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Pre-treatment of ferulic acid esterases immobilized on MNPs to enhance the extraction of ferulic acid from defatted rice bran in presence of ultrasound
Author’s Accepted Manuscript Pre-Treatment of Ferulic Acid Esterases Immobilized on MNPs to Enhance the extraction of Ferulic Acid from Defatted Rice Bran in presence of Ultrasound Sagar M. Gadalkar, Virendra K. Rathod www.elsevier.com/locate/bab
PII: DOI: Reference:
S1878-8181(16)30414-5 http://dx.doi.org/10.1016/j.bcab.2017.03.016 BCAB534
To appear in: Biocatalysis and Agricultural Biotechnology Received date: 7 November 2016 Revised date: 22 February 2017 Accepted date: 25 March 2017 Cite this article as: Sagar M. Gadalkar and Virendra K. Rathod, Pre-Treatment of Ferulic Acid Esterases Immobilized on MNPs to Enhance the extraction of Ferulic Acid from Defatted Rice Bran in presence of Ultrasound, Biocatalysis and Agricultural Biotechnology, http://dx.doi.org/10.1016/j.bcab.2017.03.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pre-Treatment of Ferulic Acid Esterases Immobilized on MNPs to Enhance the extraction of Ferulic Acid from Defatted Rice Bran in presence of Ultrasound
Sagar M. Gadalkar, Virendra K. Rathod*
Department of Chemical Engineering, Institute of Chemical Technology, Matunga, Mumbai-400019, India.
*Corresponding Authors: Email ID: [email protected], Phone: 91-22-33612020, Fax: 91-22-24145614
Abstract Ferulic acid (FA) extraction is a tough task due to its linkage with the hemicellulose, which is difficult to break. An ultrasound assisted enzymatic pre-treatment method was established to improve the yield of FA. Defatted rice bran (DFRB) was pre-treated using ferulic acid esterase (FAE) immobilized on magnetic Nanoparticles (MNPs) in presence of ultrasound. Single factors for pre-treatment were studied to achieve maximum FA yield. Further the central composite design (CCD) was determined and under predicted optimum condition maximum yield was achieved up to 3.69 mg/g of DFRB. Optimum parameters were DFRB to solvent ratio 1:32 (w/v), temperature 42.2°C, enzyme loading 2.82% and sonication power 84.85 W. The FA yield by conventional enzymatic method was 3.14 mg/g after 4h whereas the yield was enhanced to 3.69 mg/g with pre-treatment time reduced to 1h by ultrasound
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assisted enzymatic treatment. Total phenolic content was 3.86 mg GAE/g and DPPH scavenging activity was found to be 98.5%. Use of MNPs for the immobilization increased the reusability of enzyme with retained activity up to 82% after 5th cycle. GA
Keywords: Ferulic acid esterase, ultrasound, ferulic acid, pre-treatment, immobilization, Magnetic nanoparticles.
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1. Introduction The reutilization of by-products and minimization of waste obtained from agricultural and food processing, should be focused either due to disposal issue or to utilize it for valuable products. Recently consumer favours the natural extract over a synthetic compounds for food ingredients, pharmaceutical and in personal care formulations, due to latent adverse consequences of synthetic compound on health, which may include carcinogenicity (de Paiva et al., 2013). The variety of value added compounds in the waste and by-products such as proteins, polyphenols and sugars from biological sources were studied (Gadalkar et al., 2016; Gasparotto et al., 2015; Max et al., 2009). Amongst them, several reports are available on utilization of waste for the extraction of phytochemicals. Phytochemical exhibits the wide range of therapeutic activities such as, anticancer, antidiabetic, photo protective antimicrobial, radical scavenging activity and anti-inflammatory (de Paiva et al., 2013). There are several reports available on phenolic compound extraction, such as p-coumaric acid and FA solubilisation by alkaline hydrolysis (Max et al., 2009; Mussatto et al., 2007), FA extraction from corn bran (Zhao et al., 2014), ultrasound assisted extraction of capsaicin from pepper (Barbero et al., 2008). Among all the phenols, FA is one of the well-known for its good antioxidant property (Graf, 1992), FA has also been reported to have many physiological functions, including antimicrobial, anti-inflammatory, anti-thrombosis, and anti-cancer activities (Ou and Kwok, 2004). FA is the most abundant hydroxyl-cinnamic acid found in plant cell walls. Extraction of FA, from numerous agricultural sources (Table 1) was carried out, by using various methods such as alkaline hydrolysis (Buranov and Mazza, 2009; Max et al., 2009), ultrasound assisted (Sun and Wang, 2008), enzyme assisted (Bonnin et al., 2002; Laszlo et al., 2006), enzymatic ultrasound assisted (Monks et al., 2013), Ionic liquids (Yan-Ying et al., 2007), microwave assisted (Liu et al., 2006), enzyme assisted supercritical fluid extraction
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(Mushtaq et al., 2015). Even though, several studies have been reported earlier for extraction, they suffer from various drawbacks. Traditionally the extraction of FA was carried out by alkaline extraction method which not only generates waste water but also produce impure FA. Novel techniques such as, high power ultrasound assisted extraction (UAE) is less, due to degradation of FA and extraction of unwanted compounds in the extract, while use of free enzymes restrict the reusability as well as direct contact of organic solvents to the enzyme denaturants the enzyme. Therefore, the application MNP immobilized enzyme can offer the selectivity and reusability of enzyme and use of ultrasound can enhance the rate of extraction by increasing porosity of rice bran. Present study offers an alternative way of exploiting rice bran for the extraction of FA. Ferulic acid is covalently linked by ester linkages to polysaccharides and ester bonds to lignin in plant cell walls (Ishii, 1994) as shown in Fig. A.1. Ferulic acid esterase (FAE) can selectively hydrolyses the ester linkage of FA with xylan fragment. FAE in free form was restricted the reusability due to recovery issue of enzyme. Magnetic Nanoparticles (MNPs) also offers the larger surface area to attach the enzyme and due to its nano size, it can approach target site with less hindrance. MNPs permits the recovery of enzyme by magnetic separation, which makes the process cost effective. Ultrasound offers fast and efficient method for pre-treatment as compared to conventional method and it represents a highly efficient and modest method with reduced energy, time and solvent consumption. Ultrasound pretend important role in mutilation of rice bran surface, attributable to exposes larger surface area of rice bran to enzyme. Therefore, the hydrolysis of the ester linkage is done more effectively than enzymatic batch process. Mechanical waves formed by ultrasound also assist to break the product substrate complex, which further increases the availability of enzyme (Waghmare and Rathod, 2016). Optimization of operating parameters of enzymatic assisted ultrasound treatment were carried out by full factorial Central Composite design (CCD).
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Enzyme combined with ultrasound treatment can be effectively utilized for the FA production from other biomasses, such as raw rice bran, wheat bran, and corncob. 2. Materials and methods 2.1 Materials Rice bran was obtained from Nashik, India. Standard FA was procured from SRL chemicals Mumbai, India. FAE was obtained as a gift sample from Novozymes, India. All the chemical was purchased from Loba chemie, SDFCL and Himedia India. 2.2. Experimental method 2.2.1. Preparation of Magnetic Nano particle (MNPs), functionalization and Enzyme immobilization Preparation of MNPs was carried out according to earlier reported method (Nadar and Rathod, 2015) and the surface of these particles was coated with 3-aminopropyl trimethoxysilane (APTES) by a salinization reaction in order to obtain amino functionalized magnetic particles. The amino functionalized MNPs (5 mg) was mixed with 1 mL of free FAE (5 mg protein content) in sodium acetate buffer (0.1 M, pH 5.5) and kept in shaker at 150 rpm for 30 min at room temperature (32°C ±2). Then the functional cross linker glutaraldehyde was added to above mixture and kept for shaking at 150 rpm for 4 h at R.T. (32°C ±2). After cross linking, immobilized magnetic FAE were separated using magnet and washed three times with sodium acetate buffer and kept for drying overnight at room temperature. The enzyme activity for free (activity 20 U) and immobilized (activity 16 U) FAE was measured (He et al., 2015a). 2.2.2. Optimization of pre-treatment parameter and extraction of FA Defatting of rice bran was carried out for 3 h in a stirred vessel using rice bran to hexane ratio 1:3 and temperature 50°C. Resultant mixture was centrifuged with 10000×g and hexane layer was removed. DFRB cake was recovered and dried in oven at 60°C for 12 h. 5
Pre-treatment of DFRB was performed in flat bottom glass reactor of 100 mL capacity with diameter of 4.5 cm and height 10 cm along with six pitched bladed glass turbines used for agitation. Typically, 1 g of DFRB was mixed with DI water (21 mL) in glass reactor and stirred for 60 min with MNP immobilized enzyme FAE (2 %) at 30°C in a sonication bath with power of 60 W at frequency 22 kHz (dual frequency ultrasound cleaning bath (Model 6.5l200 H, Dakshin, India) of internal dimensions 230 mm × 1500 mm × 150 mm approximately. At lower frequency (22 kHz) scattering is minimum and propagation of sound wave is proper than the higher frequency (40 kHz), which allows easy cavitation at lower frequency, resulting in increasing yield (Kulkarni and Rathod, 2014). Therefore lower frequency (22 kHz) was used for the pre-treatment. Four transducers fixed at the bottom of ultrasound bath arranged in zig zag position. Cylindrical flat bottom glass reactor with a mentioned dimensions kept at height of 0.025 m from bottom of ultrasound bath. This rector position in ultrasound bath was optimized by Kulkarni et al. for mapping of an ultrasonic bath (Kulkarni and Rathod, 2014). After pre-treatment, the added enzyme was separated out with the help of strong magnet and the remaining mixture containing DFRB and water was further used for extraction of FA. The extraction of FA was performed in a stirred vessel with optimized conditions as, temperature (30±2°C), 30% ethanol and extraction time of 60 min. The mixture was centrifuged at 8000×g for 10 min (Remi- revolutionary high speed centrifuge, India). Concentration of FA was calculated by measuring its absorbance at 321 nm (Chemito spectroscan UV 2700 double beam visible spectrophotometer, India), against a standard curve of FA. Effect of different pre-treatment parameters i.e., pre-treatment time (15 to 180 min), enzyme loading (1 to 5%), DFRB to solvent (1:7, to 1:56), extraction temperature (30°C to 60°C), sonication power (60 W to120 W) and frequency (22 kHz) on the extraction of the FA
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from DFRB has been studied and optimized. Fig. 1 showed the process flow of pre-treatment and extraction steps.
2.2.3. Content of ferulic acid in DFRB To know the approximate content of ferulic acid in DFRB, alkaline extraction was carried out according to method reported by Tilay et al. 2008 with some modification. DFRB 2g was fed into flat bottom glass reactor (100 mL) and 2M NaOH was added in 1:30 ratio (w/v) to saponified the DFRB. Resulting mixture was stirred for 24 h at 40°C at 400 rpm. Approximately, 0.001 g of sodium hydrogen sulfite was added to the mixture to avoid the oxidation of FA. The mixture was centrifuged and the supernatant was analysed by UV spectrophotometer against standard curve. Approximate content of ferulic acid in DFRB determined was 4.82±0.2mg/g. 2.2.4. Experimental design The effect of four independent variables i.e. enzyme loading (A, %), solvent ratio /g DFRB (B, mL) (w/v), extraction temperature (C, °C) and sonication power (D, W) on response variable yield (Y) of FA (mg/g of DFRB) was investigated using central composite design (Table 2). Table 3 showed that each independent variable at three levels -1, 0 and +1 and rotatory α values were taken to design the experiment. The deviation in FA yield (mg/g of DFRB) according to the four retained variables A, B, C and D was evaluated using a polynomial second degree model specified in subsequent equation: ∑
∑
∑
(2)
Where,
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Yk is the response function (Ferulic acid yield, Y mg/g, β0 is a constant, βi, βii and βij are linear quadratic and interactive coefficient of the model correspondingly. Levels of independent variables were denoted as Xi and Xj.
2.3. Analytical method 2.3.1. Characterization of FA extract Total phenolic content was determined by the Folin–Ciocalteu colorimetric method (Khoddami et al., 2013). The absorbance was observed using a UV/Vis spectrophotometer at 760 nm. Gallic acid solution was used as a standard and total phenolic contents was expressed as milligrams of gallic acid equivalent (mg GAE) per g of dry weight. Radical scavenging activity (%) was measured according to DPPH assay (Wu et al., 2014). Spectrophotometric measurements was done at 517 nm using Jasco double beam spectrophotometer. Activity was calculated using formula,
2.3.2. Morphological study of RB Samples were prepared to compare the effect of extraction process. The first sample witnessed for crude RB without treatment, second is residual RB of enzyme and ultrasound treatment with the optimized conditions. These samples were directly coated with platinum before being observed by scanning electron microscopy, scans at 5 kV at a magnification of ×1000 (Jeol, Scanning Electron Microscope, USA). 3. Results and discussion 3.1. Optimization of pre-treatment by single parameter 3.1.1. Effect of pre-treatment time on FA yield
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Pre-treatment time was an important parameter that would influence the extraction efficiency as well as the activity of enzyme. Due to combined effect of ultrasound and enzyme, the process time was reduced from 4h to 1h. Pre-treatment studies were carried out for various time intervals, ranging from 15 to 180 min using ultrasound and enzyme followed by the extraction. Fig. 2 showed that as the pre-treatment time increases the extraction also increases till 60 min. However, no significant change has been obtained for the higher extraction time and maximum extraction of 2.9 mg/g of DFRB was obtained at 60 min pretreatment time. Initially, at lower pre-treatment time, FA yield was lower and it was attributed to minimum time requirement of an ultrasound to make the material surface porous and further diffusion of enzyme through the material to reach the target site. Cavitation produces physical effects such as liquid circulation currents and turbulence that have an influence on the solid RB surface, which accelerate the process by increasing penetrability of enzyme through RB surface to hydrolyse the ester linkage. Further increase in pre-treatment time, increase in yield was gradual up to 180 min (3.1 mg/g of DFRB). Thus, 60 min pretreatment time was considered to be optimum and used for further experiments. Thus, 60 min pre-treatment time was considered to be optimum and used for further experiments. 3.1.2. Effect of DFRB to Solvent Ratio on FA yield The efficiency of extraction was enhanced by various parameters, among these, solute (DFRB) to solvent ratio during pre-treatment is one of the crucial parameters. Various DFRB to solvent ratios (w/v) (1:7 to 1:56) were studied to maximise the extraction yield of FA at temperature 30°C, sonication power 60 W, enzyme loading 2% and extraction time 1 h. In presented study (Fig. 3a) it was observed that higher ratio results in higher extraction yield, which was directly related to effect of pre-treatment, in higher amount of solvent influence of sonication and enzyme was maximum due to low viscosity and higher enzyme mobility. This can be attributed to the fact that the cellulose-hemicellulose is extremely recalcitrant and do
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not efficiently approached by FAE to hydrolyse ester bond between FA and arabinose sugar of xylan fragment, and hence higher the solvent, more dilute is the reaction mixture therefore solute in per unit area of solvent was decreased, resulting in less crowding. Because of less crowding of this lignocellulosic biomass, enzyme can approach to the maximum target sites of solute. Another factor that governs the mobility of enzyme is viscosity of solution and higher viscosity reduces the mobility of the molecules. Therefore, higher amount of solvent may offered greater mobility to the enzyme. Additionally, sonication increases the porosity of solute surface by the liquid jet form by ultrasound wave, which allows the enzyme to approach the site of action (J. Mason et al., 2011). The similar trend was observed in earlier report that, the higher amount of solvent leads to the higher yield of polyphenols (Ćujić et al., 2016). Solute to solvent ratio 1:21, 1:28 and 1:35 showed extraction yield as 2.84±0.1, 3.01±0.12 and 3.23±0.09 mg/g respectively. Results specified that, lower solvent volume showed lower FA yield, it may be due to lower amount of solvent in solute enhance the viscosity of the solution and high viscosity may decreased the cavitation effect. Therefore, the highest extraction yield was observed in ratio 1:42 (3.41±0.13 mg/g). Above solute to solvent ratio (1:42) resulted into decrease in FA yield. This may because the Solute to solvent ratio increases beyond 1:28 the reduction in yield was found, because of the ultrasound energy dispersion less in solvent and the growth of dissolved impurities such as protein, polysaccharide etc. which the dissolution of FA (Al-dhabi et al., 2017). Economically, lower ratio will be favourable for the extraction. Therefore, 1:28 ratio has been selected for further optimization study. 3.1.3. Effect of enzyme loading on FA yield FAE selectively targets the ester linkage of FA and arabinose sugar or xylan residue, hence selective extraction of FA takes place. Cost of enzyme governs the economy of the 10
process hence, amount of enzyme used in the process is significantly important and immobilization of enzyme on MNPs was carried out to recycle the enzyme. Enzyme immobilized MNPs readily approaches the target site due to its smaller size, which is quite difficult for enzyme immobilized on beads. To get the optimal enzyme loading, experiments were carried out with 1% to 5% enzyme loading at temperature 30°C, DFRB to solvent ratio 1:28, sonication power 60 W and pre-treatment time 1h. From Fig. 3b it is observed that the yield was increased with an increase in enzyme loading and amongst these 2% and 3% show 3.06±0.05mg/g and 3.22±0.01 mg/g yield respectively. Further increase in enzyme loading resulted in marginal increase in FA yield, which means 2% is the minimal enzyme required for the better activity. Requirement of enzyme was less in ultrasound process than conventional enzymatic process, because ultrasound helps in breaking enzyme-product complex. This attributed to increased availability of enzyme in the mixture, hence more activity. To ensure requirement of enzyme, further studies were performed with 3% enzyme loading. 3.1.4. Effect of temperature on FA yield Temperature has an optimistic effect on the extraction of phenolic compound from natural sources. Temperature may improve the yield of extraction by different properties such as: viscosity, diffusivity, solubility and surface tension (Boonkird et al., 2008). In presented work, three factors were depend on the temperature such as activity of enzyme, solubility of FA and power dissipation of ultrasound. Enzyme activity is higher at certain temperature because, increasing the temperature of a system decreases the viscosity of the reaction mass, which results in higher mobility of enzyme, further it might help to form more enzymesubstrate complex due to decrease in mass transfer resistance (Waghmare and Rathod, 2016). FA yield was higher at 45°C and further increase in temperature gradual decrease in yield was observed. Therefore, reduction in FA yield was because of lowering in cavitation effect
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at higher temperature. Enzyme stability did not affect by higher temperature as it is reported that the temperature stability of FAE increased up to 60°C upon immobilization on MNPs (He et al., 2015b). On the other hand, solubility of FA in water was increased with rise in temperature, but at the same time, ultrasound restricts the use of higher temperature, since it lowers the surface tension of solvent, which reduces the effect of sonication (Waghmare et al., 2015). Highest enzyme activity was observed (Fig. 3c) at temperature 45°C, DFRB to solvent ratio 1:28, sonication power 60 W, enzyme loading 3% and pre-treatment time 1h. FA yield determined at aforementioned temperature 3.46±0.12 mg/g of DFRB. Therefore, 45°C set as an optimized temperature for the FA extraction. 3.1.5. Effect of sonication power on FA yield Sonication power varied from 30 to 120 W for the pre-treatment to attain optimal power. It has been observed Fig. 3d that power of 75W showed 3.60±0.05 mg/g yield of FA and it also provided lower degradation and leaching of enzyme as compared with higher sonication power at temperature 45°C, DFRB to solvent ratio 1:28, enzyme loading 3% and pre-treatment time 1h. Ultrasound acting an important role to incite a formation of cavitation bubble subjected to fast adiabatic compression and expansion, which rises the local temperature and pressure. This results in an increase the porosity of the surface of DFRB, due to which FAE MNPs can easily get in and approach the hemicellulose fragment. Fig. 7 showed the SEM images of morphology of cell of DFRB before and after extraction. It can clearly be seen that ultrasound assistance leads to damage of rice bran surface, which exposes larger surface area of rice bran to enzyme. Enzyme gets into the cell wall and hydrolyses the ester linkage more effectively than the normal batch process. Mechanical waves formed by ultrasound also assist to break the product substrate complex, which further increases the availability of enzyme, hence the yield. Further increase in sonication power may cause 12
polysaccharide depolymerisation and aggregation, which will result in a decrease in the yield (Chen et al., 2012), also it withdraws the cell debris such as oil, protein and enzyme in the solution. It may affect the enzyme activity by competing or blocking active site of enzyme. At higher sonication power range, acoustic cavitation produced hydroxyl radicals, leading to chemical decomposition of FA, because it is very susceptible to the radical moiety, which is further accountable for lowering in the yield of FA. Therefore, use of lower power (75 W) was chosen over high power. The total ultrasound power dissipation was determined by calorimetric analysis (Raskar et al., 2014), by varying sonication power at constant frequency (22 kHz) and it was observed that at sonication power 50, 75, 100 and 120 W dissipated power was 41.4, 43.2, 46.4 and 57.1 W respectively. Intensity of ultrasound was examines by considering power dissipated at 50, 75, 100 and 120 W and it was found to be 6592.35, 6878.97, 7389.53, and 9093.58 Wm-2 respectively. 3.3. Optimization of pre-treatment parameter by response surface methodology 3.3.1. Response surface methodology Experiments were carried out according to central composite full factorial model to study the combine effect of various parameters on FA yield. Multiple regression analysis was used to explore the statistical significance of variable and their interaction. By multiple regression analysis on the experimental data, following equation in coded value was obtained. FA Yield Y (mg/g DFRB) = 3.66 + 0.13A + 0.20B + 0.041C + 0.084D - 0.056AB -0.012AC-0.018AD–6.8×10-3BC0.021BD–3.1×10-3CD-0.052A2-0.085B2-0.050C2-0.038D2
(3)
Equation gives an empirical relationship of the tested variable and response. It showed the linear factors and interaction among the factor having effect on yield of FA. Ftest and P-test were calculated using ANOVA to check the statistical significance of the regression model shown in Table 4. The F-value of 366.63 with low probability P value 13
(<0.0001) indicates high significance of the model. The coefficient of determination (R2) was 0.9971 indicating significance of model. Values of "Prob > F" less than 0.05 indicate model terms are significant. The value of adjusted determination coefficient (adjusted R2= 0.99) established that the model was highly significant. Coefficient of variation (CV%=0.55) clearly showed high degree of precision and good pact of reliability of experimental value. In this case all the terms in the model are significant model terms. Values greater than 0.1 indicate the model terms are not significant. The "Lack of Fit F-value" of 3.30 implies the Lack of Fit is not significant relative to the pure error. There is a 9.98% chance that a "Lack of Fit F-value" this large could occur due to noise. Non-significant lack of fit is good, therefore the model is valid. Fig. A.2 showed the regular probability plot of residuals. These values are normally scattered among straight line, hence conforming the statistical analysis. Residual and the predicted values were correlated (Fig. A.3) for the FA extraction. It can be seen from Fig. A.3 the residuals are scattered randomly, it proposes that the variance of original observation is constant for all experimental runs. Fig. A.4 presented the actual value versus predicted value, which illustrates that, both values are in the range of operating parameters. 3.3.2. Response surface analysis Regression function was graphically represented by three dimensional (3D) response surface and two dimensional (2D) contour. Extraction yield of FA at different condition by keeping two variable constant were represented in the Fig. 4. 3D response surface Fig. 4(a) showed the effect of enzyme loading and solvent ratio on FA yield at fixed pre-treatment time. FA yield increased eventually with increase in enzyme loading and solvent ratio, this effect is very significant as shown in Table 4 ANOVA (p<0.05). Starting from 2% enzyme loading and solvent ratio 28 mL, yield improved tremendously up to 3.5% enzyme loading and 31.5 mL solvent ratio, further increase in
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variable FA yield increased gradually. At enzyme loading 2% and solvent ratio 21 mL, FA yield was lowest, it may be due to less mobility of enzyme and higher solute concentration. FA yield increased at due to effective mass transfer at higher ratio and enzyme can easily approach to the ester bond between hemicellulose and ferulic acid. Extraction of FA from corn bran with alkaline-ethanol aqueous solution has been reported with 8.47 mg/g yield by (Zhao et al., 2014). Fig. 4(b) shows that the 3D response surface and 2D contour plot by varying enzyme loading and temperature at fixed pre-treatment time. Plot showed that the yield was governed by both, enzyme loading and temperature. FA yield increases with an increase in enzyme loading and temperature. Enzyme showed minimal activity at lower temperature range 40°C to 45°C and enzyme activity is increased with an increase in temperature from 45°C to 50°C, but at higher temperature, intensity of cavitation reduces. On the other hand, higher temperature felicitates the solubility of FA in solvent, thus higher temperature also showed positive effect on FA yield (Galvan D’Alessandro et al., 2012). Thus overall effect of temperature showed slight negative effect on yield of FA, but higher temperature was increased the yield of FA to 3.7 mg/g of DFRB. 3D response surface Fig. 4(c) showed the interaction of enzyme loading and sonication power. FA yield was increased with increase in the power and enzyme loading. Higher (3-4%) enzyme loading offers more numbers of enzyme MNPs per unit area in mixture, thus observed better activity. At 3% enzyme loading, yield (3.62 mg/g) increased significantly up to 90 W. There is significant effect (p<0.05) of power this can verified from Table 4 ANOVA. The P-valve for power is < 0.01. 3D response surface Fig. 5(d) clearly indicates the solvent ratio and temperature implies the moderate effect on the yield of FA. It can be observed that response (FA yield) did not significantly increase with an increase in the temperature and solvent ratio. At lower temperature and solvent ratio, FA yield was 2.5 mg/g DFRB and it was increased (3.72 mg/g)
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with the solvent ratio (31.5-35mL). The combine effect of these variable was not significant, that can be seen in ANOVA table 4, with higher p-value (<0.1742). The effect of solvent ratio and sonication power on the yield of FA was showed in Fig. 5(e) 3D response surface, it can be clearly seen that, solvent ratio has a major contribution on extraction yield of FA. The viscosity of solution decreases with increase in solvent ratio, which enhances the cavitation effect. Therefore, in higher solvent ratio (30-35 mL) and sonication power, FA yield (3.5 mg/g) was greater. Fig. 5(e) also displayed that the power around 75 to 80 W showed higher amount of FA extraction. 3D response surface Fig. 5(f) indicates, temperature and power implies negative effect on the yield of FA. This effect is very significant with a p<0.005 shown in Table 4. This might be due to lowering in sonication effect, as a temperature increases. At higher temperature, surface tension decreases, which affects the bubble formation and collapse (Santos et al., 2009). It is attributed to the cavitation phenomena, at higher temperature surface tension of solvent decreases, hence cavitation bubble explodes with lower intensity. Though the combine effect showed negative impact but the overall yield (3.62 mg/g) was slightly increased as the solubility of FA rises with temperature in DI water. 3.4. Optimization of process Predicted model was validated by performing an experiment under predicted conditions as DFRB to solvent ratio 1:32 (w/v), temperature 42.2°C, enzyme loading 2.82% and sonication power 84.85 W. The FA yield obtained and the predicted value was 3.69 mg/g and 3.71 mg/g respectively. The experimental values show a close resemblance to predicted values hence the model is considered to be significant. 3.5. Comparison of conventional enzymatic pre-treatment and ultrasound assisted enzymatic pre-treatment
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Pre-treatment study was performed with conventional enzymatic process and optimized ultrasound assisted enzymatic treatment. Operating parameters of conventional process were same, except use of ultrasound treatment. Fig. 6 showed that the conventional enzymatic pretreatment showed around 3.16 mg/g of DFRB after 6h. Ultrasound accelerates the process and yield was increased up to 3.63 mg/g of DFRB in 1h. Therefore it can attributed that, ultrasound increases the porosity of material and assists in breaking of enzyme-product complex. Mechanical shock waves as well as liquid jets were good enough to renewal the enzyme surface and also increase the diffusion of enzyme to the target sites of substrates producing more interaction and hydrolysed the ester bond (Waghmare and Rathod, 2016). 3.6. Morphological study of conventional enzymatic treated and ultrasound assisted enzymatic treated RB Fig. 7 showed the morphological images of RB cell surface at a magnification of ×1000, it can be clearly seen from Fig. 7a that the cell of defatted RB is arranged hexagonally and intact to each other. Therefore it was difficult to approach by enzyme in intact cells due to limited porosity. The tremendous effect of the cavitation on cell structure observed in residual RB recovered after the UAE method. Application of ultrasound increase the porosity of RB cells (Fig.7b) due to which FAE MNPs can easily approach the target site. 3.7. Characterization of extract Phenolic content observed was 1.97±1.44 GAE/g.
Presented studies showed
improved phenolic content around 3.86±0.10 GAE/g, it may be due to combine effect of ultrasound and enzyme. DPPH antioxidant assay is based on free radical scavenging activity of phenols that produces a violet solution in ethanol (Foti et al., 2004). The % antioxidant activity profile was detected around 98.5±0.5% over conventional process (82.6±0.5%), which was in agreement with total phenolic content. HPLC Chromatogram of standard and crude extract of ferulic acid is presented in fig. 8. 17
3.8. Enzyme reusability study Enzyme reusability is the main concern in the process, because it directly affects the economy of the process. Thus, reusability study was critically essential for the economic feasibility of reaction. Study was carried out with the optimized conditions obtained from CCD method. Enzyme was recovered from the process with help of magnet. Recovered enzyme nano particle was than washed twice with water and centrifuged under 10000×g for 10 min and dry at room temperature. The recovered enzyme was used for further batch of extraction and yield was calculated, same procedure was repetitively used for further three successive cycles and extraction yield was calculated. First cycle yield was considered as a 100% activity of enzyme and accordingly the calculation of yields for next cycles was carried out. The yields obtained after second, third, fourth and fifth cycle were 95, 92, 87 and 82% respectively (Fig. 9). After 5th cycle enzyme loses almost 20% of its activity, which was not feasible for the process. Enzyme activity in this study is retained tremendously, that may be due to lower time of sonication treatment and absence of ethanol during pre-treatment study. 4. Conclusion The pre-treatment by means of combined effect of ultrasound and enzyme readily extract the FA from DFRB. Implementation of CCD for pre-treatment, lower down the enzyme loading and FA yield was enhanced with a reduced time than conventional method. The optimized operating parameters unveiled the highest yield of FA 3.69 mg/g of DFRB. Established process is rapid and efficient method for extraction of FA. Optimized parameters of ultrasound assisted enzymatic extraction displayed around 77% yield after 1h as compared to around 65% after 4h in the case of conventional enzymatic process. Pre-treatment showed 1.2 fold increase in the yield of FA with lower extraction time. Recovery of enzyme by immobilized on MNPs can be the cost effective process, therefore enzyme can be used effectively up to five cycles. The outcome of presented work would be beneficial for the
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economical purpose. This process can be applied for the other natural compound extraction from verity of raw material.
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Reference Al-dhabi, N.A., Ponmurugan, K., Maran, P., 2017. Ultrasonics Sonochemistry Development and validation of ultrasound-assisted solid-liquid extraction of phenolic compounds from waste spent coffee grounds. Ultrason. - Sonochemistry 34, 206–213. Barbero, G.F., Liazid, a, Palma, M., Barroso, C.G., 2008. Ultrasound-assisted extraction of capsaicinoids from peppers. Talanta 75, 1332–7. Bonnin, E., Saulnier, L., Brunel, M., Marot, C., Lesage-Meessen, L., Asther, M., Thibault, J.F., 2002. Release of ferulic acid from agroindustrial by-products by the cell walldegrading enzymes produced by Aspergillus niger I-1472. Enzyme Microb. Technol. 31, 1000–1005. Boonkird, S., Phisalaphong, C., Phisalaphong, M., 2008. Ultrasound-assisted extraction of capsaicinoids from Capsicum frutescens on a lab- and pilot-plant scale. Ultrason. Sonochem. 15, 1075–1079. Buranov, A.U., Mazza, G., 2009. Extraction and purification of ferulic acid from flax shives, wheat and corn bran by alkaline hydrolysis and pressurised solvents. Food Chem. 115, 1542–1548. Chen, R., Li, S., Liu, C., Yang, S., Li, X., 2012. Ultrasound complex enzymes assisted extraction and biochemical activities of polysaccharides from Epimedium leaves. Process Biochem. 47, 2040–2050. Ćujić, N., Šavikin, K., Janković, T., Pljevljakušić, D., Zdunić, G., Ibrić, S., 2016. Optimization of polyphenols extraction from dried chokeberry using maceration as traditional technique. Food Chem. 194, 135–42. de Paiva, L.B., Goldbeck, R., dos Santos, W.D., Squina, F.M., 2013. Ferulic acid and derivatives: Molecules with potential application in the pharmaceutical field. Brazilian J. 20
Pharm. Sci. 49, 395–411. Foti, M.C., Daquino, C., Geraci, C., 2004. Electron-Transfer Reaction of Cinnamic Acids and Their Methyl Esters with the DPPH. Radical in Alcoholic Solutions. J. Org. Chem. 69, 2309–2314. Gadalkar, S.M., Gogate, P.R., Rathod, V.K., 2016. Recovery of Proteins from Rice Mill Industry Waste (Rice Bran) Using Alkaline or NaCl-Assisted Alkaline Extraction Processes. J. Food Process Eng. doi:10.1111/jfpe.12430 Galvan D’Alessandro, L., Kriaa, K., Nikov, I., Dimitrov, K., 2012. Ultrasound assisted extraction of polyphenols from black chokeberry. Sep. Purif. Technol. 93, 42–47. Gasparotto, J.M., Werle, L.B., Mainardi, M.A., Foletto, E.L., Kuhn, R.C., Jahn, S.L., Mazutti, M.A., 2015. Ultrasound-assisted hydrolysis of sugarcane bagasse using cellulolytic enzymes by direct and indirect sonication. Biocatal. Agric. Biotechnol. 4, 480–485. Graf, E., 1992. Antioxidant potential of ferulic acid. Free Radic. Biol. Med. 13, 435–448. He, F., Zhang, S., Liu, X., 2015a. Immobilization of feruloyl esterases on magnetic nanoparticles and its potential in production of ferulic acid. J. Biosci. Bioeng. 120, 330– 334. He, F., Zhang, S., Liu, X., 2015b. Immobilization of feruloyl esterases on magnetic nanoparticles and its potential in production of ferulic acid. J. Biosci. Bioeng. 120, 330– 334. Ishii, T., 1994. Feruloyl oligosaccharides from cell walls of suspension-cultured spinach cells and sugar beet pulp. Plant Cell Physiol. 35, 701–4. J. Mason, T., Chemat, F., Vinatoru, M., 2011. The Extraction of Natural Products using Ultrasound or Microwaves. Curr. Org. Chem. 15, 237–247. 21
Khoddami, A., Wilkes, M.A., Roberts, T.H., 2013. Techniques for analysis of plant phenolic compounds. Molecules 18, 2328–2375. Kulkarni, V.M., Rathod, V.K., 2014. Mapping of an ultrasonic bath for ultrasound assisted extraction of mangiferin from Mangifera indica leaves. Ultrason. Sonochem. 21, 606– 611. Laszlo, J. a., Compton, D.L., Li, X.L., 2006. Feruloyl esterase hydrolysis and recovery of ferulic acid from jojoba meal. Ind. Crops Prod. 23, 46–53. Liu, Z., Wang, J., Shen, P., Wang, C., Shen, Y., 2006. Microwave-assisted extraction and high-speed counter-current chromatography purification of ferulic acid from Radix Angelicae sinensis. Sep. Purif. Technol. 52, 18–21. Max, B., Torrado, A.M., Moldes, A.B., Converti, A., Domínguez, J.M., 2009. Ferulic acid and p-coumaric acid solubilization by alkaline hydrolysis of the solid residue obtained after acid prehydrolysis of vine shoot prunings: Effect of the hydroxide and pH. Biochem. Eng. J. 43, 129–134. Monks, L.M., Rigo, A., Mazutti, M.A., Vladimir Oliveira, J., Valduga, E., 2013. Use of chemical, enzymatic and ultrasound-assisted methods for cell disruption to obtain carotenoids. Biocatal. Agric. Biotechnol. 2, 165–169. Mushtaq, M., Sultana, B., Anwar, F., Adnan, A., Rizvi, S.S.H., 2015. Enzyme-assisted supercritical fluid extraction of phenolic antioxidants from pomegranate peel. J. Supercrit. Fluids. 104, 122-131. Mussatto, S.I., Dragone, G., Roberto, I.C., 2007. Ferulic and p-coumaric acids extraction by alkaline hydrolysis of brewer’s spent grain. Ind. Crops Prod. 25, 231–237. Nadar, S.S., Rathod, V.K., 2016. Magnetic macromolecular cross linked enzyme aggregates (CLEAs) of glucoamylase. Enzyme Microb. Technol. 83, 78-87. 22
Ou, S., Kwok, K.C., 2004. Ferulic acid: Pharmaceutical functions, preparation and applications in foods. J. Sci. Food Agric. 84, 1261–1269. Raskar, H.D., Avhad, D.N., Rathod, V.K., 2014. Chemical Engineering and Processing : Process Intensification Ultrasound assisted production of daunorubicin : Process intensification approach. Chem. Eng. Process. Process Intensif. 77, 7–12. Salleh, N. H. M.; Daud, M. Z. M.; Arbain, D.; Ahmad, M. S.; Ismail, K. S. K., 2011. Optimization of alkaline hydrolysis of paddy straw for ferulic acid extraction. Ind. Crops Prod., 34 (3), 1635–1640. Santos, H.M., Lodeiro, C., Capelo-Martinez, J.-L., 2009. The Power of Ultrasound, in: Ultrasound in Chemistry: Analytical Applications. pp. 1–16. Sun, Y., Wang, W., 2008. Ultrasonic extraction of ferulic acid from Ligusticum chuanxiong. J. Chinese Inst. Chem. Eng. 39, 653–656. Tilay, A., Bule, M., Kishenkumar, J. & Annapure, U. 2008. Preparation of ferulic acid from agricultural wastes: Its improved extraction and purification. Journal of Agricultural and Food Chem., 56, 7644–7648. Waghmare, G. V., Rathod, V.K., 2016. Ultrasound assisted enzyme catalyzed hydrolysis of waste cooking oil under solvent free condition. Ultrason. Sonochem. 32, 60–67. Waghmare, G. V., Vetal, M.D., Rathod, V.K., 2015. Ultrasound assisted enzyme catalyzed synthesis of glycerol carbonate from glycerol and dimethyl carbonate. Ultrason. Sonochem. 22, 311–316. Wu, H., Zhu, J., Diao, W., Wang, C., 2014. Ultrasound-assisted enzymatic extraction and antioxidant activity of polysaccharides from pumpkin (Cucurbita moschata). Carbohydr. Polym. 113, 314–324. Yan-Ying, Y., Wei, Z., Shu-Wen, C., 2007. Extraction of Ferulic Acid and Caffeic Acid with
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Ionic Liquids. Chinese J. Anal. Chem. 35, 1726–1730. Zhao, S., Yao, S., Ou, S., Lin, J., Wang, Y., Peng, X., Li, A., Yu, B., 2014. Preparation of ferulic acid from corn bran: Its improved extraction and purification by membrane separation. Food Bioprod. Process. 92, 309–313.
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Table Caption Table 1. Extraction of ferulic acid by various methods from different raw material sources Table 2. Experimental values and coded levels of the independent variables used for the CCD Table 3. Experimental design for the optimization of extraction of FA from DFRB using Central Composite Rotatable Design of RSM Table 4. ANOVA for response surface quadratic model for Ferulic acid extraction
25
Table 1. Extraction of ferulic acid by various methods from different raw material sources
Source
Extraction method
FA yield (mg/g) Reference
Paddy straw
Alkaline
8.17
Salleh et al., 2011
Radix Angelicae sinensis
Microwave/ethanol
1.33
Liu et al., 2006
Flax Shives
Pressurised-Alkaline
0.18
Buranov et al., 2011
Corn bran
Pressurised-Alkaline
25.10
Buranov et al., 2011
Wheat bran
Alkaline
3.91
Buranov et al., 2011
Jojoba meal
Enzymatic
6.7
Laszlo et al., 2006
26
Table 2. Experimental values and coded levels of the independent variables used for the CCD Level Independent variable
Symbols
Unit
Low (−1)
Intermediate (0)
High (1)
Enzyme loading Solvent Ratio
A B
% mL/g
2 21
3 28
4 35
Temperature
C
°C
40
45
50
Sonication power
D
W
60
75
90
27
Table 3. Experimental design for the optimization of extraction of FA from DFRB using Central Composite Rotatable Design of RSM Run Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Enzyme Solvent Temperature loading Ratio (°C) (%) (mL) 2 4 2 4 2 4 2 4 2 4 2 4 2 4 2 4 1 5 3 3 3 3 3 3 3 3 3 3 3 3
21 21 35 35 21 21 35 35 21 21 35 35 21 21 35 35 28 28 14 42 28 28 28 28 28 28 28 28 28 28
40 40 40 40 50 50 50 50 40 40 40 40 50 50 50 50 45 45 45 45 35 55 45 45 45 45 45 45 45 45
Power (W)
FA Yield (mg/g)
60 60 60 60 60 60 60 60 90 90 90 90 90 90 90 90 75 75 75 75 75 75 45 105 75 75 75 75 75 75
2.89 3.29 3.42 3.66 3 3.35 3.53 3.68 3.1 3.46 3.61 3.7 3.22 3.55 3.69 3.76 3.17 3.73 2.92 3.71 3.37 3.54 3.32 3.69 3.64 3.66 3.65 3.63 3.65 3.64
28
Table 4. ANOVA for response surface quadratic model for Ferulic acid extraction Source
Sum of Squares
DF
Mean Square
F Value
p-value Prob > F
Model
1.908
14
0.136
366.63
< 0.0001
A
0.403
1
0.403
1084.32
< 0.0001
B
0.948
1
0.948
2550.77
< 0.0001
C
0.041
1
0.041
109.88
< 0.0001
D
0.168
1
0.168
452.93
< 0.0001
AB
0.050
1
0.050
133.20
< 0.0001
AC
0.002
1
0.002
6.07
0.0263
AD
0.005
1
0.005
14.14
0.0019
BC
0.001
1
0.001
2.03
0.1742
BD
0.007
1
0.007
18.31
0.0007
CD
0.000
1
0.000
0.42
0.5265
A2
0.073
1
0.073
196.21
< 0.0001
2
B
0.200
1
0.200
537.12
< 0.0001
C2
0.069
1
0.069
186.81
< 0.0001
D2
0.039
1
0.039
105.52
< 0.0001
Residual
0.006
15
0.000
Lack of
0.005
10
0.000
0.001
5
0.000
1.913
29
3.30
0.0998
Fit Pure Error Cor Total
29
Fig. Caption Fig. 1. Process flow diagram of pre-treatment and extraction of ferulic acid using sonication treatment and immobilized enzyme MNPs Fig. 2. Effect of pre-treatment time on FA extraction (mg/g of DFRB) in DFRB to solvent 1:14 at Temperature 30°C, sonication power 60 W, enzyme loading 2% Fig. 3. Effect of i) Solute to solvent, ii) Enzyme loading, iii) Temperature and iv) Sonication power on FA yield Fig. 4. Response surface plot showing the effect of (a) Enzyme loading and Solvent ratio, (b) Enzyme loading and Temperature and (c) Enzyme loading and Sonication power on extraction yield of Ferulic acid (mg/ g of RB) Fig. 5. Response surface plot showing the effect of (d) Temperature and solvent ratio, (e) Solvent ratio and Sonication power and (f) Sonication power and Temperature on extraction yield of Ferulic acid (mg/ g of RB) Fig. 6. Effect of conventional enzymatic pre-treatment and ultrasound assisted enzymatic pretreatment on yield of FA Fig. 7.SEM images at magnification ×1000 (a) Untreated DFRB (b) UAE and enzyme treated RB Fig. 8. HPLC chromatogram of standard and crude extract of ferulic acid Fig. 9. Reusability of Immobilized enzyme MNPs with number of cycle
30
Rice Bran Defatting Defatted Rice Bran
Pre-treatment
Parameter Optimization Enzyme Loading Sonication Temperature Ratio
Enzyme MNP
DI Water
Magnet Recycle Enzyme MNP
Stirring
Stirring
Extraction
EtOH
Centrifugation
Supernatant Extract
RB Residue
Analysis
FA Yield (mg/g)
DPPH Assay
Total phenol content
Fig. 1. Process flow diagram of pre-treatment and extraction of ferulic acid using sonication treatment and immobilized enzyme MNPs
31
4
FA mg/g DFRB
3.5 3 2.5 2 1.5 1 0.5 0 0
20
40
60
80 100 120 140 160 180 200 Time(min)
Fig. 2. Effect of pre-treatment time on FA extraction (mg/g of DFRB) in DFRB to solvent 1:14 at Temperature 30°C, sonication power 60 W, enzyme loading 2%
32
a)
b)
c)
d)
Fig. 3. Effect of a) Solute to solvent, b) Enzyme loading, c) Temperature and d) Sonication power on FA yield
33
Fig. 4. Response surface plot showing the effect of (a) Enzyme loading and Solvent ratio, (b) Enzyme loading and Temperature and (c) Enzyme loading and Sonication power on extraction yield of Ferulic acid (mg/ g of RB)
34
Fig. 5. Response surface plot showing the effect of (d) Temperature and solvent ratio, (e) Solvent ratio and Sonication power and (f) Sonication power and Temperature on extraction yield of Ferulic acid (mg/ g of RB)
35
4.5 4
FA mg/g DFRB
3.5 3 2.5 2 1.5 Conventional Enzymatic
1
Ultrasound + Enzymatic
0.5 0 0
50
100
150
200 250 300 Time (min)
350
400
450
Fig. 6. Effect of conventional enzymatic pre-treatment and ultrasound assisted enzymatic pretreatment on yield of FA
36
a)
b)
Fig. 7. SEM images at magnification ×1000 (a) Untreated DFRB (b) UAE and enzyme treated RB
37
Fig. 8. HPLC chromatogram of standard and crude extract of ferulic acid
38
100
90
FA Yield (%)
80
70
60
50 1
2
3 Number of cycle
4
5
Fig. 9. Reusability of Immobilized enzyme MNPs with number of cycle
Research highlights
Combined effect of ultrasound and enzyme, enhanced the FA yield with minimum time
Enzyme was used in pre-treatment method, to avoid its denaturation in contact with ethanol
Use of ferulic acid esterase immobilized magnetic nanoparticles allows the reusability of enzyme
Obtained FA acid showed good radical scavenging activity and total phenolic content
39
PII: DOI: Reference:
S1878-8181(16)30414-5 http://dx.doi.org/10.1016/j.bcab.2017.03.016 BCAB534
To appear in: Biocatalysis and Agricultural Biotechnology Received date: 7 November 2016 Revised date: 22 February 2017 Accepted date: 25 March 2017 Cite this article as: Sagar M. Gadalkar and Virendra K. Rathod, Pre-Treatment of Ferulic Acid Esterases Immobilized on MNPs to Enhance the extraction of Ferulic Acid from Defatted Rice Bran in presence of Ultrasound, Biocatalysis and Agricultural Biotechnology, http://dx.doi.org/10.1016/j.bcab.2017.03.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Pre-Treatment of Ferulic Acid Esterases Immobilized on MNPs to Enhance the extraction of Ferulic Acid from Defatted Rice Bran in presence of Ultrasound
Sagar M. Gadalkar, Virendra K. Rathod*
Department of Chemical Engineering, Institute of Chemical Technology, Matunga, Mumbai-400019, India.
*Corresponding Authors: Email ID: [email protected], Phone: 91-22-33612020, Fax: 91-22-24145614
Abstract Ferulic acid (FA) extraction is a tough task due to its linkage with the hemicellulose, which is difficult to break. An ultrasound assisted enzymatic pre-treatment method was established to improve the yield of FA. Defatted rice bran (DFRB) was pre-treated using ferulic acid esterase (FAE) immobilized on magnetic Nanoparticles (MNPs) in presence of ultrasound. Single factors for pre-treatment were studied to achieve maximum FA yield. Further the central composite design (CCD) was determined and under predicted optimum condition maximum yield was achieved up to 3.69 mg/g of DFRB. Optimum parameters were DFRB to solvent ratio 1:32 (w/v), temperature 42.2°C, enzyme loading 2.82% and sonication power 84.85 W. The FA yield by conventional enzymatic method was 3.14 mg/g after 4h whereas the yield was enhanced to 3.69 mg/g with pre-treatment time reduced to 1h by ultrasound
1
assisted enzymatic treatment. Total phenolic content was 3.86 mg GAE/g and DPPH scavenging activity was found to be 98.5%. Use of MNPs for the immobilization increased the reusability of enzyme with retained activity up to 82% after 5th cycle. GA
Keywords: Ferulic acid esterase, ultrasound, ferulic acid, pre-treatment, immobilization, Magnetic nanoparticles.
2
1. Introduction The reutilization of by-products and minimization of waste obtained from agricultural and food processing, should be focused either due to disposal issue or to utilize it for valuable products. Recently consumer favours the natural extract over a synthetic compounds for food ingredients, pharmaceutical and in personal care formulations, due to latent adverse consequences of synthetic compound on health, which may include carcinogenicity (de Paiva et al., 2013). The variety of value added compounds in the waste and by-products such as proteins, polyphenols and sugars from biological sources were studied (Gadalkar et al., 2016; Gasparotto et al., 2015; Max et al., 2009). Amongst them, several reports are available on utilization of waste for the extraction of phytochemicals. Phytochemical exhibits the wide range of therapeutic activities such as, anticancer, antidiabetic, photo protective antimicrobial, radical scavenging activity and anti-inflammatory (de Paiva et al., 2013). There are several reports available on phenolic compound extraction, such as p-coumaric acid and FA solubilisation by alkaline hydrolysis (Max et al., 2009; Mussatto et al., 2007), FA extraction from corn bran (Zhao et al., 2014), ultrasound assisted extraction of capsaicin from pepper (Barbero et al., 2008). Among all the phenols, FA is one of the well-known for its good antioxidant property (Graf, 1992), FA has also been reported to have many physiological functions, including antimicrobial, anti-inflammatory, anti-thrombosis, and anti-cancer activities (Ou and Kwok, 2004). FA is the most abundant hydroxyl-cinnamic acid found in plant cell walls. Extraction of FA, from numerous agricultural sources (Table 1) was carried out, by using various methods such as alkaline hydrolysis (Buranov and Mazza, 2009; Max et al., 2009), ultrasound assisted (Sun and Wang, 2008), enzyme assisted (Bonnin et al., 2002; Laszlo et al., 2006), enzymatic ultrasound assisted (Monks et al., 2013), Ionic liquids (Yan-Ying et al., 2007), microwave assisted (Liu et al., 2006), enzyme assisted supercritical fluid extraction
3
(Mushtaq et al., 2015). Even though, several studies have been reported earlier for extraction, they suffer from various drawbacks. Traditionally the extraction of FA was carried out by alkaline extraction method which not only generates waste water but also produce impure FA. Novel techniques such as, high power ultrasound assisted extraction (UAE) is less, due to degradation of FA and extraction of unwanted compounds in the extract, while use of free enzymes restrict the reusability as well as direct contact of organic solvents to the enzyme denaturants the enzyme. Therefore, the application MNP immobilized enzyme can offer the selectivity and reusability of enzyme and use of ultrasound can enhance the rate of extraction by increasing porosity of rice bran. Present study offers an alternative way of exploiting rice bran for the extraction of FA. Ferulic acid is covalently linked by ester linkages to polysaccharides and ester bonds to lignin in plant cell walls (Ishii, 1994) as shown in Fig. A.1. Ferulic acid esterase (FAE) can selectively hydrolyses the ester linkage of FA with xylan fragment. FAE in free form was restricted the reusability due to recovery issue of enzyme. Magnetic Nanoparticles (MNPs) also offers the larger surface area to attach the enzyme and due to its nano size, it can approach target site with less hindrance. MNPs permits the recovery of enzyme by magnetic separation, which makes the process cost effective. Ultrasound offers fast and efficient method for pre-treatment as compared to conventional method and it represents a highly efficient and modest method with reduced energy, time and solvent consumption. Ultrasound pretend important role in mutilation of rice bran surface, attributable to exposes larger surface area of rice bran to enzyme. Therefore, the hydrolysis of the ester linkage is done more effectively than enzymatic batch process. Mechanical waves formed by ultrasound also assist to break the product substrate complex, which further increases the availability of enzyme (Waghmare and Rathod, 2016). Optimization of operating parameters of enzymatic assisted ultrasound treatment were carried out by full factorial Central Composite design (CCD).
4
Enzyme combined with ultrasound treatment can be effectively utilized for the FA production from other biomasses, such as raw rice bran, wheat bran, and corncob. 2. Materials and methods 2.1 Materials Rice bran was obtained from Nashik, India. Standard FA was procured from SRL chemicals Mumbai, India. FAE was obtained as a gift sample from Novozymes, India. All the chemical was purchased from Loba chemie, SDFCL and Himedia India. 2.2. Experimental method 2.2.1. Preparation of Magnetic Nano particle (MNPs), functionalization and Enzyme immobilization Preparation of MNPs was carried out according to earlier reported method (Nadar and Rathod, 2015) and the surface of these particles was coated with 3-aminopropyl trimethoxysilane (APTES) by a salinization reaction in order to obtain amino functionalized magnetic particles. The amino functionalized MNPs (5 mg) was mixed with 1 mL of free FAE (5 mg protein content) in sodium acetate buffer (0.1 M, pH 5.5) and kept in shaker at 150 rpm for 30 min at room temperature (32°C ±2). Then the functional cross linker glutaraldehyde was added to above mixture and kept for shaking at 150 rpm for 4 h at R.T. (32°C ±2). After cross linking, immobilized magnetic FAE were separated using magnet and washed three times with sodium acetate buffer and kept for drying overnight at room temperature. The enzyme activity for free (activity 20 U) and immobilized (activity 16 U) FAE was measured (He et al., 2015a). 2.2.2. Optimization of pre-treatment parameter and extraction of FA Defatting of rice bran was carried out for 3 h in a stirred vessel using rice bran to hexane ratio 1:3 and temperature 50°C. Resultant mixture was centrifuged with 10000×g and hexane layer was removed. DFRB cake was recovered and dried in oven at 60°C for 12 h. 5
Pre-treatment of DFRB was performed in flat bottom glass reactor of 100 mL capacity with diameter of 4.5 cm and height 10 cm along with six pitched bladed glass turbines used for agitation. Typically, 1 g of DFRB was mixed with DI water (21 mL) in glass reactor and stirred for 60 min with MNP immobilized enzyme FAE (2 %) at 30°C in a sonication bath with power of 60 W at frequency 22 kHz (dual frequency ultrasound cleaning bath (Model 6.5l200 H, Dakshin, India) of internal dimensions 230 mm × 1500 mm × 150 mm approximately. At lower frequency (22 kHz) scattering is minimum and propagation of sound wave is proper than the higher frequency (40 kHz), which allows easy cavitation at lower frequency, resulting in increasing yield (Kulkarni and Rathod, 2014). Therefore lower frequency (22 kHz) was used for the pre-treatment. Four transducers fixed at the bottom of ultrasound bath arranged in zig zag position. Cylindrical flat bottom glass reactor with a mentioned dimensions kept at height of 0.025 m from bottom of ultrasound bath. This rector position in ultrasound bath was optimized by Kulkarni et al. for mapping of an ultrasonic bath (Kulkarni and Rathod, 2014). After pre-treatment, the added enzyme was separated out with the help of strong magnet and the remaining mixture containing DFRB and water was further used for extraction of FA. The extraction of FA was performed in a stirred vessel with optimized conditions as, temperature (30±2°C), 30% ethanol and extraction time of 60 min. The mixture was centrifuged at 8000×g for 10 min (Remi- revolutionary high speed centrifuge, India). Concentration of FA was calculated by measuring its absorbance at 321 nm (Chemito spectroscan UV 2700 double beam visible spectrophotometer, India), against a standard curve of FA. Effect of different pre-treatment parameters i.e., pre-treatment time (15 to 180 min), enzyme loading (1 to 5%), DFRB to solvent (1:7, to 1:56), extraction temperature (30°C to 60°C), sonication power (60 W to120 W) and frequency (22 kHz) on the extraction of the FA
6
from DFRB has been studied and optimized. Fig. 1 showed the process flow of pre-treatment and extraction steps.
2.2.3. Content of ferulic acid in DFRB To know the approximate content of ferulic acid in DFRB, alkaline extraction was carried out according to method reported by Tilay et al. 2008 with some modification. DFRB 2g was fed into flat bottom glass reactor (100 mL) and 2M NaOH was added in 1:30 ratio (w/v) to saponified the DFRB. Resulting mixture was stirred for 24 h at 40°C at 400 rpm. Approximately, 0.001 g of sodium hydrogen sulfite was added to the mixture to avoid the oxidation of FA. The mixture was centrifuged and the supernatant was analysed by UV spectrophotometer against standard curve. Approximate content of ferulic acid in DFRB determined was 4.82±0.2mg/g. 2.2.4. Experimental design The effect of four independent variables i.e. enzyme loading (A, %), solvent ratio /g DFRB (B, mL) (w/v), extraction temperature (C, °C) and sonication power (D, W) on response variable yield (Y) of FA (mg/g of DFRB) was investigated using central composite design (Table 2). Table 3 showed that each independent variable at three levels -1, 0 and +1 and rotatory α values were taken to design the experiment. The deviation in FA yield (mg/g of DFRB) according to the four retained variables A, B, C and D was evaluated using a polynomial second degree model specified in subsequent equation: ∑
∑
∑
(2)
Where,
7
Yk is the response function (Ferulic acid yield, Y mg/g, β0 is a constant, βi, βii and βij are linear quadratic and interactive coefficient of the model correspondingly. Levels of independent variables were denoted as Xi and Xj.
2.3. Analytical method 2.3.1. Characterization of FA extract Total phenolic content was determined by the Folin–Ciocalteu colorimetric method (Khoddami et al., 2013). The absorbance was observed using a UV/Vis spectrophotometer at 760 nm. Gallic acid solution was used as a standard and total phenolic contents was expressed as milligrams of gallic acid equivalent (mg GAE) per g of dry weight. Radical scavenging activity (%) was measured according to DPPH assay (Wu et al., 2014). Spectrophotometric measurements was done at 517 nm using Jasco double beam spectrophotometer. Activity was calculated using formula,
2.3.2. Morphological study of RB Samples were prepared to compare the effect of extraction process. The first sample witnessed for crude RB without treatment, second is residual RB of enzyme and ultrasound treatment with the optimized conditions. These samples were directly coated with platinum before being observed by scanning electron microscopy, scans at 5 kV at a magnification of ×1000 (Jeol, Scanning Electron Microscope, USA). 3. Results and discussion 3.1. Optimization of pre-treatment by single parameter 3.1.1. Effect of pre-treatment time on FA yield
8
Pre-treatment time was an important parameter that would influence the extraction efficiency as well as the activity of enzyme. Due to combined effect of ultrasound and enzyme, the process time was reduced from 4h to 1h. Pre-treatment studies were carried out for various time intervals, ranging from 15 to 180 min using ultrasound and enzyme followed by the extraction. Fig. 2 showed that as the pre-treatment time increases the extraction also increases till 60 min. However, no significant change has been obtained for the higher extraction time and maximum extraction of 2.9 mg/g of DFRB was obtained at 60 min pretreatment time. Initially, at lower pre-treatment time, FA yield was lower and it was attributed to minimum time requirement of an ultrasound to make the material surface porous and further diffusion of enzyme through the material to reach the target site. Cavitation produces physical effects such as liquid circulation currents and turbulence that have an influence on the solid RB surface, which accelerate the process by increasing penetrability of enzyme through RB surface to hydrolyse the ester linkage. Further increase in pre-treatment time, increase in yield was gradual up to 180 min (3.1 mg/g of DFRB). Thus, 60 min pretreatment time was considered to be optimum and used for further experiments. Thus, 60 min pre-treatment time was considered to be optimum and used for further experiments. 3.1.2. Effect of DFRB to Solvent Ratio on FA yield The efficiency of extraction was enhanced by various parameters, among these, solute (DFRB) to solvent ratio during pre-treatment is one of the crucial parameters. Various DFRB to solvent ratios (w/v) (1:7 to 1:56) were studied to maximise the extraction yield of FA at temperature 30°C, sonication power 60 W, enzyme loading 2% and extraction time 1 h. In presented study (Fig. 3a) it was observed that higher ratio results in higher extraction yield, which was directly related to effect of pre-treatment, in higher amount of solvent influence of sonication and enzyme was maximum due to low viscosity and higher enzyme mobility. This can be attributed to the fact that the cellulose-hemicellulose is extremely recalcitrant and do
9
not efficiently approached by FAE to hydrolyse ester bond between FA and arabinose sugar of xylan fragment, and hence higher the solvent, more dilute is the reaction mixture therefore solute in per unit area of solvent was decreased, resulting in less crowding. Because of less crowding of this lignocellulosic biomass, enzyme can approach to the maximum target sites of solute. Another factor that governs the mobility of enzyme is viscosity of solution and higher viscosity reduces the mobility of the molecules. Therefore, higher amount of solvent may offered greater mobility to the enzyme. Additionally, sonication increases the porosity of solute surface by the liquid jet form by ultrasound wave, which allows the enzyme to approach the site of action (J. Mason et al., 2011). The similar trend was observed in earlier report that, the higher amount of solvent leads to the higher yield of polyphenols (Ćujić et al., 2016). Solute to solvent ratio 1:21, 1:28 and 1:35 showed extraction yield as 2.84±0.1, 3.01±0.12 and 3.23±0.09 mg/g respectively. Results specified that, lower solvent volume showed lower FA yield, it may be due to lower amount of solvent in solute enhance the viscosity of the solution and high viscosity may decreased the cavitation effect. Therefore, the highest extraction yield was observed in ratio 1:42 (3.41±0.13 mg/g). Above solute to solvent ratio (1:42) resulted into decrease in FA yield. This may because the Solute to solvent ratio increases beyond 1:28 the reduction in yield was found, because of the ultrasound energy dispersion less in solvent and the growth of dissolved impurities such as protein, polysaccharide etc. which the dissolution of FA (Al-dhabi et al., 2017). Economically, lower ratio will be favourable for the extraction. Therefore, 1:28 ratio has been selected for further optimization study. 3.1.3. Effect of enzyme loading on FA yield FAE selectively targets the ester linkage of FA and arabinose sugar or xylan residue, hence selective extraction of FA takes place. Cost of enzyme governs the economy of the 10
process hence, amount of enzyme used in the process is significantly important and immobilization of enzyme on MNPs was carried out to recycle the enzyme. Enzyme immobilized MNPs readily approaches the target site due to its smaller size, which is quite difficult for enzyme immobilized on beads. To get the optimal enzyme loading, experiments were carried out with 1% to 5% enzyme loading at temperature 30°C, DFRB to solvent ratio 1:28, sonication power 60 W and pre-treatment time 1h. From Fig. 3b it is observed that the yield was increased with an increase in enzyme loading and amongst these 2% and 3% show 3.06±0.05mg/g and 3.22±0.01 mg/g yield respectively. Further increase in enzyme loading resulted in marginal increase in FA yield, which means 2% is the minimal enzyme required for the better activity. Requirement of enzyme was less in ultrasound process than conventional enzymatic process, because ultrasound helps in breaking enzyme-product complex. This attributed to increased availability of enzyme in the mixture, hence more activity. To ensure requirement of enzyme, further studies were performed with 3% enzyme loading. 3.1.4. Effect of temperature on FA yield Temperature has an optimistic effect on the extraction of phenolic compound from natural sources. Temperature may improve the yield of extraction by different properties such as: viscosity, diffusivity, solubility and surface tension (Boonkird et al., 2008). In presented work, three factors were depend on the temperature such as activity of enzyme, solubility of FA and power dissipation of ultrasound. Enzyme activity is higher at certain temperature because, increasing the temperature of a system decreases the viscosity of the reaction mass, which results in higher mobility of enzyme, further it might help to form more enzymesubstrate complex due to decrease in mass transfer resistance (Waghmare and Rathod, 2016). FA yield was higher at 45°C and further increase in temperature gradual decrease in yield was observed. Therefore, reduction in FA yield was because of lowering in cavitation effect
11
at higher temperature. Enzyme stability did not affect by higher temperature as it is reported that the temperature stability of FAE increased up to 60°C upon immobilization on MNPs (He et al., 2015b). On the other hand, solubility of FA in water was increased with rise in temperature, but at the same time, ultrasound restricts the use of higher temperature, since it lowers the surface tension of solvent, which reduces the effect of sonication (Waghmare et al., 2015). Highest enzyme activity was observed (Fig. 3c) at temperature 45°C, DFRB to solvent ratio 1:28, sonication power 60 W, enzyme loading 3% and pre-treatment time 1h. FA yield determined at aforementioned temperature 3.46±0.12 mg/g of DFRB. Therefore, 45°C set as an optimized temperature for the FA extraction. 3.1.5. Effect of sonication power on FA yield Sonication power varied from 30 to 120 W for the pre-treatment to attain optimal power. It has been observed Fig. 3d that power of 75W showed 3.60±0.05 mg/g yield of FA and it also provided lower degradation and leaching of enzyme as compared with higher sonication power at temperature 45°C, DFRB to solvent ratio 1:28, enzyme loading 3% and pre-treatment time 1h. Ultrasound acting an important role to incite a formation of cavitation bubble subjected to fast adiabatic compression and expansion, which rises the local temperature and pressure. This results in an increase the porosity of the surface of DFRB, due to which FAE MNPs can easily get in and approach the hemicellulose fragment. Fig. 7 showed the SEM images of morphology of cell of DFRB before and after extraction. It can clearly be seen that ultrasound assistance leads to damage of rice bran surface, which exposes larger surface area of rice bran to enzyme. Enzyme gets into the cell wall and hydrolyses the ester linkage more effectively than the normal batch process. Mechanical waves formed by ultrasound also assist to break the product substrate complex, which further increases the availability of enzyme, hence the yield. Further increase in sonication power may cause 12
polysaccharide depolymerisation and aggregation, which will result in a decrease in the yield (Chen et al., 2012), also it withdraws the cell debris such as oil, protein and enzyme in the solution. It may affect the enzyme activity by competing or blocking active site of enzyme. At higher sonication power range, acoustic cavitation produced hydroxyl radicals, leading to chemical decomposition of FA, because it is very susceptible to the radical moiety, which is further accountable for lowering in the yield of FA. Therefore, use of lower power (75 W) was chosen over high power. The total ultrasound power dissipation was determined by calorimetric analysis (Raskar et al., 2014), by varying sonication power at constant frequency (22 kHz) and it was observed that at sonication power 50, 75, 100 and 120 W dissipated power was 41.4, 43.2, 46.4 and 57.1 W respectively. Intensity of ultrasound was examines by considering power dissipated at 50, 75, 100 and 120 W and it was found to be 6592.35, 6878.97, 7389.53, and 9093.58 Wm-2 respectively. 3.3. Optimization of pre-treatment parameter by response surface methodology 3.3.1. Response surface methodology Experiments were carried out according to central composite full factorial model to study the combine effect of various parameters on FA yield. Multiple regression analysis was used to explore the statistical significance of variable and their interaction. By multiple regression analysis on the experimental data, following equation in coded value was obtained. FA Yield Y (mg/g DFRB) = 3.66 + 0.13A + 0.20B + 0.041C + 0.084D - 0.056AB -0.012AC-0.018AD–6.8×10-3BC0.021BD–3.1×10-3CD-0.052A2-0.085B2-0.050C2-0.038D2
(3)
Equation gives an empirical relationship of the tested variable and response. It showed the linear factors and interaction among the factor having effect on yield of FA. Ftest and P-test were calculated using ANOVA to check the statistical significance of the regression model shown in Table 4. The F-value of 366.63 with low probability P value 13
(<0.0001) indicates high significance of the model. The coefficient of determination (R2) was 0.9971 indicating significance of model. Values of "Prob > F" less than 0.05 indicate model terms are significant. The value of adjusted determination coefficient (adjusted R2= 0.99) established that the model was highly significant. Coefficient of variation (CV%=0.55) clearly showed high degree of precision and good pact of reliability of experimental value. In this case all the terms in the model are significant model terms. Values greater than 0.1 indicate the model terms are not significant. The "Lack of Fit F-value" of 3.30 implies the Lack of Fit is not significant relative to the pure error. There is a 9.98% chance that a "Lack of Fit F-value" this large could occur due to noise. Non-significant lack of fit is good, therefore the model is valid. Fig. A.2 showed the regular probability plot of residuals. These values are normally scattered among straight line, hence conforming the statistical analysis. Residual and the predicted values were correlated (Fig. A.3) for the FA extraction. It can be seen from Fig. A.3 the residuals are scattered randomly, it proposes that the variance of original observation is constant for all experimental runs. Fig. A.4 presented the actual value versus predicted value, which illustrates that, both values are in the range of operating parameters. 3.3.2. Response surface analysis Regression function was graphically represented by three dimensional (3D) response surface and two dimensional (2D) contour. Extraction yield of FA at different condition by keeping two variable constant were represented in the Fig. 4. 3D response surface Fig. 4(a) showed the effect of enzyme loading and solvent ratio on FA yield at fixed pre-treatment time. FA yield increased eventually with increase in enzyme loading and solvent ratio, this effect is very significant as shown in Table 4 ANOVA (p<0.05). Starting from 2% enzyme loading and solvent ratio 28 mL, yield improved tremendously up to 3.5% enzyme loading and 31.5 mL solvent ratio, further increase in
14
variable FA yield increased gradually. At enzyme loading 2% and solvent ratio 21 mL, FA yield was lowest, it may be due to less mobility of enzyme and higher solute concentration. FA yield increased at due to effective mass transfer at higher ratio and enzyme can easily approach to the ester bond between hemicellulose and ferulic acid. Extraction of FA from corn bran with alkaline-ethanol aqueous solution has been reported with 8.47 mg/g yield by (Zhao et al., 2014). Fig. 4(b) shows that the 3D response surface and 2D contour plot by varying enzyme loading and temperature at fixed pre-treatment time. Plot showed that the yield was governed by both, enzyme loading and temperature. FA yield increases with an increase in enzyme loading and temperature. Enzyme showed minimal activity at lower temperature range 40°C to 45°C and enzyme activity is increased with an increase in temperature from 45°C to 50°C, but at higher temperature, intensity of cavitation reduces. On the other hand, higher temperature felicitates the solubility of FA in solvent, thus higher temperature also showed positive effect on FA yield (Galvan D’Alessandro et al., 2012). Thus overall effect of temperature showed slight negative effect on yield of FA, but higher temperature was increased the yield of FA to 3.7 mg/g of DFRB. 3D response surface Fig. 4(c) showed the interaction of enzyme loading and sonication power. FA yield was increased with increase in the power and enzyme loading. Higher (3-4%) enzyme loading offers more numbers of enzyme MNPs per unit area in mixture, thus observed better activity. At 3% enzyme loading, yield (3.62 mg/g) increased significantly up to 90 W. There is significant effect (p<0.05) of power this can verified from Table 4 ANOVA. The P-valve for power is < 0.01. 3D response surface Fig. 5(d) clearly indicates the solvent ratio and temperature implies the moderate effect on the yield of FA. It can be observed that response (FA yield) did not significantly increase with an increase in the temperature and solvent ratio. At lower temperature and solvent ratio, FA yield was 2.5 mg/g DFRB and it was increased (3.72 mg/g)
15
with the solvent ratio (31.5-35mL). The combine effect of these variable was not significant, that can be seen in ANOVA table 4, with higher p-value (<0.1742). The effect of solvent ratio and sonication power on the yield of FA was showed in Fig. 5(e) 3D response surface, it can be clearly seen that, solvent ratio has a major contribution on extraction yield of FA. The viscosity of solution decreases with increase in solvent ratio, which enhances the cavitation effect. Therefore, in higher solvent ratio (30-35 mL) and sonication power, FA yield (3.5 mg/g) was greater. Fig. 5(e) also displayed that the power around 75 to 80 W showed higher amount of FA extraction. 3D response surface Fig. 5(f) indicates, temperature and power implies negative effect on the yield of FA. This effect is very significant with a p<0.005 shown in Table 4. This might be due to lowering in sonication effect, as a temperature increases. At higher temperature, surface tension decreases, which affects the bubble formation and collapse (Santos et al., 2009). It is attributed to the cavitation phenomena, at higher temperature surface tension of solvent decreases, hence cavitation bubble explodes with lower intensity. Though the combine effect showed negative impact but the overall yield (3.62 mg/g) was slightly increased as the solubility of FA rises with temperature in DI water. 3.4. Optimization of process Predicted model was validated by performing an experiment under predicted conditions as DFRB to solvent ratio 1:32 (w/v), temperature 42.2°C, enzyme loading 2.82% and sonication power 84.85 W. The FA yield obtained and the predicted value was 3.69 mg/g and 3.71 mg/g respectively. The experimental values show a close resemblance to predicted values hence the model is considered to be significant. 3.5. Comparison of conventional enzymatic pre-treatment and ultrasound assisted enzymatic pre-treatment
16
Pre-treatment study was performed with conventional enzymatic process and optimized ultrasound assisted enzymatic treatment. Operating parameters of conventional process were same, except use of ultrasound treatment. Fig. 6 showed that the conventional enzymatic pretreatment showed around 3.16 mg/g of DFRB after 6h. Ultrasound accelerates the process and yield was increased up to 3.63 mg/g of DFRB in 1h. Therefore it can attributed that, ultrasound increases the porosity of material and assists in breaking of enzyme-product complex. Mechanical shock waves as well as liquid jets were good enough to renewal the enzyme surface and also increase the diffusion of enzyme to the target sites of substrates producing more interaction and hydrolysed the ester bond (Waghmare and Rathod, 2016). 3.6. Morphological study of conventional enzymatic treated and ultrasound assisted enzymatic treated RB Fig. 7 showed the morphological images of RB cell surface at a magnification of ×1000, it can be clearly seen from Fig. 7a that the cell of defatted RB is arranged hexagonally and intact to each other. Therefore it was difficult to approach by enzyme in intact cells due to limited porosity. The tremendous effect of the cavitation on cell structure observed in residual RB recovered after the UAE method. Application of ultrasound increase the porosity of RB cells (Fig.7b) due to which FAE MNPs can easily approach the target site. 3.7. Characterization of extract Phenolic content observed was 1.97±1.44 GAE/g.
Presented studies showed
improved phenolic content around 3.86±0.10 GAE/g, it may be due to combine effect of ultrasound and enzyme. DPPH antioxidant assay is based on free radical scavenging activity of phenols that produces a violet solution in ethanol (Foti et al., 2004). The % antioxidant activity profile was detected around 98.5±0.5% over conventional process (82.6±0.5%), which was in agreement with total phenolic content. HPLC Chromatogram of standard and crude extract of ferulic acid is presented in fig. 8. 17
3.8. Enzyme reusability study Enzyme reusability is the main concern in the process, because it directly affects the economy of the process. Thus, reusability study was critically essential for the economic feasibility of reaction. Study was carried out with the optimized conditions obtained from CCD method. Enzyme was recovered from the process with help of magnet. Recovered enzyme nano particle was than washed twice with water and centrifuged under 10000×g for 10 min and dry at room temperature. The recovered enzyme was used for further batch of extraction and yield was calculated, same procedure was repetitively used for further three successive cycles and extraction yield was calculated. First cycle yield was considered as a 100% activity of enzyme and accordingly the calculation of yields for next cycles was carried out. The yields obtained after second, third, fourth and fifth cycle were 95, 92, 87 and 82% respectively (Fig. 9). After 5th cycle enzyme loses almost 20% of its activity, which was not feasible for the process. Enzyme activity in this study is retained tremendously, that may be due to lower time of sonication treatment and absence of ethanol during pre-treatment study. 4. Conclusion The pre-treatment by means of combined effect of ultrasound and enzyme readily extract the FA from DFRB. Implementation of CCD for pre-treatment, lower down the enzyme loading and FA yield was enhanced with a reduced time than conventional method. The optimized operating parameters unveiled the highest yield of FA 3.69 mg/g of DFRB. Established process is rapid and efficient method for extraction of FA. Optimized parameters of ultrasound assisted enzymatic extraction displayed around 77% yield after 1h as compared to around 65% after 4h in the case of conventional enzymatic process. Pre-treatment showed 1.2 fold increase in the yield of FA with lower extraction time. Recovery of enzyme by immobilized on MNPs can be the cost effective process, therefore enzyme can be used effectively up to five cycles. The outcome of presented work would be beneficial for the
18
economical purpose. This process can be applied for the other natural compound extraction from verity of raw material.
19
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Table Caption Table 1. Extraction of ferulic acid by various methods from different raw material sources Table 2. Experimental values and coded levels of the independent variables used for the CCD Table 3. Experimental design for the optimization of extraction of FA from DFRB using Central Composite Rotatable Design of RSM Table 4. ANOVA for response surface quadratic model for Ferulic acid extraction
25
Table 1. Extraction of ferulic acid by various methods from different raw material sources
Source
Extraction method
FA yield (mg/g) Reference
Paddy straw
Alkaline
8.17
Salleh et al., 2011
Radix Angelicae sinensis
Microwave/ethanol
1.33
Liu et al., 2006
Flax Shives
Pressurised-Alkaline
0.18
Buranov et al., 2011
Corn bran
Pressurised-Alkaline
25.10
Buranov et al., 2011
Wheat bran
Alkaline
3.91
Buranov et al., 2011
Jojoba meal
Enzymatic
6.7
Laszlo et al., 2006
26
Table 2. Experimental values and coded levels of the independent variables used for the CCD Level Independent variable
Symbols
Unit
Low (−1)
Intermediate (0)
High (1)
Enzyme loading Solvent Ratio
A B
% mL/g
2 21
3 28
4 35
Temperature
C
°C
40
45
50
Sonication power
D
W
60
75
90
27
Table 3. Experimental design for the optimization of extraction of FA from DFRB using Central Composite Rotatable Design of RSM Run Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Enzyme Solvent Temperature loading Ratio (°C) (%) (mL) 2 4 2 4 2 4 2 4 2 4 2 4 2 4 2 4 1 5 3 3 3 3 3 3 3 3 3 3 3 3
21 21 35 35 21 21 35 35 21 21 35 35 21 21 35 35 28 28 14 42 28 28 28 28 28 28 28 28 28 28
40 40 40 40 50 50 50 50 40 40 40 40 50 50 50 50 45 45 45 45 35 55 45 45 45 45 45 45 45 45
Power (W)
FA Yield (mg/g)
60 60 60 60 60 60 60 60 90 90 90 90 90 90 90 90 75 75 75 75 75 75 45 105 75 75 75 75 75 75
2.89 3.29 3.42 3.66 3 3.35 3.53 3.68 3.1 3.46 3.61 3.7 3.22 3.55 3.69 3.76 3.17 3.73 2.92 3.71 3.37 3.54 3.32 3.69 3.64 3.66 3.65 3.63 3.65 3.64
28
Table 4. ANOVA for response surface quadratic model for Ferulic acid extraction Source
Sum of Squares
DF
Mean Square
F Value
p-value Prob > F
Model
1.908
14
0.136
366.63
< 0.0001
A
0.403
1
0.403
1084.32
< 0.0001
B
0.948
1
0.948
2550.77
< 0.0001
C
0.041
1
0.041
109.88
< 0.0001
D
0.168
1
0.168
452.93
< 0.0001
AB
0.050
1
0.050
133.20
< 0.0001
AC
0.002
1
0.002
6.07
0.0263
AD
0.005
1
0.005
14.14
0.0019
BC
0.001
1
0.001
2.03
0.1742
BD
0.007
1
0.007
18.31
0.0007
CD
0.000
1
0.000
0.42
0.5265
A2
0.073
1
0.073
196.21
< 0.0001
2
B
0.200
1
0.200
537.12
< 0.0001
C2
0.069
1
0.069
186.81
< 0.0001
D2
0.039
1
0.039
105.52
< 0.0001
Residual
0.006
15
0.000
Lack of
0.005
10
0.000
0.001
5
0.000
1.913
29
3.30
0.0998
Fit Pure Error Cor Total
29
Fig. Caption Fig. 1. Process flow diagram of pre-treatment and extraction of ferulic acid using sonication treatment and immobilized enzyme MNPs Fig. 2. Effect of pre-treatment time on FA extraction (mg/g of DFRB) in DFRB to solvent 1:14 at Temperature 30°C, sonication power 60 W, enzyme loading 2% Fig. 3. Effect of i) Solute to solvent, ii) Enzyme loading, iii) Temperature and iv) Sonication power on FA yield Fig. 4. Response surface plot showing the effect of (a) Enzyme loading and Solvent ratio, (b) Enzyme loading and Temperature and (c) Enzyme loading and Sonication power on extraction yield of Ferulic acid (mg/ g of RB) Fig. 5. Response surface plot showing the effect of (d) Temperature and solvent ratio, (e) Solvent ratio and Sonication power and (f) Sonication power and Temperature on extraction yield of Ferulic acid (mg/ g of RB) Fig. 6. Effect of conventional enzymatic pre-treatment and ultrasound assisted enzymatic pretreatment on yield of FA Fig. 7.SEM images at magnification ×1000 (a) Untreated DFRB (b) UAE and enzyme treated RB Fig. 8. HPLC chromatogram of standard and crude extract of ferulic acid Fig. 9. Reusability of Immobilized enzyme MNPs with number of cycle
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Rice Bran Defatting Defatted Rice Bran
Pre-treatment
Parameter Optimization Enzyme Loading Sonication Temperature Ratio
Enzyme MNP
DI Water
Magnet Recycle Enzyme MNP
Stirring
Stirring
Extraction
EtOH
Centrifugation
Supernatant Extract
RB Residue
Analysis
FA Yield (mg/g)
DPPH Assay
Total phenol content
Fig. 1. Process flow diagram of pre-treatment and extraction of ferulic acid using sonication treatment and immobilized enzyme MNPs
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4
FA mg/g DFRB
3.5 3 2.5 2 1.5 1 0.5 0 0
20
40
60
80 100 120 140 160 180 200 Time(min)
Fig. 2. Effect of pre-treatment time on FA extraction (mg/g of DFRB) in DFRB to solvent 1:14 at Temperature 30°C, sonication power 60 W, enzyme loading 2%
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a)
b)
c)
d)
Fig. 3. Effect of a) Solute to solvent, b) Enzyme loading, c) Temperature and d) Sonication power on FA yield
33
Fig. 4. Response surface plot showing the effect of (a) Enzyme loading and Solvent ratio, (b) Enzyme loading and Temperature and (c) Enzyme loading and Sonication power on extraction yield of Ferulic acid (mg/ g of RB)
34
Fig. 5. Response surface plot showing the effect of (d) Temperature and solvent ratio, (e) Solvent ratio and Sonication power and (f) Sonication power and Temperature on extraction yield of Ferulic acid (mg/ g of RB)
35
4.5 4
FA mg/g DFRB
3.5 3 2.5 2 1.5 Conventional Enzymatic
1
Ultrasound + Enzymatic
0.5 0 0
50
100
150
200 250 300 Time (min)
350
400
450
Fig. 6. Effect of conventional enzymatic pre-treatment and ultrasound assisted enzymatic pretreatment on yield of FA
36
a)
b)
Fig. 7. SEM images at magnification ×1000 (a) Untreated DFRB (b) UAE and enzyme treated RB
37
Fig. 8. HPLC chromatogram of standard and crude extract of ferulic acid
38
100
90
FA Yield (%)
80
70
60
50 1
2
3 Number of cycle
4
5
Fig. 9. Reusability of Immobilized enzyme MNPs with number of cycle
Research highlights
Combined effect of ultrasound and enzyme, enhanced the FA yield with minimum time
Enzyme was used in pre-treatment method, to avoid its denaturation in contact with ethanol
Use of ferulic acid esterase immobilized magnetic nanoparticles allows the reusability of enzyme
Obtained FA acid showed good radical scavenging activity and total phenolic content
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