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Glyceridic and Unsaponifiable Components of Microencapsulated Sacha Inchi (Plukenetia huayllabambana L. and Plukenetia volubilis L.) Edible Oils

Nancy A. Chasquibol 1, Gabriela Gallardo 2, Raquel B. Gómez-Coca 3, Diego Trujillo 3, Wenceslao Moreda 3 and M. Carmen Pérez-Camino 3,*

1 Center of Studies and Innovation of Functional Foods (CEIAF)-Faculty of Industrial Engineering, Institute of Scientific Research, IDIC, University of Lima, Avda. Javier Prado Este, 4600 Surco, Lima 15023, Peru; Nchasquibol@ulima.edu.pe

2 National Institute of Industrial Technology, INTI- Av. Gral. Paz 5445, San Martín, Buenos Aires B1650WAB, Argentina; ggallar@inti.gob.ar

3 Department of Characterization and Quality of Lipids, Instituto de la Grasa-CSIC, Ctra. Sevilla-Utrera km 1, Campus University Pablo de Olavide. Bg. 46, E-41013 Sevilla, Spain; raquel.coca@ig.csic.es (R.B.G.-C.); dtg.7@hotmail.com (D.T.); wmoreda@ig.csic.es (W.M.)

* Correspondence: mcperezcamino@ig.csic.es

Received: 7 November 2019; Accepted: 10 December 2019; Published: 12 December 2019

Abstract: Sacha inchi (Plukenetia huayllabambana L. and Plukenetia volubilis L.) edible oils were microencapsulated and the lipid fraction of the microparticles was characterized. Hi-cap®, Capsule®, Arabic gum, and the binary combination of Arabic gum + maltodextrin and the ternary combination of Arabic gum + maltodextrin + whey protein isolate, were used as coating materials for the encapsulation process using spray-drying. The surface and the total oils obtained from the microparticles were evaluated in terms of fatty acid composition, minor glyceride polar compounds, polymers, oxidized triglycerides, diglycerides, monoglycerides, and free fatty acids, along with their unsaponifiable components, sterols, and tocopherols. Differences between the original oils and the microencapsulated ones were determined. The most remarkable results included the presence of polymers when there were none in the original oils, the slight loss in ω3-fatty acids, up to 6%, the loss in tocopherols, in some of the cases around 30%, the maintaining of the phytosterol in their initial levels and the presence of cholesterol in the oils encapsulated with whey protein isolate.

Keywords: analytical methods; coating materials; microencapsulation; omega-3; phytosterols; sacha inchi oils; tocopherols



1. Introduction

Since its emergence in the thirties, the microencapsulation process continues as an innovative technique for use in very different fields to those for which it was originally developed. The pharmaceutical industry made its first industrial developments by launching promising new applications that have been growing ever since with the appearance of new materials and processes. The food industry has also found a great tool in microencapsulation for solving problems, which are mainly related to preservation, even though in this field the advances have not turned out to be as spectacular as they have been in other areas. Actually, when it comes to food microencapsulation the main objectives are the protection of active principles, essential oils, and flavor so that they can be available with all their properties for a longer period of time [1]. In the particular case of



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polyunsaturated oils, although with some limitations related to shelf life, depending on the coating material, the encapsulation process helps to slow down the oxidation process [2]. Currently we can find many suitable dairy preparations for infants, which contain EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) with no fishy taste or rancidity problems during their sell-by date. Walnut oil is another polyunsaturated oil that has been successfully microencapsulated using maltodextrin combined with hydroxypropyl methylcellulose as wall materials and spray-drying [3]. Carneiro et al. [4] encapsulated linseed oils with four types of wall materials in order to determine how to maximize the encapsulation efficiency and to minimize lipid oxidation. Gallardo et al. [5] also microencapsulated linseed oil by spray-drying and applied it to bread manufacturing. Microencapsulated ω3-fatty acids have been incorporated into many other food preparations such as chocolate, powdered drink mixes, instant coffees, and teas, increasing the bioavailability of ω3-fatty acids and increasing its original shelf life [6]. Recently, promising valuable manuscripts on chia oil (>60% linolenic acid) encapsulations have been published [7–12].

The sacha inchi seeds Plukenetia huayllabambana L. and the most commonly commercialized variety Plukenetia volubilis L. yield highly appreciated edible oils with a special green-fruit smell and a particularly high content in polyunsaturated fatty acids. The fatty acid composition is considered of great importance for human health as it is linked to the prevention of cardiovascular diseases and high blood pressure. ω-3-linolenic acid (Ln) is the main fatty acid in both sacha inchi oil varieties, with percentages over 51% and 48% in P. huayllabambana and P. volubilis, respectively, followed by α-linoleic (L) and oleic (O) acids [13–16]. This special fatty acid composition makes these oils very vulnerable to oxidation, developing a rancid smell, which affects the organoleptic characteristics of the foods containing them. In this context, sacha inchi oils (SIOs) are good candidates to be processed with the aid of this technique in order to add them to food preparations while keeping their highly valuable compositions. Thus, at the beginning of this decade the first research on the microencapsulation process for the SIOs appeared and the research and publications continue to date [17–20].

Maltodextrin (MD) and/or Arabic gum (AG) are the most widely used coating materials. However, new ones like corn zein [17], whey protein isolate (WPI) [21], ovalbumin, pectin, and xanthan gum [18] are being tested. Regarding the formation of the final microencapsulated products, in the particular case of the studies with sacha inchi oils, spray-drying is the preferred system over other types of drying [20]. A freeze-drying system is also used [22]. Recently the use of spray drying and spray chilling technologies were combined to obtain microparticles loaded with sacha inchi oil with a double shell [23]. Da Silva Soares et al. [24] presented the characterization of microparticles of sacha inchi P. volubilis oil obtained by the complex coacervation of ovalbumin and sodium alginate.

Most of the research carried out on microencapsulation focuses on the process itself and the search for the best materials. The physical properties of the microparticles obtained are thoroughly studied with the latest and most precise techniques, as well as their oxidative stability. However, few papers address the issue of how this process affects the oil itself, or its major and minor components [25]. What is more, only a small number of research groups address the encapsulation process from the point of view of the oil components alone [26–29].

The aim of this study was to evaluate how the encapsulation process affects the ω-3-fatty acid composition and the minor glyceridic and unsaponifiable components of virgin sacha inchi, P. huayllabambana and P. volubilis, oils (SIHO and SIVO, respectively) once they have been microencapsulated by spray-drying using different coating materials.

2. Materials and Methods

2.1. Raw Material

The sacha inchi seeds (P. huayllabambana L. and P. volubilis L. varieties) were collected in the Rodriguez de Mendoza area (Peru). The oils from the two varieties (SIHO and SIVO) were obtained



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by a cold-press system in a pilot plant in the Center of Studies and Innovation of Functional Foods (CEIAF) from Lima University. They were kept at 4 ◦C in the dark until use.

The coating materials, MD, AG, and WPI were obtained from Frutarom-Perú S.A. Also, two chemically n-octenyl succinic andydrid (OSAN)-modified starches, Capsul® TA (derived from Tapioca starch, (Capsul)) and Hi-cap® 100 TM (derived from waxy maize, (Hi-cap)) were tested, both (Capsul and Hi-cap) supplied by Ingredion. These wall materials were chosen based on their availability in the study area, their price, and their efficiency in other similar processes.

2.2. Solvents and Reactants

Ethanol, diethyl ether, hexane, chloroform, anhydrous sodium sulphate, potassium hydroxide, sulphuric acid, and ammonium hydroxide, were of analytical grade and were purchased from Merck-Spain (Merck Group, Darmstadt, Germany). Silica cartridges for solid-phase extraction (SPE 6 cc/1 g) were from Varian (EA Middelburg, The Netherlands). Tetrahydrofuran (HPLC grade) was supplied by VWR International (West Chester, PA, USA). Pyridine was from Sigma-Aldrich and BSTFA-TMCS (N,O-Bis (trimethylsilyl) trifluoroacetamide-Trimethylsilyl chloride) (99:1, v/v) a derivatizing reagent for GC was from TCI, Co., LTD, Tokyo, Japan.

2.3. Preparation of Sacha Inchi Oil Microparticles by Spray-Drying

The microencapsulation experiments were carried out using five coating materials with each sacha inchi oil ecotype.

For SIHO the samples were: 1. SIHO + Hi-cap, 2. SIHO + Capsul, 3. SIHO + AG, 4. Binary blend: SIHO + AG + MD, and 5. Ternary blend: SIHO + AG-MD + WPI.

For SIVO the samples were: 1. SIVO + Hi-cap, 2. SIVO + Capsul, 3. SIVO + AG, 4. Binary blend: SIVO + AG + MD, and 5. Ternary blend: SIVO + AG-MD + WPI.

Solutions were prepared with the coating materials and distilled water according to specifications given by the provider: MD, Capsul, and Hi-cap were kept at the controlled temperature of 25 ◦C until completely dissolved. For AG, the mixture was stirred overnight and for WPI the mixture was heated at 80 ◦C in order to partially denature the protein and open the globular chains. The total solid concentration was fixed at 27%. Sacha inchi oil was then added to the coating material solution at a concentration of 20% with respect to total solids. Emulsions were formed using a Silverson homogenizer L5M-A operating at 9000 rpm for 10 min at a very low temperature. The resultant mixture was used in the spray-drying procedure.

The spray-drying process was carried out by a laboratory scale spray-dryer (mini spray-dryer Büchi, B-290 Switzerland with a nozzle atomization system of 0.7 mm diameter) operated at an air inlet and outlet temperature of 150 and 80 ◦C, respectively, and a flow rate of 55 mL·min−1, fed by a peristaltic pump operating at a flow rate of 12 mL·min−1. The dried powders collected were stored in opaque hermetic bags at −15 ◦C for further analysis.

2.4. Extraction of the Surface and Total Oil

From the dried powders, the surface oil (SO) and total oil (TO; oil trapped inside the microparticle core plus surface oil) were obtained in order to study their compositions. The SO is the oil which did not react with the solid matrix and can be easily obtained via organic solvents. Thus, in the spray-dried powder, the SO was determined gravimetrically by immersing an aliquot into hexane (1:10 w/v) for ten minutes at room temperature. Next, the solution was filtered through a Whatman no. 1 filter paper containing anhydrous sodium sulphate and evaporated using a rotary evaporator until dryness followed by a soft nitrogen stream until constant weight [30]. The data of the SO and the sacha inchi oil added (SIO) for encapsulation, served to determine the efficacy of the encapsulation (EE) by using the equation: EE (%) = (SIO − SO) × 100/SIO. Where SIO is the oil used for the encapsulation process and SO the surface oil obtained in the aliquot of the microparticles.



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The TO of the microparticle samples were extracted through a basic digestion following a standardized procedure [31] with slight modifications: an aliquot of 5 ± 0.01 g of microparticles was added to 40 mL of deionized water heated at 65 ◦C. After stirring, 8 mL of 30% NH4OH were added and the admixture was stirred at 65 ◦C for 15 min. After cooling at room temperature, the lipids were extracted by three liquid–liquid extractions with the following sequence of solvents: a first extraction with ethanol (20 mL) followed by diethyl ether (50 mL) and then hexane (50 mL); a second extraction with diethyl ether (25 mL), hexane (25 mL), and ethanol (10 mL); and a third extraction with diethyl ether (25 mL) and hexane (25 mL). If some emulsions appeared 10 mL of ethanol were used. The extracts were filtered through a Whatman n. 1 filter paper containing anhydrous Na2SO4, and subsequently taken to a rotary evaporator to remove the solvent. Next, the samples were brought to constant weight using a stream of nitrogen.

2.5. Analytical Determinations

The physicochemical characteristics of the microparticles, particle size distribution and morphology, along with oxidative stability and their shelf life at 25 ◦C were already described [21].

In the present work, we focused on the chemical changes that the whole microencapsulation variable process (humidity, temperature, oxygen exposition, and wall materials) would produce on the oil components of the microparticles, determining the SO and the TO.

The initial SIOs (SIHO and SIVO) and the oils extracted from the microparticles (the SO and the TO) were characterized by the determinations detailed below: fatty acid composition, determined as fatty acid methyl esters (FAMEs); glyceridic polar compounds, namely triglycerides polymers (TG-P), oxidized triglycerides (ox-TG), diglycerides (DG), monoglycerides (MG), free fatty acids (FFA); and two of the main components of the unsaponifiable matter (tocopherols and sterols).

2.5.1. Fatty Acid Composition Analysis

FAMEs were prepared according to the IUPAC Standard Method [32]. The oil samples in hexane (50 mg·2 mL−1) were transesterified using 250 µL KOH/MeOH 2 N and the FAMEs formed were analyzed using a 7890B Agilent gas chromatograph (Agilent Technologies, USA) equipped with a SP2380 polar capillary column (poly (90% biscyanopropyl–10% cyanopropylphenyl-siloxane, 60 m × 0.25 mm i.d.; 0.20 µm film thickness) and a flame ionization detector (FID). The injector and detector temperatures were maintained at 225 and 250 ◦C, respectively. Hydrogen was used as carrier gas at a flow rate of 1.0 mL·min−1. The oven temperature was set at 165 ◦C and increased to 230 ◦C at 3 ◦C·min−1 maintaining this temperature for 2 minutes. The injection volume was 1 µL.

2.5.2. Minor Glyceride Polar Compounds Analysis

The total quantity and the distribution of the minor glyceride polar compounds in the original SIOs and in the microencapsulated, both the superficial and the total one, were determined by high performance size exclusion chromatography (HPSEC) [26,28]. Previously, the oil fraction to be quantified was isolated using silica-solid phase extraction (Si-SPE). One hundred milligrams of the oil sample were weighed with mg precision and introduced into the Si-SPE column (previously conditioned with 6 mL hexane) using hexane (1 mL × 3 times) and separated into two fractions. The first fraction [unaltered triacylglycerols (TG)] was eluted with 15 mL of an hexane:diethyl ether (87:13, v/v) mixture and discarded. Subsequently, a second fraction (containing minor glyceride polar compounds) was eluted with 15 mL diethyl ether, which, after evaporation until dryness, was re-dissolved with 1 mL tetrahydrofuran (THF).

The separation and quantification of the individual glyceride polar compounds were performed by HPSEC using a chromatograph equipped with a HP1100 pump (Hewlett-Packard 1050 series), a Rheodyne 7725i injector (10 µL sample loop), and a refractive index detector (HP 1037 A, Agilent Technologies, Palo Alto, CA, USA). The separation was performed on a 100 Å column (25 cm × 0.77 cm i.d.) packed with porous, highly cross-linked styrene-divinylbenzene copolymers (5 µm film



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thickness; Agilent Technologies, Palo Alto, CA, USA). THF was used as the mobile phase at a flow rate of 0.7 mL·min−1. Quantitation of individual and total polar compounds was achieved using an external standard of pure TG in THF at concentrations of 0.1–2 mg·mL−1.

2.5.3. Tocopherol Analysis

Tocopherols were determined according to the IUPAC Standard Method 2432 [33]. Oil solutions in hexane at concentrations of 5 mg·mL−1 were prepared and analyzed by HPLC fitted with a Si-column (250 mm × 4 mm i.d.; 4 µm particle size). The elution solvent was a mixture of hexane:2-propanol (99:1, v/v) at a flow rate of 1 mL·min−1. Detection was done by fluorescence (RF-10AXL Shimadzu fluorescence detector, Shimadzu, Columbia, MD, USA), setting excitation and emission at 290 and 330 nm wave lengths, respectively. For quantitative determinations, standards of tocopherols in hexane at concentrations of 4–6 µg·mL−1 were prepared and injected.

2.5.4. Sterol Composition Analysis

A faster analytical method for the determination of phytosterols in small amounts of seed oils was applied [34]. This new procedure was followed with slight modifications mainly concerning the separation by an aminopropyl cartridge, a step, which was avoided in our case. Briefly: 50 mg of the oil samples, (SIOs, the surface ones, and total ones), were weighed with mg precision in a 20 mL test tube with screw cap and 10 µL of the internal standard of α-cholestanol (1 mg·mL−1 in chloroform) were added. The solvent was evaporated under a nitrogen steam. Subsequently, 5 mL of MeONa (3% wt/v) were added and the tube was placed in a bath at 80 ◦C for 30 min. Next, 5 mL of the admixture H2SO4 in MeOH (4% v/v) were added and maintained for 30 min at the same temperature. The lipid fraction was extracted three times with hexane (2 mL each), washed two times with water (2 mL each), and treated with anhydrous sodium sulfate to eliminate traces of water. The solution was evaporated until dryness under nitrogen, derivatized with 500 µL of the silylating reagent: BSTFA-TMCS:Pyridine1:1 (v/v) and analyzed by GC. The gas chromatograph (Agilent 6890N, Palo Alto, CA, USA) was equipped with a fused silica low-polarity capillary column (poly (5% diphenyl−95% dimethyl) siloxane, 30 m × 0.25 mm i.d. × 0.25 µm film thickness), and FID. The oven program was isothermally set at 260 ◦C with a 1:50 split ratio. Hydrogen was used as carrier gas at a 1 mL·min−1 flow rate. The temperature of the injector and detector was 300 ◦C.

2.6. Data Analysis

All the data were the average of three determinations that were statistically analyzed using the software Statistica 8.0 (StatSoft). Mean analysis was performed using Duncan’s procedure at p ≤ 0.05.

3. Results and Discussion

Since the SIOs are considered a good source of essential fatty acids, (α-L and ω3-Ln), it would be of great interest to incorporate them into food products, such as bakery products, energy drinks, infant formulations, snacks, etc. Therefore, it becomes necessary to preserve them on the one hand, and on the other to facilitate their inclusion as food ingredients. A good way to achieve this is to do it microencapsulated form. So, their fatty acid composition could remain intact for a longer period of time and, it would also ensure that the product does not evolve towards notes of rancidity that may be rejected by consumers. In this way, it is necessary to know the most appropriate coating materials and how the microencapsulation process affects the oil composition, in terms of both changes in the ω3-fatty acid and also the minor components of high value such as sterols and tocopherols.

The obtaining of the SO by immersing the microparticle samples in hexane and the TO by alkaline digestion were done with special care, avoiding high temperatures during the evaporation of solvents. Exposure to air was also avoided by protecting it under nitrogen flow to guarantee that the changes were due to the encapsulation process and not to the process of obtaining the oil samples.



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According to Table 1, the SO percentages were between 0.5% and 7.7% for SIHO and between 0.7% and 7.6% for SIVO. For both oils the wall materials Capsul and Hi-cap presented lower surface oil and consequently a higher encapsulation efficiency (EE). On the contrary, the ternary blend AG + MD + WPI gave the highest surface oil and consequently the lowest EE for both oils.



Table 1. Surface oil (SO) obtained by immersion with hexane, and encapsulation efficiency (EE).



Oil



Wall Material



SO (%w/w)



EE (%)



P. huayllabambana P. volubilis



Hi-cap Capsul AG AG + MD AG+ MD + WPI Hi-cap Capsul AG AG + MD AG+ MD + WPI



1.3 ± 0.1 f 0.5 ± 0.1 h 5.4 ± 0.1 f 4.8 ± 0.1 e 7.7 ± 0.1 a

0.7 ± 0.1 g,h 0.8 ± 0.1 g

6.5 ± 0.1 b 6.0 ± 0.1 c 7.6 ± 0.1 a



93.3 ± 0.5 C 97.6 ± 0.5 A 73.0 ± 1.5 E 75.8 ± 0.9 D 61.1 ± 0.6 G 96.5 ± 0.7 A,B 96.0 ± 0.7 B 67.4 ± 0.5 F 67.9 ± 0.5 F 61.7 ± 0.6 G



Different letters, indicate significant difference values at p < 0.05 (n = 3; lower case letters for SO and capital letters for EE).



These data cannot be due to or explained by the physical size of the particles, since, for both SIOs, the particles with the ternary blend (AG + MD + WPI) presented the largest sizes (6.1–9.8 µm for SIHO and SIVO, respectively) [21] and, consequently, the lowest surface/volume ratio. On the contrary, Capsul, also for both SIOs, was the wall material with the smallest particle size (2.5 and 1.2 for SIHO and SIVO, respectively, data not included in the above-mentioned paper) showing the majority of the population collapsed structures.

3.1. Fatty Acid Composition

The fatty acids detected in the samples of SIOs under study comprised the saturated group, which included the acids C16:0, C17:0, C18:0, and C20:0; the monounsaturated acids determined were C16:1ω9, C16:1ω7, C17:1, C18:1ω9, C18:1ω7, and C20:1ω9; and the polyunsaturated acids were C18:2ω6 and C18:3ω3. The data of the fatty acid composition of the initial SIOs are included in Table 2, where the differences between the two sacha inchi oil varieties can be observed. The oils from P. huayllabambana showed a higher unsaturation level than those from P. volubilis, having more than half its fatty acids formed by ω3-linolenic acid (58.12%) followed by ω6-linoleic (>25%) and ω9-oleic (7.95%). These data are in agreement with the requirement of the technical regulation of Peru (NTP) for sacha inchi oils of the P. huayllabambana variety, where minimum levels of oleic (7.9%), linoleic (24.0%) and linolenic (55.0%) acids are required [35]. In relation to P. volubilis, the results of the fatty acid composition obtained for this oil were also in agreement with the requirements of NTP for this variety (oleic acid >8.9%, linoleic acid >32.1% and linolenic acid >44.7%). Many researchers consider a diet healthy when it contains a lipid fraction rich in ω3-fatty acids [36]. Thus, a lower ratio of ω6-/ω3-fatty acids would be desirable in order to reduce the risk of many of the chronic diseases of high prevalence in developing countries [37]. The exact value for the ω6/ω3-fatty acid ratio is given in many papers, but certain studies indicate that the optimal ratio may vary depending on the disease under consideration.



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Table 2. Fatty acid composition of sacha inchi oils before and after the encapsulation process (total and superficial oils extracted from microencapsulation with five wall materials). Data are expressed as percentages of total fatty acids.



Samples



C16:0



C16:1



C17:0



C17:1



Fatty Acid Composition (%)



C18:0



C18:1



C18:2



C18:3



C20:0



C20:1



Trans



P. huayllabambana Initial oil Total encapsulated oils SIHO + Hi-cap SIHO + Capsul SIHO + AG SIHO + AG + MD SIHO + AG + MD + WPI Surface oils SIHO + Hi-cap SIHO + Capsul SIHO + AG SIHO+ AG + MD SIHO + AG + MD + WPI P. volubilis

Initial oil Total encapsulated oils SIVO + Hi-cap SIVO + Capsul SIVO + AG SIVO + AG + MD SIV O+ AG + MD + WPI Surface oils SIVO + Hi-cap SIVO + Capsul SIVO + AG SIVO + AG + MD SIVO + AG + MD + WPI



4.50 ± 0.24 0.07 ± 0.01 0.06 ± 0.01 0.04 ± 0.01 1.75 ± 0.05 7.95 ± 0.30 26.10 ± 0.50 58.12 ± 0.55 a 0.31 ± 0.15 0.29 ± 0.15 nd *

4.58 ± 0.23 0.05 ± 0.01 0.05 ± 0.01 0.04 ± 0.01 1.77 ± 0.09 7.88 ± 0.30 26.57 ± 0.50 58.61 ± 0.56 a 0.31 ± 0.15 0.28 ± 0.15 nd * 4.82 ± 0.25 0.03 ± 0.01 0.04 ± 0.01 0.04 ± 0.01 1.84 ± 0.09 8.57 ± 0.30 26.56 ± 0.50 57.58 ± 0.52 a 0.34 ± 0.15 0.30 ± 0.14 nd * 4.57 ± 0.25 0.06 ± 0.01 0.07 ± 0.01 0.04 ± 0.01 2.08 ± 0.08 8.62 ± 0.30 28.52 ± 0.50 55.46 ± 0.55 b 0.30 ± 0.15 0.28 ± 0.14 nd * 5.20 ± 0.23 0.03 ± 0.01 0.07 ± 0.01 0.05 ± 0.01 1.78 ± 0.09 11.08 ± 0.30 25.56 ± 0.52 55.60 ± 0.54 b 0.30 ± 0.15 0.22 ± 0.15 nd * 5.38 ± 0.24 0.04 ± 0.01 0.06 ± 0.01 0.04 ± 0.01 1.94 ± 0.09 12.38 ± 0.30 25.21 ± 0.53 54.50 ± 0.55 b 0.31 ± 0.15 0.22 ± 0.14 nd *

4.65 ± 0.24 0.07 ± 0.01 0.06 ± 0.02 0.04 ± 0.00 1.70 ± 0.09 7.85 ± 0.35 26.20 ± 0.51 58.02 ± 0.55 a 0.31 ± 0.15 0.29 ± 0.15 nd * 4.55 ± 0.23 0.07 ± 0.02 0.06 ± 0.02 0.04 ± 0.00 1.75 ± 0.09 7.90 ± 0.36 26.40 ± 0.50 58.00 ± 0.56 a 0.33 ± 0.16 0.29 ± 0.10 nd * 4.52 ± 0.23 0.09 ± 0.01 0.08 ± 0.02 0.04 ± 0.01 2.13 ± 0.09 8.35 ± 0.35 28.98 ± 0.50 55.29 ± 0.54 b 0.33 ± 0.15 0.23 ± 0.11 nd * 4.65 ± 0.24 0.06 ± 0.01 0.07 ± 0.02 0.04 ± 0.01 1.73 ± 0.09 7.84 ± 0.35 26.41 ± 0.58 58.71 ± 0.55 a 0.30 ± 0.14 0.23 ± 0.12 nd * 4.72 ± 0.25 0.07 ± 0.01 0.10 ± 0.01 0.04 ± 0.01 1.83 ± 0.09 8.07 ± 0.40 26.41 ± 0.58 58.26 ± 0.54 a 0.32 ± 0.14 0.22 ± 0.10 nd *

3.95 ± 0.21 0.06 ± 0.01 0.09 ± 0.01 0.05 ± 0.01 3.05 ± 0.15 9.90 ± 0.35 34.42 ± 0.58 47.88 ± 0.44 A,B 0.32 ± 0.15 0.28 ± 0.15 nd *

4.06 ± 0.24 0.06 ± 0.01 0.09 ± 0.01 0.05 ± 0.01 3.06 ± 0.16 10.34 ± 0.34 34.27 ± 0.58 47.66 ± 0.45 B 0.33 ± 0.15 0.28 ± 0.15 nd * 4.43 ± 0.23 0.08 ± 0.02 0.09 ± 0.01 0.05 ± 0.01 3.07 ± 0.16 12.04 ± 0.36 33.52 ± 0.58 46.18 ± 0.45 D 0.31 ± 0.15 0.28 ± 0.15 nd * 4.21 ± 0.23 0.10 ± 0.02 0.79 ± 0.01 0.05 ± 0.01 3.34 ± 0.16 9.85 ± 0.35 33.91 ± 0.58 47.18 ± 0.41 C 0.39 ± 0.01 0.23 ± 0.15 nd * 4.10 ± 0.23 0.06 ± 0.02 0.05 ± 0.01 0.11 ± 0.01 3.12 ± 0.16 10.03 ± 0.35 34.27 ± 0.58 47.77 ± 045 B 0.32 ± 0.01 0.23 ± 0.15 nd * 4.67 ± 0.23 0.09 ± 0.02 0.09 ± 0.01 0.05 ± 0.01 3.16 ± 0.16 11.44 ± 0.36 33.55 ± 0.58 46.40 ± 0.46 C,D 0.32 ± 0.01 0.28 ± 0.15 nd *

4.09 ± 0.20 0.09 ± 0.02 0.09 ± 0.01 0.09 ± 0.01 3.06 ± 0.01 10.45 ± 0.29 34.33 ± 0.56 47.38 ± 0.44 B,C 0.32 ± 0.15 0.27 ± 0.15 nd * 4.09 ± 0.20 0.09 ± 0.02 0.09 ± 0.01 0.09 ± 0.01 3.08 ± 0.01 9.42 ± 0.25 34.66 ± 0.56 48.06 ± 0.43 A,B 0.31 ± 0.15 0.28 ± 0.15 nd * 3.99 ± 0.20 0.09 ± 0.02 0.09 ± 0.01 0.09 ± 0.01 3.04 ± 0.01 9.25 ± 0.25 34.73 ± 0.54 48.47 ± 0.45 A 0.30 ± 0.16 0.21 ± 0.15 nd * 3.95 ± 0.20 0.06 ± 0.02 0.09 ± 0.01 0.09 ± 0.01 3.05 ± 0.01 9.28 ± 0.25 34.70 ± 0.54 48.48 ± 0.44 A 0.32 ± 0.15 0.21 ± 0.15 nd * 4.18 ± 0.20 0.06 ± 0.01 0.09 ± 0.01 0.09 ± 0.01 3.10 ± 0.01 10.16 ± 0.29 34.22 ± 0.55 47.71 ± 0.45 A,B 0.34 ± 0.15 0.28 ± 0.15 nd *



Different letters for the major fatty acid (ω3-Ln) indicate significant difference values at p < 0.05 (n = 3; lower case letters for P. huayllabambana oil (SIHO) and capital letters for P. volubilis oil (SIVO)).nd *: not detected.



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After the extraction of the microencapsulated oils, both from the surface and from the total microparticles, in the case of the P. huayllabambana variety (Table 2) a slight reduction in the Ln acid percentages was observed (mainly in the total oil fraction) compared to the initial oil. Thus, the encapsulation of P. huayllabambana with the ternary coating material (AG + MD + WPI) showed a value of 54.50% for Ln, which represented a 6.2% loss during the encapsulation procedure. The rest of the coating material losses during the encapsulation process varied between 4.7% and 0%.

In the case of the surface or non-encapsulated oil the loss was only appreciated when the encapsulation was done with AG as coating material (5%). In the oils obtained from the particles done with the P. volubilis variety, the decrease in the Ln percentage was not so evident, although the highest losses (around 3%) were observed for the surface oil encapsulated with Capsul as wall material.

Ln is the major and most abundant unsaturated acid in these oils and the first to be altered in the thermal conditions used in the spray-dryer equipment. Although the maximum temperature was not excessively high and the time was short, it may be that during the drying processes there was a greater shear effect, which makes the maximum temperature at some points higher than the average indicated during the passage through the spry-dryer. Under these conditions Ln is the acid most likely to be altered. It is important to note that concentrations below 1% for minor fatty acids, saturated or unsaturated, (C16:1, C17:0, and C17:1) were also possible to quantify in the encapsulated samples, and it was therefore deduced that they were not lost during the encapsulation process.

Regarding the trans fatty acid isomers (from oleic, linoleic, and linolenic acids), it is well known that they are produced from their cis-unsaturated counterparts during oil exposure at high temperatures as well as during the hydrogenation process in the presence of hydrogen and metal-catalysts [38]. The quantification can be done in the same chromatogram and conditions as for the rest of the fatty acids since they are eluted just before their corresponding cis-isomers. After the study in detail of the fatty acid chromatograms of all of the samples, the initial and the encapsulated oils, surface, or total ones, no peaks corresponding to the presence of trans fatty acids were present. That meant that during the whole encapsulation process, including the spray-drying stage, isomerization phenomena did not take place. Several papers have focused on the encapsulation feasibility of a very specific group of polyunsaturated fatty acids, conjugated linoleic acid (CLA). CLA contains a very high proportion of trans-fatty acid isomers with the C10-trans-C12-cis isomer often being the most prevalent [27,39,40] and, no report has highlighted an increase in the amount of the trans-isomers. What is more, it was pointed out that the composition of the trans-CLA isomer proportion was reduced significantly in microencapsulated oil samples with respect to the initial oil samples with the consequent increase in the saturated fatty acids, as each fatty acid methyl ester is expressed as a percentage on the total ones [27].

3.2. Minor Glyceride Polar Compound Composition

The so-called minor glyceride polar compounds comprise several families of compounds of very different molecular size, which is why they can be separated easily and quantified by HPSEC. TG-P, oxTG, DG, MG, and FFA elute from the chromatographic column in the opposite order of their molecular size. Those compounds correspond to three main alterations, which the oils exposed to high temperature suffer, atmospheric oxygen, and humidity. The corresponding alterations are thermic, oxidative, and hydrolytic, and can take place even during the oil extraction processes. These compounds were quantified by HPSEC with a refractive index detector using pure TGs as external standards and assuming the same response factor for all of them. Figure 1 includes two HPSE-chromatograms corresponding to one of the initial oils and to the total oil obtained after encapsulation. As can be observed, ox-TG, DG, MG, and FFA were detected in the original oils while the same ones, together with TG-P, were present in the encapsulated ones. Table 3 shows the data for the results of the thermal alteration (TG-P) in the first column, the oxidative alterations (ox-TG) in the second one, and diglycerides in the third one. Monoglycerides and free fatty acids were added to have data for the hydrolytic alteration (DG + MG + FA). The last column corresponds to total glyceride polar compounds



Foods 2019, 8, 671 Foods 2018, 7, x FOR PEER REVIEW



9 of 18 9 of 19



(TPCd)aatas othbetasiunmedofof rthaenythgroeoedaletderibatleioonisl.[4T1h].eFToPr Cthveaelnuceaspwsuelraetevdersyamlopwle, sath2e.6T%PCansdho2w.3e%d fsoigrntihfiecant initiadliPff.ehrueanyclelasbwamitbhanthaeairndorPig. vinoalulboiliils,owilsit,hanthdeithwigahseisnt vagalrueems (e4n5tmwgit·hg−d1)abtaeionbgtatihnoesde foobrtaninyegdofoodr the edibloeilosilen[4c1a]p. sFuolrathede ewnictahpAsuGla. ted samples the TPC showed significant differences with their original oils, with the highest values (45 mg·g−1) being those obtained for the oils encapsulated with AG.



A



B



FigurFeig1u. rHeig1h. pHerifgohrmpaenrcfeorsmizeanexceclussiizoen (eHxcPlSuEsi)ocnhro(HmPatSoEg)racmhrsoomf mationgorragmlysceorfidempinoolarr cgolmycpeoriudnedspolar from.co(Am)pSoIuHnOdsanfrdom(B.)(Ato)taSlIHoiOl fraonmd (mBi)ctrooteanlcoaipl fsruolmatemdiScIrHoeOn.cRapt s~u8la.2teTdGS-IPH; ORt. R~t8~.78o.2xT-TGG-;PR; Rt ~t ~98.1.7 oxDG; RTtG~; R9.t7~–99..61 MDGG;; RRtt ~~ 99..79–F9F.6AM. G; Rt ~ 9.9 FFA.



As caTnabblee o3.bPsoelravrecdominpFoiugnudrsei1nAto,tfaoluarndpesuakpserafipcpiaelaorielsdexcotrrarcetsepdofnrodminmgitcorooexnTcaGp,sDulGat,eMd oGil,saonfdsaFcFhAa for the twioncshaich(Pa.inhuchayilolaiblsamubnadnearasntuddPy.. vTohluebrieliws) aws intho pfirveesewnaclel omfaTteGr-iaPls(raentedntinionthteimineit8ia, linoitlhseuosreidgifnoarl

oil) meaneinngcatphsautladtuiornin. g their extraction only a cold temperature was used and that they actually were



crude or virgin oils. The major polar compounds for the two oil varieties corresponded to the hydrolytic



alteration,



whSicahmwplaes



18.2



mg·g−1



oil for SIHOMainndor1G6.l5ycmergid·ge−P1oflaorrCSoIVmOpo. uAnfdtser(mthge·ge−1n) capsulation



TG-P



ox-TG



DG+MG+FA



Total PC



procesPs.ohnueayolflathbaemmboasntaremarkable results was the presence of TG-P (retention time ~8.2, Figure 1B) in



all theInoiitlisaal mOiplles (Table 3), which meannds*that the temp7e.8ra±t0u.r1e4 used in t1h8e.2st±e0p.1o5f spray-d2r6y.0in±g0.w21ais



sufficiTenottafloErnicnaiptisautilnatgedthOeiplsolymerization of the oils. HPSEC has been successfully used to carry out



studieSsIHonOm+ iHcrio-ceanpcapsulated oil [291]..1N±e0v.0e2rtheless, it w13a.7s±n0o.t11specificall1y9.h1i±gh0.l1ig6hted tha3t3p.9ol±y0m.2e6rfs



were fSoIrHmOed+ Cdauprsinulg the encapsulatio2n.0p±r0o.c0e3ss. This as1p1e.c7t±is0.n12ot so obvi1o9u.1s,±s0in.1c5e the inv3e2s.t8ig±a0t.i3o2nhs



conduScItHedO w+ AitGh refined oils that alr5e.a5d±y0.c0o6ntained T2G0.5di±m0.e1r5s or poly1m9.e1r±s 0o.1ri5ginated 4d5u.1ri±n0g.3t4hae



deodoSrIiHzaOti+oAn Gsta+gMeDof the refining p4r.4oc±e0s.s0.7Far from c2o0m.3 p±a0r.2in6g this pr1o9c.e3s±s0w.1i5th what h44a.p0p±e0n.3s6ina



an oilSrIeHfiOni+nAgGpr+oMceDss+, WmPicIroencaps3u.0la±t0io.0n5 will bring17i.m7 ±po0.r1t2ant benefi1t9s.1b±u0t.1a5lso oil de3g9.r8a±da0.t3io1nc .



TherebSyu,rtfhaeceoObtialsined results indicated that by starting from oils without polymers in their composition,



polymSeIHrizOa+tioHni-ctaopok place and TG-0P.6w±e0r.0e1present in1b8.o2t±h0o.1i0l fractions1,9.t3h±e 0e.1n5capsulate3d8.1a±n0d.2t2hde



SIHO + Capsul



0.6 ± 0.05



11.0 ± 0.14



19.4 ± 0.17



31.0 ± 0.30 g



tneomnp-eenSSrIIcaHHatupOOrs++eu.AAlaTGGthe+edMvoaDnluese,srfeograTrdGl-ePsswo00e..f68ret±±hi00en..00ct53hoeatriannggme aot22fe520r...11i6a±±–l 500s...i512n00mcegi·tgs−f1oor11mil99,..a23fto±±iro00nP..11.47ihsucalyolslaeblya44m25re..b10laa±±nta00e..da2388ntdboa



0.2–0.9SIHmOg·+g−A1Go+ilM, fDor+ PW. PvIolubilis. D1.i0ff±er0e.0n3ces were o1b7s.0er±v0e.1d1 for total1a9.n0d± s0u.1r6face oils3i7n.0b±o0t.h24oef



them,Pw. vitohlutbhielitsotal being slightly more polymerized than the surface ones. In relation to oxidative

degradInaittiioalnOs i(lox-TG), the values for thnde i*nitial oils wer6e.57±.08.1a0nd 6.5 mg·1g6−.51±(0S.I1H6 O vs. SI2V3.O0 )±. 0A.2f6teGr

encapsTuoltaatlioennc, atphseuselacteodmOpiolus nds experimented an increase in their values and for all the core materials with sStaIVtiOsti+caHli-sciagpnificance with resp0e.4ct±t0o.2t6heir initial o8.i3ls±. 0S.t2a6tistical diff17e.r0e±nc0e.1s4were als2o5.o7b±ta0i.3n5edF betweSeInVOsu+rfCaacpesaunl d encapsulated o0.i5l ±fr0a.c2t6ions in tota8l.1p±ol0a.2r0compoun1d7s.4, ±be0i.1n6g slightly26h.0ig±h0e.2r8inF



the forSmIVeOr +foArGboth types of oils. Wo0r.k9 i±n0g.2w6 ith dried m11i.c9r±o0e.n1c2apsulated17fi.4s±h0o.1il5s, Velasc3o0.e2t±a0l.2[84B2,C]



establiSsIhVeOd+imAGpo+rtManDt differences in t0h.2e±o0x.i2d6ation betw1e0e.5n±fr0e.2e6and enca1p7s.u4l±at0e.1d6oil fracti2o8n.1s±fo0r.2t8hDe



SIVO + AG + MD + WPI



0.7 ± 0.26



8.8 ± 0.26



17.4 ± 0.13



26.9 ± 0.24 E



Surface Oils



SIVO + Hi-cap



0.3 ± 0.26



11.3 ± 0.26



17.3 ± 0.16



28.9 ± 0.30 D



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first time. In their work, the authors highlighted the differences in the heterogeneous aspects of lipid oxidation in dried microencapsulated oils.



Table 3. Polar compounds in total and superficial oils extracted from microencapsulated oils of sacha inchi (P. huayllabambana and P. volubilis) with five wall materials and in the initial oils used for encapsulation.



Samples



TG-P



Minor Glyceride Polar Compounds (mg·g−1)



ox-TG



DG + MG + FA



Total PC



P. huayllabambana

Initial Oil Total Encapsulated Oils SIHO + Hi-cap SIHO + Capsul SIHO + AG SIHO + AG + MD SIHO + AG + MD + WPI Surface Oils SIHO + Hi-cap SIHO + Capsul SIHO + AG SIHO + AG + MD SIHO + AG + MD + WPI P. volubilis

Initial Oil Total encapsulated Oils SIVO + Hi-cap SIVO + Capsul SIVO + AG SIVO + AG + MD SIVO + AG + MD + WPI Surface Oils SIVO + Hi-cap SIVO + Capsul SIVO + AG SIVO + AG + MD SIVO + AG + MD + WPI



nd *

1.1 ± 0.02 2.0 ± 0.03 5.5 ± 0.06 4.4 ± 0.07 3.0 ± 0.05

0.6 ± 0.01 0.6 ± 0.05 0.6 ± 0.05 0.8 ± 0.03 1.0 ± 0.03

nd *

0.4 ± 0.26 0.5 ± 0.26 0.9 ± 0.26 0.2 ± 0.26 0.7 ± 0.26

0.3 ± 0.26 0.9 ± 0.26 0.4 ± 0.26 0.6 ± 0.26 0.5 ± 0.26



7.8 ± 0.14

13.7 ± 0.11 11.7 ± 0.12 20.5 ± 0.15 20.3 ± 0.26 17.7 ± 0.12

18.2 ± 0.10 11.0 ± 0.14 25.1 ± 0.20 22.1 ± 0.10 17.0 ± 0.11

6.5± 0.10

8.3 ± 0.26 8.1 ± 0.20 11.9 ± 0.12 10.5 ± 0.26 8.8 ± 0.26

11.3 ± 0.26 12.6 ± 0.14 14.4 ± 0.26 14.9 ± 0.26 13.4 ± 0.26



18.2 ± 0.15

19.1 ± 0.16 19.1 ± 0.15 19.1 ± 0.15 19.3 ± 0.15 19.1 ± 0.15

19.3 ± 0.15 19.4 ± 0.17 19.3 ± 0.17 19.2 ± 0.14 19.0 ± 0.16

16.5± 0.16

17.0 ± 0.14 17.4 ± 0.16 17.4 ± 0.15 17.4 ± 0.16 17.4 ± 0.13

17.3 ± 0.16 17.4 ± 0.17 17.4 ± 0.15 17.4 ± 0.11 17.1 ± 0.18



26.0 ± 0.21 i

33.9 ± 0.26 f 32.8 ± 0.32 h 45.1 ± 0.34 a 44.0 ± 0.36 a 39.8 ± 0.31 c

38.1 ± 0.22 d 31.0 ± 0.30 g 45.0 ± 0.38 a 42.1 ± 0.28 b 37.0 ± 0.24 e

23.0 ± 0.26 G

25.7 ± 0.35 F 26.0 ± 0.28 F 30.2 ± 0.28 B,C 28.1 ± 0.28 D 26.9 ± 0.24 E

28.9 ± 0.30 D 30.9 ± 0.29 B 32.2 ± 0.30 A 32.9 ± 0.32 A 31.0 ± 0.29 B



Different letters, for total PC, indicate significant difference values at p < 0.05 (n = 3). Lower case letters for SIHO and capital letters for SIVO. nd *: not detected.



In relation to hydrolysis (DG + MG + FA), no differences were observed among the encapsulation materials, but somehow it was higher than in the initial oils.



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3.3. Tocopherol Composition



Tocopherols are ones of the most powerful natural antioxidants used as food additives [43]. Sacha inchi oil is the vegetable oil with one of the highest tocopherol contents, with γ- and δ-tocopherols being the main or unique species [14,44]. With respect to the regulations on this parameter, the NTP [35] included the value of 1900 mg·kg−1 as the minimum tocopherol content in sacha inchi oils, regardless of the variety.

In the oils under study, the total tocopherol concentrations were 2660 and 4393 mg·kg−1 for P. huayllabambana and P. volubilis, respectively (Table 4). They showed a γ/δ-tocopherol ratio of 2.1 and 1.7 for SIHO and SIVO, respectively. Figure 2A,B showed that after the encapsulation process the oils suffered a loss of 15–30% in the total tocopherol concentration for the surface and total oils and with respect to the original samples of SIHO. SIVO losses with respect to the original oil were in the range of 28–24% for total and surface oils, respectively. The data included in Table 4 indicate FtFohooadodsts2t20h01e188, ,7lo7, ,xsxsFeFOsORRwPPEeEErEReRRmREEVaVIiEnIEWlWy due in γ-tocopherol so that the γ/δ-tocopherol ratio decrease1d111aooffft1e19r9 encapsulation. For P. huayllabambana the ratio medium value after the encapsulation process was 1.6 in pbprorotophpoforrrtaitcoiotninotnhthsa,anwnδhδ-et-otroeccaoospphithewerroaolslf1ofo.r0rbfboootrhtPh.ooviliosllsuabanindlidst,htchalaettasrsuluycchihnedefffiefcecactttiwnwgaastshmamtootrhreeeiniγnt-ettenonscseoepfofhorerrSoSIlVIVlOoOssthethsaanwnfeofrorer SiSnIHIHhOiOg.h. TeThrheperhohipgigohhreterirosnstattabhbialiinltiytδy-otof fcδoδ-pt-ohtocecoroopplhhfeoerroolblowtwhitihtohilrsreesaspnpedeccttht taottosγuγ-t-cothoccoeoffppehhceterrowol al issisminionraeagigrnreteeeemnmseennftot wrwSitiIhtVhOaa rtrehecacenennftoltylrypSpIuHubbOlilsi.shThehededshstiutgudhdyeyrwwshtaheberireleitthytheoefsstδat-abtboiliciltoiytpyhoofefrtohthleewddiitfihffefrereresenpntetmcmtotoloelecγcu-utloalacrrospsppheeccrieoieslsiosofiftnotoaccogoprephehemerroeolnslts(wα(α-it,-h,γγ-aaranencddenδδ-t)l-y)aapgguaaibnilnsistthcecodonntsittniunuduoyouuwsshhheeearaetitntihnggeasatttadbdifilffiefteryreenonftttthetememdppieffereraarteutunrretesms(o1(10le00c0–u–1l18a80r0s°°pCCe))cwiweasasosefevtvoaalculouapathetededro[4[l4s55](.]α.T-T,hhγeeaaaunutdhthoδor-rs)sa[4[g45a5]i]nccosotnncccloulnudtdieneddutohthuaasttδhδ-et-aototciconopgphhaeterrodolislffswewreeernretemtmeomorrepeesstrataabtbulelreethstha(an1n0t0ht–he1eγ8γ0--a◦anCnd)dαwα-as-sppeeecvciaeielsus. .ated [45]. The authors [45] concluded that δ-tocopherols were more stable than the γ- and α-species.



AA 1232132131230550505005050505



BB 2231133132125050500505500550



LLososs is innTTococoopphhererolosls( (%)%) LLososs is innTTococoopphhererolosls( (%)%)



and FFiigguuSrrueer2f2a..cLLeooosssisl.iinn ttooccoopphheerroollss((%%))aafftteerrtthhee eennccaappssuullaattiioonn pprroocceesssseess:: ((AA)) PP..hhuuaayyllllaabbaammbbaannaaooiillssaanndd((BB))



P. volubilis oils. Total oil and



Surface oil.



P. volubilis oils.



Total oil and



Surface oil.



With respect to the coating materials, for both oil ecotypes the lower quantity of total tocopherols after encapsulation was determined when the process was done with AG for total oils. In relation to the surface oils, losses in tocopherols were lower than for the total oil mainly in the case of the P. huayllabambana variety where encapsulation with AG suffered the smallest loss with respect to the other coating materials. With regards to the data obtained for P. volubilis no differences were observed among the different materials.



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Table 4. Tocopherols in total and superficial oils extracted from microencapsulated oils of sacha inchi (P. huayllabambana and P. volubilis) with five wall materials and in the initial oils used for encapsulation.



Tocopherols (mg·kg−1)



Total Extracted Oil



Surface Oil



α-



β-



γ-



δ-



Total



Ratio γ/δ



α-



β-



γ-



δ-



Total



P. huayllabambana



Initial oil



nd *



nd * 1811 ± 20 849 ± 15



2660 ± 45 a



2.1



SIHO + Hi-cap SIHO + Capsul



nd *



nd * 1295 ± 22 746 ± 14



2041 ± 40 d



1.7



nd *



nd * 1155 ± 25 762 ± 15



1917 ± 30 e



1.5



SIHO + AG



nd *



nd * 1104 ± 24 760 ± 14



1864 ± 35 f



1.5



SIHO + AG + MD



nd *



nd * 1204 ± 22 764 ± 14



1968 ± 35 e



1.6



SIHO + AG + MD + WPI nd *



nd * 1290 ± 23 749 ± 16



2039 ± 35 d



1.7



P. volubilis



Initial Oil



nd *



nd * 2767 ± 32 1626 ± 30 4393 ± 45 A



1.7



SIVO + Hi-cap



nd *



nd * 1596 ± 33 1585 ± 32 3181 ± 35 D



1.0



SIVO + Capsul



nd *



nd * 1699 ± 30 1558 ± 30 3257 ± 35 C



1.1



SIVO + AG



nd *



nd * 1550 ± 31 1615 ± 30 3165 ± 32 D



1.0



SIVO + AG + MD



nd *



nd * 1586 ± 30 1591 ± 30 3177 ± 33 D



1.0



SIVO + AG + MD+ WPI nd *



nd * 1765 ± 32 1581 ± 30 3346 ± 35 B



1.1



nd *



nd *



1221 ± 18 837 ± 14 2058 ± 32 d



nd *



nd *



1300 ± 17 840 ± 20



2140 ± 32 c



nd *



nd *



1375 ± 19 848 ± 15



2223 ± 36 b



nd *



nd *



1200 ± 19 848 ± 15 2048 ± 31 d



nd *



nd *



1406 ± 18 842 ± 12



2248 ± 30 b



nd *



nd *



1720 ± 31 1624 ± 30 3344 ± 35 B



nd *



nd *



1710 ± 31 1625 ± 35 3335 ± 30 B



nd *



nd *



1613 ± 33 1614 ± 32 3227 ± 33 C



nd *



nd *



1675 ± 33 1614 ± 33 3289 ± 35 C



nd *



nd *



1657 ± 31 1622 ± 34 3279 ± 35 C



For total tocopherols, different letters indicate significant difference values at p < 0.05 (lower case letters for SIHO and capital letters for SIVO). nd *: not detected.



Ratio γ/δ

1.5 1.5 1.6 1.4 1.7

1.1 1.1 1.0 1.0 1.0



after encapsulation was determined when the process was done with AG for total oils. In relation to



the surface oils, losses in tocopherols were lower than for the total oil mainly in the case of the P.



huayllabambana variety where encapsulation with AG suffered the smallest loss with respect to the



other coating materials. With regards to the data obtained for P. volubilis no differences were observed



Fooadms 2o0n19g, 8t,h6e71different materials.



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3.43..4S. tSetreorloCl oCmompopsoitsiiotnion Vegetable sterols or phytosterols are components of the unsaponifiable matter of vegetable oils

witVhegaentaibmlepsotretraonlts onruptrhityiotonsatlerroollsearaencdombepnoenfeicniatsl opfrtohpeeurtniessaptoonihfiuamblaenmhaettaelrtho.f Sveevgeertaalblsetuodilsies wthielatishpttpoaabphnlyriiomtsothepseitodnerr(totLahlnDsathtLna)puvchtehryiohttiloyoespsnttoaeeclrrhorooollsllleeehsavtaneevdlrseobbleehymnyrepieficodcecuiffhacleoinpcletgrsso,itpneaertnerodtslietedimsnetacioclrecheahufsofmeelecsastenstre,uhremaoanlldlatohbw.dsoS-edrecpveretneiarsosaintelys[s4tluie6prd,4oui7epm]sr.oeAltsoetsawintbh-l(deiLselhDenevLsdei)tlys pchrtoeowfsleepensnhtttyeyirtnooyslfteoleeaorvrodsellsasarpbgeryoensroesetendsvtuueicnffirianflcogioecindontmtaetrmsoetiaennrcoachltiiaecsluhvofebflriaecansinetedenrffstoetloscattaabocrsnthoeirdhepvitgetihooanncmh[e4oaf6lfre,ek4sce7ttte]o.rpnoAlrhsoliedtghvuheeclctlsesh,vosselouelmssptoepefrlteopwmlhelyeennvttotyeesltsdye,ersaoworlmssithe agpohsyetvoestrearloclosmsumcehrcaisalmbraargnadrsinseta, ryteodgutortm, oarrkmetilpk.roHdouwctesvseurp, pthleeminecnoterpdowraitthiopnhoyftopshteyrtoolsstesruoclhs iansto moatrhgearripnreo, dyuogctusrpt,roorcemsislekd. Hunodweervheirg, hthheeiantcworapsolriamtiiotendodf upehytotolsotsesroinlsfiunntoctoiothnearliptyrordeusuctltsinpgrofcreosmsedthe tuhndmedepietcerrrorohiceoiengrsachstaiinhpogesnaucctloaawnutidasoesintdiloaibmsnysait.teshTtderhadpetsrueoegecyeteffostoeslciopntssgrsoccitanoencnftbduaeintnicmdotiniomnsni.amaTilnhiiztteyaesiderneetpshfufhreloytcuittnosggshctaeftrnrhooebmleuacmtshteiievnoidiftmyem.tieziIcrneridoothretaihnstrcisooaeupnngsscuheal,uathAtsieeolvdunismbaeysoetf a astlr. at[e4g8]y tsoucpcreostsefcutllayndpmroapionsteadin tphheytuosseteroofl aactisvpirtay.y Inchtihlliisnsgentseec,hAnilqvuime eftoral.th[4e8]psruocdcuecsstifounllyof prmopicorsoeedntchaepussuelaotfeadslpipraidyscchoilnlitnagintiencghnpihqyuteofsoterrtohlesp. Sropdrauyc-tdiornyionfgmuicsrinoegnAcaGpsaunldatMedDlipasidws acollnmtaainteinrigals phhyatvoestaelrsoolsb.eeSnpruasye-ddrtoyifnogrmuusilnagteApGhyatonsdteMroDl masicwroaplal rmticalteesri[a4l9s].have also been used to formulate (pc<ahccsm1ytaa.i0tmmpgoT%emspphsTt)eetae,ehesssrcretttotloeheaelrrltrdrmhooo(ei5rllesei%etncem(ero(;5ro<Fol%map1ili(g.an;a<(0ur<i0%Fntsr1.iti5ec)%egs,%l3rtue)oeAs),rcrl,es)s[ol.iΔ4eltO(r9s3o7>o]Ats-(.9shst>)ta0te.9ein%rg0roOmom%)lltif)(ahno<sef(out1o<rern%u1nsd%mnt)oe,dl)iir∆n,no(i5o<lnSs-s1raIiw%SOtvsoIee)tsOs,rentewsraaaoannsewlltdosrseeeolrrΔowβpel7(re-<(β-esr1a1is-e.tv%se2oine–tas)not2,tl:se.sa0cotrsΔ%ehotre5olp)or-,l(rolea6∆eslv(0st56e%(ee,00n2rn%).o4at2,l:-s–)ss(t,3tt<ceiis.hrgg04tooim.%m4gll%ema)a.sss()tat1Ft,eaes.∆or2dtrore7–oilre-2ltconh.((a0lo3<im%sl(003(%.p)f40<,ae%%)0ms,.Δt))5aei,,l5%nryao,Δd2)nl,o74d--f ∆7c-osmtigpmouanstdesnonlo(<sp1e%c)if, iacnatdio∆n7i-savinecnlausdteerdolin(0t.h2e–3N.4T%P).reFgour ltahtiisonfa.mTailbyleof5csoummpmouanridzsesntohsepmecaiifincaretisounlts isoibntcaliundeeddfoinr tthhee sNteTrPol rdeegtuelramtiionna.tioTnabinlecl5udsiunmg mtoatarilzsetsertohles manadinβ-rseistuoslttseroobl,taaisnepderfcoernttahgeesstoefrotlhe dettoetraml sitneartoiol ncoinntcelnutdsi,ntghetostiatol ssttaenroolls/caanmdpβe-sstietrooslterraotilo, ,aasnpdertcheenctahgoelessotefrtohlectoontacel nstterarotilocnonintetnhtes,inthiteial sitooislstaannodl/cinamthpoesseteorobltariantieod, afrnodmtheencchapolseusltaetrioolnc,obnoctehnftrraotmionthien stuhrefaincietiaanl doiltshaentdotianl tohiolss.eSoabchtaaininedchi afrmoroemiolssupenecntcosctniaovtpfaeslitynuo)tl,aaswltuisobittnsehtr,aoβbnl-ostsiti(aht2lo0fs5arto6emrmaoonlutdanhste2st2sh2oue5frmfmatoacgtje·oaklragns−spdt1eefrtcoohirelessStI(o(H52t60aO.5l76%oanilaadsnn. dSdSIVa52c2O2h.28,a5%rei)mnspacghse·ckiitngoiv−ia1lelsllfycoe)odr,niwbStalIietHihnvOβesug-saebinttsaodtbasltnSeetIrioVoaillOls,, asfothlleowmeadjobryspsietcoisetsan(5o6l .a7n%dacnadmp52e.s8t%er)ola.sEisnpeaclliaelldyibhlieghvesgiteotastbalneool/iclsa,mfoplelostwereodl rbaytisoistoasrteantyopl iacnaldof caSmIOpeasntedroalm. Eosupnetceidaltloy 5h.i9ghansdito3s.7tafnoorl/ScIaHmOpeasntderSoIlVraOti,orsesapreecttyipveiclayl. of SIO and amounted to 5.9 and 3.7 for SIHO and SIVO, respectively.



A



B

Figure 3. GC profile of sterols from SIHO. (A) Sterols of the original SIHO. (B) Sterols from the total oil of microencapsulated SIHO containing WPI as coating material. Peak numbers: (1): cholesterol; (2): internal standard (α-cholestanol); (3): campesterol; (4): stigmasterol; (5): clerosterol; (6): β-sitosterol; (7): sitostanol; (8): ∆5-avenasterol; (9): ∆5,24-stigmastadienol; (10): ∆7-stigmastenol; and (11): ∆7-avenasterol.



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Table 5. Sterol concentrations in initial oils and those extracted after microencapsulation.



Sterols from Total Microencapsulated Oil



Sterols from the Surface Oil



Samples



β-sitosterol (Mayor

Phytosterol) % on Total Phytosterols



Stigmasterol/ Campesterol

Ratio



Cholesterol (mg·kg−1)



Total

Phytosterols (mg·kg−1)



β-sitosterol (Mayor

Phytosterol) % on Total Phytosterols)



Stigmasterol/ Campesterol

Ratio



Cholesterol (mg·kg−1)



Total

Phytosterols (mg·kg−1)



P. huayllabambana P. volubilis



Initial oil SIHO + Hi-cap SIHO + Capsul

SIHO + AG SIHO + AG + MD SIHO + AG + MD + WPI

Initial oil SIVO + Hi-cap SIVO + Capsul

SIVO + AG SIVO + AG + MD SIVO + AG + MD + WPI



56.7 ± 1.5 60.7 ± 3.0 61.8 ± 3.8 60.8 ± 3.1 63.7 ± 6.0 62.5 ± 4.5 52.8 ± 2.0 58.2 ± 2.5 58.6 ± 3.9 55.8 ± 3.0 56.3 ± 3.4 56.0 ± 3.2



5.9 ± 0,6 5.1 ± 0.5 4.9 ± 0.5 5.2 ± 0.5 5.1 ± 0.5 4.3 ± 0.5 3.3 ± 0.6 3.7 ± 0.5 3.8 ± 0.5 4.1 ± 0.5 3.1 ± 0.6 3.0 ± 0.7



* * * * * 1014 ± 30 * * * * * 1072 ± 32



2056 ± 97 a,b 2119 ± 90 a 2154 ± 95 a 2101 ± 92 a 2050 ± 75 a 2104 ± 90 a

2225 ± 65 A 2255 ± 65 A 2282 ± 60 A 2356 ± 70 A 2348 ± 65 A 2321 ± 55 A



62.2 ± 3.5 63.7 ± 3.5 61.6 ± 3.0 61.2 ± 3.2 61.5 ± 3.1

56.0 ± 2.9 56.0 ± 2.9 56.5 ± 3.0 55.8 ± 3.0 55.8 ± 3.0



5.2 ± 0.6 5.3 ± 0.6 5.2 ± 0.5 5.3 ± 0.5 5.4 ± 0.6

3.4 ± 0.5 3.4 ± 0.5 3.8 ± 0.5 3.9 ± 0.5 4.0 ± 0.6



*



1934 ± 75 b,c



*



1888 ± 79 c



*



1893 ± 80 c



*



1855 ± 80 c



*



1901 ± 75 c



*



2269 ± 85 A



*



2290 ± 90 A



*



2277 ± 95 A



*



2240 ± 95 A



*



2279 ± 95 A



In each column different letters indicate significant difference values at p < 0.05 (lower case letters for SIHO and capital letters for SIVO). *: <0.4% on total sterols.



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After encapsulation, the average values obtained were: 2126 and 2312 mg·kg−1 for total extracted oil, around 4% higher but without significance. For the surface oil, the average sterol values were 1894 and 2271 mg·kg−1, which was 8% lower than the initial P. huayllabambana oil and remained at almost the same value for P. volubilis. In any case, the differences were statistically significant. In all samples of original oils and encapsulated ones, cholesterol did not reach a considerable value (around 0.2% of total sterols) except for the samples encapsulated with the ternary mixture of coating material. For total oil (not for the surface one) cholesterol concentrations of 1014 and 1072 mg·kg−1 were quantified for P. huayllabambana and P. volubilis, respectively, in samples encapsulated with whey protein isolate, clearly indicating that the cholesterol found comes from the coating material (Figure 3B, Table 5).

Despite the great interest in the importance of phytosterols and the need for it to be available for use in many processes, there is still little research verifying the changes suffered by the sterols coming from edible oils after encapsulation [25]. Recently Hue et al. [50] studied some physiochemical properties of kenaf seed oil microcapsules and determined their sterol compositions. They studied the evolution of phytosterols during accelerated storage conditions (from day 0 to day 24) but changes between the initial oils and the oils obtained after the process are not discussed [50].

4. Conclusions

The encapsulation process itself alters the oil, although to a minimum extent. The major fatty acid, ω3-Ln, suffered a loss of 6.2% during the encapsulation process of SIHO (for total oil) with the ternary coating material (AG + MD + WPI). In the case of SIVO, with a lower unsaturation level, the loss was lower (3%) when Capsul was used as wall material. It is interesting to note the absence of formation of trans fatty acid isomers throughout the processes.

Regarding the minor glyceride polar components, the TG-P are absent from the original oils, as no heat was used for their extraction. In contrast, all the encapsulated oils showed quantified levels of TG-P. As expected, because of its higher unsaturation level, samples coming from SIHO presented higher TG-P levels (0.6–5.5 mg·g−1 oil) than those formed with SIVO (0.2–0.9 mg·g−1 oil).

The greatest losses in minor non-glyceride components were observed in the tocopherols, 30−15% for the total and surface SIHO, respectively. In the case of SIVO losses, they were also in the range of 28–24% for total and surface oils γ-tocopherol losses were higher than δ-tocopherol for both SIOs. For both varieties (SIHO and SIVO), the encapsulation process with AG presented the highest losses in total oil.

Regarding the sterols, the losses were not statistically significant in any cases. The most remarkable and unexpected result was the presence of cholesterol when the ternary blend (AG + MD + WPI) was employed as coating material, for both SIOs. This is clearly due to the WPI material because this sterol appears in the unique formulation, which contains WPI and WPI came from an animal source.

Author Contributions: All authors have contributed equally to this manuscript. Conceptualization, M.C.P.-C., N.A.C. and G.G.; Methodology, M.C.P.-C., N.A.C., G.G. and D.T.; Investigation and Data analysis, M.C.P.-C.; N.A.C., G.G., R.B.G.-C., D.T. and W.M.; Writing—original draft preparation, M.C.P.-C., N.A.C. and G.G.; Writing—review and editing, M.C.P.-C., N.A.C., G.G. and R.B.G.-C.

Funding: This research received no specific external funding.

Acknowledgments: The authors thank the National Program of Agricultural Innovation (PNIA), Ministry of Agriculture and Irrigation-Peru and the World Bank (Project: 020-2015-PNIA/UPMSI/IE) and i-COOP-CSIC program (project COOPB20301).

Conflicts of Interest: This article represents the authors’ own work. We are not aware of any conflict of interest.

References

1. Champagne, C.P.; Fustier, P. Microencapsulation for the improved delivery of bioactive compounds into foods. Curr. Opin. Biotech. 2007, 18, 184–190. [CrossRef] [PubMed]

2. Kaushik, P.; Dowling, K.; Barrow, C.J.; Adhikari, B. Microencapsulation of omega-3 fatty acids: A review of microencapsulation and characterization methods. J. Funct. Food 2015, 19, 868–881. [CrossRef]



Foods 2019, 8, 671



16 of 18



3. Martínez, M.L.; Curti, M.I.; Roccia, P.; Llabot, J.M.; Penci, M.C.; Bodoira, R.M.; Ribotta, P.D. Oxidative stability of walnut (Juglans regia L.) and chia (Salvia hispanica L.) oils microencapsulated by spray drying. Powder Technol. 2015, 270, 271–277. [CrossRef]

4. Carneiro, H.C.F.; Tonon, R.V.; Grosso, C.R.F.; Hubinger, M.D. Encapsulation efficiency and oxidative stability of flaxseed oil microencapsulated by spray drying using different combinations of wall materials. J. Food Eng. 2013, 115, 443–451. [CrossRef]

5. Gallardo, G.; Guida, L.; Martínez, V.; López, M.C.; Bernhardt, D.; Blasco, R. Microencapsulation of linseed oil by spray drying for functional food application. Food Res. Int. 2013, 52, 473–482. [CrossRef]

6. Comunian, T.A.; Favaro-Trindade, C.S. Microencapsulation using biopolymers as an alternative to produce food enhanced with phytosterols and omega-3 fatty acids: A review. Food Hydrocol. 2016, 61, 442–457. [CrossRef]

7. Alcântara, M.A.; de Lima, A.E.A.; Mattos Braga, A.L.; Valeriano Tonon, R.; Galdeano, M.C.; da Costa Mattos, M.; Santa Brígida, A.I.; Rosenhaim, R.; Albuquerque dos Santos, N.; Tribuzy de Magalhães Cordeiro, A.M. Influence of the emulsion homogenization method on the stability of chia oil microencapsulated by spray drying. Powder Technol. 2019, 354, 877–885. [CrossRef]

8. Maeda Rojas, V.; da Costa, L.F.; Guimarães-Inácio, A.; Leimann, F.V.; Tanamati, A.; Gozzo, Â.M.; Hernandez, R.; Barreiro, M.F.; Barros, L.; Ferreira, I.; et al. Formulation of mayonnaises containing PUFAs by the addition of microencapsulated chia seeds, pumpkin seeds and baru oils. Food Chem. 2019, 274, 220–227. [CrossRef]

9. Cortés-Camargo, S.; Acuña-Avila, P.E.; Rodríguez-Huezo, M.E.; Román-Guerrero, A.; Varela-Guerrero, V.; Pérez-Alonso, C. Effect of chia mucilage addition on oxidation and release kinetics of lemon essential oil microencapsulated using mesquite gum—Chia mucilage mixtures. Food Res. Int. 2019, 116, 1010–1019. [CrossRef]

10. Lavanya, M.N.; Kathiravan, T.; Moses, J.A.; Anandharamakrishnan, C. Influence of spray-drying conditions on microencapsulation of fish oil and chia oil. Drying Technol. 2019. [CrossRef]

11. Timilsena, Y.P.; Vongsvivut, J.; Tobin, M.J.; Adhikari, R. Investigation of oil distribution in spray-dried chia seed oil microcapsules using synchrotron-FTIR microspectroscopy. Food Chem. 2019, 275, 457–466. [CrossRef] [PubMed]

12. Rodriguez, E.S.; Julio, L.M.; Henning, C.; Diehl, B.W.K.; Tomás, M.C.; Ixtaina, V.Y. Effect of natural antioxidants on the physicochemical properties and stability of freeze-dried microencapsulated chia seed oil. J. Sci. Food Agric. 2019, 99, 1682–1690. [CrossRef] [PubMed]

13. Chirinos, R.; Pedreschi, R.; Domínguez, G.; Campos, D. Comparison of the physico-chemical and phytochemical characteristics of the oil of two Plukenetia species. Food Chem. 2015, 173, 1203–1206. [CrossRef] [PubMed]

14. Chasquibol, N.A.; del Aguila, C.; Yácono, J.C.; Guinda, A.; Moreda, W.; Gómez-Coca, R.B.; Pérez-Camino, M.C. Characterization of glyceridic and unsaponifiable compounds of sacha inchi (Plukenetia huayllabambana L.) oils. J. Agric. Food Chem. 2014, 62, 10162–11016. [CrossRef]

15. Triana-Maldonado, D.M.; Torijano-Gutiérrez, S.A.; Giraldo-Estrada, C. Supercritical CO2 extraction of oil and omega-3 concentrate from sacha inchi (Plukenetia volubilis L.) from Antioquia, Colombia. Grasas Aceites 2017, 68, e172. [CrossRef]

16. Wang, S.; Zhub, F.; Kakuda, Y. Sacha inchi (Plukenetia volubilis L.): Nutritional composition, biologicalactivity, and uses. Food Chem. 2018, 265, 316–328. [CrossRef]

17. Quispe-Condori, S.; Saldaña, M.D.A.; Temelli, F. Microencapsulation of flax oil with zein using spray and freeze drying. LWT Food Sci. Technol. 2011, 44, 1880–1887. [CrossRef]

18. Vicente, J.; de Souza Cezarino, T.; Barreto Pereira, L.J.; Pinto da Rocha, E.; Raymundo Sá, G.; Domingues Gamallo, O.; de Carvalho, M.G.; García-Rojas, E.E. Microencapsulation of sacha inchi oil using emulsion-based delivery systems. Food Res. Int. 2017, 99, 612–622. [CrossRef]

19. Sánchez-Reinoso, Z.; Gutiérrez, L.F. Effects of the emulsion composition on the physical properties and oxidative stability of sacha inchi (Plukenetia volubilis L.) oil microcapsules produced by spray drying. Food Bioproc. Technol. 2017, 10, 1354–1366. [CrossRef]

20. Pastuña-Pullutasig, A.; López-Hernández, O.; Debut, A.; Vaca, A.; Rodríguez-Leyes, E.; Vicente, R.; Gonzalez, V.; González-Sanabia, M.; Tapia-Hernández, F. Microencapsulation of oil sacha inchi (Plukenetia volubilis L.) by spray drying. Rev. Colomb. Cienc. Quimic. Farmac. 2016, 45, 422–437. [CrossRef]



Foods 2019, 8, 671



17 of 18



21. Landoni, L.; Vilca Calderón, L.; Chasquibol, N.; Pérez-Camino, M.C.; Gallardo, G. Physicochemical characterization and oxidative stability of microencapsulated sacha inchi seed oils by spray drying. Grasas Aceites 2020, 71, e387, in press.

22. Oxley, J.D. Spray cooling and spray chilling for food ingredient and nutraceutical encapsulation. In Encapsulation Technologies and Delivery Systems for Food Ingredients and Nutraceuticals; Garti, N., Mc Clements, D.J., Eds.; Elsevier: Amsterdam, The Netherlands, 2012; pp. 110–130.

23. Fadini, A.L.; Dutra Alvima, I.; Porto Ribeiro, I.; Geraldini Ruzene, L.; Bataglia da Silva, L.; Bonifácio Queiroz, M.; Rauen de Oliveira, A.M.; Maia Chaves, F.C.; Ferreira Rodrigues, R.A. Innovative strategy based on combined microencapsulation technologies for food application and the influence of wall material composition. LWT-Food Sci. Technol. 2018, 91, 345–352. [CrossRef]

24. Da Silva Soares, B.; Pinto Siqueira, R.; de Carvalho, M.G.; Juarez, V.; Garcia-Rojas, E.E. Microencapsulation of sacha inchi oil (Plukenetia volubilis L.) using complex coacervation: Formation and structural characterization. Food Chem. 2019, 298, 125045. [CrossRef] [PubMed]

25. Ng, S.K.; Jessie, L.Y.L.; Tan, C.P.; Long, K.; Nyam, K.L. Effect of accelerated storage on microencapsulated Kenaf seed oil. J. Am. Oil Chem. Soc. 2013, 90, 1023–1029. [CrossRef]

26. Encina, C.; Márquez-Ruiz, G.; Holgado, F.; Giménez, B.; Vergara, C.; Robert, P. Effect of spray-drying with organic solvents on the encapsulation, release and stability of fish oil. Food Chem. 2018, 263, 283–291. [CrossRef]

27. Holgado, F.; García-Martínez, M.C.; Velasco, J.; Ruiz-Méndez, M.V.; Márquez-Ruiz, G. Microencapsulation of conjugated linoleic acid (CLA)-rich oil with skimmed milk components protects against polymerization. J. Am. Oil Chem. Soc. 2018, 95, 1399–1408. [CrossRef]

28. Velasco, J.; Dobarganes, M.C.; Márquez, G. Oxidation of free and encapsulated oil fractions in dried microencapsulated fish oil. Grasas Aceites 2000, 51, 439–446.

29. Márquez-Ruiz, G.; Velasco, J.; Dobarganes, C. Evaluation of oxidation in dried microencapsulated fish oils by a combination of adsorption and size exclusion chromatography. Eur. Food Res. Technol. 2000, 211, 13–18. [CrossRef]

30. Sankarikutty, B.M.; Sreekumar, M.; Nrayanan, C.S.; Mathew, A.G. Studies on microcapsulation of cardamon oil by spray drying technique. J. Food Sci. Technol. 1988, 26, 352–356.

31. Richardson, G.H. Standard Methods for the Examination of Dairy Products, 15th ed.; American Public Health Association: Washington, DC, USA, 1985; p. 358.

32. IUPAC Standard Method 2.301. Standard methods for the analysis of oils, fats and derivatives. In Preparation of Fatty Acid Methyl Ester; Blackwell Scientific: Oxford, UK, 1987.

33. IUPAC Standard Method 2.432. Standard methods for the analysis of oils, fats and derivatives. In Determination of Tocopherol and Tocotrienols in Vegetable Oils and Fats by HPLC; Blackwell Scientific: Oxford, UK, 1987.

34. García-González, A.; Velasco, J.; Velasco, L.; Ruiz-Méndez, M.V. An analytical simplification for faster determination of fatty acid composition and phytosterols in seed oils. Food Anal. Method. 2018, 11, 1234–1242. [CrossRef]

35. NTP. Norma Tec´nica Peruana 151.400, Amendment. NTP 151.400, 2009; Requisitos Aceite Sacha Inchi, INDECOPI: Lima, Peru, 2014.

36. Aquilani, C.; Pérez-Palacios, T.; Sirtori, F.; Jiménez-Martín, E.; Antequera, T.; Franci, O.; Bozzi, R.; Acciaioli, A.; Pugliese, C. Enrichment of Cinta Senese burger with omega-3 fatty acids. Effect of type of addition and storage condition on quality characteristics. Grasas Aceites 2018, 69, e235. [CrossRef]

37. Ilyasoglu, H. Production of structured lipid with a low omega-6/omega-3 fatty acids ratio by enzymatic interesterification. Grasas Aceites 2017, 68, e191. [CrossRef]

38. Liu, W.; Lu, G.H. Cis-trans isomerization of unsaturated fatty acids in edible oils to prepare trans fat. Grasas Aceites 2018, 69, e268. [CrossRef]

39. Jiménez, M.; Garcia, H.S.; Beristain, C.I. Spray-drying microencapsulation and oxidative stability of conjugated linoleic acid. Europ. Food Res. Technol. 2004, 219, 588–592. [CrossRef]

40. Costa, A.M.M.; Nunes, J.C.; Lima, B.N.B.; Pedrosa, C.; Calado, V.; Torres, A.G.; Pierucci, A.P.R. Effective stabilization of CLA by microencapsulation in pea protein. Food Chem. 2015, 168, 157–166. [CrossRef] [PubMed]



Foods 2019, 8, 671



18 of 18



41. Dobarganes, M.C.; Pérez-Camino, M.C.; Márquez-Ruiz, G. High Performance Size Exclusion Chromatography of Polar Compounds in Heated and Non-Heated Fats. Fatt Sci. Technol. 1988, 90, 308–311. [CrossRef]

42. Velasco, J.; Marmesat, S.; Dobarganes, C.; Márquez-Ruiz, G. Heterogeneous Aspects of Lipid Oxidation in Dried Microencapsulated Oils. J. Agric. Food Chem. 2006, 54, 1722–1729. [CrossRef]

43. Tomassi, G.; Simano, V. An assessment of the safety of tocopherols as food additives. Food Chem. Toxicol. 1986, 24, 1051–1061. [CrossRef]

44. Chasquibol, N.A.; Gómez-Coca, R.B.; Yácono, J.C.; Guinda, A.; Moreda, W.; del Aguila, C.; Pérez-Camino, M.C. Markers of quality and genuineness of commercial extra virgin sacha inchi oils. Grasas Aceites 2016, 67, e169. [CrossRef]

45. Bruscatto, M.H.; Pestana-Bauer, V.R.; Otero, D.M.; Zambiazi, R.C. Effects of heating temperature on the tocopherol contents of chemically and physically refined rice bran oil. Grasas Aceites 2019, 70, e294. [CrossRef]

46. Deng, R. Food and Food Supplements with Hypocholesterolemic Effects. Recent Patents on Food, Nutrit. Agric. 2009, 1, 15–24. [CrossRef] [PubMed]

47. Marangoni, F.; Poli, A. Phytosterols and cardiovascular health. Pharmacol. Res. 2010, 61, 193–199. [CrossRef] [PubMed]

48. Alvim, I.D.; de Souza, F.; Koury, I.P.; Jurt, T.; Dantas, F.B.H. Use of the spray chilling method to deliver hydrophobic components: Physical characterization of microparticles. Cienc. Tecnol. Aliment. 2013, 33, 34–39. [CrossRef]

49. Di Battista, C.A.; Constenla, D.; Ramírez-Rigo, M.V.; Piña, J. The use of arabic gum, maltodextrin and surfactants in the microencapsulation of phytosterols by spray drying. Powder Technol. 2015, 286, 193–201. [CrossRef]

50. Hue, W.L.; Nyam, K.L. Physiochemical properties of kenaf seed oil microcapsules before and after freeze drying and its storage stability. Int. Food Res. J. 2018, 25, 1502–1509.

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