Volume 25, Number 3, May-June 2018
|Number of page(s)||7|
|Section||Quality - Food safety|
|Published online||31 January 2018|
Incorporation of capric acid in pumpkin seed oil by sn-1,3 regioselective lipase-catalyzed acidolysis
Incorporation d’acide caprique dans de l’huile de pépin de citrouille par acidolyse catalysée par une lipase sn-1,3 régiosélective
Instituto Superior de Engenharia, Universidade do Algarve, Campus da Penha,
2 Center for Mediterranean Bioresources and Food (MeditBio), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
* Corresponding author: firstname.lastname@example.org
Accepted: 21 December 2017
Structured lipids (SLs) are novel triacylglycerols obtained by changing the native fatty acid (FA) profiles or by the incorporation of a new desired FA in the acylglycerol backbone. These modified fats present important medical and functional properties for food applications. This work aimed to synthetize a MLM-type SL, which consists of triacylglycerols containing a medium-chain FA (M) at sn-1,3 positions and a long-chain FA (L) at sn-2 position, by acidolysis of pumpkin seed oil with capric acid, catalyzed by a commercial lipase preparation from Thermomyces lanuginosa (Lipozyme TL IM). Reactions were performed at 45 °C, in solvent-free media, at 1:2 molar ratio (pumpkin seed oil:capric acid) and a fixed amount of immobilized lipase of 5%, 10%, 15% or 20%. Incorporations of C10:0 increased with time up to 31 h (29.9 ± 0.7 mol-%) when 5% lipase load was used. Significant differences were only observed between the results obtained with 5 and 20% of biocatalyst load. The subsequent experiment was carried out with 5% lipase load, at 45 °C, 1:2 molar ratio and in the presence of n-hexane. The results showed slightly higher incorporation yields in the presence of solvent, namely at 48 h-reaction (34.7 ± 1.0 mol-%). However, since the structured lipids are to be used in food products, together with environmental and economic concerns, solvent-free systems are preferred. In this study, the synthesis of a MLM-type SL from pumpkin seed oil for food uses was well succeeded.
Les lipides structurés sont de nouveaux triacylglycérols obtenus en changeant les profils des acides gras (AG) natifs ou par l’incorporation de nouveaux AG souhaités dans le squelette glycérol. Ces graisses modifiées présentent des propriétés médicales et fonctionnelles importantes pour des applications alimentaires. Ce travail vise à synthétiser un lipide structuré de type MLM, qui consiste en des triacylglycérols contenant un AG à chaîne moyenne (M) en positions sn-1,3 et un acide gras à chaîne longue (L) en position sn-2, par acidolyse de l’huile de graine de citrouille avec l’acide caprique, catalysée par une préparation commerciale de lipase de Thermomyces lanuginosa (Lipozyme TL IM). Les réactions ont été effectuées à 45 °C, sans solvant, avec un rapport molaire de 1:2 (huile de graines de citrouille: acide caprique) et une quantité fixe de lipase immobilisée de 5 %, 10 %, 15 % ou 20 %. Avec une charge en lipase de 5 %, l’incorporation du C10:0 a augmenté avec le temps jusqu’à 31 h (29,9 ± 0,7 mol %). Des différences significatives ont été observées seulement entre les résultats obtenus avec 5 et 20 % de charge en biocatalyseur. L’expérience suivante a été réalisée avec une charge en lipase de 5 %, à 45 °C, un rapport molaire 1:2 et en présence de n-hexane. Les résultats ont montré des rendements d’incorporation légèrement supérieurs en présence de solvant, à savoir 34,7 ± 1,0 mol% après 48 h de réaction. Cependant, étant donné que les lipides structurés doivent être utilisés dans les produits alimentaires, mais également pour des préoccupations environnementales et économiques, les systèmes sans solvant sont à privilégier. Dans cette étude, la synthèse d’un lipide structuré à visée alimentaire de type MLM à partir d’huile de graines de citrouille a été réalisée avec succès.
Key words: structured lipid / acidolysis / pumpkin seed oil / capric acid / lipozyme TL IM
Mots clés : lipides structurés / acidolyse / huile de pépins de citrouille / acide caprique / lipozyme TL IM
© V. Sousa et al., Published by EDP Sciences, 2018
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
During the last decades, the demand for new, healthy and economically viable products has increased considerably. Vegetable oils extracted from fruit seeds have an important functional and nutritional role in health due to their fatty acid (FA) composition and polyunsaturation level. Structured Lipids (SLs) can be produced by synthesis or modification of natural triacylglycerols (TAG). Constituent FAs of SL are medium chain FAs and unsaturated FAs (oleic, linoleic acid, polyunsaturated FA, etc.). In fact, they are novel TAG produced by the incorporation of new FAs, or changing the positions of FAs or the FA profile from the natural state (Xu, 2000a, Ferreira-Dias et al., 2013). SLs are made to possess desired nutritional, physicochemical or textural properties for various applications in the food industry. Some interesting examples of SLs are human milk fat substitutes (Tecelão et al., 2010), cocoa butter substitutes (Matsuo et al., 1981), trans-free plastic fats for margarines (Osório et al., 2001) and low calorie fats and oils (Nunes et al., 2011a, b). The production of SL by enzymatic procedures has a great potential in the future market because of the specificity of lipases as biocatalysts (Kim and Akoh, 2015). TAG, containing a medium-chain FA (M) at sn-1 and sn-3 positions, and a long-chain FA (L) at sn-2 position, named as MLM-type TAG, present lower caloric value than conventional fats and oils of the type LLL (Smith et al., 1994; Morales-Medina et al., 2017). The importance of this SL is that it is metabolized differently compared to conventional fats and oils. In fact, this SL presents lower caloric value (21–29 kJ/g) than conventional fats and oils (38 kJ/g) due to the lower caloric content of M compared to their L counterparts (Smith et al., 1994). During digestion, M is metabolized as rapidly as glucose. Since it is not readily re-esterified into new TAG, it has little tendency to accumulate in the body as stored fat, with weight control benefits (Osborn and Akoh, 2002). Therefore, it can be used for obesity reduction. Recently, Moreira et al. (2017a, b) showed that SLs could play a major role in combating or preventing obesity and other resultant diseases without producing side effects. In addition, SLs may provide the most effective means of delivering desired FAs for nutritive or therapeutic purposes, and for treating specific diseases and metabolic problems (Osborn and Akoh, 2002, Lee et al., 2012). Lipases offer high catalytic efficiency, specificity, and selectivity by incorporation of the required acyl-group into a specific position of native TAG. In addition, the process takes place in very mild conditions that maintain unaltered the structure of the functional FA (Hita et al., 2007). Lipase-catalyzed acidolysis, in which medium chain FAs (caprylic, C8:0, or capric acids, C10:0) are used as acyl donors, and vegetable or fish oils are used as the source of glycerol backbone and long chain FAs, is one of the most commonly used methods to produce SL of the MLM-type (Ferreira-Dias et al., 2013). The sn-1,3 specific lipases have currently been used to catalyze the incorporation of medium chain FAs at the sn-1,3 positions of the TAG molecule, while the central position is preferentially occupied by unsaturated long-chain FAs, namely oleic and linoleic acids (Osborn and Akoh, 2002; Turan et al., 2006; Feltes et al., 2009; Nunes et al., 2011a, b). Acidolysis reaction presents some advantages over other lipase-catalyzed reactions, such as esterification and interesterification. In fact, for MLM production via acidolysis, the acylglycerol mostly comes from native oils, which contain essential fatty acids, like linoleic acid, with health benefits. Currently, esterification gives the highest yield compared to acidolysis. However, large-scale esterification is more expensive than acidolysis due to the high cost of fatty acids and glycerol substrates. On the other hand, interesterification produces a randomly structured medium- long-chain tricylglycerols with low content of MLM compared to acidolysis reaction (Lee et al., 2012).
Pumpkin seed is a by-product of pumpkin fruit production and contains 45% oil average (dry weight basis), which is a major advantage over a number of other vegetable seed oils. Nowadays, this oil is used as a salad oil in several countries (Austria, Slovenia and Hungary), due to its pleasant sensory properties, high nutritive value and good oxidative stability, as it has a high content of natural antioxidants, such as phenolic compounds (Tsaknis et al., 1997; Murković et al., 2004; Yu et al., 2005; Stevenson et al., 2007; Fruhwirth and Hemetter, 2008; Vujasinovic et al., 2010). Moreover, this oil has gained attention, not only as an edible oil, but also as a potential nutraceutical. In fact, it is a good source of healthy compounds, especially phenolics, tocopherols (vitamin E) and carotenoids as lipophilic antioxidants (Stevenson et al., 2007), acting as preventative agents against hypertension and carcinogenic diseases (Zuhair et al., 2000; Jian et al., 2005). Also, the Δ7-sterols are specific to pumpkin seed oil and supposed to confer to this oil a beneficial effect in the treatment and prophylaxis of the prostate gland and bladder disorders (Nakić et al., 2006). In addition, it has an interesting FA composition, with four FAs making up 98% of the total, namely palmitic acid (C16:0, 9.5–14.5%), stearic acid (C18:0, 3.1–7.4%), oleic acid (C18:1, 21.0–46.9%) and linoleic acid (C18:2, 35.6–60.8%) (Murković et al., 1996). Thus, pumpkin oil is extraordinarily rich in unsaturated FAs, which represent about 84% of the total FAs. Such health benefits mean that the effective extraction technologies are required to ensure the quality of pumpkin seed oil. Traditionally, pumpkin seed oil is obtained by pressing seeds previously roasted. However, a cold-press method using unroasted seeds is emerging because it produces oil with different and improved characteristics, namely sensory properties, such as colour, taste and odour, and FA profile (Nederal et al., 2012). In addition, this method of production preserves bioactive components, such as vitamins, provitamins, phytosterols, phospholipids and squalene, which are, together with some FAs, key pumpkin seed oil nutritional value factors (Rabrenović et al., 2014). In the literature, there is no reference regarding the enzymatic acidolysis of pumpkin seed oil with C10:0. Therefore, we have attempted to synthesize enzymatically MLM-type SLs from pumpkin seed oil for food uses. The aim of this study was:
the extraction of oil from pumpkin (Cucurbita maxima, L.) seeds by cold-pressing and its characterization;
the synthesis of a MLM-type SL, from this pumpkin seed oil enriched with C10:0, catalyzed by a sn-1,3 regioselective commercial lipase prepared from Thermomyces lanuginosa (Lipozyme TL IM);
to evaluate the effects of reaction parameters, namely, reaction time, substrates’ molar ratio, and enzyme load on capric acid incorporation.
Pumpkin (Cucurbita maxima, L.) seeds were provided by Hortomelão Produtos Hortícolas e Frutos S.A., a local industry from the Santarém region, Portugal. Capric acid (C10:0, n-decanoic acid, melting point at 31 °C), and 2’,7’-dichlorofluorescein were obtained from Fluka (Buchs, Switzerland). Fatty acid methyl ester (FAME) mix C8-C24 and FAME saturated and unsaturated standards, used for gas chromatography analysis, and lipid standard mono-, di- and triglyceride mix, used for thin layer chromatography analysis, were obtained from Sulpeco (Bellefonte, USA). Silica-Gel 60 (0.25 mm width, 20 × 20 cm) thin layer chromatography plates were purchased from Merck (Darmstadt, Germany). All solvents and reagents for analyses were of chromatographic or analytical grade. The commercial immobilized sn-1,3 regioselective lipase, Lipozyme TL IM from Thermomyces lanuginosa, was a gift from Novozymes A/S (Bagsvaerd, Denmark).
The provided frozen pumpkin seeds were thawed, washed with clean tap water and dried in a tray drier, where air at 45 °C was forced by a fan to circulate over and parallel to the surface until a moisture content of 5% (w:w) was achieved. The dried seeds were dehulled by hand and crushed in a hammer mill with a 0.75 mm sieve. The oil extraction was carried out using a screw press, Täby Press type 20 (Skeppsta Maskin AB, Sweden) equipped with an outlet nozzle of 9 mm internal diameter. The oil obtained was centrifuged during 15 minutes at 2500 rpm. Finally, the oil was stored at −20 °C in sealed tubes and protected from light until analysis. The oil extraction yield obtained by cold-pressing was expressed as the percentage of extracted oil by this method and the total oil content in the seeds, assessed by solvent extraction in a Soxhlet apparatus according to AOAC 948.22 method (2000), using n-hexane and a minimum extraction time of 8 h.
Refractive index at 25 °C (using a Abbe refractometer; Atago, 1T, Tokyo, Japan), specific gravity (using a 10 mL pycnometer at 19 °C), moisture content, acid value and peroxide value of the extracted pumpkin seed oil were determined by methods described by the AOAC (1984). Viscosity of the oil was measured using a viscometer (Brookfield, DV-II +; Middleboro, USA) equipped with a cylinder spindle. A thermostatic bath was used to control the working temperature at 21 °C. The speed of the rotating cylinder varied from 0.3 rpm to 100 rpm. All analyses were performed in triplicate and average values ± standard deviation were reported. FAME prepared by the method described by Nunes et al. (2011b) were dissolved in 100 μL of n-hexane solution and 1 μL of this solution was GC analyzed. A GC 14A gas chromatograph (Shimadzu; Kyoto, Japan) equipped with a flame ionization detector and a DB-wax capillary column (30 m × 0.32 mm i.d., d.f. = 0.50 μm) was used. Injection and detector temperatures were set at 250 °C and 260 °C, respectively. The injector was used in split mode with a ratio of 1:20. Helium was used as the carrier gas at a column head pressure of 10 kPa and a flow rate of 1 mL/min. The oven temperature programme was as follows: 110 °C for 7 min, temperature increase to 170 °C at 30 °C/min, a plateau at 170 °C for 45 min, temperature increase to 230 °C at 7.5 °C/min and a final plateau at 230 °C for 15 min. The relative content of FAME was calculated as a molar percentage on the basis of the molecular weight of each FAME.
A fixed amount of immobilized lipase, 5% (w:w of total substrates) was added to the substrate mixture consisting of 3.0 g of pumpkin seed oil extracted by the cold-press method and 1.3 g of C10:0. The molar ratio of pumpkin seed oil:FA of 1:2 used corresponds to the stoichiometric value needed for the esterification of C10:0 at the sn-1 and sn-3 positions, as the biocatalyst used was sn-1,3 selective. Lipozyme TL IM was used at its original water activity value, 0.201 (Nunes et al., 2011b). The reaction was carried out in solvent-free media, at 45 °C, in thermostatted-capped cylindrical glass vessels, under magnetic stirring at 250 rpm. In a time-course experiment, samples of 0.15 mL of reaction medium were withdrawn throughout 48 h and stored at −20 °C until analysis. In order to study the effect of lipase load in C10:0 incorporation, expressed in mol-%, 24 h acidolysis reactions were performed using different concentrations of biocatalyst (5%, 10%, 15% and 20 w:w of total substrates).
To select the best reaction media, namely solvent vs. solvent-free media, acidolysis reactions were carried out without solvent or in n-hexane media, at a ratio of total substrates:solvent of 1:1.5 (v:v), at 45 °C, in thermostated-capped cylindrical glass vessels under magnetic stirring at 250 rpm for 48 h reaction-time. Acidolysis reactions were performed in triplicate and the average values ± standard deviation were reported.
This procedure was done according to the method described by Nunes et al. (2011b). Briefly, 0.1 mL of reaction medium was solubilized in 0.9 mL of chloroform and an aliquot of this product mixture (200 μL) was separated by thin-layer chromatography (TLC) on silica gel plates and developed with n-hexane:ethyl ether:acetic acid (70:30:1.5, v:v:v). TLC plates were then air-dried and sprayed with 0.2% (w:v) 2’,7’-dichlorofluorescein in 95% ethanol and the bands were visualized under ultraviolet light at 366 nm. The various groups of compounds (TAGs, free FAs, diacylglycerols and monoacylglycerols) were identified by comparison with standards. The TAG band was scraped from TLC plates and methylated and FAME were GC analyzed as previously described in the Section 2.3. The mol-% incorporation of C10:0 into TAG was calculated according to Casas-Godoy et al. (2013) by the ratio of the moles of C10:0 in the TAG and the total moles of fatty acids in the triacylglycerols.
To investigate the presence of eventual significant differences among biocatalyst loads, ANOVA of molar incorporation values was carried out using a Tukey’s-b test. Differences were considered statistically significant at p < 0.05. All statistical analyses were performed using SPSS statistical software version 21.0 (IBM SPSS Statistics 21.0; Faro, Portugal).
The oil extraction yield obtained by cold-pressing pumpkin seeds from the Santarém region was 31.0%. This value is similar to that reported by Jiao et al. (2014) with solvent extraction (28.4%) and among the values obtained using hydrolytic enzymes (29.4–59.9%) in a microwave-assisted aqueous enzymatic extraction of oil from pumpkin seeds. Also, Mitra et al. (2009) reported a maximum extraction yield of 30.7 wt.% using supercritical carbon dioxide, nearly the same obtained in this study.
Due to the differences among the species and/or varieties of Cucurbita maxima grown in different areas of the world, physico-chemical characteristics of the extracted oil of pumpkin seeds from the Santarém region (Portugal) was undertaken before its use in the synthesis of a SL (Tab. 1). Refractive index and specific gravity values observed in the present work are in accordance with Codex Stan 210 (1999), which states a range from 1.447 to 1.477 and 0.881 to 0.929, respectively for vegetable oils. Viscosity was lower than the data reported by Tsaknis et al. (1997) of 72 ± 0.58 mPa.s for crude pumpkin seed oil from Cucurbita pepo and Cucurbita maxima. Moisture content and acid value were in accordance with Codex Stan 210 (1999), which points out maximum levels of 0.2% and 4 mg KOH/g oil. The peroxide value obtained in this study (19.008 ± 1.710) was higher than the maximum limit of 15 meq O2/kg oil regulated by Codex Stan 210 (1999), which suggests that, at the moment of analysis, the pumpkin seed oil had begun to degrade by oxidation reactions, since the high amount of linoleic acid makes it specifically prone to oxidation. Three major FAs were identified: palmitic, oleic and linoleic (Tab. 1). Stearic acid was also present but in a considerably lower concentration. A high amount of total unsaturated FAs was observed (75.9%), with linoleic acid being the FA with the highest expression (55.5%). It is worth mentioning that linoleic acid possesses favourable nutritional and physiological effects in the prevention of coronary heart diseases and cancers (Oomah et al., 2000). Similar FA profiles of pumpkin seed oil were obtained by other authors (Murković et al., 2004; Popa et al., 2010; Kulkarni et al., 2012; Rezig et al., 2012; Procida et al., 2013; Jiao et al., 2014; Rabrenović et al., 2014), where their content and proportion, depended on the pumpkin varieties, climate and cultivation conditions.
Physico-chemical properties and fatty acid profile of pumpkin seed oil from the Santarém region (Portugal).
Structured triacylglycerol was obtained by acidolysis of oil extracted from seeds of Cucurbita maxima pumpkin with C10:0, catalyzed by commercial lipase preparation from Thermomyces lanuginosa (Lipozyme TL IM). Reactions were carried out in a solvent-free media at 45 °C with 5% (w:w) of immobilized lipase and a molar ratio of oil:C10:0 of 1:2. The effect of temperature on the incorporation of C10:0 into olive oil TAG in solvent free medium was previously studied by Nunes et al. (2011a, b). According to the authors, when the temperature was set at 45 °C, the viscosity of the reaction medium was reduced and the enzyme activity was such that a 28.8 mol-% incorporation yield was achieved. Therefore, the acidolysis reaction of pumpkin seed oil with C10:0 was done at this temperature. The molar ratio substrates used (1:2) corresponds to the stoichiometric value needed for the esterification of C10:0 at the sn-1 and sn-3 positions with this sn-1,3 selective enzyme. Despite sometimes a stoichiometric excess being advantageous in promoting FA incorporation, higher levels of substrate molar ratio could also positively affect acyl migration (Zou et al., 2015). Moreover, it is not economically justifiable to use a molar ratio of substrates higher than the stoichiometric value needed. In previous studies of enrichment of olive oil with medium chain FAs using biocatalysts with 1,3-regioselectivity and similar temperature and molar ratio conditions (Nunes et al., 2011b), sn-2 positional analysis were assayed and the sn-2 positions of the SLs obtained were predominantly occupied by oleic acid as in the original virgin olive oil. Therefore, in this study carried out under the same reactional conditions, FA composition at position-2 were not assayed. Monitoring the time course in enzymatic reactions is important in order to confirm the optimal reaction time for obtaining the highest yields and minimizing the overall costs in the processing. Time-course study (Fig. 1) indicated that the incorporation of C10:0 into pumpkin seed oil increased with time, reaching a plateau at 31 h (29.9 ± 0.7 mol-%). Although, incorporation values observed after 24 h were not significantly different. The same trend was observed when Lipozyme TL IM was used in the enrichment of olive oil with this medium chain FA, showing a maximum value of 28.8 ± 3.0 mol-% at 24 h reaction time (Nunes et al., 2011b). When higher lipase loads were tested (10%, 15% and 20% w:w of total substrates), the incorporation values of C10:0 into pumpkin seed oil increased with the enzyme load, showing only significant differences when incorporation took place in the presence of 5% or 20%, 24.7 ± 3.1 or 29.6 ± 1.9 mol-%, respectively (Tab. 2). However, the incorporation increased only 1.2 times when lipase load increased from 5% to 20%. According to Soumanou et al. (2013), higher enzyme amounts can accelerate the reaction rate and increase the incorporation during acidolysis, although high acyl migration may occur under these conditions. In fact, acyl migration depends on lipase load, enzyme supports with surface charges, water content and water activity, solvent and temperature (Iwasaki and Yamane, 2000; Xu 2000a, b, 2003; Fernadez-Lafuente, 2010). Also, de Araújo et al. (2016) reported that the increase of the lipase amount over the optimal value has no significant impact on the incorporation, probably due to the enzyme aggregation, which leads to an increase in substrate diffusion inhibition, causing the saturation of the reaction rate (Abed et al., 2017). Considering the marginal increase in the incorporation yield obtained with 20% lipase load, the subsequent study was carried out in the presence of 5% of lipase to avoid acyl migration, as also reported by Xu et al. (1998). Moreover, higher lipase loads increase acidolysis process costs. The impact of the solvent on C10:0 incorporation into pumpkin seed oil by Lipozyme TL IM was investigated. In the presence of n-hexane, the incorporation of C10:0 into pumpkin seed oil increased with time up to 48 hours (34.7 ± 1.0 mol-%) (Fig. 1). However, as observed in solvent free media, there are no significant differences between the incorporation yields after 24 h reaction time. The incorporation of C10:0 followed the same trend in both conditions, but slightly higher values were obtained in the presence of n-hexane, especially at 48 h-reaction, where the relative difference between solvent-free and hexane systems is about 18%. Nevertheless, since the structured lipids are to be used in food products, together with environmental and economic concerns, solvent-free systems are preferred.
Incorporations of C10:0 into pumpkin seed oil, catalyzed by 5% (w:w of total substrates) of Lipozyme TL IM in solvent free media (●) and in the presence of n-hexane (○).
Effect of lipase load on C10:0 incorporation into pumpkin seed oil.
The physico-chemical characteristics of pumpkin seed oil and its FA profile suggest that it is a healthy oil mainly due to its rich composition in unsaturated FAs, especially oleic acid (20.4 mol-%) and linoleic acid (55.5 mol-%). The synthesis of a MLM-type SL by acidolysis of pumpkin seed oil with C10:0 catalyzed by Lipozyme TL IM was well succeeded. More precisely, 34.7 mol-% of C10:0 incorporation was obtained in the presence of n-hexane, using a 5% enzyme load of Thermomyces lanuginosa lipase, 1:2 pumpkin oil:capric acid molar ratio, at 45 °C and 48 h reaction time. The presence of high lipase loads did not considerably increase the incorporation yield of C10:0. Regarding the comparison between solvent-free and n-hexane systems, higher incorporations were observed in the presence of solvent. However, since the structured lipids are to be used in food products, solvent-free systems are preferred. Additional research on the quality of this SL can assist in efforts to achieve wide application in foods.
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Cite this article as: Sousa V, Campos V, Nunes P, Pires-Cabral P. 2018. Incorporation of capric acid in pumpkin seed oil by sn-1,3 regioselective lipase-catalyzed acidolysis. OCL 25(3): A302.
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