Aqueous enzymatic extraction of palm kernel oil

Inexpensive enzyme with high hydrolitic activity is required to bring the aqueous 21 enzymatic extraction (AEE) of seed / biomass oil into commercialization. In this study 22 a commercial digestive drug which is mainly composed of amylase and protease 23 enzymes has been applied to extract the palm kernel oil. A single factor experiment 24 evaluated the effect of the ratio palm kernel to water, concentration of digestive drug 25 used, pH, incubation time and temperature on extraction efficiency, free fatty acid 26 (FFA) content and lipid profile in form of monoglyceride, diglyceride and triglyceride. 27 A significant effect was observed for all the parameters and the highest extraction 28 efficiency of 96% with FFA content less than 0.68% was observed in the incubation 29 condition of 1:5 ratio palm kernel to water, 9% w/w of digestive drug, pH of 9, 90 min 30 of incubation time at 45  C. Green metrics assesment confirmed that the AEE 31 process is more green process than the soxhlet method. In comparison with other 32 existing extraction methods, the AEE showed better extraction efficiency against the 33 screw press and supercritical carbon dioxide methods.


Introduction 38
The world vegetable oil consumption has increased 2.3% per annum during 39 the past two decades of 21 st century, with China and Brazil as the highest 40 consumers at 30 and 24 kg/capita. Consumption is predicted to continue to 41 rise by 0.9% in the coming years (OECD et al. 2020). Currently mechanical 42 pressing and solvent based extraction methods were used in the oil industry 43 (Cheng and Rosentrater 2017). Hexane is the most popular extraction solvent 44 due to its high extraction yield and low cost (Cheng and Rosentrater 2017). 45 However, hexane is toxic, flammable and classified as a pollutant (Potrich et  Another green extraction process was using enzyme in aqueous environment. 62 The extraction efficiency of AEE on some oilseeds materials such as soybean, 63 peanut, sesame, and Moringa oleifera, as well as shrimp by-product has been 64 reported as higher (Liu et  The ground palm kernel seeds were mixed with water at investigated ratio ranging 149 from 1:3 -1:7 (w/v) and the digestive drugs was added according to explored 150 concentration (%, w/w). Next, the pH was adjusted with NaOH or HCl following 151 investigated value and incubated in the shaking incubator (Vision model VS8480SN) 152 at an appropriate temperature and time. The oils were separated from water and 153 residue using centrifuge running at 8000 rpm for 5 minutes and was stored in a 154 desiccator for analysis. The acid value was determined by the standard titration 155 method following ASTM D664. The AEE oil yield was calculated following equation 156 1. The extraction efficiency was measured based on the comparison between AEE 157 and soxhlet oil yield. 158

=
(1) 159 (2) 160 where WAEE is the weight of AEE oil extracted and Wpalm kernel is the weight of palm 161 kernel used. 162 163

Lipid content and fatty acid profile analysis 164
The lipid profile i.e. monoglyceride, diglyceride and triglyceride containing in each 165 palm kernel oil extracted was determined using gas chromatography. A 1 L of 166 sample was injected to GC Shimadzu type 2010 equipped with a capillary column 167 (length 15 m ID 0.25 mm) and a flame ionization detector with temperature set to 168 370C. The carrier gas was helium with a constant delivered flow of 30 mL/min and 169 both the injection port and detector temperature was set to 370 C. 170

Green metrics calculation 171
In order to determine how green the AEE process is, the green metrics equations 172 were adopted from previous published report (Sheldon 2018)   The amount of solvent use to extract oil is important as it affects the oil extracted 205 quantity and the following procedure for oil-solvent separation (Halim et al. 2012). 206 Zakaria and Harvey explained that, based on Fick's law of diffusion, increasing the 207 volume of solvent will increase the quantity of oil extracted (Zakaria and Harvey 208 2012). Therefore, in this study the effect of the ratio of palm kernel to water on the 209 AEE intensified extraction efficiency. The FFA content and lipid profile were 210 investigated at ratio of 1:3 to 1:7 (w/v) with pH of 9, concentration of digestive drug of 211 7% w/w, incubation time of 90 min and temperature of 45C. A ratio below than 1:3 is 212 inadequate to immerse the palm kernel seeds. Even though water cannot dissolve 213 lipids, as they have different polarity, the seed to water ratio had a significant effect 214 on all the measured variables. The extraction efficiency ranging from 57.91 ± 1.60% 215 to 82.04 ± 1.60% with ratio of 1:5 was the highest, as shown in Fig. 1A 26.16 ± 0.70%, respectively. As shown in Fig. 2  the digestive drug used. The ANOVA test revealed that the concentration of 245 digestive enzyme had significant effect on the extraction efficiency and free fatty acid 246 content. The highest extraction efficiency occurring at the weight of 9% w/w was 247 96.11 ± 2.31%. Figure 1B shows that the pattern of this parameter is in agreement 248 with some published results, which supports the conclusion that the extraction 249 efficiency increased to reach the maximum levels and decreased when increasing The same trend has obtained for the effect of the weight of digestive drug to MG, DG 256 or TG yield (Fig. 2B). The yield of each lipid profile climbed to reach 1.05 ± 0.04%, 257 3.34 ± 0.61%, and 28.37 ± 0.41%, respectively, and then dropped to 0.30 ± 0.07%, 258 1.01 ± 1.42% and 20.29 ± 0.67%, respectively. Therefore, the univariate test 259 identified a significant effect of the concentration of drug used to lipid profile. 260 261

Effect of pH 262
The effect of pH on extraction efficiency, FFA content and lipid profile were 263 determined based on different pH values from 4 to 12 with an increment of 1 value. 264 There were significant effects for all independent parameters. Furthermore, the Post-265 hoc Tukey test analysis determined that the significance occurring in extraction 266 efficiency was driven by high efficiency of >70% at pH of 6 to 9, achieving extraction 267 efficiencies of 77.39 ± 3.55%, 88.94 ± 0.71%, 90.23 ± 0.53%, and 82.04 ± 1.60%, 268 respectively. In contrast, the significance of FFA content was forced by pH of 6, 10, 269 and 12 with percentage of 0.76 ± 0.02%, 0.62 ± 0.07% and 0.57 ± 0.01%, 270 respectively. 271

272
The average extraction efficiency for this parameter was 73.76% with the highest 273 occurring at pH 8 (Fig. 1C). The FFA content ranged from 0.26 ± 0.01% to 0.76 ± 274 0.02% which is lower than the minimum standard allowed for biodiesel production 275 (Britton and Raston 2015). The highest yield of MG was extracted using pH 7 with 276 1.19 ± 0.51%, while DG yielded of 3.53 ± 1.17% at the same pH. The maximum yield 277 of TG was obtained using pH 8 with valued at 28.96 ± 1.16%. 278 279 280

Effect of incubation time 281
Another important variable that could affect the extracted oil yield is incubation time. 282 Hence five different incubation times were studied starting from 30 min to 150 min 283 using the ratio of palm kernel to water of 1:5, 7% w/w of weight of the digestive drug, 284 a temperature of 45C and a pH of 9. As shown in Figure 1D incubation temperature is preferable, as maintaining thermal conditions in 311 prolonged reaction time is cost intensive. Therefore, the effect of temperature 312 in this study was investigated ranging from room temperature to 60C. 313 A significant effect was observed in this parameter for extraction efficiency and FFA 314 content. This was mainly driven by high extraction efficiency at a temperature of 315 45C, while FFA content was significantly influenced by high concentration of 0.73 ± 316 0.08% at a temperature of 60C. The average extraction efficiency was 51.93% 317 ranging from 37.19 ± 2.13% to 82.04 ± 1.60% (Fig. 1E). The FFA content was 318 observed to increase in the increasing incubation temperature. This result supports 319 previous research as mentioned above. Average extraction yield of MG, DG and TG 320 for the effect of temperature was 0.44%, 1.55% and 16.52%, respectively. The 321 highest MG concentration of 0.65 ± 0.09% was observed at a temperature of 60C, 322 while DG and TG had highest yield at 45C of 2.78 ± 0.70% and 26.17 ± 0.70%, 323 respectively. 324 325 3.6. Reusability of the digestive drug 326 One factor for reducing production cost in enzymatic method is reusing the 327 enzyme (Santos et al. 2020). To assess the reusability of the enzymes 328 contained in a digestive drug, the incubation was set based on the condition 329 giving the highest oil yield which is ratio palm kernel to water of 1:5, weight of 330 digestive drug of 9% w/w, pH of 9, incubation time of 90 min at 45C. Figure  331 1F illustrates the extraction efficiency decrease in the second cycle and almost 332 nothing after the fifth cycle. It seems the enzymes lost contact with the cell 333 walls, as the residue from first cycle remains in the environment. Even though 334 the result of this study is similar to previous published results (Nguyen et al. 335 2020) regarding the decrease of oil yield after a second cycle, the reduced 336 yield in this study after a second cycle is too high, presumably due to the 337 mixture of enzymes used in this study compared to using a single enzyme. 338 339

Green metrics assessment 340
Environmental concern has driven the demand to determine how green the 341 chemicals process is (Curzons et al. 2001;Sheldon 2018). The term 'green 342 chemistry' was used to describe the chemical process which generates less 343 waste in the production process and avoids the use of hazardous chemicals. 344 In this study the green chemistry metrics such as environmental factor, 345 process mass intensification, solvent intensity, wastewater intensity and 346 effective mass yield were calculated following equations 3 -7 mentioned 347 above. Due to insufficient published data for other palm kernel extraction 348 methods such as screw press and supercritical carbon dioxide (SCO) 349 methods, a comparison was made using data from the extraction of linseed oil 350 (Pradhan et al. 2010). Table 2  The e-factor which determines the ratio of waste product per oil yield showed 356 that the AEE method has less 22% of waste product than the soxhlet method. 357 The use of organic solvents in soxhlet extraction is harmful, requires a vast 358 quantity of solvent, and has been identified as energy intensive for evaporation 359 solvents (Halim et al. 2012 Effective mass yield (%) 6.08 0 0 0 363 It also apparent in Table 3 that the AEE processes performed better in the 364 process of mass intensification, solvent intensity and effective mass yield than 365 the soxhlet method. However due to the use of water as a solvent, the AEE 366 method had a higher waste water intensity than the soxhlet method. The 367 screw expeller and SCO methods which have been categorized as green 368 processes (Herrero and Ibáñez 2015; Sheldon 2018) showed better green 369 metrics in all the parameters calculated. This is because no toxic solvents 370 were used and no waste water was produced. In general, the SCO method 371 has better green metrics indicator than other methods. MPa while in only 12 min 25.5% oil yield was produced using the screw press 378 process. However, the extraction efficiency, calculated based on the soxhlet-379 oil yield, of the AEE method is actually better than the other two green 380 processes. The extraction efficiency of 96% was observed in the AEE process 381 while 65% and 91% occurred from screw press and SCO methods, 382 respectively. 383 384

Conclusions 385
The utilization of a commercial digestive drug as enzyme source for the AEE 386 of palm kernel has been developed using various production variables. The 387 extraction efficiency of 96% with FFA content less than the standard required 388 for vegetable oil and biodiesel raw material was achieved in an incubation 389 condition of 1:5 ratio of palm kernel to water, 9 w/w% digestive drug, pH of 9, 390 and 90 min incubation time at 45C. In contrast, the highest TG yield was 391 observed in a pH of 8 and with 7 w/w% digestive drug. There is a significant 392 effect on all parameter studied. Even though the screw press and SCO 393 methods have better green metrics indicators, the extraction efficiency of the 394 AEE process was 31% and 5%, respectively, better than those processes. 395