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.
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. Τhe 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.
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
isomer often being the most prevalent [27
] 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 (TPC) as the sum of the three alterations. The TPC values were very low, at 2.6% and 2.3% for the initial P. huayllabambana
and P. volubilis
oils, and it was in agreement with data obtained for any good edible oil [41
]. For the encapsulated 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.
As can be observed in Figure 1
A, four peaks appeared corresponding to oxTG, DG, MG, and FFA for the two sacha inchi oils under study. There was no presence of TG-P (retention time 8, in the original oil) meaning that during 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, which was 18.2 mg·g−1
oil for SIHO and 16.5 mg·g−1
for SIVO. After the encapsulation process one of the most remarkable results was the presence of TG-P (retention time ~8.2, Figure 1
B) in all the oil samples (Table 3
), which means that the temperature used in the step of spray-drying was sufficient for initiating the polymerization of the oils. HPSEC has been successfully used to carry out studies on microencapsulated oil [29
]. Nevertheless, it was not specifically highlighted that polymers were formed during the encapsulation process. This aspect is not so obvious, since the investigations conducted with refined oils that already contained TG dimers or polymers originated during the deodorization stage of the refining process. Far from comparing this process with what happens in an oil refining process, microencapsulation will bring important benefits but also oil degradation. Thereby, the obtained results indicated that by starting from oils without polymers in their composition, polymerization took place and TG-P were present in both oil fractions, the encapsulated and the non-encapsulated ones, regardless of the coating material since its formation is closely related to temperature. The values for TG-P were in the range of 0.6–5.5 mg·g−1
oil, for P. huayllabambana
and 0.2–0.9 mg·g−1
oil, for P. volubilis
. Differences were observed for total and surface oils in both of them, with the total being slightly more polymerized than the surface ones. In relation to oxidative degradations (ox-TG), the values for the initial oils were 7.8 and 6.5 mg·g−1
(SIHO vs. SIVO). After encapsulation, these compounds experimented an increase in their values and for all the core materials with statistical significance with respect to their initial oils. Statistical differences were also obtained between surface and encapsulated oil fractions in total polar compounds, being slightly higher in the former for both types of oils. Working with dried microencapsulated fish oils, Velasco et al. [42
] established important differences in the oxidation between free and encapsulated oil fractions for the first time. In their work, the authors highlighted the differences in the heterogeneous aspects of lipid oxidation in dried microencapsulated oils.
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.
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
]. 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 2
A,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 that the losses were mainly due in γ-tocopherol so that the γ/δ-tocopherol ratio decreased after encapsulation. For P. huayllabambana
the ratio medium value after the encapsulation process was 1.6 in both fractions, whereas it was 1.0 for P. volubilis
, clearly indicating that the γ-tocopherol losses were in higher proportion than δ-tocopherol for both oils and that such effect was more intense for SIVO than for SIHO. The higher stability of δ-tocopherol with respect to γ-tocopherol is in agreement with a recently published study where the stability of the different molecular species of tocopherols (α-, γ- and δ-) against continuous heating at different temperatures (100–180 °C) was evaluated [45
]. The authors [45
] concluded that δ-tocopherols were more stable than the γ- and α-species.
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.
3.4. Sterol Composition
Vegetable sterols or phytosterols are components of the unsaponifiable matter of vegetable oils with an important nutritional role and beneficial properties to human health. Several studies established that phytosterols have hypocholesterolemic effects, and decrease serum low-density lipoprotein (LDL) cholesterol levels by reducing intestinal cholesterol absorption [46
]. As the levels of phytosterols present in food are not sufficient to achieve an effect on high cholesterol levels, some twenty years ago several commercial brands started to market products supplemented with phytosterols such as margarine, yogurt, or milk. However, the incorporation of phytosterols into other products processed under high heat was limited due to loss in functionality resulting from the deterioration caused by the processing conditions. These effects can be minimized through the use of microencapsulation as a strategy to protect and maintain phytosterol activity. In this sense, Alvim et al. [48
] successfully proposed the use of a spray chilling technique for the production of microencapsulated lipids containing phytosterols. Spray-drying using AG and MD as wall materials have also been used to formulate phytosterol microparticles [49
The three main sterols (>90%) found in SIOs were β-sitosterol (60%), stigmasterol (30%), and campesterol (5%; Figure 3
A). Other minor sterols were also present: cholesterol (<0.4%), Δ7-campesterol (<1.0%), clerosterol (<1%), sitostanol (<1%), Δ5-avenasterol (1.2–2.0%), Δ5,24-stigmastadienol (<0.5%), Δ7-stigmastenol (<1%), and Δ7-avenasterol (0.2–3.4%). For this family of compounds no specification is included in the NTP regulation. Table 5
summarizes the main results obtained for the sterol determination including total sterols and β-sitosterol, as percentages of the total sterol contents, the sitostanol/campesterol ratio, and the cholesterol concentration in the initial oils and in those obtained from encapsulation, both from the surface and the total oils. Sacha inchi oils contain substantial amounts of total sterols (2056 and 2225 mg·kg−1
for SIHO and SIVO, respectively), with β-sitosterol as the major species (56.7% and 52.8%) as in all edible vegetable oils, followed by sitostanol and campesterol. Especially high sitostanol/campesterol ratios are typical of SIO and amounted to 5.9 and 3.7 for SIHO and SIVO, respectively.
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 3
B, 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