Hichm Mechqoq,Mohmed El Ygoui,Svetln Momchilov,Foud Msnd,Noureddine El Aoud,c,*

a Department of Biology,Faculty of Sciences,Ibn Zohr University,Agadir 80000,Morocco

b Institute of Organic Chemistry with Centre of Phytochemistry,Bulgarian Academy of Sciences,Sofia 1113,Bulgaria

c Polydisciplinary Faculty of Larache,University Abdelmalek Essaadi,Tetouan,Larache 92000,Morocco

ABSTRACT Argan oil is most frequently sold as pure oil, which can be directly applied topically due to its cosmetological proprieties or ingested in order to provide several health benefits.It's also commonly mixed into a number of cosmetic products like shampoos,soaps and conditioners.In this study we aimed to improve the argan oil extraction yield and quality parameters by comparing the effects of different extraction technologies.Argan kernel oils were extracted using four methods:mechanical cold press,Soxhlet extraction with n-hexane,supercritical fluid extraction(SFE),and enzyme assisted extraction with three different enzyme solutions cellulase(cellulast),pectinase(Pectinex)and a mixture of carbohydrase enzymes(Viscozyme®).The quality parameters was evaluated by determining the acid,peroxide and iodine values as well as the extinction coefficients K232 and K270 as measures of conjugated dienes and trienes,respectively.The results showed that the highest yield(66.37%±3.3%)was obtained by enzyme assisted extraction using the carbohydrases enzymes mixture(Viscozyme®),followed by Soxhlet extraction(59.5%±3.1%)and pectinase extraction(52.03%±3.55%).All argan oils samples obtained by the different methods showed a good oxidation stability,with acid,peroxide and iodine values lower than 0.8 mg/g, 15 meq/kg and 110 g/100 g according to the official argan oil norm,respectively.The results of argan oils quality parameters demonstrated that the enzyme extracted argan oils showed low oxidation,with the following quality parameters:acid values(0.4–0.6 mg/g),iodine values (95–100 g/100 g), dienes (K232 < 2), trienes (K270 < 0.35), and peroxide values(<1.5 meq/kg).The results proved that the enzyme assisted extraction method can be applied to improve the argan oil yield without affecting the oil quality.The enzyme extraction method may be a great alternative to solvent and cold press extractions for this eco-friendly processing approach.

Keywords:Argan oil Cold press Soxhlet extraction Supercritical fluid extraction Enzyme assisted extraction

1.Introduction

The Moroccan tree,Argania spinosa(L.)Skeels(formely named A.sideroxylon Roem & Schult) is mostly found on the Moroccan arid and semi-arid southwestern areas,where it covers about 800,000 ha[1].The argan tree is appreciated for its edible oil,extracted from the kernels of the fruit.This oil is recognized worldwide for its nutritional values,and is sought by the cosmetic industry for its skin and hair hydration and anti-aging properties [2].Argan oil was prepared by women on the cooperatives following a multistep process from the fruits of the argan tree.Nowadays,argan oil is extracted using a mechanical cold press.However, some local producers still use traditional extraction method,this process has been abandoned by the Moroccan cooperatives due to its unsatisfactory yield,sanitary and storage conditions.

The argan oil is mostly composed of 99%of glycerides,95%of them are triacylglycerols,the other 4%consist of diacylglycerols,monoacylglycerols,sterol esters,phosphoipids and other polar lipid classes trace[3].Those glycerides are composed of fatty acids,the latter are represented by 80%of unsaturated fatty acids among which the oleic and linoleic acids are about 45%–50%and 34%–38%[4].The argan oil contains also carotens, polyphenols (mainly ferulic and vanillic acids 25%–30%and 7%–10%,respectively)[5,6],and sterols (schottenol 150 mg/kg and spinasterol 120 mg/kg)[7].Moreover,the qualitative and quantitative parameters of those compounds are generally affected by the extraction methods.

Several studies on argan kernel oil have shown the effects of different extraction methods such as traditional extraction,mechanical press,solvent and supercritical fluid extraction(SFE)on oil yield[7].The most commonly used method is the cold press extraction.This method presents many advantages,such as the preservation of the oil active substances(extraction temperature below 60°C),in addition of being solvent-free and giving high yield in comparison with the conventional methods.Despite those advantages, the supercritical fluid extraction technique's high production costs make its use more restrictive[8].

For decades, researchers had being challenged to find and develop new extraction technologies in order to reduce or eliminate solvent,optimize energy consumption,reduce cost and increase quality in order to reduce the impact of chemicals on the environment without altering the physico-chemical properties.The use ofenzymes has proven to be a good alternative for extraction from different fruits and vegetables, as reported in several studies performed on coconut,peanut,avocado,corn,and olive,among others,for the past two decades[9–16].However,the enzymatic extraction of argan oil has not been studied yet.

In our study, we extracted argan oils using cold press,Soxhlet,supercritical fluid and enzyme assisted extraction methods, for the latter three enzyme treatments were used,pectinase,cellulase and a mix of carbohydrases enzymes.The effect of the extraction method on the yield and quality parameters was investigated.

2.Material and methods

2.1.Sample collection

Argan(Argania spinosa(L.)Skeels)fruits were collected near Tiznite(Souss Massa area,Morocco),in the period between May and July 2019.After collection,fruits were dried and the kernels extracted manually,then stored at 4°C.

2.2.Enzyme preparations

Cell-wall degrading enzyme mixture Viscozyme®(cat.No.V2010,Merck,Germany)is a multi-enzyme complex containing a wide range of carbohydrases,including arabanase, cellulase, β-glucanase, hemicellulase, and xylanase.Pectinex Ultra SP-L(cat.No.P2611,Merck,Germany)is a pectolytic enzyme preparation from Aspergillus aculeatus.Cellulast 1.5 L(cat.No.C2730,Merck,Germany)is a cellulase ATCC 26921 produced from Trichoderma reesei.

2.3.Cold press extraction

Argan oils were extracted on a cooperative in Tiout(Taroudant, Morocco).Kernel cold-pressing was carried out using a Komet DD 85 G press apparatus(IBG Monforts Oekotec GmbH & Co.KG, Mönchengladbach, Germany)with a nozzle/screw distance of 3 mm,and a nozzle diameter of 9 mm.Briefly the argan oil kernels were weighted and put in the cold press extractor,then pressed under 45°C,the obtained oils were collected in dark bottles and stored at 4°C.

2.4.Soxhlet extraction

The argan oil solvent extraction was carried out using a Soxhlet system.For that, 150 g of argan kernels were ground into powder,put in a cartridge,and extracted with 350 mL of n-hexane(Merck,Germany)for 6 h(32 extraction cycles at 42°C),each extraction was carried out in triplicate.After extraction the solvent was removed under vacuum at 40 °C.The extracted samples were stored at 4°C in dark vials.

2.5.Supercritical fluid extraction(SFE)

The extractions were carried out with a pilot-scale supercritical fluid extractor(SFE 1–2,Separex,Champigneulles,French)designed to allow the study of a wide range of conditions.It consists of a CO2tank,a liquid CO2pump(that can deliver up to 10 kg/h),two extraction vessels(1 L and 2 L, respectively) which are both connected directly and parallel between them,three separators(with 200 mL capacity each), and a cooling system using glycol.Briefly,argan kernels were ground and put in a 1 L stainless steel basket(240 g of kernel for each extraction).The extraction was performed with CO2only, at constant temperature values of 45°C,and different pressure parameters(20,30 and 40 MPa),then the samples were collected and weighted to determinate the yields after extraction.

2.6.Enzyme assisted extraction

The extraction was carried out in batch mode using a laboratory fermenter Minifors(Infors-HT,Switzerland)with a total volume of 1500 mL.The enzymes(Cellulast,Pectinex and the Viscozyme®)were added at a concentration of 2%(V/V)separately to the 500 mL suspension of ground argan kernels/distilled water in the flask,the pH was set at the optimum value of 4.The impeller speed and the temperature were fixed at 150 r/min and 50 °C, respectively.The enzyme was allowed to act at this pH for 24 h.After the enzymatic reaction,the samples were put in a microwave oven at maximum power for 30 s, the microwave action ends the reaction by stopping the enzymes activity.Then, the argan oil was separated from the reaction medium by a bench-scale centrifugation (6000 r/min for 15 min), and water traces were removed with a funnel decantation.

2.7.Extraction yield

The extraction yield has been determined based on the initial argan kernel quantity used for the extraction, and the obtained oil quantity.The yield was calculated by the equation below:

2.8.Determination of quality parameters

Acid value(AV),peroxide and iodine values(PV and IV),UV absorption at 232 nm(K232)and 270 nm(K270)were evaluated according to standard methods of AOCS [17].Briefly,the AV was determined by titration of the oil samples diluted in an ethanol/ether (1:1, V/V) solution with ethanolic KOH and was expressed as percent of oleic acid.The PV was also measured by titration,for that 1 g of oil was dissolved in 40 mL of an acetic acid and chloroform mixture(3:2,V/V),500 μL of saturated KOH were added to the solution,and left to react for 2 min,then 30 mL of distilled water were added.The IV was determined by adding a reagent containing iodine to the oil samples.Titration of both PV and IV samples were conducted with a standard Na2S2O3solution 0.01 mol/L, PV was expressed as milliequivalents of active oxygen per kilogram of oil(meq/kg), when the IV was expressed as gram of iodine per 100 g of oil(g/100 g).The extinction coefficients K232and K270of argan oils were calculated respectively from the absorption at 232 and 270 nm,for that 1 g of oil was dissolved in 10 mL cyclohexane and the UV absorbance was measured by an UV spectrophotometer(ZUZI spectrophotometer Model 4201/50,Spain).

2.9.Statistical analysis

All experiments data were expressed as the mean±SD by measuring three independent replicates.Means were statistically compared using the STATICA (Version 6.1,Statsoft, Inc., USA) program with Student's t-test at the P < 0.05 significance level.A one-way ANOVA and Newman–Keuls multiple range test were carried out to test any significant differences between extraction techniques used at P<0.05.

3.Results and discussion

3.1.Extraction yield

Fig.1 showed the yield of the argan oils extracted with different methods.The highest yield(66.37%±3.30%)was obtained by enzyme assisted extraction with Viscosyme®.While,the yield of oils extracted by other enzymes(Pectinex and Cellulast)were 40.20%±2.70%and 52.03%±3.55%,respectively.Furthermore,the Soxhlet and cold press extractions gave yield values of 57.12%±0.10%and 49.83%±0.03%, respectively.The SFE gave three different yield values, the highest yield was 48.50% ± 0.10% under 40 MPa,followed by 37.22%±2.12%under 30 MPa,and 22.03% ± 0.50% under 20 MPa.However, the highest yield obtained with SFE still was lower than the value obtained with the cold press and Soxhlet extractions.

Fig.1.Yields of argan oils obtained by different extraction methods.

The maximum extraction yield was achieved with the carbohydrases enzyme mixture (Viscozyme®).The latter is composed of arabanase, cellulase, β-glucanase, hemicellulase,and xylanase,those enzymes allow the complete degradation of the kernels structural polymers mainly composed of carbohydrates(cellulose and sugars such as glucose,arabinose and xylose)[18].This can also explain the relatively important yield found with the cellulase enzyme treatment.The introduction of the enzyme assisted extraction for the juices and oils extractions is widely reported in the bibliography [9–16].Furthermore, this method permitted the obtention of extraction yields:avocado oil(65%)using a mixture of α-amylase and protease[10],coconut oil(68%)with a mixture of pectinase,α-amylase and protease[16],peanut oil(79.32%)with alcalase,and corn oil(93.2%)using alcalase,cellulase,xylanase and pectinase[14].These enzymes disrupt the phytoconstituents of the plant material,enhancing the release of the compounds,and the choice of enzymes to use is highly linked to the nature and constituents of the treated material[19].

On the other hand,a plenty of studies of the Soxhlet extraction method have been reported on the argan kernels oil extraction.The most recent one was published by Mouahid et al.[20],where the author used the same experimental parameters and obtained a yield of 58%.On another study,Taneva et al.[3]extended the extraction time to 8 h,and reported a yield value near to 60%.

The SFE method with CO2as extraction solvent and precise temperature and pressure parameters gave also interesting yields values, in this study the extraction parameters were similar to the ones used by Taribak et al.[21].In that paper,the authors tried different temperature and pressure parameters in order to optimize extraction process, this allowed them to increase the extraction yield to 50%.

The cold press extraction is the most used technique on the local cooperatives,due to its low cost,relatively important extraction yield and the high quality of the obtained oil[22].Charrouf[23]described the cold press extraction process of the argan oil,and mentioned a yield value of 50%,which,despite being low in comparison with the other extraction techniques,stays higher than the traditional extraction that gives only 30%of oil with poor microbiological quality[24].

The enzyme assisted extraction method offers a great oil extraction option for laboratory and semi-industrial scales,due to the high yield,short extraction time,non-use of solvents, and the good quality of product [25].In another hand,the Soxhlet extraction presents the inconvenient of using solvent,which makes it not safe for human consumption[26].Despite using the CO2for oil extraction and giving a free-solvent oil, the SFE method stays highly expensive[27].

3.2.Quality parameters of argan oils

Quality parameters of the extracted oils were evaluated through determination of acid,peroxide and iodine values,in addition to the K232and K270coefficients of extinction.Those parameters are important for assessing an oil quality.The results of the quality evaluations are summarized in Table 1.

Table 1 Chemical characteristics of argan oils obtained by different extracted methods.

Acid value(AV)and peroxide value(PV)reflects the oil quality and indicates the oil state of deterioration.The obtained oils showed acid values within the range of(0.3±0.1)mg/g and(0.6±0.2)mg/g,those results suggested that there is no significant difference between the AV of oils extracted by different methods.While, the peroxide values of the different oils were slightly different.In matter of fact the pectinase extracted oil showed the highest value with (1.3 ± 0.5) meq/kg, when the other extraction methods oils had values ranging from(0.6±0.1)meq/kg to (1.2 ± 0.2) meq/kg.Those results were similar to the ones reported by Gharby et al.[28] and Taribak et al.[21].However,the acid and peroxide values are found to be lower than 0.8 mg/g and 15 meq/kg,which according to the recommendations of the official argan oil norm defines the extra virgin argan oil label[29].

Moreover,extinction coefficients K232and K270are useful parameters to evaluate the oil state of oxidation[28].K232measure the formation of primary oxidation products(hydroperoxides),mainly conjugated dienes.The highest K232value was observed on the pectinase and the Soxhletextracted oils with (1.90 ± 0.30) and (2.00 ± 0.30), respectively, the other oil samples showed values between(1.15 ± 0.05) and (1.53 ± 0.06).While K270monitors the decomposition of hydroperoxides into secondary oxidation products.In fact, the K270value is increased by conjugated trienes, after primary oxidation of linolenic acid.The results obtained after the evaluation of argan oils samples exhibited values ranging between (0.15 ±0.06) and(0.25 ±0.01),reaching the recommendations of the official argan oil norm which define a high quality oil with K232and K270values lower than 2.5 and 0.35,respectively[29].

Iodine value is one of the most useful parameters for oil characterization,it indicates the oil unsaturation degree,the unsaturated fatty acids proved to be very important elements on human nutrition due to their biological proprieties, mainly cardiovascular protective and antidiabetic proprieties[30–32].The highest iodine value was observed in the cold press extracted oil with(103.5±0.4)g/100 g,followed by the Cellulast-,SFE-(30 MPa)and Viscozymeextracted oils with(102.1±0.7),(99.5±0.3),and(99.1± 0.5) g/100 g, respectively.The lowest value (90.0 ±0.2)g/100 g was obtained with the SFE(40 MPa)-extracted oil.Despite having different values,all the samples meet the standard fixed by the official argan oil norm to the range between 90 g/100 g and 110 g/100 g[29].

Based on the results all the argan oils meet the official argan oil norm[29],indicating that the extraction process have not affected the oil physic-chemical properties.Moreover,those findings confirmed that the extraction methods have minor effect on the oil oxidative state,the argan oil oxidative stability has been attributed to its high content in compounds such as tocopherols and carotenes[3].

4.Conclusion

In this study,enzymatic assisted method was used to extract Argania spinosa kernel oil in comparison with different extraction techniques including the cold press,Soxhlet and supercritical fluid extraction.The acid,peroxide,K232,K270and iodine values of the obtained oils were similar,while enzyme assisted extraction with Viscozyme showed the highest yield in contrast with the extraction methods used in this work.

The comparison of the obtained argan oils yields and quality parameters showed that the enzyme extraction with the carbohydrases enzyme mixture ensured the highest yield especially by the Viscozyme® enzyme, 16.6% and 9.3%higher than the cold press extraction and the Soxhlet extraction,respectively.The quality parameter evaluation of the argan oils demonstrated that all the samples had optimal quality in comparison with the Moroccanargan oil quality norm[29].The enzyme extraction technique is widely used by the food industry to extract juices and oils [10–12].And it presents a good option for argan oil extraction,especially that the Soxhlet extracted oil present the inconvenient of containing solvent traces,whereas the enzyme extracted oils have the advantage of being eco-friendly,and lead to the obtention of products suitable for human use.

The cold press extraction technique is still the most used within the cooperatives due to its benefits(good extraction yield,oils quality and stability),putting in the shade the traditional method.Despite being widely used in the industry to extract fruit juices and some sorts of oils, the enzyme assisted extraction technique had never been experimented to extract argan oils.

Our investigations showed that the enzyme assisted extraction increased the yield value to more than 65%,using the Viscozyme®enzyme mixture.Moreover,the evaluation of argan oils quality parameters demonstrated that the enzyme extracted argan oils showed low oxidation,with the following quality parameters:acid values(0.4–0.6 mg/g),iodine values(95–110 g/100 g),dienes(K232<2),trienes(K270<0.35),and peroxide values(<1.5 meq/kg).

The results obtained in the present study suggested that the enzyme assisted extraction can be scaled up and used to extract argan oils for the food and cosmetic industries.However, the optimization of the enzymatic extraction parameters (pH and temperature) or the use of other enzymes may improve the yield of extraction.This method is already used in the industry to extract fruits and may be a great alternative to solvent and cold press extractions in order to recover much more quantity of oils, the yield optimization being on demand for foods and nutraceutical industries.

Author Contributions

Hicham Mechqoq:Investigation,writing original draft;Mohamed El Yaagoubi:Contribution on discussion writing and correction;Svetlana Momchilova:Data analysis,technical and scientific support,correction;Fouad Msanda:Project supervision,scientific support;Noureddine El Aouad:Co-writing,project administration,supervision.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgement

This research was funded by the EU and project EXANDAS-H2020-MSCA-RISE-2015–“Exploitation of aromatic plants' by-products for the development of novel cosmeceuticals and food Supplements”(Grant Agreement No 691247).