GREEN MICROWAVE-ASSISTED AQUEOUS ENZYMATIC EXTRACTION OF Elateriospermum tapos SEED OILS

Abstract

Microwave-assisted aqueous enzymatic extraction (MAAEE) is a promising green technology that is fast, efficient and energy saving. The physicochemical parameters and fatty acids composition of the MAAEE-extracted oil were reportedly quite comparable with those of solvent-extracted oil and control oil, showing no significant differences (p> 0.05) among the extraction methods. However, the oxidative stability of the MAAEE obtained oil was found to be considerably improved in the comparison as evident by the determinations of chemical profile values. Moreover, the microwave-enzymatic adjuvant improved the oil extraction yield as well as the oxidation properties of the oil produced without altering its other quality attributes. The present work compared the quality characteristics of microwave-assisted aqueous enzymatic extracted Elateriospermum tapos seed oil with those of hexane-extracted oil and control oil. Interestingly, MAAEE process has achieved 12% and 16% higher extraction efficiency and concentrated omega-3 fatty acid, respectively compared to non-enzymatic microwave-assisted extraction (MAE). Although resulted in lower yield than hexane-extraction (Soxhlet), MAAEE extracted 24% higher concentrated omega-3 fatty acid with almost 99% removal of anti-nutrient amygdalin within 75 seconds. Hence the study on the interaction of extraction parameters as well as the MAAEE optimization process is sustainably feasible. Keyword: microwave assisted aqueous enzymatic extraction, seed oils, Elateriospermum tapos, amygdalin Abstrak Pengekstrakan enzim berakues yang dibantu dengan gelombang mikro (MAAEE) adalah teknologi hijau yang menjanjikan proses yang pantas, cekap dan penjimatan tenaga. Parameter fizikokimia dan komposisi asid lemak daripada minyak yang diekstrak MAAEE dilaporkan setara berbanding dengan minyak ekstrak-pelarut dan minyak kawalan serta tidak menunjukkan perbezaan ketara (p> 0.05) di antara kaedah pengekstrakan. Walau bagaimanapun, kestabilan oksidatif minyak MAAEE didapati lebih baik dalam perbandingan seperti yang ditunjukkan oleh penentuan nilai-nilai profil kimia. Tambahan pula, adjuvan gelombang mikro-berenzim meningkatkan hasil pengeluaran minyak serta ciri pengoksidaan minyak yang dihasilkan tanpa mengubah sifat-sifat kualiti yang lain. Kajian ini membandingkan ciri-ciri kualiti minyak biji Elateriospermum tapos secara kaedah pengekstrakan enzim berakues-berbantu gelombang mikro dengan minyak ekstrak-pelarut heksana dan minyak kawalan. Menariknya, proses MAAEE telah masing-masing mencapai kecekapan pengekstrakan 12% dan kepekatan asid lemak omega-3 16% lebih tinggi berbanding pengekstrakan berbantu gelombang mikro tanpa berenzim (MAE). Walaupun hasilan lebih rendah daripada pengekstrakan heksana (Soxhlet), MAAEE memperoleh asid lemak omega-3 berkepekatan 24% lebih tinggi dengan hampir 99% penyingkiran amigdalin anti-nutrien dalam 75 saat. Oleh itu, kajian mengenai interaksi parameter pengekstrakan serta proses pengoptimuman MAAEE menunjukkan kewajaran secara lestari. Kata kunci: pengekstrakan enzim berakues dibantu gelombang mikro, minyak bijian, Elateriospermum tapos, amigdalin ISSN 1394 2506 Azlan & Ida Idayu: GREEN MICROWAVE-ASSISTED AQUEOUS ENZYMATIC EXTRACTION OF Elateriospermum tapos SEED OILS 489 Introduction Microwave uses electromagnetic wave which penetrates certain materials to provide volumetric heating through ionic conduction and dipole rotation [1]. Water and aqueous based solvent system offer an increasingly crucial choice for the replacement of conventional organic solvent [2]. Therefore, the microwave assisted aqueous enzymatic extraction (MAAEE) method is a highly potential, green, fast, efficient and energy saving method which eliminates the disadvantages of conventional solvent extraction method with undesirable effect on oil quality. Moreover, the treatment of seeds with enzymes enhances the extraction of oil yield due to its hydrolyzed structural polysaccharide of the cell walls and proteins associated with the lipid bodies [3]. MAAEE had been applied for oil extraction from seed crops such as pumpkin seed [3] and Forsythia suspense seed [2]. Omega-3 fatty acid is an essential fatty acid that need to be consumed through supplements or food products due to the incapability of human body to generate the compound naturally by itself [4]. Perah or scientifically known as Elateriospermum tapos seed (ETS) is an underutilized Malaysia local plant seed that had been reported to be rich of omega-3 fatty acid (Figure 1). A research by Yong and Salimon [5] claimed that ETS contains 17.4% of alpha linolenic acid (ALA) (PubChem CID: 5280934). The current work applies MAAEE for extraction of ETS oil, quantification of the specific amount of omega-3 fatty acid concentration and its anti-nutrient, amygdalin. Figure 1. Properties and molecular structure of omega-3 fatty acid [6]. Materials and Methods Plant materials Elateriospermum tapos seed (ETS) was obtained at a local farm in Kuala Lipis, Pahang, Malaysia and was botanically identified by Biodiversity Unit, Institute of Bioscience, University Putra Malaysia (specimen voucher number SK2782/15). The seeds were manually cleaned and flooded with tap water to separate the extraneous matters (dust, vain seeds and straw from threshed seeds). The shells were removed; cleaned seeds were grinded using a laboratory grinder (MX-898M Panasonic, Malaysia) and sieved (Retsch siever, Germany) to a desired particle size and then stored at -20 °C in airtight bags until further use. The moisture content of the seeds, which were 6-7%, was determined using the infrared moisture analyser (FD-620 Kett, Japan). Reagents Cellulase from Aspergillus niger (Tokyo Chemical Industry), pectinase from Aspergillus niger (1.02U/mg) and proteinase from Aspergillus melleus (≥3 units/mg solid) from Sigma Aldrich, hexane analytical and GC grade from Qrec and Merck, Malaysia. Extraction process For Soxhlet extraction (SE) process, 5 g of ETS (0.5 mm particle sizes) were placed into each thimble and 150 mL hexane was used as a solvent. This process was carried out by refluxing each sample for 6 hours on a heating mantle. After the elapsed time, a rotary evaporator was used to evaporate the solvents. The SE extraction produced Malaysian Journal of Analytical Sciences, Vol 23 No 3 (2019): 488 494 DOI: https://doi.org/10.17576/mjas-2019-2303-13 490 48.99±0.79% of oil, which was set at 100% oil recovery in the comparison with the MAAEE in this study. Next, the samples were collected and preserved at -20 °C in sealed bottles for further analysis. For MAAEE method, a domestic microwave oven system was used (Sharp Model R-397J, Malaysia) equipped with digital control system for irradiation time and irradiation microwave power (110 W, 330W, 550 W, 770 W and 1100 W) and operates at 2.4 GHz. A mode stirring table was also included in microwave system to dissipate the microwave energy during microwave heating which is facilitate the extraction sample to heat up homogeneously. Subsequently, 5 g of grinded ETS and enzyme cocktail (cellulose: pectinase: proteinase ) ratios of 1.4:1.7:1.4 were accurately weighed and placed into a 250 mL flask with 1:5 solid to solvent ratio (based on preliminary experiment). Subsequently, the MAAEE extraction was conducted according to the design of experiment (DOE) at the following conditions; 110-1100 W irradiation power, 30-120 s extraction time, 1-5% enzyme cocktail concentration and 0.5-1.5 mm particle size with a total of 30 experiment runs. After extraction, the solution obtained from MAAEE was then centrifuged at 10000 rpm speed for 15 minutes, and oil phase was withdrawn using a micropipette. The oil was then weighed, and the extraction yield was expressed as the mass percentage ratio (oil extracted /total oil obtained by SE method, %) which result an optimal oil extraction yield of 46.12±1.48% at 110 W microwave power, 30 s extraction time, 1% enzyme cocktail concentration and 0.5 mm particle size (based on previous study). Next, the samples were collected and preserved at -20 °C in sealed bottles for further analysis. Identification and quantification of omega-3 fatty acid concentration The extracted ETS oil was converted to fatty acids methyl ester (FAMEs) prior to injection. About 1 μL of the sample was injected into GC (model Perkin Elmer Auto System XL) to analyze the omega-3 fatty acid. It was equipped with flame ionization detector and BPX 90 capillary column (60 m x 0.25 mm x 0.55 μm). The detector temperature and injector temperature were programmed at 250 °C. The oven temperature program was set at the initial 100 °C followed by ramp 1 at 20.0 °C /min to 170 °C, hold for 0.00 min, ramp 2 at 10.0 °C/min to 230 °C , hold for 7 minutes and ramp 3 at 30 °C/min to 250 °C, hold for 2 minutes. Nitrogen was used as the carrier gas. Subsequently, the peaks detected by the retention time were identified by comparing the result produced with standards under the same condition. Different concentrations (100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 ppm) had been prepared from omega-3 fatty acid (alpha linolenic acid) standard (Sigma Aldrich, Germany). The standards were injected in GC using the same condition of fatty acids analysis. The calibration curve of absorbance versus concentration graph was plotted. The concentration of extracted omega-3 was determined in mg of ω-3/g of oil using equation 1. Cω−3 = [C ×V] Woil (1) Cω-3 indicates the concentration of the extracted omega-3, mg of ω-3/g of oil; C indicates the concentration of omega-3 obtained from calibration curve, mg/mL; V indicates the volume of omega-3 fatty acid solution, mL and Woil is the weight of the extracted oil, g. FTIR analysis The chemical structure of ETS oil extracted by MAAEE, MAE and SE were determined by FTIR spectrometer (Perkin-Elmer Spectrum One) equipped with a deuterated triglycine sulphate (DTGS) as a detector and a KBr/ Germanium as a beam splitter. A fe