Biotechnology Journal International 17(4): 1-8, 2017; Article no.BJI.31140 Previously known as British Biotechnology Journal ISSN: 2231–2927, NLM ID: 101616695

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Optimization of Aqueous Enzymatic Extraction of Flat-Europe Hybrid (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 th Original Research Article Accepted 30 January 2017 Published 17th 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

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*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, , 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 produces high yields. The 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).

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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. The hydrolysis time X4), each at three different levels, were employed. The parameters chosen and sample (1 μL) was injected in split mode (split their levels were based on single factor tests. ratio 1:100). The MS conditions were: ionization The level codes are shown in Table 1. voltage of 70 eV, scanning range of 35-520 A, Centrifugal time and speed were set at 20 min and ion source temperature of 230°C. The FA and 10,000 g, respectively. There were a total of composition was reported as percentage of the 31 runs for optimizing the four individual total peak area [12-14]. parameters in the CCD. A second order polynomial equation was fitted to the 3. RESULTS AND DISCUSSION experimental data of each independent variable as given below. 3.1 Single Factor Optimization Process

3.1.1 Effect of different enzyme complexes on the oil extraction yield (2)

Selecting suitable commercial enzymes is an Where, Y is the response value, Xi and Xj are important factor that can affect the extraction the levels of variables, β0 is the constant efficiency. The oil extraction yield obtained with term, β is the coefficient of the linear terms, i different enzymes is shown in Fig. 1. Without the β is the coefficient of the quadratic terms, ii application of enzyme, the oil extraction yield and βij is the coefficient of the interaction was just 27.77%. When used alone, papain term. showed the highest extraction efficiency,

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followed by α-amylase, while cellulase showed and papain were 4% and 6% of seed weight, the lowest extraction efficiency. Using a respectively. combination of enzymes resulted in a significant increase in the oil extraction yield. Among 3.1.3 Effect of the seed/water ratio on the oil enzyme combinations, papain + α-amylase + extraction yield cellulase gave the highest extraction yield (70.33%). However, the addition of cellulase For conditions including a pH value of 6, α- seemed to promote the formation of emulsions, amylase corresponding to 4% of seed weight, and created problems during oil separation. The and papain corresponding to 6% of seed weight, combination of papain and α-amylase also gave the highest extraction yield was obtained when a high extraction yield (68.60%), and the oil the seed/water ratio was 1:5 (Fig. 2C). If the separation effect with this combination was better, amount of water was higher, the extraction yield so it was selected as the best enzyme declined. The addition of water is necessary for combination for the extraction of hazelnut oil. the aqueous enzymatic extraction of oil, but excess water would reduce the concentration of 3.1.2 Effect of the amount of α-amylase and the reaction substrates, thereby reducing the papain on the oil extraction yield efficacy of the enzyme. Therefore, the optimal seed/water ratio was set at 1:5. The amount of enzyme added is an important factor that can influence the extraction efficiency. 3.1.4 Effect of different pH on the oil The other four factors (seed/water ratio, extraction yield extraction pH, extraction temperature, and extraction time) were set at 1:4, 6, 60°C, and 4 h, Different kinds of enzymes have different optimal respectively. Fig. 2A shows that an increase in pH values [15]. Therefore, determining the the amount of α-amylase resulted in the gradual optimal pH for an enzyme complex is very increase of the extraction yield of flat-European important. The other extraction conditions hazelnut oil. The maximum extraction yield included the addition of papain and α-amylase at (68.13%) was obtained when the amount of α- a proportion of 6% and 4% of seed weight, amylase added was 4% of the seed weight. respectively, a seed/water ratio of 1:5, a However, the extraction yield did not increase hydrolysis temperature of 60.00°C, and an with further increases in the amount of enzyme. extraction time of 4 h. The effect of different pH The amount of papain tended to have a similar on the oil extraction yield is shown in Fig. 2D. effect on the oil extraction yield (Fig. 2B). A The oil extraction yield continued to increase with dosage of 6% papain, relative to seed weight, an increase in pH (4.00-6.00), reaching the peak produced the highest oil extraction yield value (73.33%) at pH 6.00, and declining at (73.43%). Considering the oil extraction yield and higher pH, which suggests that the optimal pH of cost factors, the optimum amount of α-amylase the enzyme complex was 6.00.

Fig. 1. Effect of different enzyme complexes on the oil extraction yield PC: papain and cellulase; Pα: papain and α-amylase; Cα: cellulase and α-amylase; PCα: papain, cellulase and α- amylase. Values expressed are means (N = 3) ± standard deviation. Means in each bar with different superscripts were significantly (p＜0.05) different

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3.1.5 Effect of hydrolysis temperatures on obtained when the hydrolysis temperature was the oil extraction yield 60°C. When the hydrolysis temperature increased from 60 to 70°C, the oil extraction yield Different enzymes have different optimal decreased rapidly. Thus, a hydrolysis temperatures. To study the effect of different temperature of 60°C was considered to be temperatures on the oil extraction yield, optimal for the present study. The decrease in hydrolysis was carried out at different extraction yield was observed when the temperatures (45, 50, 55, 60, 65, and 70°C), temperature exceeded 60°C may have been while the other extraction conditions included the caused by inactivation of the enzyme. addition of papain and α-amylase at levels of 6% and 4% of seed weight, respectively, a 3.1.6 Effect of different hydrolysis times on seed/water ratio of 1:5, a hydrolysis pH of 6.00, the oil extraction yield and an extraction time of 4 h. As shown in Fig. 2E, the oil extraction yield increased when the Hydrolysis time was positively correlated with the hydrolysis temperature increased from 45 to oil extraction yield (Fig. 2F). For this experiment, 60°C. The maximum yield (75.90%) was the amount of enzyme complex, seed/water ratio,

Fig. 2. Effect of different parameters on oil extraction yield Values expressed are means (N = 3) ± standard deviation

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extraction pH, and hydrolysis temperature were Fig. 3D). Fig. 3E and Fig. 3F display the 3D set at 6% papain and 4% α-amylase (relative to response surface plot and contour plot showing seed weight), 1:5 (w/v), 4.00, and 60°C, the effect of varying pH value and hydrolysis time respectively. The oil extraction yield increased as on the oil extraction yield, when a fixed the hydrolysis time increased from 2 to 4 h, and hydrolysis temperature of 59.90°C and hydrolysis the maximum oil extraction yield (73.53%) was time of 4.78 h were used. This plot indicated that obtained when the hydrolysis time was 4 h. After the maximum oil extraction yield could be this point, the oil extraction yield started to achieved when the pH value and seed/water decline. This result might be explained by the ratio were 6.13 and 1:5, respectively. stabilization of the emulsion after a long hydrolysis period, which would make it difficult to 3.2.2 Verification of the predictive model release the oil by centrifugation. Therefore, a hydrolysis time of 4 h was adopted in the present The second-order polynomial model obtained work. (Eq. 3) was utilized for RSM optimization by using Minitab 15. The maximum extraction yield 3.2 Optimization of Aqueous Enzymatic of 81.90% is predicted at optimum condition: at seed/water ratio of 1:5, pH value of 6.13, Extraction of Oil from Flat-European hydrolysis time of 4.78 h, and hydrolysis Hybrid Hazelnut by RSM temperature of 59.90°C. The suitability of the model equation for predicting the optimum 3.2.1 Interaction between independent response values was tested by using the variables selected optimal conditions. The experimental oil extraction yield value was 82.36 ± 0.42% (N = 3) The three dimensional (3D) plots and contour when applying the modified optimal conditions, plots were generated with one variable kept at its which is close to the predicted value. This optimum level, and varying the others within the indicated that the model was adequate for the experimental range and shown in Fig. 3. The oil extraction process. extraction yield in relation to seed/water ratio and pH value is shown in Fig. 3A and Fig. 3B. It was 3.2.3 Fatty acid composition analysis of flat- demonstrated that the oil extraction yield European hazelnut oil increased when the pH increased from 5.00 to 6.13, but further pH increases resulted in a The composition of flat-European hazelnut oil decrease of the oil extraction yield. The oil was analyzed by GC-MS. The results are shown extraction yield was also found to increase when in Table 1. The major fatty acid components of the seed/water ratio was reduced from 1:4 to 1:5, the flat-European hazelnut oil were oleic acid, but decreased when the ratio was further linoleic acid, linolenic acid, palmitic acid, reduced from 1:5 to 1:6. A similar effect on the oil palmitoleic acid, arachidonic acid, and Squalene. extraction yield was observed between Oleic acid was the most abundant component, seed/water ratio and hydrolysis time (Fig. 3C and accounting for 77.22% of the total fatty acids.

Table 1. Fatty acid composition of flat-European hazelnut oil

Number Components Molecular formula Molecular weight Relative content (%)

1 Benzoic acid C7H6O2 122.1214 0.01 2 Myristic acid C14H28O2 228.37 0.02 3 Pentadecanoic acid C15H30O2 242.4 0.01 4 Hexadecanoic acid C16H32O2 256.4 5.04 5 9-Hexadenoic acid C16H30O2 254.41 0.18 6 Heptadecanoic acid C17H34O2 270.45 0.04 7 Octadecanoic acid C18H36O2 284.48 2.35 8 Oleic acid C18H34O2 282.47 77.22 9 Linoleic acid C18H32O2 280.44 14.06 10 Linolenic acid C18H30O2 278.4 0.13 11 Eicosan(o)ic acid C20H40O2 312.53 0.18 12 Eicosenoic acid C20H38O2 310.51 0.44 13 Behenic Acid C22H44O2 340.58 0.02 14 Squalene C30H50 410.72 0.03

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Fig. 3. 3D plots showing the correlative effects on the oil extraction yield of seed/water ratio and pH value (A), seed/water ratio and hydrolysis time (C), hydrolysis temperature and hydrolysis time (E). Contour plots showing the correlative effects on the oil extraction yield of seed/water ratio and pH value (B), seed/water ratio and hydrolysis time (D), hydrolysis temperature and hydrolysis time (F)

Oleic acid is a monounsaturated fatty acid that is enzyme combination for the aqueous enzymatic able to reduce the total cholesterol and bad extraction of flat-European hybrid hazelnut oil. cholesterol levels in blood, but it does not reduce The best technological parameters for the the levels of good cholesterol, so it is very process were: seed/water ratio of 1:5 (w/v), pH effective for the treatment of heart and head value of 6.13, reaction temperature of 59.90°C, blood-vessel diseases [16]. Linoleic acid and reaction time of 4.78 h. With these represented 14.06% of the total fatty acids, while conditions, the oil yield was 82.36%. GC-MS was linolenic acid represented 0.13%. Unsaturated used to analyze the fatty acid composition of the fatty acids were 92.01% of the total. flat-European hybrid hazelnut oil, identifying oleic acid (77.22%), linoleic acid (14.06%), linolenic 4. CONCLUSION acid (0.13%), palmitic acid (5.04%), stearic acid (2.35%), and arachidonic acid (0.44%) as the The mixture of papain (6% of seed weight) and major compounds. The unsaturated fatty acid α-amylase (4% of seed weight) was the best content was 92.01% of the total.

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Peer-review history: The peer review history for this paper can be accessed here: http://sciencedomain.org/review-history/17872

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