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In situ lipase-catalyzed transesterification in rice bran for synthesis of fatty acid methyl ester
Industrial Crops & Products 120 (2018) 140–146
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In situ lipase-catalyzed transesterification in rice bran for synthesis of fatty acid methyl ester
T
Nakyung Choia,1, Da Som Nob,c,1, Heejin Kimc, Byung Hee Kimd, Jieun Kwake, Jeom-Sig Leee, ⁎ In-Hwan Kima,c, a
Department of Integrated Biomedical and Life Science, Graduate School, Korea University, Seoul 02841, Republic of Korea School of Environmental and Biological Sciences, Rutgers University, New Brunswick, NJ 08901, USA c Department of Public Health Sciences, Graduate School, Korea University, Seoul 02841, Republic of Korea d Department of Food and Nutrition, Sookmyung Women’s University, Seoul 04310, Republic of Korea e National Institute of Crop Science, Rural Development Administration, Suwon, Gyunggi-do 16429, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Biodiesel In situ Repeated transesterification Rice bran lipase
Fatty acid methyl ester (FAME) were synthesized via in situ lipase-catalyzed transesterification in rice bran without additional catalyst. With this method, FAME were synthesized directly from oil in rice bran by simply adding methanol, with the aid of rice bran lipase already existing in rice bran. The effects of temperature, molar ratio (oil in rice bran to methanol), and water content of the rice bran were investigated. The yield of FAME and the free fatty acid content were monitored as a function of reaction time. The optimum conditions were a temperature of 40 °C, a molar ratio of 1:6, and a water content of 12%. Under the optimum conditions, the FAME yield of 83.4 wt% was obtained after 12 days. To further increase the FAME yield, the transesterification was repeated using the rice bran obtained from the first transesterification. The oils in rice bran that could be converted to FAME were completely transformed throughout the repeated transesterification.
1. Introduction Biodiesel is defined as monoalkyl esters of long-chain fatty acids derived from vegetable oils or animal fats for use in diesel engines (Bisen et al., 2010; Robles-Medina et al., 2009). It has become an important alternative to conventional diesel fuel since it is renewable, biodegradable, non-toxic, and essentially free of sulfur and aromatics. The use of biodiesel can reduce the exhaust emissions of particulate matters and green-house gases (Körbitz, 1999). The fundamental reaction in biodiesel production is transesterification, which can be catalyzed either chemically or enzymatically. Short reaction time and high yield of biodiesel are advantages of chemical transesterification (Marchetti et al., 2007). However, it has several major disadvantages, namely, high-energy requirements, difficulties in the recovery of catalysts and glycerol, and environmental pollution associated with the generation of large volumes of wastewater (Ognjanovic et al., 2009; Salis et al., 2005). Enzymatic transesterification can overcome the problems facing conventional chemical methods without compromising their advantages. Most importantly, enzymatic transesterification is carried out under mild reaction condition, and
⁎
1
does not require any complex process for glycerol recovery or wastewater treatment (Al-Zuhair et al., 2007). Thus, enzymatic transesterification has been widely studied for the biodiesel production. However, high cost and short life span of enzymes are major drawbacks of enzymatic methods. Supercritical technology has emerged as a good alternative method for biodiesel production, since supercritical alcohols can react with refined oils efficiently without a help of catalyst (Rathore and Madras, 2007; Saka and Kusdiana, 2001). However, this method is economically inefficient due to the high-energy requirements. In situ transesterification is another effective method for the production of biodiesel. In this method, oil is not extracted from seeds or oil-bearing sources prior to the transesterification. Instead, the oilbearing material contacts with alcohol directly in the presence of a catalyst. For the catalyst, chemical catalysts such as sulfuric acid or sodium hydroxide have been commonly used for biodiesel production under in situ condition (Georgogianni et al., 2008; Harrington and D’Arcy-Evans, 1985). Several studies have investigated the synthesis of biodiesel using rice bran as a substrate via in situ transesterification with a chemical catalyst (Lei et al., 2010; Özgül-Yücel and Türkay, 2003; Shiu et al., 2010).
Corresponding author at: Department of Public Health Sciences, Graduate School, Korea University, Seoul 02841, Republic of Korea. E-mail address: [email protected] (I.-H. Kim). Nakyung Choi and Da Som No contributed equally to this research.
https://doi.org/10.1016/j.indcrop.2018.04.049 Received 2 February 2018; Received in revised form 17 April 2018; Accepted 18 April 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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without the addition of methanol to investigate the hydrolytic activity. The reaction was performed without methanol and other conditions were the same as those of first group. The yield of FAME and FFA content were investigated for the transesterification activity and hydrolytic activity, respectively.
Rice bran is a by-product obtained from the outer layer of the brown rice kernel during milling to produce white rice. It contains 12–23% crude oil, which mainly consists of triacylglycerol (TAG), depending on the rice origin (Luh et al., 1991). Immediately following the milling process, rice bran deteriorates rapidly because the lipase in the rice bran has high hydrolytic activity. Free fatty acid (FFA) resulted from hydrolysis of TAG increase acidity which contributes to the formation of an off-flavor and soapy taste, and changes in functional property. The increase in FFA is unprofitable for the oil extraction in the industrial perspective due to high refining loss (Tao et al., 1993). Thus, to prevent hydrolysis caused by rice bran lipase, stabilization of rice bran immediately after the milling has been necessary but arduous task for oil industry. Numerous researches on rice bran lipase focused on its deactivation to stabilize the rice bran. Various technologies such as heating, treatment with hydrochloric acid, exposure to microwave irradiation, treatments with chemical inhibitors, low temperature storage, and osmic heating, have been explored for deactivation of the rice bran (Prabhakar and Venkatesh, 1986; Prakash and Ramanatham, 1994; Raghavendra et al., 2007). However, the possible utilization of rice bran lipase for beneficial purposes has not been studied. The aim of this study was to investigate in situ lipase-catalyzed transesterification in rice bran for the production of fatty acid methyl ester (FAME). Surprisingly, FAME were synthesized effectively with methanol by a lipase naturally existing in the rice bran. For optimization of the reaction, the effects of temperature, molar ratio (oil in rice bran to methanol), and water content were thoroughly studied. In addition, the effect of repeated transesterification was also investigated.
2.3. In situ lipase-catalyzed transesterification in rice bran For in situ lipase-catalyzed transesterification in rice bran, rice bran (100.0 g), which contains 15.0 g of oil (17.3 mmol, based on TAG), was put into a 1 L polypropylene bottle and 3.3 g of methanol (103.7 mmol) was added. The mixture of rice bran and methanol was then shaken vigorously for 5 min until the rice bran was uniformly wetted by the methanol. The rice bran wetted with the methanol was divided in a 15 mL polypropylene bottle and each bottle was tightly closed with a screw cap to prevent methanol from leaking. These samples were incubated in an incubator (Model IB-15G; Jeio tech, Daejeon, Republic of Korea) at the desired temperature and samples in individual bottles were taken for analysis at the desired reaction time. Subsequently, the oils in the rice bran were extracted in a 250 mL flask by stirring with 100 mL of n-hexane for 1 h. The extraction was performed twice and nhexane was completely removed using a rotary evaporator at 60 °C. The oils were stored in a glass vial at −85 °C until used. The effects of temperature (20–70 °C), molar ratio of oil in rice bran to methanol (1:3-1:9), and water content (0–24%) on the in situ lipasecatalyzed transesterification in rice bran were investigated. Because the rice bran initially had a water content of 12%, samples containing 0% and 6% water were prepared by removing some of the water by freezedrying. Samples with water contents of 18% and 24% were prepared by adding appropriate volumes of water to the rice bran. The water content of rice bran was determined by the oven drying method at 105 °C according to AOAC Official Method 950.01 (2002).
2. Materials and methods 2.1. Materials The rice bran used in this study was supplied by the Korean Rural Development Administration (Suwon, Republic of Korea) and the rice cultivar was Boramchan. The rice bran prior to use was passed through a 30-mesh sieve to sift out the hull, germ, broken endosperm part, and any other impurities. The oil and water contents of rice bran were 15 wt %, and 12%, respectively. The oils in rice bran were composed of 0.7 wt % of FFA, 1.8 wt% of diacylglycerol (DAG), 92.7 wt% of TAG, and 4.8 wt% of unsaponifiable matters. The fatty acids in crude rice bran oil consisted of 0.2 wt% myristic acid, 16.5 wt% palmitic acid, 0.1 wt% palmitoleic acid, 1.4 wt% stearic acid, 42.2 wt% oleic acid, 39.1 wt% linoleic acid, and 0.5 wt% of eicosenoic acid. The rice bran was stored in sealed containers at −85 °C until used. Tricaprin (GLC-570) as an internal standard was purchased from Nu-Check Prep, Inc. (Elysian, MN, USA) and fatty acid methyl ester mixture (CRM47885) was purchased from Sigma Aldrich Korea (Seoul, Republic of Korea). The other chemicals used in this study were of analytical grade unless otherwise noted.
2.4. Analysis of products Fifty milligram of oil extracted was weighed accurately into a 5 mL volumetric flask and diluted with chloroform. Each sample solution was transferred to a vial and analyzed by a gas chromatography. Tricaprin (0.5 mg/mL) was used as an internal standard. A gas chromatography (Model 3800; Varian, Palo Alto, CA, USA) equipped with a DB-1ht capillary column (15 m × 0.25 mm i.d.; J&W Scientific, Folsom, CA, USA) and a flame ionization detector was used. The column was initially held at 120 °C for 3 min and then heated to 370 °C at a rate of 20 °C/min. The column was then held at 370 °C for 3 min. Helium was used as a carrier gas at a flow rate of 1.5 mL/min and a split ratio was 1/50. The injector and detector temperatures were set at 370 °C. The yield of FAME (wt%) was calculated as in the following equation. Yield of FAME (wt%) = a/b × 100
2.2. Effect of heat treatment on the catalytic activity of rice bran lipase
Where a is the weight of FAME in rice bran, and b is the total weight of FAME, FFA, monoacylglycerol (MAG), DAG, TAG, and unsaponifiable matters in rice bran. FFA content was measured by acid value according to AOAC Official Method 940.28 (2002) and the content of unsaponifiable matters was determined according to AOAC Official Method 933.08 (2002). All experiments were conducted in triplicate.
To verify synthesis of FAME by in situ lipase-catalyzed transesterification in rice bran, the rice bran was heated for 15 min, 30 min, and 60 min and then divided into two groups to investigate the transesterification activity for the synthesis of FAME (first group) and the hydrolytic activity for the formation of FFA (second group). As a control, unheated rice bran was incubated in the same manner. The heat treatment was carried out with rice bran sealed in a glass bottle. After the heat treatment, the rice bran was employed for determination of hydrolytic and transesterification activities. The heated rice bran as first group was incubated with the addition of methanol to investigate the transesterification activity. The incubation temperature, the molar ratio of the oil in rice bran to methanol, and the water content of the rice bran were set at 40 °C, 1:6, and 12%, respectively. The heated rice bran as second group was incubated
3. Results and discussion 3.1. Effect of heat treatment on the catalytic activity of rice bran lipase Rice bran lipase is known to have strong hydrolytic activity, which increases the FFA content of rice bran and decrease the oil extraction recovery. More than two types of the rice bran lipases have been identified, and they tend to have a regiospecificity at sn-1, 3 positions of 141
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Fig. 1. The effect of heat treatment on the in situ transesterification activity of rice bran lipase. The reaction was performed at a molar ratio of 1:6 (oil in rice bran to methanol), a temperature of 40 °C, and a water content of 12%. The yield of fatty acid methyl ester (wt%) was determined as a function of reaction time. Fatty acid methyl ester refers to FAME.
TAG as well as a substrate specificity toward shorter FA during hydrolysis (Aizono et al., 1976; Prabhu et al., 1999; Takano, 1993). In contrast to the active researches on the hydrolytic activity of rice bran lipase, there has been very little effort devoted to the utilization of the rice bran lipase in other reactions. As an innovative attempt, in situ lipase-catalyzed transesterification in rice bran was carried out using methanol and FAME were synthesized successfully from the oil in the rice bran without any additional catalyst. For the trials of first group with methanol, the unheated rice bran had very high transesterification activity and the maximum FAME yield of 83.4 wt% was achieved after 12 days (Fig. 1). Meanwhile, for the trials involving heated rice bran, the transesterification activity decreased dramatically as the heating time increased. After 15 days, the yield was only 12 wt% when the rice bran was heated for 60 min. For the trials of second group without methanol, the unheated rice bran also showed very high hydrolytic activity (Fig. 2). However, for the trials involving heated rice bran, a marked decrease of hydrolytic activity was observed and the lowest FFA content was also obtained when rice bran was heated for 60 min. These results indicated that there
Fig. 3. The effect of temperature on the yield of fatty acid methyl ester (a) and free fatty acid content (b) for the in situ lipase-catalyzed reactions in rice bran as a function of reaction time. The reaction was performed at a molar ratio of 1:6 (oil in rice bran to methanol) and a water content of 12%. Fatty acid methyl ester and free fatty acid refer to FAME and FFA, respectively.
was a high correlation between transesterification activity and hydrolytic activity of rice bran lipase. Therefore, it was verified that FAME were synthesized by the in situ lipase-catalyzed transesterification in rice bran. 3.2. Effect of temperature The reaction temperature is one of the most important parameters in enzymatic reactions. An increase in temperature can decrease mass transfer limitations by decreasing the mixture’s viscosity and enhancing the mutual solubility or diffusion processes of substrates, and can also increase interactions between enzyme particles and substrates (Xu et al., 2000). However, temperatures that are too high can induce irreversible denaturation of the enzyme protein and shorten the half-life of the enzyme activity, although some enzymes from microorganisms are stable and active at high temperatures (Turner and Vulfson, 2000). The effect of temperature on the in situ lipase-catalyzed transesterification in rice bran was determined by the yield of FAME (wt%) as a function of reaction time (Fig. 3a). The FFA content (wt%) in the reaction mixture was also monitored (Fig. 3b). During the first 9 days of the reaction, the yield of FAME increased considerably when the temperature was increased from 20 to 40 °C, but there were no further
Fig. 2. The effect of heat treatment on the in situ hydrolytic activity of rice bran lipase. The reaction was performed at a temperature of 40 °C, and a water content of 12%. The free fatty acid content (wt%) was determined as a function of reaction time. Free fatty acid refers to FFA. 142
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increase in the yields of FAME between 40 and 50 °C. However, as the reaction time increased from 9 to 12 days, the yield of FAME further increased up to 83.4 wt% at 40 °C. By contrast, the increase of FAME yield was negligible at 50 °C because of the increase of FFA content. After 6 days of reaction, the yield of FAME decreased when the temperature increased from 50 to 70 °C, while the FFA content increased. Overall, the decrease of FAME yield at the temperatures higher than 40 °C may have been caused by an increase in FFA and a decrease in transesterification activity. The control of the FFA is very important since it adversely affects the quality of the final product (Chong et al., 2007). It has been reported that optimum temperature on the hydrolytic activity of the lipase, which was isolated from defatted rice bran, was between 30 and 40 °C (Aizono et al., 1976; Prabhu et al., 1999; Rajeshwara and Prakash, 1995). In particular, in these studies, the hydrolytic activity of rice bran lipase was reduced at the temperature above 40 °C. Many studies on various lipase-catalyzed reactions have described a Ping-Pong mechanism for the inhibition of hydrolytic activity by alcohol (Bousquet-Dubouch et al., 2001; Martinelle and Hult, 1995). Thus, the hydrolytic activity of rice bran lipase can be affected by both alcohol and temperature. Our results indicated that incubation at higher temperatures leads to evaporation of methanol, which was inhibitor of rice bran lipase, and evaporation of methanol resulted in increase of FFA. In other words, methanol have more effect on hydrolytic activity of rice bran lipase than temperature. Hence, 40 °C was selected as the optimum temperature and used in subsequent experimental trials, since it represents both the maximum yield and the high transesterification rate. 3.3. Effect of molar ratio In general, alcohol can dissolve glycerol liberated from the reaction and prevent it from blocking the interface between substrates and enzymes (Rodrigues et al., 2008). However, excessive alcohol above the stoichiometric molar ratio is undesirable for enzyme activity because methanol is known to have a strong enzyme-denaturing activity. The effect of molar ratio on the in situ lipase-catalyzed transesterification in rice bran was determined by the yield of FAME (wt%) as a function of reaction time (Fig. 4a). The FFA content (wt%) in the reaction mixture was also monitored (Fig. 4b). As the molar ratio increased from 1:3 to 1:6, the yield of FAME increased gradually. However, the difference in the yields of FAME achieved between the molar ratio of 1:6 and 1:7.5 was negligible throughout the entire reaction. Furthermore, when the molar ratio further increased from 1:7.5 to 1:9, the yields of FAME decreased considerably. Methanol is widely used for synthesis of biodiesel because of its economic feasibility and accessibility in most countries (Antczak et al., 2009; Deng et al., 2005). However, the enzymatic transesterification of methanol is problematic, because lipase inhibition by methanol is stronger than that by longerchain aliphatic alcohols. (Salis et al., 2005; Tan et al., 2010). Shimada et al. reported an irreversible inactivation of enzyme caused by more than one-third of the stoichiometric amount of methanol for methanolysis of vegetable oil catalyzed by the immobilized C. antarctica lipase (Shimada et al., 2002). The enzyme inactivation is likely related to the amount of undissolved alcohol touching the enzyme in the reaction mixture. In the present study, the transesterification at a molar ratio beyond 1:7.5 implies deactivation of the rice bran lipase by the methanol. Meanwhile, the FFA content in the reaction mixture decreased substantially with an increase of molar ratio. At the molar ratio of 1:3, the FFA content increased to 25 wt% as the reaction time increased up to 15 days. However, with the molar ratio of 1:9, the FFA content remained constant under 5 wt%. This demonstrates that a hydrolysis is highly inhibited by a larger amount of methanol. With a larger amount of methanol, transesterification prevails over hydrolysis as methanol competes with water existing in the rice bran to react with the rice bran oil.
Fig. 4. The effect of molar on the yield of fatty acid methyl ester (a) and free fatty acid content (b) for the in situ lipase-catalyzed reactions in rice bran as a function of reaction time. The reaction was performed at a temperature of 40 °C and a water content of 12%. Fatty acid methyl ester and free fatty acid refer to FAME and FFA, respectively.
In summary, large amounts of methanol inhibit the transesterification by deactivating the rice bran lipase and also depress the hydrolysis. To take advantage of the mutually exclusive behaviors of methanol, the molar ratio of 1:6 (oil in rice bran to methanol) was considered as the appropriate compromise and was selected as the optimum condition. 3.4. Effect of water content It is known that reactions involving lipases require water to maintain the enzyme structure and achieve catalytic activity (Watanabe et al., 2003; Zaks and Klibanov, 1988). The use of too much water, however, can result in a shift in the reaction towards hydrolysis, and lead to an increase of FFA during transesterification (He and Shahidi, 1997; Yang et al., 2003). The effect of water content on the in situ lipase-catalyzed transesterification in rice bran was determined by the yield of FAME (wt%) as a function of reaction time (Fig. 5a). The FFA content (wt%) in the reaction mixture was also monitored (Fig. 5b). As the water content of rice bran increased from 0 to 12%, the yield of FAME increased drastically throughout the entire reaction. By contrast, when the water content further increased from 12 to 24%, the yield decreased considerably. The maximum FAME yield of 83.4 wt% was obtained at the 143
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the FAME yield was also the lowest at a water content of 0%. These results demonstrate that an appropriate amount of water is necessary to maintain the lipase activity on the transesterification. As the water content increased, the transesterification activity of the lipase for the FAME synthesis was improved, but the hydrolysis also increased. At water contents higher than 12%, the decrease of FAME yield was attributed to the fact that the rate of hydrolysis increased as water content increased, thereby leading to a concomitant increase in FFA. Water forms hydrogen bonds with functional groups in the protein molecules and affects the enzymatic reactions (Kurkal et al., 2005; Kurplus and McCammon, 1983). A critical amount of water is needed to maintain the three-dimensional form of an enzyme that is necessary for its catalytic activity. However, excess water causes enzyme inactivation and undesirable hydrolysis. Therefore, the optimum water content is a compromise between minimizing hydrolytic activity and maximizing transesterification activity. Hence, 12% was selected as the optimum water content since it represents both the maximum yield and the fastest FAME synthesis. This water content coincided with the original water content of the rice bran used in this study, implying that the rice bran lipase carries the highest activity on the transesterification with its original water content. 3.5. Repeated transesterification Under the selected optimum condition, the FAME yield of 83.4 wt% was attained via the first transesterification after 12 days. The oils in the rice bran after the first transesterification was composed of 83.4 wt% of FAME, 8.5 wt% of FFA, 1.0 wt% of monoacylglycerol (MAG), 0.7 wt% of DAG, 1.7 wt% of TAG, and 4.8 wt% of unsaponifiable matters (Table 1). Among the oils in the rice bran, only FFA, MAG, DAG, and TAG can be converted to FAME. However, these oils were still present in the rice bran after the first transesterification. Therefore, to improve the FAME yield, the transesterification was repeated using the rice bran obtained from first transesterification of 12 days. The control of water content of rice bran was one of the important factors because FFA of 8.5 wt% still remained in the rice bran after first transesterification. Thus, as a pre-treatment of the repeated transesterification, water in the rice bran after first transesterification was removed by freeze-drying and residual methanol in the rice bran was also removed during this process. Subsequently, the dried rice bran was subjected to a repeated transesterification under the optimum condition, which was the molar ratio of 1:6 (oil in rice bran to methanol), and the temperature of 40 °C. The effect of repeated transesterification was determined by the yield of FAME and the FFA content as a function of reaction time (Fig. 6a and b). As the repeated reaction progressed, a considerable increase in the yield of FAME was observed. After 6 days of repeated transesterification, the yield of FAME was increased to 95.2 wt% from 83.4 wt% for the first transesterification. Meanwhile, the FFA content showed a
Fig. 5. The effect of water content on the yield of fatty acid methyl ester (a) and free fatty acid content (b) for the in situ lipase-catalyzed reactions in rice bran as a function of reaction time. The reaction was performed at a temperature of 40 °C and a molar ratio of 1:6 (oil in rice bran to methanol). Fatty acid methyl ester and free fatty acid refer to FAME and FFA, respectively.
water content of 12% after 12 days. By contrast, a steady increase of FFA was observed when the water content was increased from 0 to 24% though the entire reaction. It is noteworthy that the FFA content at a water content of 0% remained nil throughout the entire reaction, implying that the hydrolysis was completely inhibited due to the insufficient water as a substrate. However,
Table 1 Compositions (wt%) of the oils extracted from the original rice bran, the rice bran after the first transesterification, and the rice bran after the repeated transesterification. FAMEd a
Original rice bran First transesterificationb Repeated transesterificationc a b c d e f g
e
ND 83.4 ± 3.9f 95.2 ± 1.6g
FFA
MAG
DAG
TAG
Unsaponifiable matters
0.7 ± 0.3 8.5 ± 0.2 ND
ND 1.0 ± 0.1 ND
1.8 ± 0.6 0.7 ± 0.2 ND
92.7 ± 1.3 1.6 ± 0.3 ND
4.8 ± 1.2 4.8 ± 1.2 4.8 ± 1.2
The oils in original rice bran. The oil content in the rice bran was 15 wt%. The oils in rice bran after first transesterification. The oils in rice bran after repeated transesterification. Abbreviations: FAME, fatty acid methyl ester; FFA, free fatty acid; MAG, monoacylglycerol; DAG, diacylglycerol; TAG, triacylglycerol. ND: not detected. Limit of detection: FFA, 4.6 mg/kg oil; MAG, 7.7 mg/kg oil; DAG, 4.4 mg/kg oil; TAG, 3.8 mg/kg oil. The weight of FAME recovered after the first transesterification was 12.5 g per 100 g of rice bran. The weight of FAME recovered after the repeated transesterification was 14.3 g per 100 g of rice bran. 144
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purposes. Conflicts of interest There are no conflicts of interest to declare. Acknowledgements This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project title: Evaluation of quality and processing property of longterm stored rice, Project No. PJ011037)" Rural Development Administration, Republic of Korea. References Official Methods of Analysis of AOAC International. AOAC International, Gaithersburg, MD, USA. Aizono, Y., Funatsu, M., Fujiki, Y., Watanabe, M., 1976. Purification and characterization: of rice bran lipase II. Agric. Biol. Chem. 40, 317–324. Al-Zuhair, S., Ling, F.W., Jun, L.S., 2007. Proposed kinetic mechanism of the production of biodiesel from palm oil using lipase. Process Biochem. 42, 951–960. Antczak, M.S., Kubiak, A., Antczak, T., Bielecki, S., 2009. Enzymatic biodiesel synthesis–key factors affecting efficiency of the process. Renew Energy 34, 1185–1194. 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Fig. 6. The effect of repeated transesterification on the yield of fatty acid methyl ester (a) and free fatty acid content (b) for the in situ lipase-catalyzed reactions in rice bran as a function of reaction time. The rice bran obtained from the first transesterification was employed as a substrate for repeated transesterification. Fatty acid methyl ester and free fatty acid refer to FAME and FFA, respectively.
marked decrease as the repeated reaction time increased. The FFA content decreased to 2.4 wt% after 3 days of repeated transesterification and became nil after 6 days. By contrast, for the trial conducted with a water content of 12%, neither FAME yield nor FFA content changed during the entire repeated transesterification. Consequently, the oils (FFA, MAG, DAG, and TAG) that could be converted to FAME, were completely transformed to FAME by the repeated transesterification.
4. Conclusions In situ lipase-catalyzed transesterification in rice bran was developed as a cost-effective and eco-friendly method for the production of FAME. It was verified that FAME were synthesized under in situ condition of rice bran by its own lipase. Water and methanol were crucial factors for both hydrolysis and transesterification. Too much water adversely affected the yield of FAME because it increased the FFA content. Excess methanol inhibited the transesterification activity as well as hydrolytic activity of rice bran lipase. Our strategy, which is to synthesize FAME by in situ lipase-catalyzed transesterification in rice bran, suggested a meaningful way to use rice bran for profitable 145
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Tan, T., Lu, J., Nie, K., Deng, L., Wang, F., 2010. Biodiesel production with immobilized lipase: a review. Biotechnol. Adv. 28, 628–634. Tao, J., Rao, R., Liuzzo, J., 1993. Microwave heating for rice bran stabilization. J. Microwave Power E. E. 28, 156–164. Turner, N.A., Vulfson, E.N., 2000. At what temperature can enzymes maintain their catalytic activity? Enzyme Microb. Technol. 27, 108–113. Watanabe, T., Shimizu, M., Sugiura, M., Sato, M., Kohori, J., Yamada, N., Nakanishi, K., 2003. Optimization of reaction conditions for the production of DAG using immobilized 1,3-regiospecific lipase lipozyme RM IM. J. Am. Oil Chem. Soc. 80, 1201–1207. Xu, X.B., Fomuso, L.B., Akoh, C.C., 2000. Synthesis of structured triacylglycerols by lipase-catalyzed acidolysis in a packed bed bioreactor. J. Agric. Food Chem. 48, 3–10. Yang, T., Xu, X., He, C., Li, L., 2003. Lipase-catalyzed modification of lard to produce human milk fat substitutes. Food Chem. 80, 473–481. Zaks, A., Klibanov, A.M., 1988. The effect of water on enzyme action in organic media. J. Biol. Chem. 263, 8017–8021.
Biocatalysis: towards ever greener biodiesel production. Biotechnol. Adv. 27, 398–408. Rodrigues, R.C., Volpato, G., Wada, K., Ayub, M.A.Z., 2008. Enzymatic synthesis of biodiesel from transesterification reactions of vegetable oils and short chain alcohols. J. Am. Oil Chem. Soc. 85, 925–930. Saka, S., Kusdiana, D., 2001. Biodiesel fuel from rapeseed oil as prepared in supercritical methanol. Fuel 80, 225–231. Salis, A., Pinna, M., Monduzzi, M., Solinas, V., 2005. Biodiesel production from triolein and short chain alcohols through biocatalysis. J. Biotechnol. 119, 291–299. Shimada, Y., Watanabe, Y., Sugihara, A., Tominaga, Y., 2002. Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing. J. Mol. Catal. B: Enzym. 17, 133–142. Shiu, P.-J., Gunawan, S., Hsieh, W.-H., Kasim, N.S., Ju, Y.-H., 2010. Biodiesel production from rice bran by a two-step in-situ process. Bioresour. Technol. 101, 984–989. Takano, K., 1993. Mechanism of lipid hydrolysis in rice bran. Cereal Food World 38, 695–698.
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
In situ lipase-catalyzed transesterification in rice bran for synthesis of fatty acid methyl ester
T
Nakyung Choia,1, Da Som Nob,c,1, Heejin Kimc, Byung Hee Kimd, Jieun Kwake, Jeom-Sig Leee, ⁎ In-Hwan Kima,c, a
Department of Integrated Biomedical and Life Science, Graduate School, Korea University, Seoul 02841, Republic of Korea School of Environmental and Biological Sciences, Rutgers University, New Brunswick, NJ 08901, USA c Department of Public Health Sciences, Graduate School, Korea University, Seoul 02841, Republic of Korea d Department of Food and Nutrition, Sookmyung Women’s University, Seoul 04310, Republic of Korea e National Institute of Crop Science, Rural Development Administration, Suwon, Gyunggi-do 16429, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Biodiesel In situ Repeated transesterification Rice bran lipase
Fatty acid methyl ester (FAME) were synthesized via in situ lipase-catalyzed transesterification in rice bran without additional catalyst. With this method, FAME were synthesized directly from oil in rice bran by simply adding methanol, with the aid of rice bran lipase already existing in rice bran. The effects of temperature, molar ratio (oil in rice bran to methanol), and water content of the rice bran were investigated. The yield of FAME and the free fatty acid content were monitored as a function of reaction time. The optimum conditions were a temperature of 40 °C, a molar ratio of 1:6, and a water content of 12%. Under the optimum conditions, the FAME yield of 83.4 wt% was obtained after 12 days. To further increase the FAME yield, the transesterification was repeated using the rice bran obtained from the first transesterification. The oils in rice bran that could be converted to FAME were completely transformed throughout the repeated transesterification.
1. Introduction Biodiesel is defined as monoalkyl esters of long-chain fatty acids derived from vegetable oils or animal fats for use in diesel engines (Bisen et al., 2010; Robles-Medina et al., 2009). It has become an important alternative to conventional diesel fuel since it is renewable, biodegradable, non-toxic, and essentially free of sulfur and aromatics. The use of biodiesel can reduce the exhaust emissions of particulate matters and green-house gases (Körbitz, 1999). The fundamental reaction in biodiesel production is transesterification, which can be catalyzed either chemically or enzymatically. Short reaction time and high yield of biodiesel are advantages of chemical transesterification (Marchetti et al., 2007). However, it has several major disadvantages, namely, high-energy requirements, difficulties in the recovery of catalysts and glycerol, and environmental pollution associated with the generation of large volumes of wastewater (Ognjanovic et al., 2009; Salis et al., 2005). Enzymatic transesterification can overcome the problems facing conventional chemical methods without compromising their advantages. Most importantly, enzymatic transesterification is carried out under mild reaction condition, and
⁎
1
does not require any complex process for glycerol recovery or wastewater treatment (Al-Zuhair et al., 2007). Thus, enzymatic transesterification has been widely studied for the biodiesel production. However, high cost and short life span of enzymes are major drawbacks of enzymatic methods. Supercritical technology has emerged as a good alternative method for biodiesel production, since supercritical alcohols can react with refined oils efficiently without a help of catalyst (Rathore and Madras, 2007; Saka and Kusdiana, 2001). However, this method is economically inefficient due to the high-energy requirements. In situ transesterification is another effective method for the production of biodiesel. In this method, oil is not extracted from seeds or oil-bearing sources prior to the transesterification. Instead, the oilbearing material contacts with alcohol directly in the presence of a catalyst. For the catalyst, chemical catalysts such as sulfuric acid or sodium hydroxide have been commonly used for biodiesel production under in situ condition (Georgogianni et al., 2008; Harrington and D’Arcy-Evans, 1985). Several studies have investigated the synthesis of biodiesel using rice bran as a substrate via in situ transesterification with a chemical catalyst (Lei et al., 2010; Özgül-Yücel and Türkay, 2003; Shiu et al., 2010).
Corresponding author at: Department of Public Health Sciences, Graduate School, Korea University, Seoul 02841, Republic of Korea. E-mail address: [email protected] (I.-H. Kim). Nakyung Choi and Da Som No contributed equally to this research.
https://doi.org/10.1016/j.indcrop.2018.04.049 Received 2 February 2018; Received in revised form 17 April 2018; Accepted 18 April 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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without the addition of methanol to investigate the hydrolytic activity. The reaction was performed without methanol and other conditions were the same as those of first group. The yield of FAME and FFA content were investigated for the transesterification activity and hydrolytic activity, respectively.
Rice bran is a by-product obtained from the outer layer of the brown rice kernel during milling to produce white rice. It contains 12–23% crude oil, which mainly consists of triacylglycerol (TAG), depending on the rice origin (Luh et al., 1991). Immediately following the milling process, rice bran deteriorates rapidly because the lipase in the rice bran has high hydrolytic activity. Free fatty acid (FFA) resulted from hydrolysis of TAG increase acidity which contributes to the formation of an off-flavor and soapy taste, and changes in functional property. The increase in FFA is unprofitable for the oil extraction in the industrial perspective due to high refining loss (Tao et al., 1993). Thus, to prevent hydrolysis caused by rice bran lipase, stabilization of rice bran immediately after the milling has been necessary but arduous task for oil industry. Numerous researches on rice bran lipase focused on its deactivation to stabilize the rice bran. Various technologies such as heating, treatment with hydrochloric acid, exposure to microwave irradiation, treatments with chemical inhibitors, low temperature storage, and osmic heating, have been explored for deactivation of the rice bran (Prabhakar and Venkatesh, 1986; Prakash and Ramanatham, 1994; Raghavendra et al., 2007). However, the possible utilization of rice bran lipase for beneficial purposes has not been studied. The aim of this study was to investigate in situ lipase-catalyzed transesterification in rice bran for the production of fatty acid methyl ester (FAME). Surprisingly, FAME were synthesized effectively with methanol by a lipase naturally existing in the rice bran. For optimization of the reaction, the effects of temperature, molar ratio (oil in rice bran to methanol), and water content were thoroughly studied. In addition, the effect of repeated transesterification was also investigated.
2.3. In situ lipase-catalyzed transesterification in rice bran For in situ lipase-catalyzed transesterification in rice bran, rice bran (100.0 g), which contains 15.0 g of oil (17.3 mmol, based on TAG), was put into a 1 L polypropylene bottle and 3.3 g of methanol (103.7 mmol) was added. The mixture of rice bran and methanol was then shaken vigorously for 5 min until the rice bran was uniformly wetted by the methanol. The rice bran wetted with the methanol was divided in a 15 mL polypropylene bottle and each bottle was tightly closed with a screw cap to prevent methanol from leaking. These samples were incubated in an incubator (Model IB-15G; Jeio tech, Daejeon, Republic of Korea) at the desired temperature and samples in individual bottles were taken for analysis at the desired reaction time. Subsequently, the oils in the rice bran were extracted in a 250 mL flask by stirring with 100 mL of n-hexane for 1 h. The extraction was performed twice and nhexane was completely removed using a rotary evaporator at 60 °C. The oils were stored in a glass vial at −85 °C until used. The effects of temperature (20–70 °C), molar ratio of oil in rice bran to methanol (1:3-1:9), and water content (0–24%) on the in situ lipasecatalyzed transesterification in rice bran were investigated. Because the rice bran initially had a water content of 12%, samples containing 0% and 6% water were prepared by removing some of the water by freezedrying. Samples with water contents of 18% and 24% were prepared by adding appropriate volumes of water to the rice bran. The water content of rice bran was determined by the oven drying method at 105 °C according to AOAC Official Method 950.01 (2002).
2. Materials and methods 2.1. Materials The rice bran used in this study was supplied by the Korean Rural Development Administration (Suwon, Republic of Korea) and the rice cultivar was Boramchan. The rice bran prior to use was passed through a 30-mesh sieve to sift out the hull, germ, broken endosperm part, and any other impurities. The oil and water contents of rice bran were 15 wt %, and 12%, respectively. The oils in rice bran were composed of 0.7 wt % of FFA, 1.8 wt% of diacylglycerol (DAG), 92.7 wt% of TAG, and 4.8 wt% of unsaponifiable matters. The fatty acids in crude rice bran oil consisted of 0.2 wt% myristic acid, 16.5 wt% palmitic acid, 0.1 wt% palmitoleic acid, 1.4 wt% stearic acid, 42.2 wt% oleic acid, 39.1 wt% linoleic acid, and 0.5 wt% of eicosenoic acid. The rice bran was stored in sealed containers at −85 °C until used. Tricaprin (GLC-570) as an internal standard was purchased from Nu-Check Prep, Inc. (Elysian, MN, USA) and fatty acid methyl ester mixture (CRM47885) was purchased from Sigma Aldrich Korea (Seoul, Republic of Korea). The other chemicals used in this study were of analytical grade unless otherwise noted.
2.4. Analysis of products Fifty milligram of oil extracted was weighed accurately into a 5 mL volumetric flask and diluted with chloroform. Each sample solution was transferred to a vial and analyzed by a gas chromatography. Tricaprin (0.5 mg/mL) was used as an internal standard. A gas chromatography (Model 3800; Varian, Palo Alto, CA, USA) equipped with a DB-1ht capillary column (15 m × 0.25 mm i.d.; J&W Scientific, Folsom, CA, USA) and a flame ionization detector was used. The column was initially held at 120 °C for 3 min and then heated to 370 °C at a rate of 20 °C/min. The column was then held at 370 °C for 3 min. Helium was used as a carrier gas at a flow rate of 1.5 mL/min and a split ratio was 1/50. The injector and detector temperatures were set at 370 °C. The yield of FAME (wt%) was calculated as in the following equation. Yield of FAME (wt%) = a/b × 100
2.2. Effect of heat treatment on the catalytic activity of rice bran lipase
Where a is the weight of FAME in rice bran, and b is the total weight of FAME, FFA, monoacylglycerol (MAG), DAG, TAG, and unsaponifiable matters in rice bran. FFA content was measured by acid value according to AOAC Official Method 940.28 (2002) and the content of unsaponifiable matters was determined according to AOAC Official Method 933.08 (2002). All experiments were conducted in triplicate.
To verify synthesis of FAME by in situ lipase-catalyzed transesterification in rice bran, the rice bran was heated for 15 min, 30 min, and 60 min and then divided into two groups to investigate the transesterification activity for the synthesis of FAME (first group) and the hydrolytic activity for the formation of FFA (second group). As a control, unheated rice bran was incubated in the same manner. The heat treatment was carried out with rice bran sealed in a glass bottle. After the heat treatment, the rice bran was employed for determination of hydrolytic and transesterification activities. The heated rice bran as first group was incubated with the addition of methanol to investigate the transesterification activity. The incubation temperature, the molar ratio of the oil in rice bran to methanol, and the water content of the rice bran were set at 40 °C, 1:6, and 12%, respectively. The heated rice bran as second group was incubated
3. Results and discussion 3.1. Effect of heat treatment on the catalytic activity of rice bran lipase Rice bran lipase is known to have strong hydrolytic activity, which increases the FFA content of rice bran and decrease the oil extraction recovery. More than two types of the rice bran lipases have been identified, and they tend to have a regiospecificity at sn-1, 3 positions of 141
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Fig. 1. The effect of heat treatment on the in situ transesterification activity of rice bran lipase. The reaction was performed at a molar ratio of 1:6 (oil in rice bran to methanol), a temperature of 40 °C, and a water content of 12%. The yield of fatty acid methyl ester (wt%) was determined as a function of reaction time. Fatty acid methyl ester refers to FAME.
TAG as well as a substrate specificity toward shorter FA during hydrolysis (Aizono et al., 1976; Prabhu et al., 1999; Takano, 1993). In contrast to the active researches on the hydrolytic activity of rice bran lipase, there has been very little effort devoted to the utilization of the rice bran lipase in other reactions. As an innovative attempt, in situ lipase-catalyzed transesterification in rice bran was carried out using methanol and FAME were synthesized successfully from the oil in the rice bran without any additional catalyst. For the trials of first group with methanol, the unheated rice bran had very high transesterification activity and the maximum FAME yield of 83.4 wt% was achieved after 12 days (Fig. 1). Meanwhile, for the trials involving heated rice bran, the transesterification activity decreased dramatically as the heating time increased. After 15 days, the yield was only 12 wt% when the rice bran was heated for 60 min. For the trials of second group without methanol, the unheated rice bran also showed very high hydrolytic activity (Fig. 2). However, for the trials involving heated rice bran, a marked decrease of hydrolytic activity was observed and the lowest FFA content was also obtained when rice bran was heated for 60 min. These results indicated that there
Fig. 3. The effect of temperature on the yield of fatty acid methyl ester (a) and free fatty acid content (b) for the in situ lipase-catalyzed reactions in rice bran as a function of reaction time. The reaction was performed at a molar ratio of 1:6 (oil in rice bran to methanol) and a water content of 12%. Fatty acid methyl ester and free fatty acid refer to FAME and FFA, respectively.
was a high correlation between transesterification activity and hydrolytic activity of rice bran lipase. Therefore, it was verified that FAME were synthesized by the in situ lipase-catalyzed transesterification in rice bran. 3.2. Effect of temperature The reaction temperature is one of the most important parameters in enzymatic reactions. An increase in temperature can decrease mass transfer limitations by decreasing the mixture’s viscosity and enhancing the mutual solubility or diffusion processes of substrates, and can also increase interactions between enzyme particles and substrates (Xu et al., 2000). However, temperatures that are too high can induce irreversible denaturation of the enzyme protein and shorten the half-life of the enzyme activity, although some enzymes from microorganisms are stable and active at high temperatures (Turner and Vulfson, 2000). The effect of temperature on the in situ lipase-catalyzed transesterification in rice bran was determined by the yield of FAME (wt%) as a function of reaction time (Fig. 3a). The FFA content (wt%) in the reaction mixture was also monitored (Fig. 3b). During the first 9 days of the reaction, the yield of FAME increased considerably when the temperature was increased from 20 to 40 °C, but there were no further
Fig. 2. The effect of heat treatment on the in situ hydrolytic activity of rice bran lipase. The reaction was performed at a temperature of 40 °C, and a water content of 12%. The free fatty acid content (wt%) was determined as a function of reaction time. Free fatty acid refers to FFA. 142
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increase in the yields of FAME between 40 and 50 °C. However, as the reaction time increased from 9 to 12 days, the yield of FAME further increased up to 83.4 wt% at 40 °C. By contrast, the increase of FAME yield was negligible at 50 °C because of the increase of FFA content. After 6 days of reaction, the yield of FAME decreased when the temperature increased from 50 to 70 °C, while the FFA content increased. Overall, the decrease of FAME yield at the temperatures higher than 40 °C may have been caused by an increase in FFA and a decrease in transesterification activity. The control of the FFA is very important since it adversely affects the quality of the final product (Chong et al., 2007). It has been reported that optimum temperature on the hydrolytic activity of the lipase, which was isolated from defatted rice bran, was between 30 and 40 °C (Aizono et al., 1976; Prabhu et al., 1999; Rajeshwara and Prakash, 1995). In particular, in these studies, the hydrolytic activity of rice bran lipase was reduced at the temperature above 40 °C. Many studies on various lipase-catalyzed reactions have described a Ping-Pong mechanism for the inhibition of hydrolytic activity by alcohol (Bousquet-Dubouch et al., 2001; Martinelle and Hult, 1995). Thus, the hydrolytic activity of rice bran lipase can be affected by both alcohol and temperature. Our results indicated that incubation at higher temperatures leads to evaporation of methanol, which was inhibitor of rice bran lipase, and evaporation of methanol resulted in increase of FFA. In other words, methanol have more effect on hydrolytic activity of rice bran lipase than temperature. Hence, 40 °C was selected as the optimum temperature and used in subsequent experimental trials, since it represents both the maximum yield and the high transesterification rate. 3.3. Effect of molar ratio In general, alcohol can dissolve glycerol liberated from the reaction and prevent it from blocking the interface between substrates and enzymes (Rodrigues et al., 2008). However, excessive alcohol above the stoichiometric molar ratio is undesirable for enzyme activity because methanol is known to have a strong enzyme-denaturing activity. The effect of molar ratio on the in situ lipase-catalyzed transesterification in rice bran was determined by the yield of FAME (wt%) as a function of reaction time (Fig. 4a). The FFA content (wt%) in the reaction mixture was also monitored (Fig. 4b). As the molar ratio increased from 1:3 to 1:6, the yield of FAME increased gradually. However, the difference in the yields of FAME achieved between the molar ratio of 1:6 and 1:7.5 was negligible throughout the entire reaction. Furthermore, when the molar ratio further increased from 1:7.5 to 1:9, the yields of FAME decreased considerably. Methanol is widely used for synthesis of biodiesel because of its economic feasibility and accessibility in most countries (Antczak et al., 2009; Deng et al., 2005). However, the enzymatic transesterification of methanol is problematic, because lipase inhibition by methanol is stronger than that by longerchain aliphatic alcohols. (Salis et al., 2005; Tan et al., 2010). Shimada et al. reported an irreversible inactivation of enzyme caused by more than one-third of the stoichiometric amount of methanol for methanolysis of vegetable oil catalyzed by the immobilized C. antarctica lipase (Shimada et al., 2002). The enzyme inactivation is likely related to the amount of undissolved alcohol touching the enzyme in the reaction mixture. In the present study, the transesterification at a molar ratio beyond 1:7.5 implies deactivation of the rice bran lipase by the methanol. Meanwhile, the FFA content in the reaction mixture decreased substantially with an increase of molar ratio. At the molar ratio of 1:3, the FFA content increased to 25 wt% as the reaction time increased up to 15 days. However, with the molar ratio of 1:9, the FFA content remained constant under 5 wt%. This demonstrates that a hydrolysis is highly inhibited by a larger amount of methanol. With a larger amount of methanol, transesterification prevails over hydrolysis as methanol competes with water existing in the rice bran to react with the rice bran oil.
Fig. 4. The effect of molar on the yield of fatty acid methyl ester (a) and free fatty acid content (b) for the in situ lipase-catalyzed reactions in rice bran as a function of reaction time. The reaction was performed at a temperature of 40 °C and a water content of 12%. Fatty acid methyl ester and free fatty acid refer to FAME and FFA, respectively.
In summary, large amounts of methanol inhibit the transesterification by deactivating the rice bran lipase and also depress the hydrolysis. To take advantage of the mutually exclusive behaviors of methanol, the molar ratio of 1:6 (oil in rice bran to methanol) was considered as the appropriate compromise and was selected as the optimum condition. 3.4. Effect of water content It is known that reactions involving lipases require water to maintain the enzyme structure and achieve catalytic activity (Watanabe et al., 2003; Zaks and Klibanov, 1988). The use of too much water, however, can result in a shift in the reaction towards hydrolysis, and lead to an increase of FFA during transesterification (He and Shahidi, 1997; Yang et al., 2003). The effect of water content on the in situ lipase-catalyzed transesterification in rice bran was determined by the yield of FAME (wt%) as a function of reaction time (Fig. 5a). The FFA content (wt%) in the reaction mixture was also monitored (Fig. 5b). As the water content of rice bran increased from 0 to 12%, the yield of FAME increased drastically throughout the entire reaction. By contrast, when the water content further increased from 12 to 24%, the yield decreased considerably. The maximum FAME yield of 83.4 wt% was obtained at the 143
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the FAME yield was also the lowest at a water content of 0%. These results demonstrate that an appropriate amount of water is necessary to maintain the lipase activity on the transesterification. As the water content increased, the transesterification activity of the lipase for the FAME synthesis was improved, but the hydrolysis also increased. At water contents higher than 12%, the decrease of FAME yield was attributed to the fact that the rate of hydrolysis increased as water content increased, thereby leading to a concomitant increase in FFA. Water forms hydrogen bonds with functional groups in the protein molecules and affects the enzymatic reactions (Kurkal et al., 2005; Kurplus and McCammon, 1983). A critical amount of water is needed to maintain the three-dimensional form of an enzyme that is necessary for its catalytic activity. However, excess water causes enzyme inactivation and undesirable hydrolysis. Therefore, the optimum water content is a compromise between minimizing hydrolytic activity and maximizing transesterification activity. Hence, 12% was selected as the optimum water content since it represents both the maximum yield and the fastest FAME synthesis. This water content coincided with the original water content of the rice bran used in this study, implying that the rice bran lipase carries the highest activity on the transesterification with its original water content. 3.5. Repeated transesterification Under the selected optimum condition, the FAME yield of 83.4 wt% was attained via the first transesterification after 12 days. The oils in the rice bran after the first transesterification was composed of 83.4 wt% of FAME, 8.5 wt% of FFA, 1.0 wt% of monoacylglycerol (MAG), 0.7 wt% of DAG, 1.7 wt% of TAG, and 4.8 wt% of unsaponifiable matters (Table 1). Among the oils in the rice bran, only FFA, MAG, DAG, and TAG can be converted to FAME. However, these oils were still present in the rice bran after the first transesterification. Therefore, to improve the FAME yield, the transesterification was repeated using the rice bran obtained from first transesterification of 12 days. The control of water content of rice bran was one of the important factors because FFA of 8.5 wt% still remained in the rice bran after first transesterification. Thus, as a pre-treatment of the repeated transesterification, water in the rice bran after first transesterification was removed by freeze-drying and residual methanol in the rice bran was also removed during this process. Subsequently, the dried rice bran was subjected to a repeated transesterification under the optimum condition, which was the molar ratio of 1:6 (oil in rice bran to methanol), and the temperature of 40 °C. The effect of repeated transesterification was determined by the yield of FAME and the FFA content as a function of reaction time (Fig. 6a and b). As the repeated reaction progressed, a considerable increase in the yield of FAME was observed. After 6 days of repeated transesterification, the yield of FAME was increased to 95.2 wt% from 83.4 wt% for the first transesterification. Meanwhile, the FFA content showed a
Fig. 5. The effect of water content on the yield of fatty acid methyl ester (a) and free fatty acid content (b) for the in situ lipase-catalyzed reactions in rice bran as a function of reaction time. The reaction was performed at a temperature of 40 °C and a molar ratio of 1:6 (oil in rice bran to methanol). Fatty acid methyl ester and free fatty acid refer to FAME and FFA, respectively.
water content of 12% after 12 days. By contrast, a steady increase of FFA was observed when the water content was increased from 0 to 24% though the entire reaction. It is noteworthy that the FFA content at a water content of 0% remained nil throughout the entire reaction, implying that the hydrolysis was completely inhibited due to the insufficient water as a substrate. However,
Table 1 Compositions (wt%) of the oils extracted from the original rice bran, the rice bran after the first transesterification, and the rice bran after the repeated transesterification. FAMEd a
Original rice bran First transesterificationb Repeated transesterificationc a b c d e f g
e
ND 83.4 ± 3.9f 95.2 ± 1.6g
FFA
MAG
DAG
TAG
Unsaponifiable matters
0.7 ± 0.3 8.5 ± 0.2 ND
ND 1.0 ± 0.1 ND
1.8 ± 0.6 0.7 ± 0.2 ND
92.7 ± 1.3 1.6 ± 0.3 ND
4.8 ± 1.2 4.8 ± 1.2 4.8 ± 1.2
The oils in original rice bran. The oil content in the rice bran was 15 wt%. The oils in rice bran after first transesterification. The oils in rice bran after repeated transesterification. Abbreviations: FAME, fatty acid methyl ester; FFA, free fatty acid; MAG, monoacylglycerol; DAG, diacylglycerol; TAG, triacylglycerol. ND: not detected. Limit of detection: FFA, 4.6 mg/kg oil; MAG, 7.7 mg/kg oil; DAG, 4.4 mg/kg oil; TAG, 3.8 mg/kg oil. The weight of FAME recovered after the first transesterification was 12.5 g per 100 g of rice bran. The weight of FAME recovered after the repeated transesterification was 14.3 g per 100 g of rice bran. 144
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purposes. Conflicts of interest There are no conflicts of interest to declare. Acknowledgements This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project title: Evaluation of quality and processing property of longterm stored rice, Project No. PJ011037)" Rural Development Administration, Republic of Korea. References Official Methods of Analysis of AOAC International. AOAC International, Gaithersburg, MD, USA. Aizono, Y., Funatsu, M., Fujiki, Y., Watanabe, M., 1976. Purification and characterization: of rice bran lipase II. Agric. Biol. Chem. 40, 317–324. Al-Zuhair, S., Ling, F.W., Jun, L.S., 2007. Proposed kinetic mechanism of the production of biodiesel from palm oil using lipase. Process Biochem. 42, 951–960. Antczak, M.S., Kubiak, A., Antczak, T., Bielecki, S., 2009. Enzymatic biodiesel synthesis–key factors affecting efficiency of the process. Renew Energy 34, 1185–1194. 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Fig. 6. The effect of repeated transesterification on the yield of fatty acid methyl ester (a) and free fatty acid content (b) for the in situ lipase-catalyzed reactions in rice bran as a function of reaction time. The rice bran obtained from the first transesterification was employed as a substrate for repeated transesterification. Fatty acid methyl ester and free fatty acid refer to FAME and FFA, respectively.
marked decrease as the repeated reaction time increased. The FFA content decreased to 2.4 wt% after 3 days of repeated transesterification and became nil after 6 days. By contrast, for the trial conducted with a water content of 12%, neither FAME yield nor FFA content changed during the entire repeated transesterification. Consequently, the oils (FFA, MAG, DAG, and TAG) that could be converted to FAME, were completely transformed to FAME by the repeated transesterification.
4. Conclusions In situ lipase-catalyzed transesterification in rice bran was developed as a cost-effective and eco-friendly method for the production of FAME. It was verified that FAME were synthesized under in situ condition of rice bran by its own lipase. Water and methanol were crucial factors for both hydrolysis and transesterification. Too much water adversely affected the yield of FAME because it increased the FFA content. Excess methanol inhibited the transesterification activity as well as hydrolytic activity of rice bran lipase. Our strategy, which is to synthesize FAME by in situ lipase-catalyzed transesterification in rice bran, suggested a meaningful way to use rice bran for profitable 145
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