- Email: [email protected]
Effect of ultrasound treatment on oil recovery from soybean gum by using phospholipase C
Journal of Cleaner Production 69 (2014) 237e242
Contents lists available at ScienceDirect
Journal of Cleaner Production journal homepage: www.elsevier.com/locate/jclepro
Effect of ultrasound treatment on oil recovery from soybean gum by using phospholipase C Xiaofei Jiang, Ming Chang, Xiaosan Wang, Qingzhe Jin, Xingguo Wang* State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 August 2013 Received in revised form 20 January 2014 Accepted 21 January 2014 Available online 31 January 2014
Comparative studies of enzymatic oil recovery process of water degummed soybean gum (WDSG) were carried out in mechanical-stirring (MS) and ultrasonic-assisted mechanical-stirring (UAMS) systems. The influences of ultrasonic power (0.68e1.52 W/cm3), gum/water ratio (w/w) (1/0.5e1/3.0), reaction temperature (40e65 C), enzyme dosage (200e1000 mg/kg) and reaction time (2e7 h) were investigated subsequently. A suitable ultrasonic power of 1.20 W/cm3 was determined to guarantee satisfactory oil recovery efficiency and enzyme activity. Compared to the MS system, less time, water and enzyme dosage were required in the UAMS system for the oil recovery process, and the thermal stability of phospholipase C (PLC) under ultrasound treatment was increased. Analysis of the quality of recovered oil showed that indices including phosphorus content, free fatty acid content, peroxide value, fatty acid composition, color, smell and taste could meet the requirements of crude soybean oil. However, ultrasound treatment could accelerate the primary oxidation of recovered oil due to the effect of cavitation. These results indicate that using PLC to recover oil from WDSG with ultrasound treatment is feasible and effective, and more attention should be paid on the oxidative stability of recovered oil for its further application. Ó 2014 Published by Elsevier Ltd.
Keywords: Phospholipase C Oil recovery Ultrasound Water degummed soybean gum
1. Introduction Soybean gum obtained from the water degumming process is a byproduct during the oil refining process, and the main composition of the gum is neutral oil, phospholipids and water. The gum can be easily deteriorated under high temperature, releasing unpleasant smell, and resulting in a negative impact on the environment (Van Nieuwenhuyzen, 1976). However the phospholipids contained in the gum are well recognized for excellent surface-active properties and are extensively used in food, cosmetics, pharmaceutical and chemical industries (Wu and Wang, 2003). In addition, the
Abbreviations: WDSG, water degummed soybean gum; MS, mechanical-stirring; UAMS, ultrasonic-assisted mechanical-stirring; PLC, phospholipase C; DAG, diacylglycerols; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidic acid; NPPC, p-nitrophenylphosphorylcholine; FFA, free fatty acid; PV, peroxide value; OSI, oxidative stability; FA, fatty acid composition; FID, flame ionization detector; FAME, fatty acid methyl ester; RSD, relative standard deviation. * Corresponding author. Tel./fax: þ86 510 85876799. E-mail addresses: [email protected] (X. Jiang), [email protected] (M. Chang), [email protected] (X. Wang), [email protected] (Q. Jin), [email protected] (X. Wang). 0959-6526/$ e see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jclepro.2014.01.060
water degummed soybean gum (WDSG) usually contains a substantial amount of neutral oil (30e40% on the dry basis). Therefore it is quite valuable to recover the oil from the gum, as the price of soybean oil is much higher than the soybean gum. Conventional preparation of deoiled phospholipids from the WDSG is based on acetone extraction utilizing the difference in solubility between phospholipids and triacylglycerols (TAGs) (Szuhaj, 1983). This process usually includes repeated extraction procedures consuming large amounts of acetone, which is either costly or ineffective. In addition, the acetone used in the extraction process or retained in the final products can be a potential hazard to human health (Liu et al., 2011). Therefore, it is necessary to find a more ecofriendly way to recover high-quality neutral oil from the soybean gum and obtain deoiled phospholipids. In recent years, enzymatic-assisted oil recovery process has attracted great attention as it can avoid the consumption of solvent during the whole recovery process. A previous patent (Paulitz et al., 1987) showed that enzymes could catalyze the hydrolysis of gum, facilitating the formation of an oil phase. The presence of lysophospholipids in the gum could promote the oil recovery. More recently, an oil recuperation process has been disclosed in which wet gums are mixed with an acid or a phospholipase (Kellens and De Greyt, 2013). The resulting mixture is then allowed to separate
238
X. Jiang et al. / Journal of Cleaner Production 69 (2014) 237e242
into two or more phases, the oily phase of which is then recuperated. Nevertheless, low oil recovery is one of the major challenges for this process which may be overcome by utilizing selected enzymes in combination with appropriate treatment. Phospholipase C (PLC) is a kind of hydrolytic enzyme that can hydrolyze the bond between the acylglycerol and the phosphate group of phospholipids. Accordingly, it liberates diacylglycerols (DAG) which will not be removed from the oil (Casado et al., 2012). The gum obtained by water degumming process contains appreciable amounts of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which can be released to DAG on exposure to PLC. Phospholipase-catalyzed reaction takes place at the interface between the aqueous phase containing the enzyme and the oil phase containing neutral oil and phospholipids, which means that the rate of this reaction increases when the interfacial area is increased (Dijkstra, 2010). In recent years, ultrasound is considered as an effective and non-polluting method that can accelerate heat and mass transfer during the extraction process (Chemat and Khan, 2011; Sivakumar et al., 2007). It has been successively used in oil extraction from soybeans (Li et al., 2004b), flaxseed (Zhang et al., 2008) and almond powder (Zhang et al., 2009). Besides, some researchers have used ultrasound as a kind of pretreatment during the aqueous enzymatic oil extraction process to facilitate the oil recovery (Shah et al., 2005; Sharma and Gupta, 2006). Ultrasound irradiation, a mechanical rather than an electromagnetic wave, is an alternative method to increase interfacial area in enzymatic reactions (Huang et al., 2010; Mason et al., 1996). An early study disclosed a kinetic model for enzymatic reactions by taking the interfacial area into account (Mukataka et al., 1985). Though some studies have shown that appropriate treatment of ultrasound can enhance the activity and reaction rate of some kinds of enzymes (Li et al., 2005, 2004a; Liu et al., 2008), little work has been devoted to the effect of ultrasound on the activity of PLC during the enzymatic oil recovery process of soybean gum. The objective of this study is to investigate the effect of PLC on oil recovery process of WDSG. Comparative studies were carried out in mechanical-stirring (MS) and ultrasonic-assisted mechanical-stirring (UAMS) systems to evaluate the effect of ultrasound on enzymatic oil recovery process. Quality analysis of recovered oil was conducted to estimate whether the indices of recovered oil could meet the requirements of the standards.
PLC activity was determined using NPPC as a substrate (Flieger et al., 2000). We defined 1 U/g of PLC as the amount of enzyme needed to produce 1 nM nitrophenol per minute by the hydrolysis of NPPC under appropriate conditions: 100 mL PLC was added to 2 mL of NPPC solution (10 mM NPPC, 250 mM TriseHCl (pH 7.2), 600eg/L sorbitol and 1 mM ZnCl2) in a test tube. The tube was incubated at 37 C for 30 min. Substrate hydrolysis was then quantified by measuring absorbance at 410 nm. The activity of PLC was calculated by the following equation:
2. Materials and methods
Activity ðU=gÞ ¼ 1:3636 103 A=t
2.1. Materials WDSG (water content 46.7%; phospholipids content 27.9%; oil content 28.8%) was kindly provided by Donghai Cereal & Oils Co. Ltd. (Zhangjiagang, China). PLC was prepared by our own laboratory using submerged fermentation of a genetically modified Bacillus cereus with activity assayed to be 150 U/g. It had activity on PC and PE, while it had no activity on phosphatidylinositol (PI) or phosphatidic acid (PA). p-Nitrophenylphosphorylcholine (NPPC) with purity higher than 98% was purchased from Sigma Chemical Ltd. (Shanghai, China). All other reagents of analytical grade were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China), and the solutions were prepared in deionized water. 2.2. Equipment MS experiments were carried out in a thermostatic water bath equipped with an agitation unit (IKA, Germany, model Eurostar 40 digital). The agitation unit consisted of a 3-bladed propeller-type impeller with a diameter of 75 mm (IKA, Germany, model R1389),
Fig. 1. The schematic diagram of ultrasonic equipment.
and the reaction temperature could be maintained within 0.2 C of the desired temperature. Ultrasound irradiations were carried out in a 28 kHz ultrasonic water bath, equipped with a thermostatic setup allowing irradiations to be performed within 0.5 C of the desired temperature (Huaneng, China, model HN, tank capacity 27 L). Ultrasonic waves were emitted from the bath bottom through a piezo-electric disk (d ¼ 5.87 cm). The output power of the ultrasound could be adjusted from 40 to 100% of the total power (300 W) by using a control button, and the delivered power dissipated in the medium was estimated by the calorimetric method (Li et al., 2004b).
2.3. Determination of PLC activity
(1)
where A is the absorption value in 410 nm and t (min) is the time used in the substrate hydrolysis reaction.
2.4. PLC oil recovery process in MS system WDSG (100 g) and a certain amount of water (50e300 g) were added to a beaker. The beaker was immersed in a thermostatic water bath equipped with an agitation unit (seen in Section 2.2) and the mixture was preheated to a required temperature of enzymatic reaction (40e65 C) under stirring at 500 rpm. After equilibrating for 10 min, a certain amount of PLC (200e1000 mg/ kg) was added to the mixture to initiate the enzymatic oil recovery reaction. The enzymatic reaction was conditioned at settled temperature (40e65 C) under stirring at 500 rpm. After a certain time of enzymatic treatment (2e7 h), the oil mixture was heated to 95 C for 10 min to inactivate the enzyme and then quickly centrifuged at 10 000 rpm for 10 min. The resulting mixture was separated into three phases, and the oily phase was collected and weighed for calculation of the oil recovery efficiency. All reactions were carried out in triplicate.
X. Jiang et al. / Journal of Cleaner Production 69 (2014) 237e242
2.5. PLC oil recovery process in UAMS system The first several steps of the process were carried out similar to the enzymatic oil recovery process without ultrasonic treatment (seen in Section 2.4). After the addition of enzyme, the mixture was quickly placed in an ultrasonic water bath equipped with an agitation unit (seen in Section 2.2). The schematic diagram of ultrasonic equipment was shown in Fig. 1, and the distance of reactor from tank bottom was about 10 cm. The enzymatic oil recovery process was carried out under stirring at 500 rpm in combination with the ultrasonic irradiation (120e270 W; 28 kHz) at settled temperature (40e65 C). After a certain time of ultrasonic-assisted enzymatic treatment (2e7 h), the oil mixture was heated to 95 C for 10 min to inactivate the enzyme and then quickly centrifuged at 10 000 rpm for 10 min. The resulting mixture was separated into three phases, and the oily phase was collected and weighed for calculation of the oil recovery efficiency. All reactions were carried out in triplicate. 2.6. Determination of oil recovery efficiency During the enzymatic oil recovery process, the DAG generated by PLC can also be counted as neutral oil, which means that the total mass of oil can be increased under PLC treatment. Thus it is reasonable for the oil recovery efficiency to exceed 100% under appropriate extraction conditions. The oil recovery efficiency was calculated by the following equation:
Oil recovery efficiency ð%Þ ¼ ðm1 =m2 Þ 100%
(2)
where m1 (g) is the mass of recovered oil obtained after the enzymatic oil recovery process, m2 (g) is the mass of oil contained in WDSG. 2.7. Determination of recovered oil quality indices The phosphorus content of the samples was determined according to GB/T 5537-2008 (National Standard of the People’s Republic China, 2008). The free fatty acid (FFA) content of the samples was determined in accordance with GB/T 5530-2005. The peroxide value (PV) of the samples was determined in accordance with GB/T 5538-2005. The water content of the samples was measured by Karl Fisher titration. The smell and taste of the samples were determined according to GB/T 5525-2008. The color of the samples was measured by Lovibond method (GB/T 22460-2008). The oxidative stability (OSI) of the samples was analyzed by the Rancimat method using a Metrohm 743 Rancimat (Herisau, Switzerland) instrument. Samples of 3.0 g were analyzed under a heating block of 110 C at a constant air flow of 10 L/h. The fatty acid (FA) composition of the samples was analyzed by GC equipped with a PEG-20000 capillary column (30 m 0.25 mm 0.25 mm), a flame ionization detector (FID) and nitrogen as carrier gas. The injection was performed in split mode with a split ratio of 1:50. The fatty acid methyl ester (FAME) solution was prepared by the method of BF3-CH3OH (GB/T 17376-2008). 1.0 mL of FAME solution was injected at an injector temperature of 250 C, column temperature: 100 C (3 min), 100e 180 C (20 C/min), 180 C (4 min), 180e235 C (12 C/min), 235 C (15 min), FID temperature of 250 C and carrier gas (N2) flow of 60 mL/min. The FA composition reported was based on the area response using FID. All experiments were carried out in triplicate. 2.8. Statistical analysis All experiments were performed in triplicate and the mean values and standard deviations were calculated. Statistical analysis
239
was performed using Origin 8.0 software (OriginLab Ltd., USA). One-way ANOVA was carried out by using Tukey adjustment to determine the significant difference between treatments. Significant differences were declared at P 0.05.
3. Results and discussion 3.1. The effect of ultrasonic power on oil recovery efficiency For enzymatic reactions with ultrasonic treatment, the power of ultrasound is an important parameter. The effect of ultrasonic power on different kinds of enzymes is quite different. In our study, the intensities of the output power were adjusted to 120, 150, 180, 210, 240 and 270 W. The actual values of powers dissipated in the water were measured by the calorimetric method (Li et al., 2004b) and their values were 0.68, 0.85, 1.02, 1.20, 1.36 and 1.53 W/cm3 with experimental relative standard deviation (RSD) of 10.8, 9.6, 8.3, 10.5, 9.8 and 8.7%, respectively. Fig. 2 showed the effect of ultrasonic power on the oil recovery efficiency from WDSG. In both of the two ultrasonic-assisted systems with or without mechanical stirring, the oil recovery efficiency from WDSG increased rapidly with the increase in ultrasonic power in the lower range (from 0.68 to 1.20 W/cm3), which might be attributed to the cavitation effect caused by ultrasound (Vilkhu et al., 2008). When cavitation bubbles collapse near the phase boundary of two immiscible liquids, the resultant shock wave can provide a very efficient stirring/mixing of the layers. Besides, increasing ultrasonic power in an appropriate range could promote the mass transfer of the reaction system and reduce the substrate inhibition and aggregation on the interfacial area, resulting in a finer dispersion of enzyme and gum in the mixture. When the ultrasonic power exceeded 1.20 W/cm3, a decrease of oil recovery efficiency was observed, which might be explained that too high intensity of ultrasonic power could reduce or even inactivate the enzyme activity (Chemat and Khan, 2011). These results are somewhat consistent with other researchers who indicated that ultrasonic waves of low intensity had a small effect on the mass transfer of the solution compared to the high intensity which could accelerate the mass transfer. However, too high intensity of the ultrasonic waves might lead to the disruption of the enzyme (Bezbradica et al., 2006; Sakakibara et al., 1996).
Fig. 2. Effect of ultrasound power on the oil recovery efficiency: gum/water ratio ¼ 1/ 1.5 (w/w), reaction temperature ¼ 45 C, enzyme dosage ¼ 600 mg/kg, reaction time ¼ 2 h. In ultrasonic-assisted system, no stirring (d-d); in ultrasonic-assisted mechanical-stirring system, stirring ¼ 500 rpm (dCd).
240
X. Jiang et al. / Journal of Cleaner Production 69 (2014) 237e242
Compared to the ultrasonic-assisted system without mechanical-stirring, the oil recovery efficiency of the UAMS system was much higher (Fig. 2), which meant that mechanical stirring had its own advantage in facilitating the dispersion of gum and enzyme in the mixture when the same reaction time was used. To obtain the maximal oil recovery efficiency and save the reaction time, we selected UAMS system with ultrasonic power of 1.20 W/cm3 for further studies.
3.2. The effect of ultrasound on optimum gum/water ratio The gum/water ratio (w/w) is an important factor in the enzymatic oil recovery process. The gum would be agglomerated and could not be fully dispersed in the reaction mixture if the water addition was not enough. Enough water could decrease the viscosity of the mixture, facilitate the dispersion of the gum and enzyme, and provide a larger interfacial area between the reaction phases. The gum/water ratios from 1/0.5 to 1/3 (w/w) were selected to study the effect of water amount on the oil recovery efficiency. As shown in Fig. 3, in MS system, the oil recovery efficiency increased gradually with the increase in water amount from 1/0.5 to 1/2.5 (w/w), and then its value decreased slightly with further addition of water to 1/3 (w/w). In the UAMS system, the highest oil recovery efficiency was at the gum/water ratio of 1/2 (w/w). These results might be explained that there existed a critical point of water amount in the two systems, and the substrates (gums) may already be saturated by water at the critical point, thus further addition of water had no more contribution to the oil recovery efficiency. Besides, excess of free water may dilute the concentration of the enzyme in the reaction mixture, leading to a decrease in the PLC catalytic efficiency. The optimum values of the gum/water ratios (w/w) were 1/2.5 and 1/2 for MS and UAMS systems, respectively. It meant that less water was required to saturate the substrates under ultrasonic-assisted treatment. These results might be attributed to the emulsification effect of ultrasound, which could produce more interfacial area between the reaction phases when the same water amount was added and could accelerate the mass transfer in the reaction systems (Liu et al., 2008).
Fig. 4. Effect of temperature on the oil recovery efficiency: enzyme dosage ¼ 600 mg/ kg, reaction time ¼ 2 h, stirring ¼ 500 rpm. In mechanical-stirring system, gum/oil ratio ¼ 1/2.5 (w/w) (d-d); in ultrasonic-assisted mechanical-stirring system, ultrasonic power ¼ 1.20 W/cm3, gum/oil ratio ¼ 1/2.0 (w/w) (dCd).
3.3. The effect of ultrasound on optimum temperature In our study, the effect of temperature on enzymatic oil recovery efficiency was determined within the temperature range from 40 to 65 C. As shown in Fig. 4, the oil recovery efficiency increased slightly with increasing temperature up to 55 C, and then decreased sharply when the temperature was above 65 C, which meant that most of the activity of PLC was lost at 65 C in the MS system. In the UAMS system, the optimum temperature was also observed at 55 C. However, only a slight decrease of the oil recovery efficiency occurred when the temperature was above 65 C, which meant that a majority of the activity of PLC could be remained at 65 C in the UAMS system. These results indicated that ultrasonic treatment could increase the activity and promote the thermal stability of PLC, and the effect of ultrasonic cavitation could provide full contact of PLC with substrates to produce stable enzyme-substrate intermediates, thus enhanced thermal stability of the enzyme (Wan et al., 2007). In further experiments, we selected the reaction temperature of 55 C for the two systems. 3.4. The effect of ultrasound on optimum enzyme dosage
Fig. 3. Effect of gum/water ratio (w/w) on the oil recovery efficiency: reaction temperature ¼ 45 C, enzyme dosage ¼ 600 mg/kg, reaction time ¼ 2 h, stirring ¼ 500 rpm. In mechanical-stirring system (d-d); in ultrasonic-assisted mechanical-stirring system, ultrasonic power ¼ 1.20 W/cm3 (dCd).
Fig. 5 showed the effect of enzyme dosage (relative to the mass of gum) on the oil recovery efficiency of WDSG. In the MS system, the efficiency of oil recovery increased fast when the enzyme dosage was below 800 mg/kg and showed a slight decrease when the addition of PLC exceeded 800 mg/kg. By contrast, in the UAMS system, the oil recovery efficiency continuously increased when the PLC dosage increased from 200 to 1000 mg/kg. Decrease in oil recovery efficiency along with increasing enzyme dosage was not observed in the UAMS system, which was quite different from the results obtained from the MS system. Besides, when the same amounts of PLC were added, the ultrasonic-assisted treatment could effectively recover more oil from the WDSG compared to the MS treatment. Under ideal conditions, the oil recovery efficiency should increase with the addition of enzyme dosage until maximal oil recovery efficiency was obtained, where enzyme saturated the interface, and then maintained constant at any further increasing in enzyme dosage. The different effects of enzyme dosage under the two systems might be attributed to the effect of ultrasound on
X. Jiang et al. / Journal of Cleaner Production 69 (2014) 237e242
241
Table 1 Overview of the phosphorus, FFA, water, color (R represents red color; Y represents yellow color), FA composition, OSI and PV value of recovered soybean oils after different kinds of treatments. Parameters
Recovered oils Requirements for crude soybean Stirringa oil (GB 1535-2003)
Phosphorus content (%) 0.800 Free fatty acid 4.000 [(KOH)/(mg/g)] Water content (%) 0.200 Colorc R 4.0, Y 35
Fig. 5. Effect of enzyme dosage on the oil recovery efficiency: reaction temperature ¼ 55 C, reaction time ¼ 2 h, stirring ¼ 500 rpm. In mechanical-stirring system, gum/oil ratio ¼ 1/2.5 (w/w) (d-d); in ultrasonic-assisted mechanical-stirring system, ultrasonic power ¼ 1.20 W/cm3, gum/oil ratio ¼ 1/2.0 (w/w) (w/w) (dCd).
dispersion. UAMS treatment could create a better effect on the dispersion of gum in the reaction mixture, which contributed to a more homogeneous reaction system and facilitated dispersion of PLC on the interfacial area between the phases (Mason et al., 1996; Suslick and Price, 1999). Within the range of enzyme dosage (200e 1000 mg/kg) chosen in our study, the efficiency of oil recovery increased continuously in the UAMS system, which meant that higher interfacial area and higher enzyme quantity could be accommodated with the treatment of ultrasound. However, in the MS system, the effect of mechanical stirring was not good enough to provide a fine contact of PLC to the substrates. Besides, enzyme dosage at higher concentrations is more liable to agglomeration, which will reduce the actual effective reaction area on the phases. Thus, when the addition of PLC was beyond 800 mg/kg, a slight
Fatty acid composition (%) Palmitic Stearic Oleic Linoleic Linolenic Oxidative stability (h) Peroxide value (mmol/kg)
8.0e13.5 2.5e5.4 17.7e28.0 49.8e59.0 5.0e11.0 No requirement 7.500
0.36 0.04 2.49 0.06
Ultrasound þ Stirringb 0.38 0.05d 2.53 0.04d
0.30 0.04 0.28 0.03d R ¼ 2.4, Y ¼ 12 R ¼ 2.7, Y ¼ 11
10.48 0.35 4.45 0.30 24.89 0.19 53.08 0.14 6.48 0.10 6.23 0.07 3.86 0.12
10.98 0.23d 4.93 0.22d 24.73 0.31d 52.36 0.25e 6.32 0.28d 5.68 0.09e 4.13 0.08e
a
Mechanical-stirring system: gum/water ratio ¼ 1/2.5 (w/w), reaction time ¼ 7 h. Ultrasonic-assisted mechanical-stirring system: gum/water ratio ¼ 1/2.0 (w/w), reaction time ¼ 5 h, ultrasonic power ¼ 1.20 W/cm3. PLC dosage ¼ 800 mg/kg, stirring ¼ 500 rpm, reaction temperature ¼ 55 C for the two systems. c The type of cuvette used to determine color is 133.4 mm. d No significant difference between the two treatments (P > 0.05). e Significant difference between treatments (P 0.05). b
decrease of oil recovery efficiency was observed in MS system. In consideration of the cost and oil recovery efficiency of PLC, the dosage of 800 mg/kg was chosen for further study. 3.5. The effect of ultrasound on reaction time Fig. 6 showed the effect of reaction time on the oil recovery efficiency of WDSG in MS and UAMS systems. In the MS system, the oil recovery efficiency could reach 135% after 7 h of enzymatic treatment. However, in the UAMS system, it took less than 5 h for enzymatic treatment to achieve an oil recovery efficiency of more than 145%. These results might be explained by two reasons. Firstly, the ultrasound treatment could increase the interfacial area of the reaction system and facilitate the dispersion of substrates. Secondly, ultrasound treatment might have an effect on the active center of the enzyme, providing rapid and enough combination of the enzyme with substrates (Barton et al., 1996). In consideration of the oil recovery efficiency and reaction time, we selected 7 h and 5 h as the optimum reaction times for the MS and UAMS systems, respectively. 3.6. The effect of ultrasound on the quality of recovered oil
Fig. 6. Effect of reaction time on the oil recovery efficiency: reaction temperature ¼ 55 C, enzyme dosage ¼ 800 mg/kg, stirring ¼ 500 rpm. In mechanicalstirring system, gum/oil ratio ¼ 1/2.5 (w/w) (d-d); in ultrasonic-assisted mechanical-stirring system, ultrasonic power ¼ 1.20 W/cm3, gum/oil ratio ¼ 1/2.0 (w/w) (dCd).
The quality of recovered oils under optimal conditions in MS and UAMS systems are given in Table 1. The phosphorus, FFA and water contents of the recovered oils did not show significant difference (P > 0.05) between the MS and UAMS systems and no apparent abnormal smell or taste was found in the recovered oils treated by ultrasound. The color of the recovered oils under ultrasonic treatment was a little darker than that of the oils without ultrasonic treatment. The PV value of recovered oils treated by ultrasound increased significantly compared to those without ultrasonic treatment (P 0.05), which meant that primary oxidation of recovered oils was accelerated under the treatment of ultrasound. In addition, the value of OSI showed that ultrasonic treatment could reduce the oxidative stability of recovered oils from about 6.23 h to about 5.68 h, which further proved that ultrasound could accelerate
242
X. Jiang et al. / Journal of Cleaner Production 69 (2014) 237e242
the oxidation of oils during the extraction process. The FA composition of recovered oils under ultrasonic treatment showed a slight decrease in the relative percentage of unsaturated fatty acids and an increase in the percentage of saturated fatty acids. It might be explained that unsaturated fatty acids are more susceptible to oxidation, whereas saturated fatty acids are more stable to oxidation (Li et al., 2004b). Previous articles claimed that ultrasound could increase fat and oil degradations (Chemat et al., 2004; Pingret et al., 2012, 2013). In our study, the role of ultrasound as an initiator in the oxidation of recovered oils was also observed. The reason might be explained that metals existing naturally in the WDSG could be responsible for the formation of oxy-radical species to induce the oxidation and the effect of cavitation might accelerate such kind of oxidation. More studies should be carried out to better elucidate the oxidation mechanism of oils treated by ultrasound. Though, the treatment of ultrasound might reduce the oxidative stability of recovered oils to some extent, all of the indices (phosphorus content, FFA content, PV value, FA composition, color, smell and taste) except the water content could meet the requirements of crude soybean oil (GB 1535-2003), which indicated that using PLC to recover the oil from WDSG was feasible. 4. Conclusion The present study showed that ultrasound in combination with PLC treatment was an efficient way to accelerate enzymatic oil recovery reaction rate and enhance oil recovery efficiency of WDSG. The oil recovery efficiency could achieve more than 145% within 5 h under ultrasound treatment. The optimum working temperature of PLC did not change under ultrasound treatment. However the thermostability of PLC was increased under ultrasound treatment. Besides, the water required to saturate the substrates was less under ultrasound treatment. Quality analysis of the recovered oils showed that ultrasound treatment could accelerate the primary oxidation of recovered oils due to the effect of cavitation. All indices except the water content of enzymatic recovered oils could meet the requirements of crude soybean oil (GB 1535-2003). These results indicate that using PLC to recover the oil from WDSG is feasible and ultrasound can be a clean and effective way to reduce the time, water and enzyme dosage used during the oil recovery process. Acknowledgment The work is supported by Key Projects in the National Science & Technology Pillar Program during the 12th Five-Year Plan Period of China (Contract No: 2011BAD02B03), the Fundamental Research Funds for the Central Universities (JUSRP11226) and supported by the Major State Basic Research Development Program of China (973 Program, 2012CB720802, 2012CB720806). References Barton, S., Bullock, C., Weir, D., 1996. The effects of ultrasound on the activities of some glycosidase enzymes of industrial importance. Enzym. Microb. Technol. 18, 190e194. Bezbradica, D., Mijin, D., Siler-Marinkovic, S., Knezevic, Z., 2006. The Candida rugosa lipase catalyzed synthesis of amyl isobutyrate in organic solvent and solventfree system: a kinetic study. J. Mol. Catal. B: Enzym. 38, 11e16.
Casado, V., Martin, D., Torres, C., Reglero, G., 2012. Phospholipases in food industry: a review. Methods Mol. Biol. (Clifton, N.J.) 861, 495e523. Chemat, F., Grondin, I., Costes, P., Moutoussamy, L., Sing, A., Smadja, J., 2004. High power ultrasound effects on lipid oxidation of refined sunflower oil. Ultraso. Sonochem. 11, 281e285. Chemat, F., Khan, M.K., 2011. Applications of ultrasound in food technology: processing, preservation and extraction. Ultraso. Sonochem. 18, 813e835. Dijkstra, A.J., 2010. Enzymatic degumming. Eur. J. Lipid Sci. Technol. 112, 1178e1189. Flieger, A., Gong, S., Faigle, M., Neumeister, B., 2000. Critical evaluation of p-nitrophenylphosphorylcholine (p-NPPC) as artificial substrate for the detection of phospholipase C. Enzym. Microb. Technol. 26, 451e458. Huang, J., Liu, Y., Song, Z., Jin, Q., Liu, Y., Wang, X., 2010. Kinetic study on the effect of ultrasound on lipase-catalyzed hydrolysis of soy oil: study of the interfacial area and the initial rates. Ultraso. Sonochem. 17, 521e525. Kellens, M., De Greyt, W., 2013. Enzymatic oil recuperation process. US Patent 8,435,766. Li, C., Yoshimoto, M., Ogata, H., Tsukuda, N., Fukunaga, K., Nakao, K., 2005. Effects of ultrasonic intensity and reactor scale on kinetics of enzymatic saccharification of various waste papers in continuously irradiated stirred tanks. Ultraso. Sonochem. 12, 373e384. Li, C., Yoshimoto, M., Tsukuda, N., Fukunaga, K., Nakao, K., 2004a. A kinetic study on enzymatic hydrolysis of a variety of pulps for its enhancement with continuous ultrasonic irradiation. Biochem. Eng. J. 19, 155e164. Li, H., Pordesimo, L., Weiss, J., 2004b. High intensity ultrasound-assisted extraction of oil from soybeans. Food Res. Int. 37, 731e738. Liu, F., Liu, Y., Liu, X., Shan, L., Wang, X., 2011. Preparation of deoiled soy lecithin by ultrafiltration. J. Am. Oil Chem. Soc. 88, 1807e1812. Liu, Y., Jin, Q., Shan, L., Liu, Y., Shen, W., Wang, X., 2008. The effect of ultrasound on lipase-catalyzed hydrolysis of soy oil in solvent-free system. Ultraso. Sonochem. 15, 402e407. Mason, T., Paniwnyk, L., Lorimer, J., 1996. The uses of ultrasound in food technology. Ultraso. Sonochem. 3, S253eS260. Mukataka, S., Kobayashi, T., Takahasshi, J., 1985. Kinetics of enzymatic hydrolysis of lipids in biphasic organic-aqueous systems. J. Ferment. Technol. 63, 461e466. Paulitz, B.G.A., Segers, J.C., Spits, A.J., 1987. Process relating to triglyceride oils. EP Patent 0,122,727. Pingret, D., Durand, G., Fabiano-Tixier, A.S., Rockenbauer, A., Ginies, C., Chemat, F., 2012. Degradation of edible oil during food processing by ultrasound: electron paramagnetic resonance, physicochemical, and sensory appreciation. J. Agric. Food Chem. 60, 7761e7768. Pingret, D., Fabiano-Tixier, A.S., Chemat, F., 2013. Degradation during application of ultrasound in food processing: a review. Food Control 31, 593e606. Sakakibara, M., Wang, D., Takahashi, R., Takahashi, K., Mori, S., 1996. Influence of ultrasound irradiation on hydrolysis of sucrose catalyzed by invertase. Enzym. Microb. Technol. 18, 444e448. Shah, S., Sharma, A., Gupta, M., 2005. Extraction of oil from Jatropha curcas L. seed kernels by combination of ultrasonication and aqueous enzymatic oil extraction. Bioresour. Technol. 96, 121e123. Sharma, A., Gupta, M., 2006. Ultrasonic pre-irradiation effect upon aqueous enzymatic oil extraction from almond and apricot seeds. Ultraso. Sonochem. 13, 529e534. Sivakumar, V., Ravi Verma, V., Rao, P., Swaminathan, G., 2007. Studies on the use of power ultrasound in solideliquid myrobalan extraction process. J. Clean. Prod. 15, 1813e1818. Suslick, K.S., Price, G.J., 1999. Applications of ultrasound to materials chemistry. Ann. Rev. Mater. Sci. 29, 295e326. Szuhaj, B., 1983. Lecithin production and utilization. J. Am. Oil Chem. Soc. 60, 306e 309. Van Nieuwenhuyzen, W., 1976. Lecithin production and properties. J. Am. Oil Chem. Soc. 53, 425e427. Vilkhu, K., Mawson, R., Simons, L., Bates, D., 2008. Applications and opportunities for ultrasound assisted extraction in the food industry e a review. Innov. Food Sci. Emerg. 9, 161e169. Wan, C., Huang, F., Li, W., Niu, Y., 2007. Effects of ultrasonic on removing nonhydratable phospholipids in oil by phospholipase A1. China Oils Fats 2, 33e36. Wu, Y., Wang, T., 2003. Soybean lecithin fractionation and functionality. J. Am. Oil Chem. Soc. 80, 319e326. Zhang, Q.A., Zhang, Z.Q., Yue, X.F., Fan, X.H., Li, T., Chen, S.F., 2009. Response surface optimization of ultrasound-assisted oil extraction from autoclaved almond powder. Food Chem. 116, 513e518. Zhang, Z.S., Wang, L.J., Li, D., Jiao, S.S., Chen, X.D., Mao, Z.H., 2008. Ultrasoundassisted extraction of oil from flaxseed. Sep. Purif. Technol. 62, 192e198.
Contents lists available at ScienceDirect
Journal of Cleaner Production journal homepage: www.elsevier.com/locate/jclepro
Effect of ultrasound treatment on oil recovery from soybean gum by using phospholipase C Xiaofei Jiang, Ming Chang, Xiaosan Wang, Qingzhe Jin, Xingguo Wang* State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 August 2013 Received in revised form 20 January 2014 Accepted 21 January 2014 Available online 31 January 2014
Comparative studies of enzymatic oil recovery process of water degummed soybean gum (WDSG) were carried out in mechanical-stirring (MS) and ultrasonic-assisted mechanical-stirring (UAMS) systems. The influences of ultrasonic power (0.68e1.52 W/cm3), gum/water ratio (w/w) (1/0.5e1/3.0), reaction temperature (40e65 C), enzyme dosage (200e1000 mg/kg) and reaction time (2e7 h) were investigated subsequently. A suitable ultrasonic power of 1.20 W/cm3 was determined to guarantee satisfactory oil recovery efficiency and enzyme activity. Compared to the MS system, less time, water and enzyme dosage were required in the UAMS system for the oil recovery process, and the thermal stability of phospholipase C (PLC) under ultrasound treatment was increased. Analysis of the quality of recovered oil showed that indices including phosphorus content, free fatty acid content, peroxide value, fatty acid composition, color, smell and taste could meet the requirements of crude soybean oil. However, ultrasound treatment could accelerate the primary oxidation of recovered oil due to the effect of cavitation. These results indicate that using PLC to recover oil from WDSG with ultrasound treatment is feasible and effective, and more attention should be paid on the oxidative stability of recovered oil for its further application. Ó 2014 Published by Elsevier Ltd.
Keywords: Phospholipase C Oil recovery Ultrasound Water degummed soybean gum
1. Introduction Soybean gum obtained from the water degumming process is a byproduct during the oil refining process, and the main composition of the gum is neutral oil, phospholipids and water. The gum can be easily deteriorated under high temperature, releasing unpleasant smell, and resulting in a negative impact on the environment (Van Nieuwenhuyzen, 1976). However the phospholipids contained in the gum are well recognized for excellent surface-active properties and are extensively used in food, cosmetics, pharmaceutical and chemical industries (Wu and Wang, 2003). In addition, the
Abbreviations: WDSG, water degummed soybean gum; MS, mechanical-stirring; UAMS, ultrasonic-assisted mechanical-stirring; PLC, phospholipase C; DAG, diacylglycerols; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidic acid; NPPC, p-nitrophenylphosphorylcholine; FFA, free fatty acid; PV, peroxide value; OSI, oxidative stability; FA, fatty acid composition; FID, flame ionization detector; FAME, fatty acid methyl ester; RSD, relative standard deviation. * Corresponding author. Tel./fax: þ86 510 85876799. E-mail addresses: [email protected] (X. Jiang), [email protected] (M. Chang), [email protected] (X. Wang), [email protected] (Q. Jin), [email protected] (X. Wang). 0959-6526/$ e see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jclepro.2014.01.060
water degummed soybean gum (WDSG) usually contains a substantial amount of neutral oil (30e40% on the dry basis). Therefore it is quite valuable to recover the oil from the gum, as the price of soybean oil is much higher than the soybean gum. Conventional preparation of deoiled phospholipids from the WDSG is based on acetone extraction utilizing the difference in solubility between phospholipids and triacylglycerols (TAGs) (Szuhaj, 1983). This process usually includes repeated extraction procedures consuming large amounts of acetone, which is either costly or ineffective. In addition, the acetone used in the extraction process or retained in the final products can be a potential hazard to human health (Liu et al., 2011). Therefore, it is necessary to find a more ecofriendly way to recover high-quality neutral oil from the soybean gum and obtain deoiled phospholipids. In recent years, enzymatic-assisted oil recovery process has attracted great attention as it can avoid the consumption of solvent during the whole recovery process. A previous patent (Paulitz et al., 1987) showed that enzymes could catalyze the hydrolysis of gum, facilitating the formation of an oil phase. The presence of lysophospholipids in the gum could promote the oil recovery. More recently, an oil recuperation process has been disclosed in which wet gums are mixed with an acid or a phospholipase (Kellens and De Greyt, 2013). The resulting mixture is then allowed to separate
238
X. Jiang et al. / Journal of Cleaner Production 69 (2014) 237e242
into two or more phases, the oily phase of which is then recuperated. Nevertheless, low oil recovery is one of the major challenges for this process which may be overcome by utilizing selected enzymes in combination with appropriate treatment. Phospholipase C (PLC) is a kind of hydrolytic enzyme that can hydrolyze the bond between the acylglycerol and the phosphate group of phospholipids. Accordingly, it liberates diacylglycerols (DAG) which will not be removed from the oil (Casado et al., 2012). The gum obtained by water degumming process contains appreciable amounts of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which can be released to DAG on exposure to PLC. Phospholipase-catalyzed reaction takes place at the interface between the aqueous phase containing the enzyme and the oil phase containing neutral oil and phospholipids, which means that the rate of this reaction increases when the interfacial area is increased (Dijkstra, 2010). In recent years, ultrasound is considered as an effective and non-polluting method that can accelerate heat and mass transfer during the extraction process (Chemat and Khan, 2011; Sivakumar et al., 2007). It has been successively used in oil extraction from soybeans (Li et al., 2004b), flaxseed (Zhang et al., 2008) and almond powder (Zhang et al., 2009). Besides, some researchers have used ultrasound as a kind of pretreatment during the aqueous enzymatic oil extraction process to facilitate the oil recovery (Shah et al., 2005; Sharma and Gupta, 2006). Ultrasound irradiation, a mechanical rather than an electromagnetic wave, is an alternative method to increase interfacial area in enzymatic reactions (Huang et al., 2010; Mason et al., 1996). An early study disclosed a kinetic model for enzymatic reactions by taking the interfacial area into account (Mukataka et al., 1985). Though some studies have shown that appropriate treatment of ultrasound can enhance the activity and reaction rate of some kinds of enzymes (Li et al., 2005, 2004a; Liu et al., 2008), little work has been devoted to the effect of ultrasound on the activity of PLC during the enzymatic oil recovery process of soybean gum. The objective of this study is to investigate the effect of PLC on oil recovery process of WDSG. Comparative studies were carried out in mechanical-stirring (MS) and ultrasonic-assisted mechanical-stirring (UAMS) systems to evaluate the effect of ultrasound on enzymatic oil recovery process. Quality analysis of recovered oil was conducted to estimate whether the indices of recovered oil could meet the requirements of the standards.
PLC activity was determined using NPPC as a substrate (Flieger et al., 2000). We defined 1 U/g of PLC as the amount of enzyme needed to produce 1 nM nitrophenol per minute by the hydrolysis of NPPC under appropriate conditions: 100 mL PLC was added to 2 mL of NPPC solution (10 mM NPPC, 250 mM TriseHCl (pH 7.2), 600eg/L sorbitol and 1 mM ZnCl2) in a test tube. The tube was incubated at 37 C for 30 min. Substrate hydrolysis was then quantified by measuring absorbance at 410 nm. The activity of PLC was calculated by the following equation:
2. Materials and methods
Activity ðU=gÞ ¼ 1:3636 103 A=t
2.1. Materials WDSG (water content 46.7%; phospholipids content 27.9%; oil content 28.8%) was kindly provided by Donghai Cereal & Oils Co. Ltd. (Zhangjiagang, China). PLC was prepared by our own laboratory using submerged fermentation of a genetically modified Bacillus cereus with activity assayed to be 150 U/g. It had activity on PC and PE, while it had no activity on phosphatidylinositol (PI) or phosphatidic acid (PA). p-Nitrophenylphosphorylcholine (NPPC) with purity higher than 98% was purchased from Sigma Chemical Ltd. (Shanghai, China). All other reagents of analytical grade were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China), and the solutions were prepared in deionized water. 2.2. Equipment MS experiments were carried out in a thermostatic water bath equipped with an agitation unit (IKA, Germany, model Eurostar 40 digital). The agitation unit consisted of a 3-bladed propeller-type impeller with a diameter of 75 mm (IKA, Germany, model R1389),
Fig. 1. The schematic diagram of ultrasonic equipment.
and the reaction temperature could be maintained within 0.2 C of the desired temperature. Ultrasound irradiations were carried out in a 28 kHz ultrasonic water bath, equipped with a thermostatic setup allowing irradiations to be performed within 0.5 C of the desired temperature (Huaneng, China, model HN, tank capacity 27 L). Ultrasonic waves were emitted from the bath bottom through a piezo-electric disk (d ¼ 5.87 cm). The output power of the ultrasound could be adjusted from 40 to 100% of the total power (300 W) by using a control button, and the delivered power dissipated in the medium was estimated by the calorimetric method (Li et al., 2004b).
2.3. Determination of PLC activity
(1)
where A is the absorption value in 410 nm and t (min) is the time used in the substrate hydrolysis reaction.
2.4. PLC oil recovery process in MS system WDSG (100 g) and a certain amount of water (50e300 g) were added to a beaker. The beaker was immersed in a thermostatic water bath equipped with an agitation unit (seen in Section 2.2) and the mixture was preheated to a required temperature of enzymatic reaction (40e65 C) under stirring at 500 rpm. After equilibrating for 10 min, a certain amount of PLC (200e1000 mg/ kg) was added to the mixture to initiate the enzymatic oil recovery reaction. The enzymatic reaction was conditioned at settled temperature (40e65 C) under stirring at 500 rpm. After a certain time of enzymatic treatment (2e7 h), the oil mixture was heated to 95 C for 10 min to inactivate the enzyme and then quickly centrifuged at 10 000 rpm for 10 min. The resulting mixture was separated into three phases, and the oily phase was collected and weighed for calculation of the oil recovery efficiency. All reactions were carried out in triplicate.
X. Jiang et al. / Journal of Cleaner Production 69 (2014) 237e242
2.5. PLC oil recovery process in UAMS system The first several steps of the process were carried out similar to the enzymatic oil recovery process without ultrasonic treatment (seen in Section 2.4). After the addition of enzyme, the mixture was quickly placed in an ultrasonic water bath equipped with an agitation unit (seen in Section 2.2). The schematic diagram of ultrasonic equipment was shown in Fig. 1, and the distance of reactor from tank bottom was about 10 cm. The enzymatic oil recovery process was carried out under stirring at 500 rpm in combination with the ultrasonic irradiation (120e270 W; 28 kHz) at settled temperature (40e65 C). After a certain time of ultrasonic-assisted enzymatic treatment (2e7 h), the oil mixture was heated to 95 C for 10 min to inactivate the enzyme and then quickly centrifuged at 10 000 rpm for 10 min. The resulting mixture was separated into three phases, and the oily phase was collected and weighed for calculation of the oil recovery efficiency. All reactions were carried out in triplicate. 2.6. Determination of oil recovery efficiency During the enzymatic oil recovery process, the DAG generated by PLC can also be counted as neutral oil, which means that the total mass of oil can be increased under PLC treatment. Thus it is reasonable for the oil recovery efficiency to exceed 100% under appropriate extraction conditions. The oil recovery efficiency was calculated by the following equation:
Oil recovery efficiency ð%Þ ¼ ðm1 =m2 Þ 100%
(2)
where m1 (g) is the mass of recovered oil obtained after the enzymatic oil recovery process, m2 (g) is the mass of oil contained in WDSG. 2.7. Determination of recovered oil quality indices The phosphorus content of the samples was determined according to GB/T 5537-2008 (National Standard of the People’s Republic China, 2008). The free fatty acid (FFA) content of the samples was determined in accordance with GB/T 5530-2005. The peroxide value (PV) of the samples was determined in accordance with GB/T 5538-2005. The water content of the samples was measured by Karl Fisher titration. The smell and taste of the samples were determined according to GB/T 5525-2008. The color of the samples was measured by Lovibond method (GB/T 22460-2008). The oxidative stability (OSI) of the samples was analyzed by the Rancimat method using a Metrohm 743 Rancimat (Herisau, Switzerland) instrument. Samples of 3.0 g were analyzed under a heating block of 110 C at a constant air flow of 10 L/h. The fatty acid (FA) composition of the samples was analyzed by GC equipped with a PEG-20000 capillary column (30 m 0.25 mm 0.25 mm), a flame ionization detector (FID) and nitrogen as carrier gas. The injection was performed in split mode with a split ratio of 1:50. The fatty acid methyl ester (FAME) solution was prepared by the method of BF3-CH3OH (GB/T 17376-2008). 1.0 mL of FAME solution was injected at an injector temperature of 250 C, column temperature: 100 C (3 min), 100e 180 C (20 C/min), 180 C (4 min), 180e235 C (12 C/min), 235 C (15 min), FID temperature of 250 C and carrier gas (N2) flow of 60 mL/min. The FA composition reported was based on the area response using FID. All experiments were carried out in triplicate. 2.8. Statistical analysis All experiments were performed in triplicate and the mean values and standard deviations were calculated. Statistical analysis
239
was performed using Origin 8.0 software (OriginLab Ltd., USA). One-way ANOVA was carried out by using Tukey adjustment to determine the significant difference between treatments. Significant differences were declared at P 0.05.
3. Results and discussion 3.1. The effect of ultrasonic power on oil recovery efficiency For enzymatic reactions with ultrasonic treatment, the power of ultrasound is an important parameter. The effect of ultrasonic power on different kinds of enzymes is quite different. In our study, the intensities of the output power were adjusted to 120, 150, 180, 210, 240 and 270 W. The actual values of powers dissipated in the water were measured by the calorimetric method (Li et al., 2004b) and their values were 0.68, 0.85, 1.02, 1.20, 1.36 and 1.53 W/cm3 with experimental relative standard deviation (RSD) of 10.8, 9.6, 8.3, 10.5, 9.8 and 8.7%, respectively. Fig. 2 showed the effect of ultrasonic power on the oil recovery efficiency from WDSG. In both of the two ultrasonic-assisted systems with or without mechanical stirring, the oil recovery efficiency from WDSG increased rapidly with the increase in ultrasonic power in the lower range (from 0.68 to 1.20 W/cm3), which might be attributed to the cavitation effect caused by ultrasound (Vilkhu et al., 2008). When cavitation bubbles collapse near the phase boundary of two immiscible liquids, the resultant shock wave can provide a very efficient stirring/mixing of the layers. Besides, increasing ultrasonic power in an appropriate range could promote the mass transfer of the reaction system and reduce the substrate inhibition and aggregation on the interfacial area, resulting in a finer dispersion of enzyme and gum in the mixture. When the ultrasonic power exceeded 1.20 W/cm3, a decrease of oil recovery efficiency was observed, which might be explained that too high intensity of ultrasonic power could reduce or even inactivate the enzyme activity (Chemat and Khan, 2011). These results are somewhat consistent with other researchers who indicated that ultrasonic waves of low intensity had a small effect on the mass transfer of the solution compared to the high intensity which could accelerate the mass transfer. However, too high intensity of the ultrasonic waves might lead to the disruption of the enzyme (Bezbradica et al., 2006; Sakakibara et al., 1996).
Fig. 2. Effect of ultrasound power on the oil recovery efficiency: gum/water ratio ¼ 1/ 1.5 (w/w), reaction temperature ¼ 45 C, enzyme dosage ¼ 600 mg/kg, reaction time ¼ 2 h. In ultrasonic-assisted system, no stirring (d-d); in ultrasonic-assisted mechanical-stirring system, stirring ¼ 500 rpm (dCd).
240
X. Jiang et al. / Journal of Cleaner Production 69 (2014) 237e242
Compared to the ultrasonic-assisted system without mechanical-stirring, the oil recovery efficiency of the UAMS system was much higher (Fig. 2), which meant that mechanical stirring had its own advantage in facilitating the dispersion of gum and enzyme in the mixture when the same reaction time was used. To obtain the maximal oil recovery efficiency and save the reaction time, we selected UAMS system with ultrasonic power of 1.20 W/cm3 for further studies.
3.2. The effect of ultrasound on optimum gum/water ratio The gum/water ratio (w/w) is an important factor in the enzymatic oil recovery process. The gum would be agglomerated and could not be fully dispersed in the reaction mixture if the water addition was not enough. Enough water could decrease the viscosity of the mixture, facilitate the dispersion of the gum and enzyme, and provide a larger interfacial area between the reaction phases. The gum/water ratios from 1/0.5 to 1/3 (w/w) were selected to study the effect of water amount on the oil recovery efficiency. As shown in Fig. 3, in MS system, the oil recovery efficiency increased gradually with the increase in water amount from 1/0.5 to 1/2.5 (w/w), and then its value decreased slightly with further addition of water to 1/3 (w/w). In the UAMS system, the highest oil recovery efficiency was at the gum/water ratio of 1/2 (w/w). These results might be explained that there existed a critical point of water amount in the two systems, and the substrates (gums) may already be saturated by water at the critical point, thus further addition of water had no more contribution to the oil recovery efficiency. Besides, excess of free water may dilute the concentration of the enzyme in the reaction mixture, leading to a decrease in the PLC catalytic efficiency. The optimum values of the gum/water ratios (w/w) were 1/2.5 and 1/2 for MS and UAMS systems, respectively. It meant that less water was required to saturate the substrates under ultrasonic-assisted treatment. These results might be attributed to the emulsification effect of ultrasound, which could produce more interfacial area between the reaction phases when the same water amount was added and could accelerate the mass transfer in the reaction systems (Liu et al., 2008).
Fig. 4. Effect of temperature on the oil recovery efficiency: enzyme dosage ¼ 600 mg/ kg, reaction time ¼ 2 h, stirring ¼ 500 rpm. In mechanical-stirring system, gum/oil ratio ¼ 1/2.5 (w/w) (d-d); in ultrasonic-assisted mechanical-stirring system, ultrasonic power ¼ 1.20 W/cm3, gum/oil ratio ¼ 1/2.0 (w/w) (dCd).
3.3. The effect of ultrasound on optimum temperature In our study, the effect of temperature on enzymatic oil recovery efficiency was determined within the temperature range from 40 to 65 C. As shown in Fig. 4, the oil recovery efficiency increased slightly with increasing temperature up to 55 C, and then decreased sharply when the temperature was above 65 C, which meant that most of the activity of PLC was lost at 65 C in the MS system. In the UAMS system, the optimum temperature was also observed at 55 C. However, only a slight decrease of the oil recovery efficiency occurred when the temperature was above 65 C, which meant that a majority of the activity of PLC could be remained at 65 C in the UAMS system. These results indicated that ultrasonic treatment could increase the activity and promote the thermal stability of PLC, and the effect of ultrasonic cavitation could provide full contact of PLC with substrates to produce stable enzyme-substrate intermediates, thus enhanced thermal stability of the enzyme (Wan et al., 2007). In further experiments, we selected the reaction temperature of 55 C for the two systems. 3.4. The effect of ultrasound on optimum enzyme dosage
Fig. 3. Effect of gum/water ratio (w/w) on the oil recovery efficiency: reaction temperature ¼ 45 C, enzyme dosage ¼ 600 mg/kg, reaction time ¼ 2 h, stirring ¼ 500 rpm. In mechanical-stirring system (d-d); in ultrasonic-assisted mechanical-stirring system, ultrasonic power ¼ 1.20 W/cm3 (dCd).
Fig. 5 showed the effect of enzyme dosage (relative to the mass of gum) on the oil recovery efficiency of WDSG. In the MS system, the efficiency of oil recovery increased fast when the enzyme dosage was below 800 mg/kg and showed a slight decrease when the addition of PLC exceeded 800 mg/kg. By contrast, in the UAMS system, the oil recovery efficiency continuously increased when the PLC dosage increased from 200 to 1000 mg/kg. Decrease in oil recovery efficiency along with increasing enzyme dosage was not observed in the UAMS system, which was quite different from the results obtained from the MS system. Besides, when the same amounts of PLC were added, the ultrasonic-assisted treatment could effectively recover more oil from the WDSG compared to the MS treatment. Under ideal conditions, the oil recovery efficiency should increase with the addition of enzyme dosage until maximal oil recovery efficiency was obtained, where enzyme saturated the interface, and then maintained constant at any further increasing in enzyme dosage. The different effects of enzyme dosage under the two systems might be attributed to the effect of ultrasound on
X. Jiang et al. / Journal of Cleaner Production 69 (2014) 237e242
241
Table 1 Overview of the phosphorus, FFA, water, color (R represents red color; Y represents yellow color), FA composition, OSI and PV value of recovered soybean oils after different kinds of treatments. Parameters
Recovered oils Requirements for crude soybean Stirringa oil (GB 1535-2003)
Phosphorus content (%) 0.800 Free fatty acid 4.000 [(KOH)/(mg/g)] Water content (%) 0.200 Colorc R 4.0, Y 35
Fig. 5. Effect of enzyme dosage on the oil recovery efficiency: reaction temperature ¼ 55 C, reaction time ¼ 2 h, stirring ¼ 500 rpm. In mechanical-stirring system, gum/oil ratio ¼ 1/2.5 (w/w) (d-d); in ultrasonic-assisted mechanical-stirring system, ultrasonic power ¼ 1.20 W/cm3, gum/oil ratio ¼ 1/2.0 (w/w) (w/w) (dCd).
dispersion. UAMS treatment could create a better effect on the dispersion of gum in the reaction mixture, which contributed to a more homogeneous reaction system and facilitated dispersion of PLC on the interfacial area between the phases (Mason et al., 1996; Suslick and Price, 1999). Within the range of enzyme dosage (200e 1000 mg/kg) chosen in our study, the efficiency of oil recovery increased continuously in the UAMS system, which meant that higher interfacial area and higher enzyme quantity could be accommodated with the treatment of ultrasound. However, in the MS system, the effect of mechanical stirring was not good enough to provide a fine contact of PLC to the substrates. Besides, enzyme dosage at higher concentrations is more liable to agglomeration, which will reduce the actual effective reaction area on the phases. Thus, when the addition of PLC was beyond 800 mg/kg, a slight
Fatty acid composition (%) Palmitic Stearic Oleic Linoleic Linolenic Oxidative stability (h) Peroxide value (mmol/kg)
8.0e13.5 2.5e5.4 17.7e28.0 49.8e59.0 5.0e11.0 No requirement 7.500
0.36 0.04 2.49 0.06
Ultrasound þ Stirringb 0.38 0.05d 2.53 0.04d
0.30 0.04 0.28 0.03d R ¼ 2.4, Y ¼ 12 R ¼ 2.7, Y ¼ 11
10.48 0.35 4.45 0.30 24.89 0.19 53.08 0.14 6.48 0.10 6.23 0.07 3.86 0.12
10.98 0.23d 4.93 0.22d 24.73 0.31d 52.36 0.25e 6.32 0.28d 5.68 0.09e 4.13 0.08e
a
Mechanical-stirring system: gum/water ratio ¼ 1/2.5 (w/w), reaction time ¼ 7 h. Ultrasonic-assisted mechanical-stirring system: gum/water ratio ¼ 1/2.0 (w/w), reaction time ¼ 5 h, ultrasonic power ¼ 1.20 W/cm3. PLC dosage ¼ 800 mg/kg, stirring ¼ 500 rpm, reaction temperature ¼ 55 C for the two systems. c The type of cuvette used to determine color is 133.4 mm. d No significant difference between the two treatments (P > 0.05). e Significant difference between treatments (P 0.05). b
decrease of oil recovery efficiency was observed in MS system. In consideration of the cost and oil recovery efficiency of PLC, the dosage of 800 mg/kg was chosen for further study. 3.5. The effect of ultrasound on reaction time Fig. 6 showed the effect of reaction time on the oil recovery efficiency of WDSG in MS and UAMS systems. In the MS system, the oil recovery efficiency could reach 135% after 7 h of enzymatic treatment. However, in the UAMS system, it took less than 5 h for enzymatic treatment to achieve an oil recovery efficiency of more than 145%. These results might be explained by two reasons. Firstly, the ultrasound treatment could increase the interfacial area of the reaction system and facilitate the dispersion of substrates. Secondly, ultrasound treatment might have an effect on the active center of the enzyme, providing rapid and enough combination of the enzyme with substrates (Barton et al., 1996). In consideration of the oil recovery efficiency and reaction time, we selected 7 h and 5 h as the optimum reaction times for the MS and UAMS systems, respectively. 3.6. The effect of ultrasound on the quality of recovered oil
Fig. 6. Effect of reaction time on the oil recovery efficiency: reaction temperature ¼ 55 C, enzyme dosage ¼ 800 mg/kg, stirring ¼ 500 rpm. In mechanicalstirring system, gum/oil ratio ¼ 1/2.5 (w/w) (d-d); in ultrasonic-assisted mechanical-stirring system, ultrasonic power ¼ 1.20 W/cm3, gum/oil ratio ¼ 1/2.0 (w/w) (dCd).
The quality of recovered oils under optimal conditions in MS and UAMS systems are given in Table 1. The phosphorus, FFA and water contents of the recovered oils did not show significant difference (P > 0.05) between the MS and UAMS systems and no apparent abnormal smell or taste was found in the recovered oils treated by ultrasound. The color of the recovered oils under ultrasonic treatment was a little darker than that of the oils without ultrasonic treatment. The PV value of recovered oils treated by ultrasound increased significantly compared to those without ultrasonic treatment (P 0.05), which meant that primary oxidation of recovered oils was accelerated under the treatment of ultrasound. In addition, the value of OSI showed that ultrasonic treatment could reduce the oxidative stability of recovered oils from about 6.23 h to about 5.68 h, which further proved that ultrasound could accelerate
242
X. Jiang et al. / Journal of Cleaner Production 69 (2014) 237e242
the oxidation of oils during the extraction process. The FA composition of recovered oils under ultrasonic treatment showed a slight decrease in the relative percentage of unsaturated fatty acids and an increase in the percentage of saturated fatty acids. It might be explained that unsaturated fatty acids are more susceptible to oxidation, whereas saturated fatty acids are more stable to oxidation (Li et al., 2004b). Previous articles claimed that ultrasound could increase fat and oil degradations (Chemat et al., 2004; Pingret et al., 2012, 2013). In our study, the role of ultrasound as an initiator in the oxidation of recovered oils was also observed. The reason might be explained that metals existing naturally in the WDSG could be responsible for the formation of oxy-radical species to induce the oxidation and the effect of cavitation might accelerate such kind of oxidation. More studies should be carried out to better elucidate the oxidation mechanism of oils treated by ultrasound. Though, the treatment of ultrasound might reduce the oxidative stability of recovered oils to some extent, all of the indices (phosphorus content, FFA content, PV value, FA composition, color, smell and taste) except the water content could meet the requirements of crude soybean oil (GB 1535-2003), which indicated that using PLC to recover the oil from WDSG was feasible. 4. Conclusion The present study showed that ultrasound in combination with PLC treatment was an efficient way to accelerate enzymatic oil recovery reaction rate and enhance oil recovery efficiency of WDSG. The oil recovery efficiency could achieve more than 145% within 5 h under ultrasound treatment. The optimum working temperature of PLC did not change under ultrasound treatment. However the thermostability of PLC was increased under ultrasound treatment. Besides, the water required to saturate the substrates was less under ultrasound treatment. Quality analysis of the recovered oils showed that ultrasound treatment could accelerate the primary oxidation of recovered oils due to the effect of cavitation. All indices except the water content of enzymatic recovered oils could meet the requirements of crude soybean oil (GB 1535-2003). These results indicate that using PLC to recover the oil from WDSG is feasible and ultrasound can be a clean and effective way to reduce the time, water and enzyme dosage used during the oil recovery process. Acknowledgment The work is supported by Key Projects in the National Science & Technology Pillar Program during the 12th Five-Year Plan Period of China (Contract No: 2011BAD02B03), the Fundamental Research Funds for the Central Universities (JUSRP11226) and supported by the Major State Basic Research Development Program of China (973 Program, 2012CB720802, 2012CB720806). References Barton, S., Bullock, C., Weir, D., 1996. The effects of ultrasound on the activities of some glycosidase enzymes of industrial importance. Enzym. Microb. Technol. 18, 190e194. Bezbradica, D., Mijin, D., Siler-Marinkovic, S., Knezevic, Z., 2006. The Candida rugosa lipase catalyzed synthesis of amyl isobutyrate in organic solvent and solventfree system: a kinetic study. J. Mol. Catal. B: Enzym. 38, 11e16.
Casado, V., Martin, D., Torres, C., Reglero, G., 2012. Phospholipases in food industry: a review. Methods Mol. Biol. (Clifton, N.J.) 861, 495e523. Chemat, F., Grondin, I., Costes, P., Moutoussamy, L., Sing, A., Smadja, J., 2004. High power ultrasound effects on lipid oxidation of refined sunflower oil. Ultraso. Sonochem. 11, 281e285. Chemat, F., Khan, M.K., 2011. Applications of ultrasound in food technology: processing, preservation and extraction. Ultraso. Sonochem. 18, 813e835. Dijkstra, A.J., 2010. Enzymatic degumming. Eur. J. Lipid Sci. Technol. 112, 1178e1189. Flieger, A., Gong, S., Faigle, M., Neumeister, B., 2000. Critical evaluation of p-nitrophenylphosphorylcholine (p-NPPC) as artificial substrate for the detection of phospholipase C. Enzym. Microb. Technol. 26, 451e458. Huang, J., Liu, Y., Song, Z., Jin, Q., Liu, Y., Wang, X., 2010. Kinetic study on the effect of ultrasound on lipase-catalyzed hydrolysis of soy oil: study of the interfacial area and the initial rates. Ultraso. Sonochem. 17, 521e525. Kellens, M., De Greyt, W., 2013. Enzymatic oil recuperation process. US Patent 8,435,766. Li, C., Yoshimoto, M., Ogata, H., Tsukuda, N., Fukunaga, K., Nakao, K., 2005. Effects of ultrasonic intensity and reactor scale on kinetics of enzymatic saccharification of various waste papers in continuously irradiated stirred tanks. Ultraso. Sonochem. 12, 373e384. Li, C., Yoshimoto, M., Tsukuda, N., Fukunaga, K., Nakao, K., 2004a. A kinetic study on enzymatic hydrolysis of a variety of pulps for its enhancement with continuous ultrasonic irradiation. Biochem. Eng. J. 19, 155e164. Li, H., Pordesimo, L., Weiss, J., 2004b. High intensity ultrasound-assisted extraction of oil from soybeans. Food Res. Int. 37, 731e738. Liu, F., Liu, Y., Liu, X., Shan, L., Wang, X., 2011. Preparation of deoiled soy lecithin by ultrafiltration. J. Am. Oil Chem. Soc. 88, 1807e1812. Liu, Y., Jin, Q., Shan, L., Liu, Y., Shen, W., Wang, X., 2008. The effect of ultrasound on lipase-catalyzed hydrolysis of soy oil in solvent-free system. Ultraso. Sonochem. 15, 402e407. Mason, T., Paniwnyk, L., Lorimer, J., 1996. The uses of ultrasound in food technology. Ultraso. Sonochem. 3, S253eS260. Mukataka, S., Kobayashi, T., Takahasshi, J., 1985. Kinetics of enzymatic hydrolysis of lipids in biphasic organic-aqueous systems. J. Ferment. Technol. 63, 461e466. Paulitz, B.G.A., Segers, J.C., Spits, A.J., 1987. Process relating to triglyceride oils. EP Patent 0,122,727. Pingret, D., Durand, G., Fabiano-Tixier, A.S., Rockenbauer, A., Ginies, C., Chemat, F., 2012. Degradation of edible oil during food processing by ultrasound: electron paramagnetic resonance, physicochemical, and sensory appreciation. J. Agric. Food Chem. 60, 7761e7768. Pingret, D., Fabiano-Tixier, A.S., Chemat, F., 2013. Degradation during application of ultrasound in food processing: a review. Food Control 31, 593e606. Sakakibara, M., Wang, D., Takahashi, R., Takahashi, K., Mori, S., 1996. Influence of ultrasound irradiation on hydrolysis of sucrose catalyzed by invertase. Enzym. Microb. Technol. 18, 444e448. Shah, S., Sharma, A., Gupta, M., 2005. Extraction of oil from Jatropha curcas L. seed kernels by combination of ultrasonication and aqueous enzymatic oil extraction. Bioresour. Technol. 96, 121e123. Sharma, A., Gupta, M., 2006. Ultrasonic pre-irradiation effect upon aqueous enzymatic oil extraction from almond and apricot seeds. Ultraso. Sonochem. 13, 529e534. Sivakumar, V., Ravi Verma, V., Rao, P., Swaminathan, G., 2007. Studies on the use of power ultrasound in solideliquid myrobalan extraction process. J. Clean. Prod. 15, 1813e1818. Suslick, K.S., Price, G.J., 1999. Applications of ultrasound to materials chemistry. Ann. Rev. Mater. Sci. 29, 295e326. Szuhaj, B., 1983. Lecithin production and utilization. J. Am. Oil Chem. Soc. 60, 306e 309. Van Nieuwenhuyzen, W., 1976. Lecithin production and properties. J. Am. Oil Chem. Soc. 53, 425e427. Vilkhu, K., Mawson, R., Simons, L., Bates, D., 2008. Applications and opportunities for ultrasound assisted extraction in the food industry e a review. Innov. Food Sci. Emerg. 9, 161e169. Wan, C., Huang, F., Li, W., Niu, Y., 2007. Effects of ultrasonic on removing nonhydratable phospholipids in oil by phospholipase A1. China Oils Fats 2, 33e36. Wu, Y., Wang, T., 2003. Soybean lecithin fractionation and functionality. J. Am. Oil Chem. Soc. 80, 319e326. Zhang, Q.A., Zhang, Z.Q., Yue, X.F., Fan, X.H., Li, T., Chen, S.F., 2009. Response surface optimization of ultrasound-assisted oil extraction from autoclaved almond powder. Food Chem. 116, 513e518. Zhang, Z.S., Wang, L.J., Li, D., Jiao, S.S., Chen, X.D., Mao, Z.H., 2008. Ultrasoundassisted extraction of oil from flaxseed. Sep. Purif. Technol. 62, 192e198.