- Email: [email protected]
Ultrasound irradiation promoted enzymatic alcoholysis for synthesis of monoglyceryl phenolic acids in a solvent-free system
Accepted Manuscript Ultrasound irradiation promoted enzymatic alcoholysis for synthesis of monoglyceryl phenolic acids in a solvent-free system Chunfang Xu, Haiping Zhang, Jie Shi, Mingming Zheng, Xia Xiang, Fenghong Huang, Junyong Xiao PII: DOI: Reference:
S1350-4177(17)30412-1 http://dx.doi.org/10.1016/j.ultsonch.2017.09.016 ULTSON 3864
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
12 July 2017 6 September 2017 7 September 2017
Please cite this article as: C. Xu, H. Zhang, J. Shi, M. Zheng, X. Xiang, F. Huang, J. Xiao, Ultrasound irradiation promoted enzymatic alcoholysis for synthesis of monoglyceryl phenolic acids in a solvent-free system, Ultrasonics Sonochemistry (2017), doi: http://dx.doi.org/10.1016/j.ultsonch.2017.09.016
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 proof before it is published in its final 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.
Ultrasound irradiation promoted enzymatic alcoholysis for synthesis of monoglyceryl phenolic acids in a solvent-free system Chunfang Xua1, Haiping Zhanga1, Jie Shia, Mingming Zhengab*, Xia Xiangab, Fenghong Huanga*, Junyong Xiaob a
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Oil crops
and Lipids Process Technology National & Local Joint Engineering Laboratory, Key Laboratory of Oilseeds processing, Ministry of Agriculture, Hubei Key Laboratory of Lipid Chemistry and Nutrition, Wuhan 430062, China b
Functional Oil Laboratory Associated by Oil Crops Research Institute, Chinese
Academy of Agricultural Sciences and Infinite (China) Co. LTD, Guangzhou 51000, China
1. These authors contributed equally to this work.
1
Abstract Monoglyceryl phenolic acids (MPAs) were known as the natural hydrophilic antioxidants which could be used in different fields such as food, pharmaceutical, cosmetic etc. A novel enzymatic route of MPAs synthesis by the alcoholysis of phenolic acid ethyl esters with glycerol under ultrasound irradiation in solvent free system was developed. Optimization of reaction parameters shows that a high conversion of above 97.4% can be obtained under the following conditions: phenolic acid ethyl esters to glycerol molar ratio of 1:10, with 6% catalyst (Novozym 435), at 60OC and 200 rpm, with ultrasound input of 250 W, at 20 kHz frequency. Compared to the conventional stirring method, the activation energy for phenolic acid ethyl esters conversion was decreased from 65.0 kJ/mol to 32.1 kJ/mol under ultrasound promotion; the apparent kinetic constant (Vm/Km) increased above 1.2-folds; the lipase amount decreased to 50%; the time required for the maximum conversion reduced up to 3-folds without damaging the lipase activity, which is the fastest report for enzymatic synthesis of MPAs.
Keywords: Ultrasound irradiation, enzymatic alcoholysis, monoglyceryl phenolic acids, solvent-free, antioxidation
2
Introduction Phenolic acids are secondary plant metabolites widely existed dispersed in fruits, vegetables and cereals [1,2]. Due to their it antioxidant capacity, phenolic acids usually act as reducing agents, hydrogen donators, and singlet oxygen quenchers [3]. More important, phenolic acids exhibit potential health benefits and pharmacological properties including UV absorbant, anti-inflammatory, anti-carcinogenic and neuroprotective effects [4-6]. However, phenolic acids have low solubility and stability in water or oil system, which limits their application in food processing and cosmetic industries significantly [7,8]. An effective method for expanding application of phenolic acids is to modify their molecular structure and enhance for enhancing the hydrophobic or hydrophilic properties [9-11]. Usually, it can be achieved through enzymatic derivatization by esterifying the carboxylic acid group with long-chain alcohols or glycerol, which should could obtain an amphiphilic molecule without losing its original functional properties [9,10]. As natural derivatives of ferulic acid, monoferuloyl glycerol (MFG) and diferuloyl glycerol (DFG) have been used as UV absorbers and antioxidants in many applications [14,15]. Due to the trace amount of MFG and DFG (<0.1%) in nature [8], it is meaningful to synthesis of glyceryl phenolic acids with different phenolic acids source. Presently, It is time-consuming to produce these modified phenolic acid compounds presently by the lipase-catalyzed transesterification reactions. usually take long time to produce these modified phenolic acid compounds. Furthermore, High 3
enzyme amount, high reaction temperature, long reaction times and low conversions often occurred due to the number of organic solvents capable of solubilizing adequate amounts of both polar phenolic acids and non-polar substrates is limited. high enzyme amount, high reaction temperature, long reaction times and low conversions often occurred. Furthermore, high vacuum was usually indispensable to remove the byproduct including ethanol or water during the whole reaction course [16], which and the complex reaction devices is always required requires complex reaction devices. Therefore, it is still a challenge to develop alternative strategy to improve the efficiency of enzymatic phenolic acids derivatization of phenolic acids. Ultrasound irradiation is an environmentally friendly method and has gained popularity and potential for use in applications in organic chemistry and in biotechnology [17,18]. Considering the violent collapse of enormous bubbles simultaneously, tremendous generation of heat and pressure occur, which could be helpful to remove the byproduct like ethanol without vacuum. The micro jet was is also formed which helps to create turbulence in the reaction system [19]. Based on the effects mentioned above, ultrasound can accelerate chemical transformation, improve yields and produce novel chemical reactions compared with tradition methods. Ultrasound could improve the enzyme performance, increase the product yield and make reaction condition more moderate. It is especially suitable for enzymatic heterogeneous systems using immiscible substrates and catalysts [20-25]. Ultrasonic bath was commonly used to provide the ultrasound irradiation. However, it is difficult to realize production on a large scale by the limit of the device 4
volume. Furthermore, only a fraction of the ultrasound energy could transfer to the reaction system, because of the indirect transmittal mode. In contrast, ultrasound carrying out with microtip probe could be more effective in enzymatic reactions. Recently, ultrasound irradiation has been used to accelerate the enzymatic reactions such as esterification of phytosterol, flavonoids and methanol with fatty acids in organic solvent [20,21,24]. However, the application of this novel ultrasound mode to enzymatic reactions has been less extensively studied. The main objective of this study is to investigate the enzymatic alcoholysis of phenolic acid ethyl esters with glycerol under microtip probe ultrasound irradiation in a solvent free system. A sequential experimental strategy was carried out to systematically evaluate the effect of substrates concentration, phenolic acid ethyl esters to glycerol molar ratio, ultrasonic power on the conversion and enzyme activity. To the best of our knowledge, concerning the enzymatic alcoholysis of phenolic acid ethyl esters with glycerol under ultrasonic irradiation, a systematic study for accelerating the synthesis as well as the kinetic and thermodynamics studies concerning the enzymatic alcoholysis of phenolic acid ethyl esters with glycerol under ultrasonic irradiation has not been reported.
2． ．Experimental 2.1. Materials Ethyl ferulate (EF), Ethyl caffeate (EC) were purchased from Aladdin Reagent (Shanghai, China). Glycerol (99%, dehydration using an activated 3Å molecular sieve 5
before being used), ethanol (95%) and glacial acetic acid (HPLC grade) were purchased from Tianjin Guoyao Chemical Co., Ltd. (Tianjin, China). Novozym 435 (immobilized on polyacrylic resin) from Candida antarctica were purchased from Novozymes (Bagsvaerd, Denmark). Methanol (HPLC grade) was purchased from Merck KGaA (Darmstadt, Germany). 2.2. Equipment Experiments were carried out in a 100 mL beaker with thermostatic water bath with magnetic stirrer (Guohua Instrument Co., Ltd., Changzhou, China) and microtip probe (diameter of 13 mm) connected to a multi-frequency sonochemistry generator (Chengdu Jiuzhou Ultrasonic Technology Co., Chengdu, China) The ultrasonic unit has an power output of 100-300 W. An SHIMADZU LC-M20A series HPLC system was used to monitor the progress of the reactions.
2.3. Ultrasound irradiation synthesis and purification of monoglyceryl phenolic acids A typical enzymatic alcoholysis was conducted by combining 0.3 mol of glycerol, a certain amount of phenolic acid ethyl esters (0.9-5.4 mol) and lipase (2%-12% of the total weight of the substrate). The mixtures were added into a 100 mL beaker which would be placed in a thermostatic oil bath with microtip probe for ultrasound enhancement and magnetic agitation at 250 rpm for a certain time. During the reactions, samples were withdrawn from the mixture every certain time to analyze for reaction progress. All analyzes were performed in triplicate and the results were 6
reported as the mean standard deviation. Following monoglyceryl phenolic acids preparation, immobilized lipase and molecular sieves were was filtered from the mixture; One mL liquid raw product was extracted with 3 mL water for three times to remove the excess glycerol. After centrifuge at 5000 rpm for 5 min, the white powder was recovered and then dried by freeze-drying. The obtained monoglyceryl phenolic acids were subject to HPLC analyzed for purity.
2.4. Kinetics study Taking into account the results obtained in the experiments, the Arrhenius equation were acquired from the plot of reaction rate against temperature. The Michaelis-Menten equation was got in a certain amount of glycerol with various phenolic acid ethyl ester concentrations at the optimized condition. The initial reaction rates (v0, mol/ (L min)) were determined through the yield-time profiles corresponding to the first 12 min of the reactions (20.0% or less phenolic acid ethyl esters conversion), where the profiles were found to be approximately linear. Then the activation energies (Ea, kJ/mol), the maximum reaction rate (Vm, mol/ (L min)) and Michaelis–Menten constants (Km, mol/L) could be calculated through the equations.
2.5. Reuse of the lipase At the end of each round of reaction, the immobilized lipase was separated from the substrate by vacuum filtration and washed with 95% ethanol for twice 7
subsequently. After drying in vacuum at room temperature the lipase can be reused in a subsequent new reaction.
2.6. Qualitative and quantitative analysis of monoglyceryl phenolic acids and diglyceryl phenolic acids The products were analyzed by HPLC with a C18 reverse phase column (5 µm, 250 mm×4.6 mm) fitted with a photodiode array detector (SPD-M20A) at 35 oC, and eluted with a binary gradient of solvent A (water, 0.5% v/v glacial acetic acid) and solvent B (methanol) at 1 mL/min. The elution sequence consisted, consecutively, of a linear gradient from 20% (v/v) B to 100% B (v/v) over 30 min, followed by 100% B for 2 min, then to 20% B for 8 min. The elution was monitored at 325 nm. The purified products were analyzed using a hybrid, triple quadrupole/linear ion trap mass spectrometer, API 4000 Q-Trap, with an APCI interface performed in positive mode. CUR (curtain gas) pressure: 137.9 kPa; NC (nebulizer current): 27.58 kPa; TEM (temperature): 450◦C; Scan mode: EMS; Scan rate: 4000 u/s; GS1 (ion source gas 1) pressure: 344.75 kPa; DP (declustering potential): 90 V; CE (collision energy): 35 V; mass range: 100-1000 m/z. The products were characterized by 1H NMR spectroscopy using D6-DMSO (0.01% TMS) as a solvent on 600 MHz frequency of Bruker instrument. 2.7. DPPH radicals scavenging ability detection The reaction mixture consisting of 2.5 mL of 0.1 mM DPPH in methanol and 500 µL of sample (1.0 mM in methonal) was incubated at room temperature for 30 8
min. The radicals scavenging activity was measured at 517 nm against pure methanol (blank) using a UV-vis spectrophotometer (DU800, USA) in a 1 cm quartz cell.
3. Results and discussion 3.1. Optimization of the ultrasonic conditions Ultrasonic power is a critical factor that influencing ultrasound irradiation reactions. Ultrasound of relative higher intensity could have more obvious effect on the mass transfer of the solution, thus accelerate the enzymatic-catalyzed reactions. On the contrary, too high intensity of ultrasound could cause the inactivation of enzymes [26]. The influence of ultrasound power on EF conversion and monoferuloyl glycerol (MFG) yield were investigated in the range of 100 W-300 W with the frequency was kept at 20 kHz invariably. As shown in Fig.1, the conversion presented rapid increase with increasing power in the range of 100 W-250 W, and an obvious decrease of conversion was observed when the power exceeded 250 W. MFG yield showed the same variation trend with EF conversion, as more than 92% EF transferred into MFG. The maximum EF conversion and MFG yield reached 98% and 91% respectively in 250 W. It ascribed to the fact that ultrasonic irradiation caused a decrease in the particle size of the immobilized lipase and consequently increased the catalytic surface area, thus increase enzyme and substrate molecule collision frequency [27]. However, too high ultrasonic intensities were reported to reduce or even inactivate the enzyme activity [28]. Considering the EF conversion, stability of lipase and energy consuming, 250 W was selected to study the characteristics of lipase-catalyzed 9
reactions in the following experiments. In order to investigate the ultrasound irradiation time on the conversion of EF, the reaction continue with mechanic agitation under the same condition after a certain ultrasonic irradiation time from 1 h to 4 h. The total reaction time is kept at 4 h. As shown in Fig. 2, the EF conversion and MFG yield presented rapid increase with the increase of ultrasonic irradiation time from 0 h to 4 h. This was because ultrasound could help to reduce particle size of substrate and immobilized lipase, which would be useful to reduce mass transfer limitations. The irradiation time was longer than our previous reports [20, 21], this was due to the high viscosity of glycerol and low solubility of EF in glycerol. When the ultrasonic time was 3 h, the EF conversion has already reached 97%, and the MFG yield almost kept constant with further prolong the reaction time. Thus, both the ultrasound irradiation time and the total reaction time were kept at 3 h.
3.2. Effect of reaction temperature The conversion, solubility of the phenolic acids and the stability as well as activity of the lipase are strongly related to the reaction temperature. The effect of reaction temperature on EF conversion and relative products yields were investigated at different temperatures ranging from 50oC to 75oC. As shown in Fig.3a, under mechanical stirring, the conversion increased with the rising of the temperature from 50oC to 75 oC. Under ultrasound irradiation, the rising of the temperature from 50 o
C-65oC resulted in an increase of EF conversion. It might be due to the fact that 10
relative high temperature can reduce mass transfer limitations, increase the solubility of substrate and enhance the activity of lipase. However, a sharp decrease in EF conversion was observed when the temperature was further increased above 65oC. It might be ascribed to two main reasons, on one hand, excessive high temperature lead to the deactivation of the lipase; on the other hand, the increase of temperature affects the bubble formation and collapse caused by ultrasound cavitation, thus the effect of ultrasound appeared to be weaker at higher temperature [29]. Fig.3b showed the influence of temperature on monoferuloyl glycerol (MFG) and diferuloyl glycerol (DFG) yields under ultrasonic irradiation. MFG and DFG reached the maximum yield at 60oC and 65 oC, respectively. In order to obtain more target product (MFG), 60 oC was selected for the enzymatic alcoholysis under ultrasonic irradiation. The optimized temperature under ultrasound irradiation (60 oC) was much lower than that of conventional stirring (75 oC). It might be ascribe to the fact that the ultrasonic cavitation leaded to huge energy release during the process of the bubble formation and collapse. The enzymatic reaction became much easier than that under conventional stirring which needed more energy from external environment.
3.3. Effect of substrate molar ratio The effect of molar ratio of EF to glycerol on the conversion of EF under ultrasound irradiation and mechanical stirring was evaluated ranging from 1:3 to 1:18. As shown in Fig.4a, as the molar ratio changed from 1:3 to 1:10, EF conversion increased from 53.9% to 97.5%. Then, the conversion was almost maintained at the 11
same level when the molar ratio varied from 1:10-1:18. This phenomenon attribute to the high viscosity of the reaction mixture, mass transfer resistance, and strong electron-donating and steric hindrance effects [30]. The proposed critical points were 1:10 and 1:18 for ultrasound irradiation and stirring, respectively, which means that the glycerol amount required under ultrasonic condition is only half of that under stirring condition. The result might be benefit from the dispersion effect of ultrasound, which would produce more contact area between EF and glycerol and accelerate mass transfer of the substrates. Fig.4b showed the effect of substrate molar ratio on MFG and DFG yields. As same as EF conversion, MFG yield showed the same change trend. While DFG reached the highest yield of 7.6% at the molar ratio of 1:6, because the relatively abundant EF could continue react with MFG. 3.4. Effect of enzyme dosage The dependency between the EF conversion and relative products yields with enzyme load was investigated. The enzyme dosage was varied from 2% to 12% according to the weight of the total substrates which were held constant. As shown in Fig.5a, the EF conversion was drastically raised with the increasing of the enzyme load from 2% to 6% under ultrasound, this was ascribed to the reason that the increasing of enzyme load can provide more active sites, increase the probability of enzyme-substrate collision, and enhance higher reaction rate [31]. However, when the dosage further increased to 12%, it showed the same variation trend with 6%, which could be due to biocatalyst agglomeration and possible diffusional problems [32]. According to Sun’s report [30], excessive amounts of enzyme would lead to more 12
byproduct ferulic acid being formed. The enzyme dosage needed (6%) under ultrasound was only half of that under conventional stirring (12%), the result further illustrated that ultrasound irradiation could reduce mass transfer limitations and enhance reaction efficiency significantly. Thus, 12% and 6% enzyme was selected for stirring reaction and ultrasonic reaction in the further experiment, respectively. As shown in Fig.5b, the yield variation trend of MFG and DFG were almost opposite, when enzyme dosage was 6%, MFG yield could got 91.6% and DFG yield was 4.3%.
3.5. Reusability of lipase The reusability of immobilized enzymes which can reduce the cost of enzymatic reactions is rather important for practical application [20]. The study of enzyme reusability was carried out in the presence and absence of ultrasound irradiation. After each reaction, the lipase was separated from the reaction system by filtration and washed with 95% ethanol subsequently, which could effectively remove the residue on the lipase, and release the enzyme activity sites. As shown in Fig. 6, EF conversion was higher with ultrasound irradiation than that with conventional stirring only. In the meanwhile, the initial apparent activity of lipase was about 6-fold higher with ultrasound irradiation than that with conventional stirring. The result further confirmed that the ultrasonic could enhance the conversion and the apparent activity of lipase simultaneously. But after 10 recycles, the EF conversions and apparent activities of lipase under ultrasound irradiation and conventional stirring was were dropped to 51.8%, 36.1% and 40.3 µmol/(g×min), 4.4 µmol/(g×min), respectively. 13
Furthermore, the downward trends of EF conversion in presence and in absence of ultrasound irradiation were almost the same. This result confirmed that ultrasound irradiation could enhance the conversion of enzymatic synthesis of phenolic glycerides without essential damaging to the lipase activity. Compared with conventional methods, ultrasound could improve the enzyme performance, increase the product yield and make reaction condition more moderate.
3.6. Mathematical modeling of the reaction kinetics. The initial reaction rates, defined as the initial EF conversion per unit time (V0, mol/ (L min)), was calculated from six experimental points of EF conversion. The Arrhenius law can be described as follows: lnV0 = ln A − Ea/(R×T)
(1)
where A= the Arrhenius constant, Ea= the activation energy, R= the gas constant, and T= absolute temperature (K) Two plots on behalf of two different reaction conditions of lnV0 versus 1/T shows good linearity at the reciprocal temperatures (Fig. 7b). From the slopes, it is possible to calculate the activation energy. As the feruloyl of EF has electron-donating and steric hindrance effects, it can hinder the compatible reaction of EF to some extent, which makes the activation energy under stirring (65.0 kJ/mol) much higher. But under ultrasound irradiation, ultrasound can reduce the particle size and increase the substrate-enzyme interface area through its high cavitation energy, which allows substrate to access active sites more easily. Then the value of Ea under 14
ultrasound could decrease to 32.1 kJ/mol. As a result, the free energy barrier for ultrasound irradiation is considerably lower as compared to stirring, which means the reaction became much easier with ultrasound irradiation. In this study, the concentration of glycerol was much larger than the other reaction substrate, thus EF and product MFG and DFG could be considered unchanged. The enzymatic alcoholysis in this study can be seen as a “pseudo-first-order” reaction. In addition, EF concentration was controlled in a low level so that enzyme activity would not be inhibited by any substrate. The initial reaction rate can be expressed as: V0=Vm[S]/(Km+[S])
(2)
V0 is the initial reaction rate; Vm is the maximum reaction rate; [S] is the initial concentration of EF; Km is Michaelis constant for EF. And Eq. (2) can be transferred to the Lineweaver-Burk form: 1/V0=Km/Vm*1/[S]+1/Vm
(3)
Initial reaction rates were calculated from the linear portion of the concentration-time profiles (Fig.7a), and the kinetic parameters can be obtained by non-linear regression analysis for the above model [32]. The results could be seen in Table 1. The alcoholysis reaction of ethyl ferulate under ultrasound irradiation, showed increase in the Km (1.06 mol/L) value by 4-fold and Vmax (0.037 mol/(L×min)) value by 5-fold as compared to conventional stirring system, which showed similar tendency with the esterification reaction of D-isoascorbyl palmitate under ultrasound [33]. According to Kuo [34], the value of apparent kinetic constant Vm/Km is often 15
recognized as a suitable evaluation parameter of enzyme performance. The Vm/Km value of ultrasound was 1.2 times higher than that of conventional stirring, it in another aspect showed that ultrasound could improve the enzyme performance thus increase the product yield.
3.7． ．Enzymatic alcoholysis of different phenolic acid ethyl esters with glycerol A comparative study was done on alcoholysis of glycerol with different phenolic acid esters under mechanical stirring and ultrasound irradiation. As shown in Table 2, the conversion of EF and EC using ultrasonic irradiation were significantly higher than that of mechanical stirring. It is noteworthy that the alcoholysis rate under ultrasonic irradiation was above 3 times as that using mechanical stirring, which makes the fastest record for enzymatic synthesis of MPAs. The results suggest that ultrasonic irradiation can be used to accelerate the alcoholysis rate of glycerol with a series of phenolic acid esters with relatively high conversion. Benefit from the generation of heat and high pressure during collapse of bubbles, ultrasonic irradiation could remove the byproduct (enthanol) under normal pressure, which could further accelerate the reaction and increase the conversion. Moreover, the ultrasound irradiation is responsible for increasing interaction between the substrate and lipase by increasing the collisions, micro-stirring, and improved contact [18].
3.8. Characterization of the prepared MPGs The enzymatic alcoholysis products were monitored by HPLC. The liquid 16
chromatograms of ethyl ferulate, ethyl caffeate and corresponding monoglyceryl phenolic acids obtained under optimal conditions are shown in Fig. 8. It is evident that the enzymatic alcoholysis of ethyl phenolic acids could significantly increase the hydrophilic of the product, which could be expressed as the relative shorter retention time. Not any byproduct (ferulic acid and caffee acid) was found from the chromatography. HPLC analysis revealed the presence of a highly pure (>99%) compound after purification, residual ethyl phenolic acids and glyceride were not detected in the chromatography (Fig. 8a1, 8b3). In addition, APCI-MS and NMR was also carried out to characterize the molecular structure. The result confirmed that only a phenolic acid molecular was acylated to the framework of glyceride. In detail, monoglyceryl ferulic acid and monoglyceryl caffeic acid had their molecular ion peaks [M+1]+ at m/z 269 and 255 and [M+Na]+ at m/z 291 and 277, respectively. The NMR data of the alcoholysis product of glycerol with ethyl ferulate was listed in Table 3 and Fig. S1 and the deductive structure was were shown in Fig. 8a. The structure of 1-glyceryl ferulate could be confirmed from the higher chemical shift from the primary hydroxyl (C-5’ in Fig. 8a) of glycerol were compared to those for hydroxyls of C-2’ and C-3’[35].
3.9. DPPH radicals scavenging activity The DPPH radicals scavenging activity was compared with those of phenolic acids, ethyl phenolic acid, tocopherol and TBHQ. As shown in Table 4, the DPPH 17
scavenging activities of monoglyceryl ferulic acid and monoglyceryl caffeic acid were above 92.6%, which were comparable to tocopherol and TBHQ. The monoglyceryl phenolic acids also showed comparable scavenging ability with the corresponding phenolic acids and ethyl phenolic acids, which prove that alcoholysis modification did not affect the original antioxidation activity of the phenolic acids. 4. Conclusion In this study, an efficient enzymatic route for MPAs synthesis by the alcoholysis of EF and glycerol under ultrasound irradiation was developed. The results showed that a promising strategy to overcome mass transfer limitations arising from the use of phenolic acid ester and glyceride as the substrate. Compared with mechanical stirring, the temperature, amount of glycerol, enzyme dosage and reaction time needed were much less under ultrasonic irradiation. The overall alcoholysis reaction rate in the ultrasonic enhancement process (3-12 h) was almost 3-fold as that in the stirring process (6-36 h) without essential damaging to the lipase activity. The apparent kinetic constants showed that ultrasonic enhancement could improve the enzyme performance and the affinity between the substrate and enzyme. Considering the high efficient, mild condition and facile operation, the novel protocol can potentially be explored for other functional lipids modification in the future.
Acknowledgements This work was supported by the National Natural Science Foundation of China (31371843, 31671820), the Basic Applied Research Project of Wuhan City 18
(2016020101010095),
the
program
of
China
Scholarships
Council
(No.
201603250035), Agricultural Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2013-OCRI) and the Director Fund of Oil Crops Research Institute (1610172014006).
References 1. A. Crozier, I. B. Jaganath, M. N. Clifford, Dietary phenolics: chemistry,
bioavailability and effects on health, Cheminform. 40 (2009) 1001-1043. 2. C. Castelluccio, G. P. Bolwell, C. Gerrish, C. Riceevans, Differential distribution
of ferulic acid to the major plasma constituents in relation to its potential as an antioxidant, Biochem. J. 316 (1996) 691-694. 3. P. G. Pietta, Flavonoids as antioxidants, J. Nat. Prod. 63 (2000) 1035-1042. 4. W. Y. Huang, Y. Z. Cai, Y. Zhang, Natural phenolic compounds from medicinal
herbs and dietary plants: potential use for cancer prevention, Nutr. Cancer. 62 (2010) 1-20. 5. C. T. Ho, Phenolic Compounds in Food, ACS Symp. Ser. 1992. 6. T. M. S. Silva, F. P. D. Santos, A. Evangelista-Rodrigues, E. M. S. D. Silva, G. S.
D. Silva, Phenolic compounds, melissopalynological, physicochemical analysis and antioxidant activity of jandaíra (Melipona subnitida) honey, J. Food. Compos. Anal. 29 (2013) 10-18. 7. H. Stamatis, V. Sereti, F. N. Kolisis, Enzymatic synthesis of hydrophilic and
hydrophobic derivatives of natural phenolic acids in organic media, J. Mol. Catal. B-Enzym. 11 (2001) 323-328. 19
8. M. C. Figueroaespinoza, P. Villeneuve, Phenolic acids enzymatic lipophilization, J.
Agr. Food. Chem. 53 (2005) 2779-2787. 9. S. Gholivand, O. Lasekan, C. P. Tan, F. Abas, L. S. Wei, Comparative study of the
antioxidant activities of some lipase-catalyzed alkyl dihydrocaffeates synthesized in ionic liquid, Food Chem. 224 (2017) 365-371. 10. Y. Shi, Y. Wu, X. Lu, Y. Ren, Q. Wang, C. Zhu, D. Yu, H. Wang, Lipase-catalyzed
esterification of ferulic acid with lauryl alcohol in ionic liquids and antibacterial properties in vitro against three food-related bacteria, Food Chem. 220 (2017) 249-256 11. P. D. Tomke, V. K. Rathod, Ultrasound assisted lipase catalyzed synthesis of
cinnamyl acetate via transesterification reaction in a solvent free medium, Ultrason. Sonochem. 27 (2015) 241-246. 12. T. J. Ha, K. Nihei, I. Kubo, Lipoxygenase inhibitory activity of octyl gallate, J.
Agr. Food. Chem. 52 (2004) 3177-3181. 13. K. Nihei, A. Nihei, I. Kubo, Molecular design of multifunctional food additives:
antioxidative antifungal agents, J. Agr. Food. Chem. 52 (2004) 5011-5020. 14. D. L. Compton, J. A. Laszlo, K. O. Evans, Antioxidant properties of feruloyl
glycerol derivatives, Ind. Crop. Prod.36 (2012) 217-221. 15. K. Warner, J. A. Laszlo, Addition of ferulic acid, ethyl ferulate, and feruloylated
monoacyl- and diacylglycerols to salad oils and frying oils, J. Am. Oil. Chem. Soc. 82 (2005) 647-652. 16. S. D. Sun, S. Zhu, Y. L. Bi, Solvent-free enzymatic synthesis of feruloylated 20
structured lipids by the transesterification of ethyl ferulate with castor oil, Food. Chem. 5 (2014) 292-295. 17. T. J. Mason, Ultrasound in synthetic organic chemistry, Chem. Soc. Rev. 26 (1997)
443-451. 18. S. R. Bansode, V. K. Rathod, An investigation of lipase catalysed sonochemical
synthesis: A review, Ultrason. Sonochem.38 (2017) 503-529. 19. S. R. Bansode, V. K. Rathod, Ultrasound assisted lipase catalysed synthesis of
isoamyl butyrate, Process. Biochem. 49 (2014) 1297-1303. 20. M. M. Zheng, L. Wang, F. H. Huang, L. Dong, P. M. Guo, Q. C. Deng, W. L. Li, C.
Zheng, Ultrasonic pretreatment for lipase-catalyed synthesis of phytosterol esters with different acyl donors, Ultrason. Sonochem. 19 (2012) 1015-1020. 21. M. M. Zheng, L. Wang, F. H. Huang, P. M. Guo, F. Wei, Q. C. Deng, C. Zheng, C.
Y. Wan, Ultrasound irradiation promoted lipase-catalyzed synthesis of flavonoid esters with unsaturated fatty acids, J. Mol. Catal. B-Enzym. 95 (2013) 82-88. 22. C. Li, M. Yoshimoto, H. Ogata, N. Tsukuda, K. Fukunaga, K. Nakao, Effects of
ultrasonic intensity and reactor scale on kinetics of enzymatic saccharification of various waste papers in continuously irradiated stirred tanks, Ultrason. Sonochem. 12 (2005) 373. 23. S. B. More, J. T. Waghmare, P. R. Gogate, Ultrasound pretreatment as a novel
approach for intensification of lipase catalyzed esterification of tricaprylin, Ultrason. Sonochem. 36 (2017) 253-261. 24. C. M. Trentin, R. P. Scherer, C. D. Rosa, H. Treichel, D. Oliveira, J. V. Oliveira, 21
Continuous lipase-catalyzed esterification of soybean fatty acids under ultrasound irradiation, Bioprocess Biosyst. Eng. 37 (2014) 841-847. 25. J. Wang, S. Wang, Z. Li, S. Gu, X. Wu, F. Wu, Ultrasound irradiation accelerates
the lipase-catalyzed synthesis of methyl caffeate in an ionic liquid, J. Mol. Catal. B-Enzym. 111 (2015) 21-28. 26. Y. X. Liu, Q. Z. Jin, L. Shan, Y. F. Liu, W. Shen, X. G. Wang, The effect of
ultrasound on lipase-catalyzed hydrolysis of soy oil in solvent-free system, Ultrason. Sonochem. 15 (2008) 402-407. 27. S. L. Liu, X. Y. Dong, F. Wei, X. Wang, X. Lv, Juan Z, L. Wu, S. Y. Quek, H.
Chen, Ultrasonic pretreatment in lipase-catalyzed synthesis of structured lipids with high 1,3-dioleoyl-2-palmitoylglycerol content, Ultrason. Sonochem. 23 (2015) 100. 28. Y. Peng, M. U. Qing, W. Wei, Y. Xiong, J. W. Liu, Z. X. Lu, T. Wu, H. Z. Zhao,
Synthesis of Ascorbyl Fatty Acid Esters from Lard by Ultrasound-assisted Lipase Catalysis, Food Science, 32 (2011) 101-105. 29. S. D. Sun, X. W. Chen, Y. L. Bi, J. N. Chen, G. L. Yang, W. Liu, Functionalized
ionic liquid-catalyzed 1-feruloyl-sn-glycerol synthesis, J. Am. Oil. Chem. Soc. 91 (2014) 759-765. 30. E. L. Soo, A. B. Salleh, M. Basri, R. N. Z. A. Rahman, K. Kamaruddin, Response
surface methodological study on lipase-catalyzed synthesis of amino acid surfactants, Process Biochem. 39 (2004) 1511-1518. 31. G. D. Yadav, S. Devendran, Lipase catalyzed synthesis of cinnamyl acetate via 22
transesterification in non-aqueous medium, Process. Biochem. 47 (2012) 496-502. 32. J. H. Huang, Y. F. Liu, Q. Z. Jin, X. J. Wu, X. G. Wang, Z. H. Song,
Enzyme-Catalyzed Synthesis of Monoacylglycerols Citrate: Kinetics and Thermodynamics, J. Am. Oil. Chem. Soc. 89 (2012) 1627-1632. 33. F. J. Cui, H. X. Zhao, W. J. Sun, Z. Wei, S. L. Yu, Q. Zhou, Y. Dong, Ultrasound
assisted
lipase-catalyzed
synthesis
of
D-isoascorbyl
palmitate:
process
optimization and kinetic evaluation, Chem. Cent. J. 7 (2013) 180-190. 34. C. H. Kuo, F. W. Hsiao, J. H. Chen, C. W. Hsieh, Y. C. Liu, C. J. Shieh, Kinetic
aspects of ultrasound-accelerated lipase catalyzed acetylation and optimal synthesis of 4'-acetoxyresveratrol, Ultrason. Sonochem. 20 (2013) 546-552. 35. M. Tsuchiyama, T. Sakamoto, T. Fujita, S. Murata, H. Kawasaki, Esterification of
ferulic acid with polyols using a ferulic acid esterase from Aspergillus niger, Biochim. Biophys. Acta. 1760 (2006) 1071-1079.
23
Figure Captions Fig. 1. Effect of ultrasonic power on EF conversion and MFG yield. The ultrasonic frequency was 20 kHz, ultrasonic time was 4 h; the molar ratio of EF to glycerol was 1:10 (mol: mol), the enzyme dosage is 10% by total weight of substrates, 200 rpm, 60℃.
Fig. 2. Effect of ultrasonic time on EF conversion and MFG yield. The reaction condition was same as that described in Fig.1 except the ultrasonic power was 250 W.
Fig. 3. Effect of temperature on EF conversion under ultrasound irradiation and conventional stirring (a), effect of temperature on MFG and DFG yields under ultrasound irradiation (b). Ultrasound irradiation condition: 250 W, 20 kHz, 3 h. The molar ratio of EF to glycerol was 1:10 (mol: mol), the enzyme dosage is 10% by total weight of substrates, 200 rpm.
Fig. 4. Effect of substrate molar ratio on EF conversion under ultrasound irradiation and conventional stirring (a); effect of substrate molar ratio on MFG and DFG yields under ultrasound irradiation (b). The reaction conditions were same as that described in Fig.3 except the substrate molar ratio.
Fig. 5. Effect of enzyme dosage on EF conversion under ultrasound irradiation and conventional stirring (a), effect of enzyme dosage on MFG and DFG yield under ultrasound irradiation (b). The reaction condition was same as that described in Fig.4 24
except the enzyme dosage.
Fig. 6. Reuse of the lipase for enzymatic transeaterification of EF in presence and absence of ultrasound irradiation.
Fig. 7. Lineweaver-Burk plot of the reciprocal of initial reaction rate and EF concentrations (a) and Arrhenius plot of the reciprocal of temperature and the logarithm of initial reaction rate (b).
Fig. 8. Liquid chromatograms of alcoholysis products (a1, b3) and purified monoglyceryl ferulic acid (a2) and purified monoglyceryl caffeic acid (b4). Peaks: (1) monoglyceryl ferulic acid; (2) diglyceryl ferulic acid; (3) ethyl ferulate; (4) monoglyceryl caffeic acid; (5) diglyceryl caffeic acid; (6) ethyl caffeate.
25
26
27
28
29
30
31
32
33
Table 1. Kinetic parameters for the enzymatic alcoholysis of EF with glycerol in solvent-free system. Values Kinetic parameters Ultrasound
Stirring
Vmax (mol/(L×min))
0.037±0.004
0.0061±0.0008
Km (mol/L)
1.06±0.11
0.21±0.03
Vm/Km (min-1)
0.035±0.004
0.029±0.004
The values are the means±standard errors of three experiments.
34
Table 2. The alcoholysis of glycerol with ethyl ferulate and ethyl caffeate under ultrasonic irradiation and mechanical stirring Ultrasound
Phenolic acid ethyl esters Ethyl ferulate (EF) Ethyl caffeate (EC)
Stirring Conversion
Time (h)
Conversion (%)
Time (h)
3
98.5
9
86.7
12
97.4
36
63.1
(%)
The ultrasonic enhancement condition: 250 W, 20 kHz, 60oC, 1:10 (EF/EC: glycerol, mol: mol) and the enzyme dosage was 6% by total weight of substrates, 200 rpm; the conventional stirring condition: 75 oC, 1:18 (EF/EC: glycerol, mol: mol) and the enzyme dosage was 12% by total weight of substrates, 200 rpm.
35
Table 3. 1H-NMR spectral data for the alcoholysis product of glycerol with ethyl ferulate in DMSO-d6 H Position Glycerol
Ferulic acid
Phenolic hydroxyl Alcoholic hydroxyl
1H-NMR δH (integral, mult., JHz) 4.18 (2H，dd，11.1, 3.8) 4.05 (1H，s) 3.74 (2H，dd，11.0, 6.6) 6.50 (1H，d, 15.9) 7.59 (1H，d,15.9) 7.14 (1H，d,7.9) 6.82 (1H，d, 8.1) 3.85 (3H，s) 7.34 (1H，s) 9.62 (1H，s) 4.93 (1H，s) 4.68 (1H，s)
5’ 3’ 2’ 6’ 7’ 8’ 9’ 11’ 12’ 10’ 4’ 1’
36
Table 4． ． Comparison of the scavenge abilities of DPPH radicals Compound
DPPH scavenging activity (%)
Monoglyceryl ferulic acid
92.6
Ferulic acid
95.5
Ethyl ferulate
93.8
Monoglyceryl caffeic acid
94.6
Caffeic acid
95.1
Ethyl caffeate
94.8
Tocopherol
96.8
TBHQ
91.7
37
Highlight:
Make the fastest record for enzymatic synthesis of monoglyceryl phenolic acids. Ultrasonic irradiation increased the alcoholysis rate up to 3-fold, decreased the enzyme dosage by 50%. Ultrasonic irradiation decreased Ea up to1-fold and increased Vm/Km above 1.2-folds. Monoglyceryl phenolic acids could be produced with different acyl donors.
38
39
S1350-4177(17)30412-1 http://dx.doi.org/10.1016/j.ultsonch.2017.09.016 ULTSON 3864
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
12 July 2017 6 September 2017 7 September 2017
Please cite this article as: C. Xu, H. Zhang, J. Shi, M. Zheng, X. Xiang, F. Huang, J. Xiao, Ultrasound irradiation promoted enzymatic alcoholysis for synthesis of monoglyceryl phenolic acids in a solvent-free system, Ultrasonics Sonochemistry (2017), doi: http://dx.doi.org/10.1016/j.ultsonch.2017.09.016
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 proof before it is published in its final 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.
Ultrasound irradiation promoted enzymatic alcoholysis for synthesis of monoglyceryl phenolic acids in a solvent-free system Chunfang Xua1, Haiping Zhanga1, Jie Shia, Mingming Zhengab*, Xia Xiangab, Fenghong Huanga*, Junyong Xiaob a
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Oil crops
and Lipids Process Technology National & Local Joint Engineering Laboratory, Key Laboratory of Oilseeds processing, Ministry of Agriculture, Hubei Key Laboratory of Lipid Chemistry and Nutrition, Wuhan 430062, China b
Functional Oil Laboratory Associated by Oil Crops Research Institute, Chinese
Academy of Agricultural Sciences and Infinite (China) Co. LTD, Guangzhou 51000, China
1. These authors contributed equally to this work.
1
Abstract Monoglyceryl phenolic acids (MPAs) were known as the natural hydrophilic antioxidants which could be used in different fields such as food, pharmaceutical, cosmetic etc. A novel enzymatic route of MPAs synthesis by the alcoholysis of phenolic acid ethyl esters with glycerol under ultrasound irradiation in solvent free system was developed. Optimization of reaction parameters shows that a high conversion of above 97.4% can be obtained under the following conditions: phenolic acid ethyl esters to glycerol molar ratio of 1:10, with 6% catalyst (Novozym 435), at 60OC and 200 rpm, with ultrasound input of 250 W, at 20 kHz frequency. Compared to the conventional stirring method, the activation energy for phenolic acid ethyl esters conversion was decreased from 65.0 kJ/mol to 32.1 kJ/mol under ultrasound promotion; the apparent kinetic constant (Vm/Km) increased above 1.2-folds; the lipase amount decreased to 50%; the time required for the maximum conversion reduced up to 3-folds without damaging the lipase activity, which is the fastest report for enzymatic synthesis of MPAs.
Keywords: Ultrasound irradiation, enzymatic alcoholysis, monoglyceryl phenolic acids, solvent-free, antioxidation
2
Introduction Phenolic acids are secondary plant metabolites widely existed dispersed in fruits, vegetables and cereals [1,2]. Due to their it antioxidant capacity, phenolic acids usually act as reducing agents, hydrogen donators, and singlet oxygen quenchers [3]. More important, phenolic acids exhibit potential health benefits and pharmacological properties including UV absorbant, anti-inflammatory, anti-carcinogenic and neuroprotective effects [4-6]. However, phenolic acids have low solubility and stability in water or oil system, which limits their application in food processing and cosmetic industries significantly [7,8]. An effective method for expanding application of phenolic acids is to modify their molecular structure and enhance for enhancing the hydrophobic or hydrophilic properties [9-11]. Usually, it can be achieved through enzymatic derivatization by esterifying the carboxylic acid group with long-chain alcohols or glycerol, which should could obtain an amphiphilic molecule without losing its original functional properties [9,10]. As natural derivatives of ferulic acid, monoferuloyl glycerol (MFG) and diferuloyl glycerol (DFG) have been used as UV absorbers and antioxidants in many applications [14,15]. Due to the trace amount of MFG and DFG (<0.1%) in nature [8], it is meaningful to synthesis of glyceryl phenolic acids with different phenolic acids source. Presently, It is time-consuming to produce these modified phenolic acid compounds presently by the lipase-catalyzed transesterification reactions. usually take long time to produce these modified phenolic acid compounds. Furthermore, High 3
enzyme amount, high reaction temperature, long reaction times and low conversions often occurred due to the number of organic solvents capable of solubilizing adequate amounts of both polar phenolic acids and non-polar substrates is limited. high enzyme amount, high reaction temperature, long reaction times and low conversions often occurred. Furthermore, high vacuum was usually indispensable to remove the byproduct including ethanol or water during the whole reaction course [16], which and the complex reaction devices is always required requires complex reaction devices. Therefore, it is still a challenge to develop alternative strategy to improve the efficiency of enzymatic phenolic acids derivatization of phenolic acids. Ultrasound irradiation is an environmentally friendly method and has gained popularity and potential for use in applications in organic chemistry and in biotechnology [17,18]. Considering the violent collapse of enormous bubbles simultaneously, tremendous generation of heat and pressure occur, which could be helpful to remove the byproduct like ethanol without vacuum. The micro jet was is also formed which helps to create turbulence in the reaction system [19]. Based on the effects mentioned above, ultrasound can accelerate chemical transformation, improve yields and produce novel chemical reactions compared with tradition methods. Ultrasound could improve the enzyme performance, increase the product yield and make reaction condition more moderate. It is especially suitable for enzymatic heterogeneous systems using immiscible substrates and catalysts [20-25]. Ultrasonic bath was commonly used to provide the ultrasound irradiation. However, it is difficult to realize production on a large scale by the limit of the device 4
volume. Furthermore, only a fraction of the ultrasound energy could transfer to the reaction system, because of the indirect transmittal mode. In contrast, ultrasound carrying out with microtip probe could be more effective in enzymatic reactions. Recently, ultrasound irradiation has been used to accelerate the enzymatic reactions such as esterification of phytosterol, flavonoids and methanol with fatty acids in organic solvent [20,21,24]. However, the application of this novel ultrasound mode to enzymatic reactions has been less extensively studied. The main objective of this study is to investigate the enzymatic alcoholysis of phenolic acid ethyl esters with glycerol under microtip probe ultrasound irradiation in a solvent free system. A sequential experimental strategy was carried out to systematically evaluate the effect of substrates concentration, phenolic acid ethyl esters to glycerol molar ratio, ultrasonic power on the conversion and enzyme activity. To the best of our knowledge, concerning the enzymatic alcoholysis of phenolic acid ethyl esters with glycerol under ultrasonic irradiation, a systematic study for accelerating the synthesis as well as the kinetic and thermodynamics studies concerning the enzymatic alcoholysis of phenolic acid ethyl esters with glycerol under ultrasonic irradiation has not been reported.
2． ．Experimental 2.1. Materials Ethyl ferulate (EF), Ethyl caffeate (EC) were purchased from Aladdin Reagent (Shanghai, China). Glycerol (99%, dehydration using an activated 3Å molecular sieve 5
before being used), ethanol (95%) and glacial acetic acid (HPLC grade) were purchased from Tianjin Guoyao Chemical Co., Ltd. (Tianjin, China). Novozym 435 (immobilized on polyacrylic resin) from Candida antarctica were purchased from Novozymes (Bagsvaerd, Denmark). Methanol (HPLC grade) was purchased from Merck KGaA (Darmstadt, Germany). 2.2. Equipment Experiments were carried out in a 100 mL beaker with thermostatic water bath with magnetic stirrer (Guohua Instrument Co., Ltd., Changzhou, China) and microtip probe (diameter of 13 mm) connected to a multi-frequency sonochemistry generator (Chengdu Jiuzhou Ultrasonic Technology Co., Chengdu, China) The ultrasonic unit has an power output of 100-300 W. An SHIMADZU LC-M20A series HPLC system was used to monitor the progress of the reactions.
2.3. Ultrasound irradiation synthesis and purification of monoglyceryl phenolic acids A typical enzymatic alcoholysis was conducted by combining 0.3 mol of glycerol, a certain amount of phenolic acid ethyl esters (0.9-5.4 mol) and lipase (2%-12% of the total weight of the substrate). The mixtures were added into a 100 mL beaker which would be placed in a thermostatic oil bath with microtip probe for ultrasound enhancement and magnetic agitation at 250 rpm for a certain time. During the reactions, samples were withdrawn from the mixture every certain time to analyze for reaction progress. All analyzes were performed in triplicate and the results were 6
reported as the mean standard deviation. Following monoglyceryl phenolic acids preparation, immobilized lipase and molecular sieves were was filtered from the mixture; One mL liquid raw product was extracted with 3 mL water for three times to remove the excess glycerol. After centrifuge at 5000 rpm for 5 min, the white powder was recovered and then dried by freeze-drying. The obtained monoglyceryl phenolic acids were subject to HPLC analyzed for purity.
2.4. Kinetics study Taking into account the results obtained in the experiments, the Arrhenius equation were acquired from the plot of reaction rate against temperature. The Michaelis-Menten equation was got in a certain amount of glycerol with various phenolic acid ethyl ester concentrations at the optimized condition. The initial reaction rates (v0, mol/ (L min)) were determined through the yield-time profiles corresponding to the first 12 min of the reactions (20.0% or less phenolic acid ethyl esters conversion), where the profiles were found to be approximately linear. Then the activation energies (Ea, kJ/mol), the maximum reaction rate (Vm, mol/ (L min)) and Michaelis–Menten constants (Km, mol/L) could be calculated through the equations.
2.5. Reuse of the lipase At the end of each round of reaction, the immobilized lipase was separated from the substrate by vacuum filtration and washed with 95% ethanol for twice 7
subsequently. After drying in vacuum at room temperature the lipase can be reused in a subsequent new reaction.
2.6. Qualitative and quantitative analysis of monoglyceryl phenolic acids and diglyceryl phenolic acids The products were analyzed by HPLC with a C18 reverse phase column (5 µm, 250 mm×4.6 mm) fitted with a photodiode array detector (SPD-M20A) at 35 oC, and eluted with a binary gradient of solvent A (water, 0.5% v/v glacial acetic acid) and solvent B (methanol) at 1 mL/min. The elution sequence consisted, consecutively, of a linear gradient from 20% (v/v) B to 100% B (v/v) over 30 min, followed by 100% B for 2 min, then to 20% B for 8 min. The elution was monitored at 325 nm. The purified products were analyzed using a hybrid, triple quadrupole/linear ion trap mass spectrometer, API 4000 Q-Trap, with an APCI interface performed in positive mode. CUR (curtain gas) pressure: 137.9 kPa; NC (nebulizer current): 27.58 kPa; TEM (temperature): 450◦C; Scan mode: EMS; Scan rate: 4000 u/s; GS1 (ion source gas 1) pressure: 344.75 kPa; DP (declustering potential): 90 V; CE (collision energy): 35 V; mass range: 100-1000 m/z. The products were characterized by 1H NMR spectroscopy using D6-DMSO (0.01% TMS) as a solvent on 600 MHz frequency of Bruker instrument. 2.7. DPPH radicals scavenging ability detection The reaction mixture consisting of 2.5 mL of 0.1 mM DPPH in methanol and 500 µL of sample (1.0 mM in methonal) was incubated at room temperature for 30 8
min. The radicals scavenging activity was measured at 517 nm against pure methanol (blank) using a UV-vis spectrophotometer (DU800, USA) in a 1 cm quartz cell.
3. Results and discussion 3.1. Optimization of the ultrasonic conditions Ultrasonic power is a critical factor that influencing ultrasound irradiation reactions. Ultrasound of relative higher intensity could have more obvious effect on the mass transfer of the solution, thus accelerate the enzymatic-catalyzed reactions. On the contrary, too high intensity of ultrasound could cause the inactivation of enzymes [26]. The influence of ultrasound power on EF conversion and monoferuloyl glycerol (MFG) yield were investigated in the range of 100 W-300 W with the frequency was kept at 20 kHz invariably. As shown in Fig.1, the conversion presented rapid increase with increasing power in the range of 100 W-250 W, and an obvious decrease of conversion was observed when the power exceeded 250 W. MFG yield showed the same variation trend with EF conversion, as more than 92% EF transferred into MFG. The maximum EF conversion and MFG yield reached 98% and 91% respectively in 250 W. It ascribed to the fact that ultrasonic irradiation caused a decrease in the particle size of the immobilized lipase and consequently increased the catalytic surface area, thus increase enzyme and substrate molecule collision frequency [27]. However, too high ultrasonic intensities were reported to reduce or even inactivate the enzyme activity [28]. Considering the EF conversion, stability of lipase and energy consuming, 250 W was selected to study the characteristics of lipase-catalyzed 9
reactions in the following experiments. In order to investigate the ultrasound irradiation time on the conversion of EF, the reaction continue with mechanic agitation under the same condition after a certain ultrasonic irradiation time from 1 h to 4 h. The total reaction time is kept at 4 h. As shown in Fig. 2, the EF conversion and MFG yield presented rapid increase with the increase of ultrasonic irradiation time from 0 h to 4 h. This was because ultrasound could help to reduce particle size of substrate and immobilized lipase, which would be useful to reduce mass transfer limitations. The irradiation time was longer than our previous reports [20, 21], this was due to the high viscosity of glycerol and low solubility of EF in glycerol. When the ultrasonic time was 3 h, the EF conversion has already reached 97%, and the MFG yield almost kept constant with further prolong the reaction time. Thus, both the ultrasound irradiation time and the total reaction time were kept at 3 h.
3.2. Effect of reaction temperature The conversion, solubility of the phenolic acids and the stability as well as activity of the lipase are strongly related to the reaction temperature. The effect of reaction temperature on EF conversion and relative products yields were investigated at different temperatures ranging from 50oC to 75oC. As shown in Fig.3a, under mechanical stirring, the conversion increased with the rising of the temperature from 50oC to 75 oC. Under ultrasound irradiation, the rising of the temperature from 50 o
C-65oC resulted in an increase of EF conversion. It might be due to the fact that 10
relative high temperature can reduce mass transfer limitations, increase the solubility of substrate and enhance the activity of lipase. However, a sharp decrease in EF conversion was observed when the temperature was further increased above 65oC. It might be ascribed to two main reasons, on one hand, excessive high temperature lead to the deactivation of the lipase; on the other hand, the increase of temperature affects the bubble formation and collapse caused by ultrasound cavitation, thus the effect of ultrasound appeared to be weaker at higher temperature [29]. Fig.3b showed the influence of temperature on monoferuloyl glycerol (MFG) and diferuloyl glycerol (DFG) yields under ultrasonic irradiation. MFG and DFG reached the maximum yield at 60oC and 65 oC, respectively. In order to obtain more target product (MFG), 60 oC was selected for the enzymatic alcoholysis under ultrasonic irradiation. The optimized temperature under ultrasound irradiation (60 oC) was much lower than that of conventional stirring (75 oC). It might be ascribe to the fact that the ultrasonic cavitation leaded to huge energy release during the process of the bubble formation and collapse. The enzymatic reaction became much easier than that under conventional stirring which needed more energy from external environment.
3.3. Effect of substrate molar ratio The effect of molar ratio of EF to glycerol on the conversion of EF under ultrasound irradiation and mechanical stirring was evaluated ranging from 1:3 to 1:18. As shown in Fig.4a, as the molar ratio changed from 1:3 to 1:10, EF conversion increased from 53.9% to 97.5%. Then, the conversion was almost maintained at the 11
same level when the molar ratio varied from 1:10-1:18. This phenomenon attribute to the high viscosity of the reaction mixture, mass transfer resistance, and strong electron-donating and steric hindrance effects [30]. The proposed critical points were 1:10 and 1:18 for ultrasound irradiation and stirring, respectively, which means that the glycerol amount required under ultrasonic condition is only half of that under stirring condition. The result might be benefit from the dispersion effect of ultrasound, which would produce more contact area between EF and glycerol and accelerate mass transfer of the substrates. Fig.4b showed the effect of substrate molar ratio on MFG and DFG yields. As same as EF conversion, MFG yield showed the same change trend. While DFG reached the highest yield of 7.6% at the molar ratio of 1:6, because the relatively abundant EF could continue react with MFG. 3.4. Effect of enzyme dosage The dependency between the EF conversion and relative products yields with enzyme load was investigated. The enzyme dosage was varied from 2% to 12% according to the weight of the total substrates which were held constant. As shown in Fig.5a, the EF conversion was drastically raised with the increasing of the enzyme load from 2% to 6% under ultrasound, this was ascribed to the reason that the increasing of enzyme load can provide more active sites, increase the probability of enzyme-substrate collision, and enhance higher reaction rate [31]. However, when the dosage further increased to 12%, it showed the same variation trend with 6%, which could be due to biocatalyst agglomeration and possible diffusional problems [32]. According to Sun’s report [30], excessive amounts of enzyme would lead to more 12
byproduct ferulic acid being formed. The enzyme dosage needed (6%) under ultrasound was only half of that under conventional stirring (12%), the result further illustrated that ultrasound irradiation could reduce mass transfer limitations and enhance reaction efficiency significantly. Thus, 12% and 6% enzyme was selected for stirring reaction and ultrasonic reaction in the further experiment, respectively. As shown in Fig.5b, the yield variation trend of MFG and DFG were almost opposite, when enzyme dosage was 6%, MFG yield could got 91.6% and DFG yield was 4.3%.
3.5. Reusability of lipase The reusability of immobilized enzymes which can reduce the cost of enzymatic reactions is rather important for practical application [20]. The study of enzyme reusability was carried out in the presence and absence of ultrasound irradiation. After each reaction, the lipase was separated from the reaction system by filtration and washed with 95% ethanol subsequently, which could effectively remove the residue on the lipase, and release the enzyme activity sites. As shown in Fig. 6, EF conversion was higher with ultrasound irradiation than that with conventional stirring only. In the meanwhile, the initial apparent activity of lipase was about 6-fold higher with ultrasound irradiation than that with conventional stirring. The result further confirmed that the ultrasonic could enhance the conversion and the apparent activity of lipase simultaneously. But after 10 recycles, the EF conversions and apparent activities of lipase under ultrasound irradiation and conventional stirring was were dropped to 51.8%, 36.1% and 40.3 µmol/(g×min), 4.4 µmol/(g×min), respectively. 13
Furthermore, the downward trends of EF conversion in presence and in absence of ultrasound irradiation were almost the same. This result confirmed that ultrasound irradiation could enhance the conversion of enzymatic synthesis of phenolic glycerides without essential damaging to the lipase activity. Compared with conventional methods, ultrasound could improve the enzyme performance, increase the product yield and make reaction condition more moderate.
3.6. Mathematical modeling of the reaction kinetics. The initial reaction rates, defined as the initial EF conversion per unit time (V0, mol/ (L min)), was calculated from six experimental points of EF conversion. The Arrhenius law can be described as follows: lnV0 = ln A − Ea/(R×T)
(1)
where A= the Arrhenius constant, Ea= the activation energy, R= the gas constant, and T= absolute temperature (K) Two plots on behalf of two different reaction conditions of lnV0 versus 1/T shows good linearity at the reciprocal temperatures (Fig. 7b). From the slopes, it is possible to calculate the activation energy. As the feruloyl of EF has electron-donating and steric hindrance effects, it can hinder the compatible reaction of EF to some extent, which makes the activation energy under stirring (65.0 kJ/mol) much higher. But under ultrasound irradiation, ultrasound can reduce the particle size and increase the substrate-enzyme interface area through its high cavitation energy, which allows substrate to access active sites more easily. Then the value of Ea under 14
ultrasound could decrease to 32.1 kJ/mol. As a result, the free energy barrier for ultrasound irradiation is considerably lower as compared to stirring, which means the reaction became much easier with ultrasound irradiation. In this study, the concentration of glycerol was much larger than the other reaction substrate, thus EF and product MFG and DFG could be considered unchanged. The enzymatic alcoholysis in this study can be seen as a “pseudo-first-order” reaction. In addition, EF concentration was controlled in a low level so that enzyme activity would not be inhibited by any substrate. The initial reaction rate can be expressed as: V0=Vm[S]/(Km+[S])
(2)
V0 is the initial reaction rate; Vm is the maximum reaction rate; [S] is the initial concentration of EF; Km is Michaelis constant for EF. And Eq. (2) can be transferred to the Lineweaver-Burk form: 1/V0=Km/Vm*1/[S]+1/Vm
(3)
Initial reaction rates were calculated from the linear portion of the concentration-time profiles (Fig.7a), and the kinetic parameters can be obtained by non-linear regression analysis for the above model [32]. The results could be seen in Table 1. The alcoholysis reaction of ethyl ferulate under ultrasound irradiation, showed increase in the Km (1.06 mol/L) value by 4-fold and Vmax (0.037 mol/(L×min)) value by 5-fold as compared to conventional stirring system, which showed similar tendency with the esterification reaction of D-isoascorbyl palmitate under ultrasound [33]. According to Kuo [34], the value of apparent kinetic constant Vm/Km is often 15
recognized as a suitable evaluation parameter of enzyme performance. The Vm/Km value of ultrasound was 1.2 times higher than that of conventional stirring, it in another aspect showed that ultrasound could improve the enzyme performance thus increase the product yield.
3.7． ．Enzymatic alcoholysis of different phenolic acid ethyl esters with glycerol A comparative study was done on alcoholysis of glycerol with different phenolic acid esters under mechanical stirring and ultrasound irradiation. As shown in Table 2, the conversion of EF and EC using ultrasonic irradiation were significantly higher than that of mechanical stirring. It is noteworthy that the alcoholysis rate under ultrasonic irradiation was above 3 times as that using mechanical stirring, which makes the fastest record for enzymatic synthesis of MPAs. The results suggest that ultrasonic irradiation can be used to accelerate the alcoholysis rate of glycerol with a series of phenolic acid esters with relatively high conversion. Benefit from the generation of heat and high pressure during collapse of bubbles, ultrasonic irradiation could remove the byproduct (enthanol) under normal pressure, which could further accelerate the reaction and increase the conversion. Moreover, the ultrasound irradiation is responsible for increasing interaction between the substrate and lipase by increasing the collisions, micro-stirring, and improved contact [18].
3.8. Characterization of the prepared MPGs The enzymatic alcoholysis products were monitored by HPLC. The liquid 16
chromatograms of ethyl ferulate, ethyl caffeate and corresponding monoglyceryl phenolic acids obtained under optimal conditions are shown in Fig. 8. It is evident that the enzymatic alcoholysis of ethyl phenolic acids could significantly increase the hydrophilic of the product, which could be expressed as the relative shorter retention time. Not any byproduct (ferulic acid and caffee acid) was found from the chromatography. HPLC analysis revealed the presence of a highly pure (>99%) compound after purification, residual ethyl phenolic acids and glyceride were not detected in the chromatography (Fig. 8a1, 8b3). In addition, APCI-MS and NMR was also carried out to characterize the molecular structure. The result confirmed that only a phenolic acid molecular was acylated to the framework of glyceride. In detail, monoglyceryl ferulic acid and monoglyceryl caffeic acid had their molecular ion peaks [M+1]+ at m/z 269 and 255 and [M+Na]+ at m/z 291 and 277, respectively. The NMR data of the alcoholysis product of glycerol with ethyl ferulate was listed in Table 3 and Fig. S1 and the deductive structure was were shown in Fig. 8a. The structure of 1-glyceryl ferulate could be confirmed from the higher chemical shift from the primary hydroxyl (C-5’ in Fig. 8a) of glycerol were compared to those for hydroxyls of C-2’ and C-3’[35].
3.9. DPPH radicals scavenging activity The DPPH radicals scavenging activity was compared with those of phenolic acids, ethyl phenolic acid, tocopherol and TBHQ. As shown in Table 4, the DPPH 17
scavenging activities of monoglyceryl ferulic acid and monoglyceryl caffeic acid were above 92.6%, which were comparable to tocopherol and TBHQ. The monoglyceryl phenolic acids also showed comparable scavenging ability with the corresponding phenolic acids and ethyl phenolic acids, which prove that alcoholysis modification did not affect the original antioxidation activity of the phenolic acids. 4. Conclusion In this study, an efficient enzymatic route for MPAs synthesis by the alcoholysis of EF and glycerol under ultrasound irradiation was developed. The results showed that a promising strategy to overcome mass transfer limitations arising from the use of phenolic acid ester and glyceride as the substrate. Compared with mechanical stirring, the temperature, amount of glycerol, enzyme dosage and reaction time needed were much less under ultrasonic irradiation. The overall alcoholysis reaction rate in the ultrasonic enhancement process (3-12 h) was almost 3-fold as that in the stirring process (6-36 h) without essential damaging to the lipase activity. The apparent kinetic constants showed that ultrasonic enhancement could improve the enzyme performance and the affinity between the substrate and enzyme. Considering the high efficient, mild condition and facile operation, the novel protocol can potentially be explored for other functional lipids modification in the future.
Acknowledgements This work was supported by the National Natural Science Foundation of China (31371843, 31671820), the Basic Applied Research Project of Wuhan City 18
(2016020101010095),
the
program
of
China
Scholarships
Council
(No.
201603250035), Agricultural Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2013-OCRI) and the Director Fund of Oil Crops Research Institute (1610172014006).
References 1. A. Crozier, I. B. Jaganath, M. N. Clifford, Dietary phenolics: chemistry,
bioavailability and effects on health, Cheminform. 40 (2009) 1001-1043. 2. C. Castelluccio, G. P. Bolwell, C. Gerrish, C. Riceevans, Differential distribution
of ferulic acid to the major plasma constituents in relation to its potential as an antioxidant, Biochem. J. 316 (1996) 691-694. 3. P. G. Pietta, Flavonoids as antioxidants, J. Nat. Prod. 63 (2000) 1035-1042. 4. W. Y. Huang, Y. Z. Cai, Y. Zhang, Natural phenolic compounds from medicinal
herbs and dietary plants: potential use for cancer prevention, Nutr. Cancer. 62 (2010) 1-20. 5. C. T. Ho, Phenolic Compounds in Food, ACS Symp. Ser. 1992. 6. T. M. S. Silva, F. P. D. Santos, A. Evangelista-Rodrigues, E. M. S. D. Silva, G. S.
D. Silva, Phenolic compounds, melissopalynological, physicochemical analysis and antioxidant activity of jandaíra (Melipona subnitida) honey, J. Food. Compos. Anal. 29 (2013) 10-18. 7. H. Stamatis, V. Sereti, F. N. Kolisis, Enzymatic synthesis of hydrophilic and
hydrophobic derivatives of natural phenolic acids in organic media, J. Mol. Catal. B-Enzym. 11 (2001) 323-328. 19
8. M. C. Figueroaespinoza, P. Villeneuve, Phenolic acids enzymatic lipophilization, J.
Agr. Food. Chem. 53 (2005) 2779-2787. 9. S. Gholivand, O. Lasekan, C. P. Tan, F. Abas, L. S. Wei, Comparative study of the
antioxidant activities of some lipase-catalyzed alkyl dihydrocaffeates synthesized in ionic liquid, Food Chem. 224 (2017) 365-371. 10. Y. Shi, Y. Wu, X. Lu, Y. Ren, Q. Wang, C. Zhu, D. Yu, H. Wang, Lipase-catalyzed
esterification of ferulic acid with lauryl alcohol in ionic liquids and antibacterial properties in vitro against three food-related bacteria, Food Chem. 220 (2017) 249-256 11. P. D. Tomke, V. K. Rathod, Ultrasound assisted lipase catalyzed synthesis of
cinnamyl acetate via transesterification reaction in a solvent free medium, Ultrason. Sonochem. 27 (2015) 241-246. 12. T. J. Ha, K. Nihei, I. Kubo, Lipoxygenase inhibitory activity of octyl gallate, J.
Agr. Food. Chem. 52 (2004) 3177-3181. 13. K. Nihei, A. Nihei, I. Kubo, Molecular design of multifunctional food additives:
antioxidative antifungal agents, J. Agr. Food. Chem. 52 (2004) 5011-5020. 14. D. L. Compton, J. A. Laszlo, K. O. Evans, Antioxidant properties of feruloyl
glycerol derivatives, Ind. Crop. Prod.36 (2012) 217-221. 15. K. Warner, J. A. Laszlo, Addition of ferulic acid, ethyl ferulate, and feruloylated
monoacyl- and diacylglycerols to salad oils and frying oils, J. Am. Oil. Chem. Soc. 82 (2005) 647-652. 16. S. D. Sun, S. Zhu, Y. L. Bi, Solvent-free enzymatic synthesis of feruloylated 20
structured lipids by the transesterification of ethyl ferulate with castor oil, Food. Chem. 5 (2014) 292-295. 17. T. J. Mason, Ultrasound in synthetic organic chemistry, Chem. Soc. Rev. 26 (1997)
443-451. 18. S. R. Bansode, V. K. Rathod, An investigation of lipase catalysed sonochemical
synthesis: A review, Ultrason. Sonochem.38 (2017) 503-529. 19. S. R. Bansode, V. K. Rathod, Ultrasound assisted lipase catalysed synthesis of
isoamyl butyrate, Process. Biochem. 49 (2014) 1297-1303. 20. M. M. Zheng, L. Wang, F. H. Huang, L. Dong, P. M. Guo, Q. C. Deng, W. L. Li, C.
Zheng, Ultrasonic pretreatment for lipase-catalyed synthesis of phytosterol esters with different acyl donors, Ultrason. Sonochem. 19 (2012) 1015-1020. 21. M. M. Zheng, L. Wang, F. H. Huang, P. M. Guo, F. Wei, Q. C. Deng, C. Zheng, C.
Y. Wan, Ultrasound irradiation promoted lipase-catalyzed synthesis of flavonoid esters with unsaturated fatty acids, J. Mol. Catal. B-Enzym. 95 (2013) 82-88. 22. C. Li, M. Yoshimoto, H. Ogata, N. Tsukuda, K. Fukunaga, K. Nakao, Effects of
ultrasonic intensity and reactor scale on kinetics of enzymatic saccharification of various waste papers in continuously irradiated stirred tanks, Ultrason. Sonochem. 12 (2005) 373. 23. S. B. More, J. T. Waghmare, P. R. Gogate, Ultrasound pretreatment as a novel
approach for intensification of lipase catalyzed esterification of tricaprylin, Ultrason. Sonochem. 36 (2017) 253-261. 24. C. M. Trentin, R. P. Scherer, C. D. Rosa, H. Treichel, D. Oliveira, J. V. Oliveira, 21
Continuous lipase-catalyzed esterification of soybean fatty acids under ultrasound irradiation, Bioprocess Biosyst. Eng. 37 (2014) 841-847. 25. J. Wang, S. Wang, Z. Li, S. Gu, X. Wu, F. Wu, Ultrasound irradiation accelerates
the lipase-catalyzed synthesis of methyl caffeate in an ionic liquid, J. Mol. Catal. B-Enzym. 111 (2015) 21-28. 26. Y. X. Liu, Q. Z. Jin, L. Shan, Y. F. Liu, W. Shen, X. G. Wang, The effect of
ultrasound on lipase-catalyzed hydrolysis of soy oil in solvent-free system, Ultrason. Sonochem. 15 (2008) 402-407. 27. S. L. Liu, X. Y. Dong, F. Wei, X. Wang, X. Lv, Juan Z, L. Wu, S. Y. Quek, H.
Chen, Ultrasonic pretreatment in lipase-catalyzed synthesis of structured lipids with high 1,3-dioleoyl-2-palmitoylglycerol content, Ultrason. Sonochem. 23 (2015) 100. 28. Y. Peng, M. U. Qing, W. Wei, Y. Xiong, J. W. Liu, Z. X. Lu, T. Wu, H. Z. Zhao,
Synthesis of Ascorbyl Fatty Acid Esters from Lard by Ultrasound-assisted Lipase Catalysis, Food Science, 32 (2011) 101-105. 29. S. D. Sun, X. W. Chen, Y. L. Bi, J. N. Chen, G. L. Yang, W. Liu, Functionalized
ionic liquid-catalyzed 1-feruloyl-sn-glycerol synthesis, J. Am. Oil. Chem. Soc. 91 (2014) 759-765. 30. E. L. Soo, A. B. Salleh, M. Basri, R. N. Z. A. Rahman, K. Kamaruddin, Response
surface methodological study on lipase-catalyzed synthesis of amino acid surfactants, Process Biochem. 39 (2004) 1511-1518. 31. G. D. Yadav, S. Devendran, Lipase catalyzed synthesis of cinnamyl acetate via 22
transesterification in non-aqueous medium, Process. Biochem. 47 (2012) 496-502. 32. J. H. Huang, Y. F. Liu, Q. Z. Jin, X. J. Wu, X. G. Wang, Z. H. Song,
Enzyme-Catalyzed Synthesis of Monoacylglycerols Citrate: Kinetics and Thermodynamics, J. Am. Oil. Chem. Soc. 89 (2012) 1627-1632. 33. F. J. Cui, H. X. Zhao, W. J. Sun, Z. Wei, S. L. Yu, Q. Zhou, Y. Dong, Ultrasound
assisted
lipase-catalyzed
synthesis
of
D-isoascorbyl
palmitate:
process
optimization and kinetic evaluation, Chem. Cent. J. 7 (2013) 180-190. 34. C. H. Kuo, F. W. Hsiao, J. H. Chen, C. W. Hsieh, Y. C. Liu, C. J. Shieh, Kinetic
aspects of ultrasound-accelerated lipase catalyzed acetylation and optimal synthesis of 4'-acetoxyresveratrol, Ultrason. Sonochem. 20 (2013) 546-552. 35. M. Tsuchiyama, T. Sakamoto, T. Fujita, S. Murata, H. Kawasaki, Esterification of
ferulic acid with polyols using a ferulic acid esterase from Aspergillus niger, Biochim. Biophys. Acta. 1760 (2006) 1071-1079.
23
Figure Captions Fig. 1. Effect of ultrasonic power on EF conversion and MFG yield. The ultrasonic frequency was 20 kHz, ultrasonic time was 4 h; the molar ratio of EF to glycerol was 1:10 (mol: mol), the enzyme dosage is 10% by total weight of substrates, 200 rpm, 60℃.
Fig. 2. Effect of ultrasonic time on EF conversion and MFG yield. The reaction condition was same as that described in Fig.1 except the ultrasonic power was 250 W.
Fig. 3. Effect of temperature on EF conversion under ultrasound irradiation and conventional stirring (a), effect of temperature on MFG and DFG yields under ultrasound irradiation (b). Ultrasound irradiation condition: 250 W, 20 kHz, 3 h. The molar ratio of EF to glycerol was 1:10 (mol: mol), the enzyme dosage is 10% by total weight of substrates, 200 rpm.
Fig. 4. Effect of substrate molar ratio on EF conversion under ultrasound irradiation and conventional stirring (a); effect of substrate molar ratio on MFG and DFG yields under ultrasound irradiation (b). The reaction conditions were same as that described in Fig.3 except the substrate molar ratio.
Fig. 5. Effect of enzyme dosage on EF conversion under ultrasound irradiation and conventional stirring (a), effect of enzyme dosage on MFG and DFG yield under ultrasound irradiation (b). The reaction condition was same as that described in Fig.4 24
except the enzyme dosage.
Fig. 6. Reuse of the lipase for enzymatic transeaterification of EF in presence and absence of ultrasound irradiation.
Fig. 7. Lineweaver-Burk plot of the reciprocal of initial reaction rate and EF concentrations (a) and Arrhenius plot of the reciprocal of temperature and the logarithm of initial reaction rate (b).
Fig. 8. Liquid chromatograms of alcoholysis products (a1, b3) and purified monoglyceryl ferulic acid (a2) and purified monoglyceryl caffeic acid (b4). Peaks: (1) monoglyceryl ferulic acid; (2) diglyceryl ferulic acid; (3) ethyl ferulate; (4) monoglyceryl caffeic acid; (5) diglyceryl caffeic acid; (6) ethyl caffeate.
25
26
27
28
29
30
31
32
33
Table 1. Kinetic parameters for the enzymatic alcoholysis of EF with glycerol in solvent-free system. Values Kinetic parameters Ultrasound
Stirring
Vmax (mol/(L×min))
0.037±0.004
0.0061±0.0008
Km (mol/L)
1.06±0.11
0.21±0.03
Vm/Km (min-1)
0.035±0.004
0.029±0.004
The values are the means±standard errors of three experiments.
34
Table 2. The alcoholysis of glycerol with ethyl ferulate and ethyl caffeate under ultrasonic irradiation and mechanical stirring Ultrasound
Phenolic acid ethyl esters Ethyl ferulate (EF) Ethyl caffeate (EC)
Stirring Conversion
Time (h)
Conversion (%)
Time (h)
3
98.5
9
86.7
12
97.4
36
63.1
(%)
The ultrasonic enhancement condition: 250 W, 20 kHz, 60oC, 1:10 (EF/EC: glycerol, mol: mol) and the enzyme dosage was 6% by total weight of substrates, 200 rpm; the conventional stirring condition: 75 oC, 1:18 (EF/EC: glycerol, mol: mol) and the enzyme dosage was 12% by total weight of substrates, 200 rpm.
35
Table 3. 1H-NMR spectral data for the alcoholysis product of glycerol with ethyl ferulate in DMSO-d6 H Position Glycerol
Ferulic acid
Phenolic hydroxyl Alcoholic hydroxyl
1H-NMR δH (integral, mult., JHz) 4.18 (2H，dd，11.1, 3.8) 4.05 (1H，s) 3.74 (2H，dd，11.0, 6.6) 6.50 (1H，d, 15.9) 7.59 (1H，d,15.9) 7.14 (1H，d,7.9) 6.82 (1H，d, 8.1) 3.85 (3H，s) 7.34 (1H，s) 9.62 (1H，s) 4.93 (1H，s) 4.68 (1H，s)
5’ 3’ 2’ 6’ 7’ 8’ 9’ 11’ 12’ 10’ 4’ 1’
36
Table 4． ． Comparison of the scavenge abilities of DPPH radicals Compound
DPPH scavenging activity (%)
Monoglyceryl ferulic acid
92.6
Ferulic acid
95.5
Ethyl ferulate
93.8
Monoglyceryl caffeic acid
94.6
Caffeic acid
95.1
Ethyl caffeate
94.8
Tocopherol
96.8
TBHQ
91.7
37
Highlight:
Make the fastest record for enzymatic synthesis of monoglyceryl phenolic acids. Ultrasonic irradiation increased the alcoholysis rate up to 3-fold, decreased the enzyme dosage by 50%. Ultrasonic irradiation decreased Ea up to1-fold and increased Vm/Km above 1.2-folds. Monoglyceryl phenolic acids could be produced with different acyl donors.
38
39