Partial hydrolysis of soybean oil by phospholipase A1 (Lecitase Ultra)

Food Chemistry 121 (2010) 1066–1072

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Partial hydrolysis of soybean oil by phospholipase A1 (Lecitase Ultra) Yong Wang a,b, Mouming Zhao a,*, Keke Song b, Lili Wang b, Shuze Tang b, William W. Riley b a b

College of Light Industry and Food Science, South China University of Technology, Guangzhou 510641, China Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China

a r t i c l e

i n f o

Article history: Received 20 July 2009 Received in revised form 2 December 2009 Accepted 25 January 2010

Keywords: Partial hydrolysis Soybean oil Phospholipase A1 (Lecitase Ultra)

a b s t r a c t The partial hydrolysis of soybean oil, as catalysed by phospholipase A1 (Lecitase Ultra) in a solvent-free system, was investigated in this study. The optimal pH and temperature for the partial hydrolysis of soybean oil by phospholipase A1 (Lecitase Ultra) were 6.8 and 40 °C, respectively. Phospholipase A1 (Lecitase Ultra) displayed good stability over a range of pH values from 4.7 to 7.4, and at temperatures below 60 °C. Phospholipase A1 (Lecitase Ultra) is postulated to possess sn-1,3-position regiospecificity towards triacylglycerols (TAGs), based on the identification of the fatty acids released from TAGs following partial hydrolysis by swine pancreatic lipase (SPL) and phospholipase A1 (Lecitase Ultra). Alternative TAG hydrolysis routes for phospholipase A1 (Lecitase Ultra) are postulated, and several reaction equilibrium and rate constants were determined. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Phospholipase A1 (PLA1) (E.C. 3.1.1.32) represents a very diverse sub-group of phospholipase isoenzymes with 1-acyl hydrolytic activity. PLA1 displays broad substrate specificity and also harbors some lipase activity, considering the sequence similarity of PLA1 to other lipases (Guo, Vikbjerg, & Xu, 2005). Recently, a new enzyme preparation with phospholipase A1 activity, trade name Lecitase Ultra, has been patented and made available commercially (Bojsen et al., 2007). According to information from the supplier, the product mixture is obtained by the fusion of lipase genes from Thermomyces lanuginosa and phospholipase genes from Fusarium oxysporum (Fernandez-Lorente et al., 2008). This new enzyme combines the stability of the T. lanuginosa lipase enzyme and the activity of the F. oxysporum enzyme. Lecitase Ultra is a carboxylic ester hydrolase which displays activity towards both phospholipids and triacylglycerols (TAGs) (Slizyte, Rustad, & Storro, 2005). Major commercial uses include the degumming of vegetable oils and the improvement of lecithin emulsification properties (Yang, Zhou, Yang, Wang, & Wang, 2008). The mechanisms and the kinetics involved in triacylglycerol (TAG) hydrolysis to produce fatty acids from vegetable oils, including soybean oil, by the action of various lipases, have been studied extensively (Freitas, Bueno, Perez, Santos, & de Castro, 2007; Liu et al., 2008; Primozic, Habulin, & Knez, 2003; Ramachandran, Al-

* Corresponding author. Address: College of Light Industry and Food Science, South China University of Technology, Wushan Rd. 381, Guangzhou 510641, China. Tel.: +86 20 87113914; fax: +86 20 87114954. E-mail address: [email protected] (M. Zhao). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.01.051

Zuhair, Fong, & Gak, 2006; Ting, Tung, Giridhar, & Wu, 2006). The intent has been to optimise the hydrolytic conditions and identify possible enhancing technologies to increase the yield of fatty acids. Partial hydrolysis of TAGs is an alternative process for the production of diacylglycerols (DAGs), which are regarded as functional oils and a partial substitute for TAGs in the diet (Cheong et al., 2007). DAGs are usually produced enzymatically, through esterification and glycerolysis. In lipase-catalysed esterification, DAGs are synthesised through the esterification of FA to glycerol, with the simultaneous removal of water (Berger, Laumen, & Schneider, 1992; Lo, Baharin, Tan, & Lai, 2004; Watanabe et al., 2003). Glycerolysis, however, involves the trans-esterification of an acyl moiety from TAGs to monoacylglycerols (MAGs) formed during the reaction (Kristensen, Xu, & Mu, 2005a, 2005b). Due to the low activity of lipase in the non-aqueous phase and the high viscosity of glycerol during esterification and glycerolysis, the yield of DAGs is quite low and the monoacylglycerol (MAG) by-product is far more abundant. Hence, partial hydrolysis of TAGs to produce DAGs enzymatically is more attractive than are esterification and glycerolysis, due to an adequate supply of the primary feedstock (vegetable oils and animal fats) and the inexpensive reactant (water). Soybean oil is one of the most abundant vegetable oils in the world. Its relatively high content of unsaturated fatty acids, stability during various cooking applications, and low price make it a valuable part of the food chain and human diet. Compared to commercial immobilised lipases, Lecitase Ultra demonstrates excellent potential for application within the food industry due to its comparative advantages of low cost and high enzyme activity. The partial hydrolysis of soybean oil catalysed by Lecitase Ultra to produce DAG-enriched oils, therefore, is a promising alternative technology for the production of functional oils.

Y. Wang et al. / Food Chemistry 121 (2010) 1066–1072

However, the use of Lecitase Ultra for the partial hydrolysis of soybean oil to produce DAG-enriched oil has received little attention to date. We previously reported on a process to produce DAG-enriched oil from soybean oil, catalysed by phospholipase A1 (Lecitase Ultra) (Wang, Zhao, Ou, & Song, 2009). However, the principles involved in the partial hydrolysis of soybean oil by phospholipase A1 (Lecitase Ultra) were not discussed. In the present study, the regiospecificity of PLA1 (Lecitase Ultra) towards TAGs and the reaction route of the partial hydrolysis of soybean oil have been investigated. Furthermore, the optimal conditions for the partial hydrolysis of soybean oil, such as pH and temperature, were also studied. The results obtained will be useful in understanding the mechanisms involved in the partial hydrolysis of vegetable oils by phospholipase A1 (Lecitase Ultra) and in determining the way to control production of the desired reaction products. 2. Materials and methods 2.1. Materials Refined soybean oil was purchased from Donghai Cereal and Oil Co., Ltd. (Zhangjiagang, Jiangsu, China). Commercial phospholipase A1 (Lecitase Ultra) was obtained from Novozymes A/S (Bagsvaerd, Denmark), while swine pancreatic lipase (SPL) was purchased from Sigma (St. Louis, MO, USA). Soybean oil diacylglycerol standards were prepared by column chromatography separation after molecular distillation, as described below. Fifty (50) grams of soybean oil were hydrolysed by phospholipase A1 (Lecitase Ultra) at a reaction temperature of 45 °C, an enzyme load of 30 U/g (of the oil mass), and a reaction time of 8 h. After the reaction was complete, the mixture was allowed to settle for 60 min, in order to separate it into two layers, namely, the non-polar upper layer (NUPL) and the lower aqueous layer. The NUPL was molecularly distilled at 160 °C to remove the released fatty acids. The residue of the first distillation was distilled again at 215 °C, and the distillate was collected as diacylglycerol oil (DO). One (1.0) g DO was dissolved in 20 ml of hexane, and the solution was then loaded into a silica column (u1.5 cm  30 cm). TAGs were eluted with 250 ml of hexane, and DAGs were eluted with 400 ml of ethyl ether/hexane (3:7, v/v). The DAG standard was collected after the solvents were evaporated by a rotary evaporator under a vacuum. The purity of the standard was analysed by thin layer chromatography (TLC) and HPLC/ESI/MS (as described in Section 2.6). The standard was developed by TLC on silica gel plates (SIL GF254, 20 cm  20 cm  0.25 mm; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), using benzene:diethyl ether:acetic ether:acetic acid (80:10:10:0.2, v/v/v/v) as the mobile phase. After visualisation by iodine vapour, only DG fractions were evident.

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where AVt and AV0 were the acid values of the NUPL after the reaction and the acid value of soybean oil before the reaction, respectively: m was the weight of the oil layer (5.0 g); V was the volume of the mixture; and t was the reaction time (10 min). The optimal reaction temperature was determined by adding soybean oil to 100.0 ml aliquots of sodium phosphate buffer (0.25 mM, pH 6.8) at 30, 35, 40, 45, 50, 65 and 70 °C. 2.3. Stability of PLA1 (Lecitase Ultra) The stability of PLA1 (Lecitase Ultra) across a wide range of pH values was assayed as follows: PLA1 (Lecitase Ultra) (0.2 ml) was mixed with 0.8 ml of sodium phosphate buffer (0.25 mM) at pH 0.7, 1.7, 2.6, 3.7, 4.7, 5.7, 6.6, 7.4, 8.4, 9.4, 10.4, 11.3 or 12.5, and the various mixtures were stored at 4 °C for 2 h. A 0.5 ml aliquot of each mixture was withdrawn to determine the initial velocity, in a reaction medium of sodium phosphate buffer (0.25 mM) at pH 6.8 and 40 °C. The stability of PLA1 (Lecitase Ultra) by temperature was assayed as follows: PLA1 (Lecitase Ultra) (1.0 ml) was incubated in a water bath at temperatures of 4, 25, 40, 45, 50, 55, 60, 70, 75 or 80 °C for 30 min, and then a 0.1 ml aliquot of enzyme was withdrawn to determine the initial velocity with sodium phosphate buffer (0.25 mM) at pH 6.8 and 40 °C. 2.4. Regiospecificity of PLA1 (Lecitase Ultra) Soybean oil (50.0 g) was partially hydrolysed by PLA1 (0.1 ml), at a water content of 20.0 g and at 40 °C for 4 h. After the reaction was complete, the mixture was allowed to settle into two layers – the NUPL and the lower aqueous layer. The released free fatty acids of the NUPL were distilled by molecular distillation at an evaporator temperature of 160 °C, and the composition of fatty acids was analysed by GC. A controlled partial hydrolysis experiment, using swine pancreatic lipase (SPL), was conducted over a 4 h time period at 37 °C. The free fatty acids released by SPL were collected by molecular distillation and analysed by GC. 2.5. Partial hydrolysis of soybean oil Soybean oil (50.0 g) was partially hydrolysed by PLA1 (Lecitase Ultra) (0.1 ml) in 20.0 g of water for 0.5, 1.0, 2.0, 4.0 6.0 or 8.0 h, at 40, 45 and 50 °C, using a water bath shaker (approximately 150 rpm). After the reactions were completed, each mixture was allowed to settle into two layers, and the DAG content of the NUPL layer was analysed by RP-HPLC, and the glycerol content of the aqueous layer was analysed by GC. A control reaction was also carried out for 8 h at 30 °C, also in 20.0 g of water.

2.2. Optimal pH and temperature values for partial hydrolysis 2.6. Measurement of equilibrium constants The optimal pH value for the partial hydrolysis of soybean oil by PLA1 (Lecitase Ultra) was determined as follows: soybean oil (5.0 g) was added to 100.0 ml aliquots of sodium phosphate buffer (0.25 mM), adjusted to pH 5.7, 6.4, 6.6, 6.8, 7.0, 7.2 and 7.6. After 0.10 ml PLA1 (Lecitase Ultra) was added to each solution, the mixture was placed in a water bath shaker (approximately 180 rpm) for 10 min at 40 °C. The mixture was then centrifuged at 11,200g for 5 min to separate it into two layers. The acid value of the upper oil layer was determined by titration of free fatty acids with a 0.1 M KOH solution. The initial reaction velocity (Vi) of the released free fatty acids was calculated using the following equation:

Vi ¼

ðAVt  AV0 Þ=56:11  m V t

ð1Þ

Fifty (50.0) grams of a substrate mixture (20.2 g soybean oil, 25.0 g soybean fatty acids, 3.0 g glycerol, 2.2 g water) and 0.1 ml of PLA1 (Lecitase Ultra) were placed in a screw-capped 250 ml flask. The flask was shaken (approximately 150 rpm) at 40 °C. After 5 days, an aliquot of 2.0 ml of the reaction mixture was withdrawn and allowed to settle for 2 h. The acid value of the NUPL was determined by titration with a 0.1 M KOH solution. The reaction was stopped when the acid value was found to be nearly constant (on the seventh day). 2.7. Analysis of diacylglycerol content by RP-HPLC and HPLC/ESI/MS The diacylglycerol content of the NUPL was determined by reverse-phase high performance liquid chromatography (RP-HPLC),

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using Agilent 1100 series (Agilent Technologies Inc., Palo Alto, CA, USA). Samples were dissolved in the mobile phase (acetonitrile/ isopropanol, 56:44, v/v) at a concentration of 10.0 mg/ml, filtered through a 0.45 lm nylon membrane filter, and 20.0 ll of the sample were injected onto a Diamonsil C18 5 lm column (150  4.6 mm i.d.) (Dikma Technologies Inc., Tianjin, China) with an UV detector (set to read at 210 nm); (flow rate = 1.0 ml/min). The DAG peaks in the standard, which did not co-elute with the sample TAGs and MAGs, were selected for construction of calibration curves based on areas, and the DAG content results were given as the weight percentage of total acylglycerols. The composition of the DAG standard was identified by HPLC/ ESI/MS. Samples were diluted to a concentration of 1.0 mg/ml in the solvent (acetonitrile/isopropanol, 56:44, v/v), and were then injected onto a Pinnacle II C18 5 lm column (150  2.1 mm i.d.) (Restek Corp., Bellefonte, PA USA), in an Aglient 1100 HPLC. The column was connected to a 4000 QTRAP mass spectrometer (Applied Biosystems Inc., Foster City, CA, USA) with an atmospheric pressure ion source to sample positive ions from the electro-spray interface. Formic acid (0.5%), in acetonitrile/isopropanol (55:45, v/ v), was added to improve the ionisation of DAGs. The mass spectra, between 300 and 1200 m/z, were obtained at an ion scan rate of 5500 am/s. 2.8. Diacylglycerol fraction analysis 1,3-DAG and 1,2-DAG components of the NUPL preparations were isolated by thin layer chromatography (TLC) on silica gel plates (SIL GF254, 20 cm  20 cm, 0.25 mm), provided by Qingdao Haiyang Chemical Co., Ltd. (Qingdao, Shandong, China), using benzene/diethyl ether/acetic acid (80:10:10:0.2, v/v/v/v) as the mobile phase. 1,3-DAG fraction (RF = 0.87) and 1,2-DAG fraction (RF = 0.78) were scraped away following visualisation by iodine vapour and extracted with diethyl ether (2  3 ml). Samples of DAGs were then filtered through a 0.45 lm nylon membrane filter to remove impurities, and 20.0 ll samples were injected onto a Lichrosorb Si-60 5 lm column (250  4.6 mm i.d., Grace Division, IL, USA) and separated by HPLC (LC20 AT, Shimadzu Inc., Kyoto, Japan) with a UV detector (210 nm). The mobile phase was n-hexane/98% aqueous isopropanol (99.6:0.4, v/v) with a flow rate of 1.0 ml/min. The ratio of sn-1,3 DAG and sn-1,2(2,3)-DAG were calculated from the peak area data. 2.9. Fatty acid composition analysis The fatty acid composition of soybean oil and the released fatty acids of the NUPL were analysed by gas chromatography (GC 900A, Shanghai Kechuang Chromatograph Instrument Co., Ltd., Shanghai, China). The gas chromatograph was equipped with a capillary column (HP-5, 30 m  0.32 mm  0.25 lm; Agilent Technologies Inc., Palo Alto, CA, USA), a flame ionization detector (FID), and N2 carrier gas. The injection was performed in split mode with a split ratio of 80:1. Four (4.0) grams samples of soybean oil were dissolved in 40.0 ml methanol and then 0.5 ml 1.0 M methanolic KOH was added. After a 10 min reaction period at the boiling point of methanol, n-hexane (20 ml) and water (40 ml) were added, and the mixture was then transferred to a separatory funnel. The upper organic phase was dried over anhydrous Na2SO4, and concentrated under a stream of nitrogen for analysis. A 4.0 g sample of the released fatty acids was esterified with 50.0 ml of 1.0 M methanolic H2SO4 for 15 min at the boiling point of methanol, and then partitioned between n-hexane (30 ml) and water (100 ml) in a separatory funnel. The upper organic phase was dried and concentrated for analysis. A fatty acid methyl ester (FAME) solution (1 ll) was injected at an injector temperature of 240 °C, column temperature of 195 °C, FID temperature of 240 °C and carrier gas (N2) flow of 60 ml/min.

The fatty acid composition percentage was based on the area response, using a FID detector. 2.10. Glycerol content analysis The glycerol content of the lower aqueous layer was analysed using a gas chromatograph (GC 900A, Shanghai Kechuang Chromatograph Instrument Co., Ltd., Shanghai, China) equipped with a capillary column (SPB™-5, 30 m  0.32 mm  0.25 lm; Supelco Inc., Bellefonte, PA, USA) with N2 carrier gas, at a flow rate of 80 ml/min, an injection volume of 1 ll, and a FID detector. The injector, column and detector temperatures were 290, 210 and 190 °C, respectively. Glycerol was characterised by a single peak with a retention time of 1.70 min. The concentration of free glycerol was determined by calibration with a standard aqueous glycerol solution by area response (0.01–1% glycerol, w/v). 2.11. Statistical analysis Analyses for DAGs, fatty acid profiles, and glycerol and acid values were done in triplicate, with data reported as the means ± standard deviations. One-way ANOVA was performed using SPSS 14 statistical software (SPSS Inc., Chicago, IL). Differences were considered to be significant at p 6 0.05, according to Duncan’s Multiple Range Test. 3. Results and discussion 3.1. Optimal pH and temperature values for partial hydrolysis The effect of pH on the initial velocity of soybean oil partial hydrolysis by PLA1 (Lecitase Ultra) is shown in Fig. 1a. The range of pH values used for this assay was selected according to the manufacturer’s recommendation. The initial velocity increased with increasing pH and reached a maximum of 7.46 ± 0.11  102 mmol l1 min1 at pH 6.8. However, initial velocity decreased with increasing pH when the pH was over 6.8. PLA1 (Lecitase Ultra) was more likely to hydrolyse phospholipids at low pH, which is why citric acid is used to adjust pH when PLA1 (Lecitase Ultra) is applied during the degumming of vegetable oils (Yang et al., 2008). TAGs proved to be better substrates for PLA1 (Lecitase Ultra) when hydrolysis reactions occurred under neutral and alkaline conditions (Bojsen et al., 2007). One unit of PLA1 (Lecitase Ultra) enzyme activity in this study was defined as the amount of enzyme that was necessary to hydrolyse 1 lmol of fatty acid from TAGs per minute at pH 6.8 and 40 °C. Lecitase Ultra activity using these assay criteria was 771.72 ± 36.80 U/ml (n = 3). Claims for Lecitase Ultra activity have reached 10,000 U/ml for the hydrolysis of the carboxyl group of butyl laurate. Due to differences in the substrate and the measurement conditions involved, the enzyme activity of Lecitase Ultra for the partial hydrolysis of soybean oil was markedly lower. The optimal pH for the partial hydrolysis of soybean oil by PLA1 (Lecitase Ultra) was 6.8 (near neutrality), which means that buffer might not be needed under commercial conditions in the partial hydrolysis of vegetable oils. The effect of temperature on the initial velocity of soybean oil partial hydrolysis by PLA1 (Lecitase Ultra) is shown in Fig. 1b. The maximum initial velocity of partial hydrolysis was reached at 7.32 ± 0.13  102 mmol l1 min1 at 40 °C. Initial velocity decreased rapidly when the temperature exceeded 50 °C. The elevated reaction temperature improved the solubility of oil in water and decreased the viscosity of the reaction mixture, thereby enhancing mass transfer within the reaction. The elevated reaction temperature increased the probability of molecular collision and

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0.07 0.06 0.05 0.04 0.03 0.02 0.01 0

5

5.5

6

6.5

7

7.5

0.08

-1

0.08

b

0.07

-1

-1

-1

Initial velocity mmol.L .min )

a

Initial velocity mmol.L .min )

Y. Wang et al. / Food Chemistry 121 (2010) 1066–1072

0.06 0.05 0.04 0.03 0.02 0.01 0

8

25

35 45 55 Reaction temperature

2

4

6

8

10

12

14

pH value

Initial velocity mmol.L .min )

0

-1

d

0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

0.06

-1

-1 -1

c

Initial velocity mmol.L .min )

pH value

0.05

65

75

0.04 0.03 0.02 0.01 0

0

20

40 60 Temperature

80

100

)

Fig. 1. (a) Effect of pH value on the initial velocity of soybean oil hydrolysis by phospholipase A1 (Lecitase Ultra) at a reaction temperature of 40 °C. (b) Effect of temperature on the initial velocity of soybean oil hydrolysis by phospholipase A1 (Lecitase Ultra) at pH 6.8. (c) Effect of treated pH value on stability of phospholipase A1 (Lecitase Ultra) expressed as initial velocity at a reaction temperature of 40 °C. (d) Effect of temperature on thermal stability of phospholipase A1 (Lecitase Ultra) expressed as initial velocity at pH 6.8.

contact with the interfacial area, which improved the efficiency of catalysis (Al-Zuhair, Hasan, & Ramachandran, 2003). However, a high reaction temperature caused irreversible enzyme denaturation, which decreased enzyme activity (Primozic et al., 2003). So, the optimal temperature for the reaction needed to be a compromise between these two effects of high temperature. The optimal temperature for the hydrolysis of palm olein by Lipozyme TL IM (Thermomyces lanuginosa lipase) was reported to be 55 °C (Chew et al., 2008), which was slightly higher than that determined for PLA1 (Lecitase Ultra) in this study. 3.2. Stability of PLA1 (Lecitase Ultra) The results for the Lecitase Ultra stability studies relative to pH and temperature are shown in Fig. 1c. PLA1 (Lecitase Ultra) displayed good stability over a range of pH values from 4.7 to 7.4, which was in accordance with the optimal pH recommended by the producer. The initial velocity decreased rapidly at pH values below 3.0 and over 10.0. Extreme pH values denatured protein and caused irreversible enzyme denaturation. Furthermore, ion dissociation at the active site of the enzyme occurred at a relatively mild pH value, which caused some reversible enzyme denaturation. By incubation of the enzyme at the optimal pH, the enzyme returned to normal activity. The phospholipase A1 thermal stability results (Lecitase Ultra) at different temperatures are presented in Fig. 1d. PLA1 (Lecitase Ultra) displayed good stability at a temperature below 45 °C, and the initial velocity of partial hydrolysis was nearly constant. However, the initial velocity decreased very rapidly at a temperature above 60 °C. After treatment at 70 °C for 30 min, the initial velocity was only half of what it was when treated below 45 °C. When treated at a high enough temperature for a long enough period of time,

some PLA1 (Lecitase Ultra) was irreversibly denatured, and activity was lost completely. The aim of the study to determine the optimal pH and temperature for PLA1 (Lecitase Ultra) differed from the goal of the PLA1 (Lecitase Ultra) stability studies over a range of pH values and temperatures. The goal of the former study was to establish the optimal conditions for the partial hydrolysis of soybean oil by the enzyme, while the goal of the latter was to determine the inherent properties of the enzyme by measuring initial velocity under optimal conditions following treatment at different pH values and temperatures. A rather narrow range of pH values and temperatures was, by necessity, selected for the former study, given the practical considerations in the hydrolysis of soybean oil. However, the pH values and the temperatures selected for the latter investigation were relatively extreme in order to adequately study the stability of PLA1 (Lecitase Ultra) under these extreme conditions. 3.3. Regiospecificity of PLA1 (Lecitase Ultra) and reaction routes The identities of the released free fatty acids from soybean oil hydrolyzed by PLA1 (Lecitase Ultra) and swine pancreatic lipase (SPL) are provided in Table 1. The amount of palmitic acid released after partial hydrolysis of soybean oil with PLA1 and SPL were significantly higher (p 6 0.05) than other fatty acids, whereas the amount of linoleic acid released was significantly less (p 6 0.05). In natural fats and oils, saturated fatty acids are often found in the sn-1 position of TAGs, unsaturated fatty acids, such as linoleic acid, often occupy the sn-2 position, while some special fatty acids, such as linolenic acid, are often found in the sn-3 position. Since SPL is known to possess sn-1,3-specificity, it was not unexpected that a higher content of saturated fatty acids would be found in the total pool of fatty acids released by SPL. However, the content

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Table 1 Composition of free fatty acids released from soybean oil hydrolysed by phospholipase A1 (Lecitase Ultra) and swine pancreatic lipase (as control).a,b

Palmitic acid Stearic acid Oleic acid Linoleic acid Linolenic acid

Soybean oil fatty acids

Released fatty acids (PLA)

Released fatty acids (SPL, as control)

10.57 ± 0.20a 0.94 ± 0.05 25.77 ± 0.58a 57.82 ± 1.51c 4.90 ± 0.97a

13.61 ± 0.24b nd 28.28 ± 2.15a 52.79 ± 2.09b 5.32 ± 0.28a

14.72 ± 0.34c nd 32.36 ± 0.94b 47.77 ± 0.44a 5.13 ± 0.16a

Released fatty acids (PLA1) with different acid values (mg KOH/g) 25.10 ± 0.26

56.45 ± 0.37

106.58 ± 0.84

16.96 ± 0.82c nd 17.60 ± 1.17a,b 59.06 ± 0.78a 6.27 ± 0.35b

15.28 ± 0.14b nd 14.87 ± 0.48a 64.04 ± 0.48b 5.73 ± 0.10b

11.65 ± 0.59a nd 19.17 ± 3.02b 63.72 ± 2.67b 5.06 ± 0.33a

Abbreviations used: PLA1, phospholipase A1; SPL, swine pancreatic lipase; nd, not detected. a,b,c Values in the same line with different letters are significantly (p 6 0.05) different, n = 3.

of linolenic acid released from soybean oil was not significantly different (p 6 0.05), since linolenic acid often takes the sn-3 position. Since Lecitase Ultra is obtained from the fusion of genes from both Thermomyces lanuginosa lipase and Fusarium oxysporum phospholipase (Fernandez-Lorente et al., 2008), we postulate that it may possess sn-1,3-specificity towards TAGs. The commercial Thermomyces lanuginosa lipase (Lipozyme TL IM), which shows sn1,3-specificity towards TAGs, has been used for trans-esterification and glycerolysis in the production of DAGs (Cheong et al., 2007; Kristensen et al., 2005a, 2005b). However, there were some differences in the composition of the released fatty acids between PLA1 and SPL. The reason for this may be the difference in the partial hydrolysis reaction route taken by these two enzymes. For the hydrolysis of TAGs by SPL, the main enzyme for the digestion of oils in the small intestine, dietary oils are hydrolysed primarily into 1,2-DAG and free fatty acids over a 2–6 h period, and these products are absorbed quickly by the intestinal villi. Hence, the fatty acids released by the partial hydrolysis of soybean oil by SPL are primarily from the sn-1,3-position of TAGs. Nevertheless, PLA1 (Lecitase Ultra) partial hydrolysis reaction routes have been proposed as follows (see Fig. 2): TAGs are first partially hydrolysed into 1,2-DAG and fatty acids by PLA1 (Lecitase Ultra), followed by the sequential hydrolysis of 1,2-DAG into 2MAG, which converts into 1-MAG by acyl migration. Finally, 1MAG is hydrolysed into glycerol, following route 2 (Fig. 2). However, some 1,2-DAG converts into 1,3-DAG by acyl migration, which is then sequentially hydrolysed into 1-MAG, and finally into glycerol, following route 1. The results of the composition of released free fatty acids collected by molecular distillation at 130 °C from soybean oil with

different acid values are described in Table 1. The saturated fatty acid content of the released fatty acids, with acid values of 25.10 ± 0.26 mg KOH/g and 106.58 ± 0.84 mg KOH/g, were 16.96 ± 0.82 wt.% and 11.65 ± 0.59 wt.%, respectively. We postulate that, in the initial stage of the reaction, the hydrolysis of TAG followed route 2, due to the sn-1,3-specificity of PLA1 (Lecitase Ultra), releasing fatty acids with a high level of saturation from the sn-1,3position of TAG. In the second stage, the partial hydrolysis reaction simultaneously followed route 1, due to the acyl migration of 1,2DAG into 1,3-DAG, resulting in the release of more unsaturated fatty acids from the sn-2-position (acyl migration). In the final stage of the reaction, route 1 was the primary route followed, since 1,3-DAG was the favoured substrate over 1,2-DAG by PLA1 (Lecitase Ultra), with sn-1,3-specificity. Watanabe et al. (2003) proposed reaction routes for fatty acid esterification with glycerol to produce DAGs by 1,3-regiospecfic lipase Lipozyme RM IM, which were reversible reactions involving the hydrolysis of TAGs. 3.4. Equilibrium constants and kinetics The equilibrium constants in Fig. 2 were calculated as follows:

½1; 2-DAG½FFA ½TAG½H2 O ½FA½2-MAG K2 ¼ k3=k4 ¼ ½1; 2-DAG½H2 O ½1; 3-DAG K3 ¼ k5=k6 ¼ ½1; 2-DAG K1 ¼ k1=k2 ¼

Fig. 2. Reaction routes for phospholipase A1 (Lecitase Ultra) catalysed TAG hydrolysis.

ð2Þ ð3Þ ð4Þ

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½FA½1-MAG ½1; 3-DAG½H2 O ½FA½Glycerol K5 ¼ k9=k10 ¼ ½H2 O½1-MAG ½1-MAG K6 ¼ k11=k12 ¼ ½2-MAG K4 ¼ k7=k8 ¼

ð5Þ ð6Þ ð7Þ

where K1–K6 were the equilibrium constants, k1–k12 were the rate constants in the reaction, and [ ] denoted the respective concentrations. Because no MAG was detected by RP-HPLC and identified by HPLC/ESI/MS in the NUPL in this work, the equilibrium constants K2, K6 and K5 were combined into the new equilibrium constant K20 = K2  K5  K6, which referred to the equilibrium constant for the hydrolysis of 1,2-DAG into glycerol and fatty acid. In the same manner, the equilibrium constants K4 and K5 were combined into the equilibrium constant K40 = K4  K5, which referred to the equilibrium constant for the hydrolysis of 1,3-DAG into glycerol and fatty acid. The equilibrium constants for the partial hydrolysis of TAG by phospholipase A1 (Lecitase Ultra) at 40 °C are shown in Table 2. The equilibrium constant for the acyl migration of 2-MAG to 1-MAG (K6) was reported to be approximately 10 (Laszlo, Compton, & Vermillion, 2008), so the value of K4/K2 at 6.19 indicated that 1,3DAG was more easily hydrolysed by PLA1 (Lecitase Ultra) than was 1,2-DAG. The results of the study on glycerol content in the aqueous layer, acid value, DAG content and the ratio of 1,3-DAG to 1,2DAG of the NUPL are shown in Table 3. The temperature from 40 °C to 50 °C was suitable for the partial hydrolysis of soybean oil. Hence, the glycerol contents, acid values and DAG contents between 40 °C and 45 °C were very close. The maximum DAG content in the NUPL was 26.51 ± 0.32 wt.% at reaction time 8 h and at 40 °C.

Table 2 Equilibrium constant for each phospholipase A1 (Lecitase Ultra) catalysed TAG hydrolysis step at 40 °C. Equilibrium constant

Value

K1 K20 = K2  K6  K5 K3 K40 = K4  K5

0.22 3.52 1.61 2.18

The tendency of the concentration of the reactants and the products at these three reaction temperatures were nearly the same. The concentration of TAG decreased with increasing reaction time, whereas that of DAG increased with reaction time at the initial stage of the reaction, but reached a steady-state condition quickly and for an extended period of time. A reduction in DAG concentrations was observed after a reaction time of >6 h at 45 °C and 50 °C. The trend in the ratio of 1,3-DAG to 1,2-DAG versus reaction time was also the same at different temperatures, namely, an increase at the initial stage (1.25 at 1 h and at 45 °C) due to acyl migration of 1,2-DAG to 1,3-DAG; a period of constancy (nearly 1.0) due to hydrolysis of DAGs by two simultaneous metabolic routes; and a decrease following a long reaction time (0.53 at 8 h and at 45 °C) due to the preferred hydrolysis of 1,3-DAG. Changes in the ratio of 1,3-DAG to 1,2-DAG versus reaction time confirmed the hypothetical reaction route for the partial hydrolysis of TAG by PLA1 (Lecitase Ultra). The hydrolysis of TAG by PLA1 (Lecitase Ultra) is a typical consecutive reaction, in which 1,3-DAG, 1,2-DAG, 1-MAG and 2-MAG are intermediate products. In a consecutive reaction, the tendency is for the concentration of intermediate products to increase in the initial stages of the reaction, and then remain constant for an extended period of time, before finally decreasing to near zero. The steady-state concentration of intermediate products depends on the rate constants for the reaction(s) involved (Atkins, 1982). No MAGs were detected in the reaction, which suggests that k9 was much greater than k7. The constant concentration of DAG was about 20 wt.% during the reaction, which indicates that k1, k3 and k7 are of the same relative order of magnitude. According to steady-state theory for intermediate products in a consecutive reaction, the reaction velocity of TAG was calculated according to the following equation:

V ¼

d½TAG ¼ k1½H2 O½TAG dt

ð8Þ

The concentration of water was nearly constant, since water was in excess at the initial stage of the hydrolysis in this study. Hence, the rate constant k1 was calculated by integration according to the following equation:

k1 ¼

ln½TAG0  ln½TAGt t½H2 O

ð9Þ

Table 3 Concentration of glycerol in the water phase, and concentration of diacylglycerol (DAG) and acid value of the upper non-polar layer of soybean oil catalysed by phospholipase A1 (Lecitase Ultra) at different temperaturesa,b. Reaction time (h) 0.5

1

2

4

6

8

0.46 ± 0.01b 0.40 ± 0.02a 0.38 ± 0.01a

0.69 ± 0.04b 0.63 ± 0.07b 0.46 ± 0.04a

1.22 ± 0.05a 1.19 ± 0.05a 1.23 ± 0.12a

1.38 ± 0.05a 1.29 ± 0.04a 1.41 ± 0.09a

1.98 ± 0.02b 1.93 ± 0.05b 1.69 ± 0.07a

2.12 ± 0.14a 2.28 ± 0.04a 2.17 ± 0.18a

Acid value (mg KOH/g) 40 °C 27.69 ± 1.45b 45 °C 29.53 ± 0.01c 50 °C 23.98 ± 0.02a

38.46 ± 0.53b 40.78 ± 0.11c 28.01 ± 0.87a

55.58 ± 0.06c 49.65 ± 0.12b 48.04 ± 0.32a

57.48 ± 0.12b 52.53 ± 0.38a 53.77 ± 1.57a

63.80 ± 1.03b 65.45 ± 0.25c 58.91 ± 0.67a

68.99 ± 1.03c 66.72 ± 0.59b 64.23 ± 0.59a

DAG content (wt.%) 40 °C 21.50 ± 0.36c 45 °C 16.75 ± 0.23b 50 °C 15.45 ± 0.95a

22.52 ± 0.06c 18.55 ± 0.10b 16.65 ± 0.30a

24.21 ± 0.21c 21.01 ± 0.44b 18.42 ± 0.26a

24.12 ± 0.27c 20.45 ± 0.03b 18.87 ± 0.11a

24.49 ± 0.17c 20.39 ± 0.05b 18.06 ± 0.11a

26.51 ± 0.32c 18.65 ± 0.31a 19.26 ± 0.01b

Ratio of 1,3-DAG to 1,2-DAG 40 °C 1.29 45 °C 1.08 50 °C 1.01

0.93 1.25 1.09

0.81 1.05 1.24

0.97 1.00 0.86

0.97 0.71 0.87

0.67 0.53 0.64

Glycerol (wt.%) 40 °C 45 °C 50 °C

a b

Values in same column of each item with different letters are significantly (p 6 0.05) different, n = 3. Values for Ratio of 1,3-DAG to 1,2-DAG are means of three replicates.

1072

Y. Wang et al. / Food Chemistry 121 (2010) 1066–1072

Table 4 Rate constants for TAG hydrolysis at different reaction temperaturesa. k1 40 °C 45 °C 50 °C a

and 2009B080701063) the Ministry of Science and Technology of the People’s Republic of China under grant 2006BAD27B04.

k2 1

1

1.23  10–5 l mol s 1.18  105 mol1 s1 8.30  106 mol1 s1

5.59  105 l mol1 s1 5.36  105 mol1 s1 3.77  105 mol1 s1

Values are the means of three replicates.

where [TAG]0 and [TAG]t were the concentrations of TAG at the initial and the selected reaction times, respectively. The values for the rate constants k1 and k2 are shown in Table 4. The rate constant for k1 at 40 °C was the greatest among those determined at the three temperatures, which is consistent with the results seen in Fig. 1a. In addition, the highest content of DAG in the NUPL at the 8 h reaction time point was also observed at 40 °C. If DAG is the desired final reaction product for functional food applications, then consideration should be given to controlling the k1 to k3 + k7 ratio by selecting the proper reaction temperature. This is critical to the rate constant of the reaction. The rate constants k3 to k12 were not determined because of the complexity of the equations. A control experiment for the partial hydrolysis of soybean oil at 30 °C was carried out under the same conditions. The DAG content in the NUPL, with an acid value of 52.64 ± 0.58 mg KOH/g, was 25.58 ± 1.32 wt.%, which was higher than that in the NUPL with the same acid value at 40 °C. This demonstrated that the temperature had different effects on the rate constants at each reaction step. It appeared that a higher concentration of DAG in the NUPL, with the same acid value, was achieved at a lower reaction temperature. 4. Conclusion The optimal pH and temperature values for the partial hydrolysis of soybean oil by PLA1 (Lecitase Ultra) were 6.8 and 40 °C, respectively. PLA1 (Lecitase Ultra) displayed good stability over a pH range of 4.7–7.4 and temperatures below 60 °C. PLA1 (Lecitase Ultra) possessed 1,3-regiospecficity towards TAGs during the partial hydrolysis of soybean oil. The reaction routes for the partial hydrolysis of soybean oil by PLA1 (Lecitase Ultra) were postulated as follows: in the initial stage of the reaction, the hydrolysis of TAG followed route 2, due to the sn-1,3-selectivity of PLA1 (Lecitase Ultra), releasing fatty acids with a high content of saturation from the sn-1,3-position of TAG. In the second stage, the partial hydrolysis reaction simultaneously followed route 1, through the acyl migration of accumulated 1,2-DAG into 1,3-DAG, which was hydrolysed to release more unsaturated fatty acids from the sn-2-position (also due to acyl migration). In the final stage of the reaction, route 1 was primarily followed, and 1,3-DAG was the favoured substrate for PLA1 over 1,2-DAG, with sn-1,3-selectivity. The careful selection of reaction temperature and reaction time were critical to achieving a high content of the desired DAG end-product. Acknowledgement This research project was supported by the Science and Technology Council of Guangdong Province (under grant 2008A01090003

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