Biotechnology Journal International 17(4): 1-8, 2017; Article no.BJI.31140 Previously known as British Biotechnology Journal ISSN: 2231–2927, NLM ID: 101616695 SCIENCEDOMAIN international www.sciencedomain.org Optimization of Aqueous Enzymatic Extraction of Flat-Europe Hybrid Hazelnut (Corylus heterophylla Fisch. × C. avellana L.) Oil and Analysis of Its Components Chunmao Lv1*, Na Liu1, Xianjun Meng1, Changying Lu1 and Xiao Li1 1College of Food Science, Shenyang Agricultural University, Shenyang 110866, P. R. China. Authors’ contributions This work was carried out in collaboration between all authors. Author CL designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Authors NL, CL and XM managed the analyses of the study. Author XL managed the literature searches. All authors read and approved the final manuscript. Article Information DOI: 10.9734/BJI/2017/31140 Editor(s): (1) Ghousia Begum, Toxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad, India. Reviewers: (1) Gyula Oros, PPI HAS, Budapest, Hungary. (2) Lorna T. Enerva, Polytechnic University of the Philippines, Philippines. Complete Peer review History: http://www.sciencedomain.org/review-history/17872 Received 22nd December 2016 Accepted 30th January 2017 Original Research Article th Published 17 February 2017 ABSTRACT Aim: The research aimed to evaluate the technological factors of the composite enzymatic extraction of Flat-European hybrid hazelnut oil, and analyze the fatty acid composition, to lay the foundations for the industrialized production of this oil. Study Design: Based on single factor tests, the best conditions for the aqueous enzymatic extraction of the oil determined by response surface analysis. Place and Duration of Study: Food Science College, Shenyang Agricultural University between March 2014 and December 2015. Methodology: The composition of Flat-European hybrid hazelnut was determined using GC-MS. Results: Selection of suitable commercial enzymes is an important factor that can affect the extraction efficiency. Based on single factor tests, best conditions for the aqueous enzymatic extraction of the oil, determined by response surface analysis, were as follows: pH 6.13, enzymatic hydrolysis temperature of 59.9°C, dose of papain equal to 6% of seed weight, dose of α-amylase _____________________________________________________________________________________________________ *Corresponding author: E-mail: [email protected], [email protected]; Lv et al.; BJI, 17(4): 1-8, 2017; Article no.BJI.31140 equal to 4% of seed weight, seed/water ratio of 1:5 (w/v), and enzyme hydrolysis time of 4.78 hours. By means of GC-MS analysis oleic acid (77.22%), linoleic acid (14.06%), linolenic acid (0.13%), palmitic acid (5.04%), stearic acid (2.35%), and arachidonic acid (0.44%) were identified as the major compounds of the flat-European hybrid hazelnut oil. Conclusion: The mixture of papain (6% of seed weight) and α-amylase (4% of seed weight) was the best enzyme combination for the aqueous enzymatic extraction of flat-European hybrid hazelnut oil. The best technological parameters for the oil extraction process were: seed/water ratio of 1:5 (w/v), pH value of 6.13, reaction temperature of 59.90°C, and reaction time of 4.78 h. With these conditions, the oil yield was 82.36%, where unsaturated fatty acids were 92% of the total. Keywords: Flat-European hybrid hazelnut; aqueous enzymatic extraction; hazelnut oil; response surface analysis. 1. INTRODUCTION are prevented, because of the mild extraction conditions, oil and protein are simultaneously Edible oil is an important component of the daily isolated. Although the aqueous enzymatic diet of many people. The consumption of high extraction of oil from corn, peanut, rice bran, and quality edible oil is also good for health. Hazelnut rapeseed has been studied [4-8], reports on has a high oil content, with unsaturated fatty hazelnut oil extraction are rare. Considering the acids representing more than 90% of the oil [1]. oil extraction yield and economic factors, this Oleic acid is the most abundant component of study aimed to evaluate the technological factors hazelnut oil, representing about 72%-82% of the of the composite enzymatic extraction of flat- total, which is the same proportion as in olive oil. European hybrid hazelnut oil, and analyze the Oleic acid is very effective for the treatment of fatty acid composition, to lay the foundations for heart and head blood-vessel diseases. Hazelnut the industrialized production of this oil. oil is rich in vitamins, β-sitosterol, carbolic acid and other special components [2]; it contains 2. MATERIALS AND METHODS paclitaxel, the world's highest-selling anti-cancer drug. Hazelnut oil also has a high economic and 2.1 Materials nutritional value. Flat-European hazelnut seeds were acquired Flat-European hybrid hazelnut is a new hazelnut from three professional production cooperatives variety, which was produced as the hybrid (Yang, Benxi, China). Papain (400 U/mg) and α- between the European hazelnut and a wild amylase (≥ 4000 U/mg, from Bacillus subtilis) hazelnut from northeast China. Flat-European were obtained from Dingguo Biochemical Co. hybrid hazelnut has a big pip with high kernel (Beijing, China). Cellulase (≥ 35 U/mg, from ratio and the tree produces high yields. The fruit Trichoderma) was purchased from Guoyao is oval or elliptic, plump, bright and clean with Biochemical Co. (Beijing, China). Nineteen kinds good flavor quality. Flat-European hybrid of grain fatty acid methyl ester were provided by hazelnut is cold-resistant, and is the only Supelco (Bellefonte, PA, USA). All other reagents hazelnut variety of large fruit that can resist were of analytical grade unless otherwise temperatures below -35°C. Therefore, oil specified. extracted from flat-European hybrid hazelnut has bright prospects as functional edible oil. 2.2 Selection of Suitable Commercial Enzyme and Hydrolysis Conditions Aqueous enzymatic extraction of oil is an emerging technology that employs mechanical The dehulled hazelnut seeds were crushed by a and biological methods to destroy the cell walls grinder. The material that passed through 40- of the seeds and uses water as the extraction mesh sieve was used for the extraction. Ground and separation medium to release the oil from seeds (10 g) were mixed with distilled water at a the seeds. With this process, it is possible to ratio of 1:4 w/v. The mixture was boiled for 5 min eliminate not only the use of hexane but also and allowed to cool down to room temperature. some refining steps [3]. Hence, this is an The pH was then adjusted to 5.00 with 0.5 M environmentally friendly and safe alternative to NaOH and 0.5 M HCl [9]. The enzyme(s), either solvent extraction, because problems like alone or in combination (papain, α-amylase, flammability and the risk of explosion are avoided, cellulase), were then added to the mixture to a and the generation of toxic organic volatile gases final proportion of 2% of the seeds’ weight (v/w). 2 Lv et al.; BJI, 17(4): 1-8, 2017; Article no.BJI.31140 The mixture was then placed in a water bath at Minitab version 15.1.0.0 (Minitab Inc., 60°C for 4 h. The temperature was then Pennsylvania, USA) was used for regression and increased to 90°C and kept at this temperature graphical analyses of the data. After analyzing for 10 min, cooled down to room temperature, the collected data, the three-dimensional and centrifuged at 10,000 g for 20 min. The oil response surface curves and contour plots were phase and emulsion phase were collected drawn by the software and used for further separately. The emulsion phase was further investigations. The quality of the polynomial centrifuged to separate the oil. Oil samples were model equation was judged statistically by the carefully collected with a pipette, combined and coefficient of determination R2. The significance weighed. A suitable enzyme (or combination of level was set at p < 0.05. Their significance was enzymes) was selected based on the efficiency evaluated by analysis of variance (ANOVA). of oil recovery. The selected enzyme, a combination of papain and α-amylase, gave the All experimental treatments were performed at best effect (Fig. 1) and was subsequently applied least two times, with triplicate samples for each in the single factor optimization process. The experiment. The values reported are the mean ± effects of enzyme concentration (1-10% v/w), SD. Statistical analysis of ANOVA was done incubation time (2 to 8 h), temperature (45-70°C), using SPSS ver. 17.0 for Windows (SPSS pH of reaction (4.00-9.00), and seed/water ratio Institute, Cary, N.C.). The equation of extraction (1:3-1:7, w/v) were analyzed. Controls lacking oil yield is listed below. Total oil in the seed was enzymes were used for all experiments. measured by the Soxhlet extraction method. 2.4 Analysis of Fatty Acid (FA) (1) Composition 2.3 Experimental Design and Statistical Samples were analyzed using an Agilent 6890 Analysis GC equipped with an Agilent 5973 mass selective detector, a 7694 auto-sampler and a Response Surface Methodology analysis (RSM) split-splitless injector. The GC was linked to an was used with the central composite design MS Chem Station HP vs. C.00·07. A Trace TR- (CCD) to investigate the effect of enzyme FAME (Thermo, Texas, USA) column (30 m × 0.25 mm, film thickness 0.25 μm) was used. treatment on the amount of oil extraction yield -1 (yield, defined as the mass of oil (g) per mass of Flow rate was 0.90 mL min . The injector and hazelnut seeds (100 g)) [10,11]. Four interface were set at 150°C and 230°C, respectively. The oven temperature was independent parameters (seed/water ratio X1, pH programmed to rise from 80°C to 240°C (8°C value X2, hydrolysis temperature X3, and min-1), and was held at 240°C for 5 min.