed to the high stability of W/O emulsion (McClements, 2004McClements, D. J. (2004). Food emulsions : principles, practices, and techniques (2nd ed.). Boca Raton: CRC Press. http://dx.doi.org/10.1201/9781420039436. ), and could be interesting for application of this emulsion in dressing sauces, for example, even if this product must be thermal treated until 60 °C because it is very stable in this condition.

4 Conclusions

This study showed that it is feasible to encapsulate the PLHE in a stable W/O emulsion system using PGPR as emulsifier and ultrasound equipment to homogenize. The W/O emulsions showed small and very homogeneous droplet sizes and a very high physical stability and Newtonian behavior. Overall, these properties were affected by the concentrations of the emulsifier (PGPR) and phase ratios. In addition, the analyzed physical properties of the more stable primary emulsion have shown that it can have potential applications in the food, pharmaceutical and cosmetic industry.

Acknowledgements

The authors acknowledge the São Paulo State Research Foundation (FAPESP) for the grants (2013/07914-8 and 2016/18788-1) and the Brazilian National Council for Scientific and Technological Development (CNPq) for the Research fellowship of Paulo J.A. Sobral (30.0799/2013-6). This study was financed in part by the “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil” (CAPES) - Finance Code 001 (PhD fellowships of Larissa Tessaro).

  • Practical Application: A very stable W/O emulsion containing antioxidant extract was developed.

References

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Publication Dates

  • Publication in this collection
    11 June 2021
  • Date of issue
    2022

History

  • Received
    07 Dec 2020
  • Accepted
    17 Mar 2021
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