Food Chemistry 145 (2014) 796–801

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Synthesis and evaluation of odour-active methionyl esters of fatty acids via esterification and transesterification of butter oil Cheng Li a, Jingcan Sun b, Caili Fu b, Bin Yu c, Shao Quan Liu b,⇑, Tianhu Li a, Dejian Huang b,⇑ a

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore 637371, Singapore Food Science and Technology Program, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore c Firmenich Asia Pte Ltd, Tuas, Singapore 638377, Singapore b

a r t i c l e

i n f o

Article history: Received 7 May 2013 Received in revised form 14 August 2013 Accepted 28 August 2013 Available online 7 September 2013 Keywords: Methionyl ester Lipase Sulfur-containing flavours

a b s t r a c t Methionol-derived fatty acid esters were synthesised by both chemical and lipase catalysed esterification between fatty acids and methionol. Beneficial effects of both methods were compared qualitatively and quantitatively by GC–MS/GC-FID results. And the high acid and heat stability of our designed methionyl esters meet the requirement of the food industry. Most importantly, the sensory test showed that fatty acid carbon-chain length had an important effect on the flavour attributes of methionyl esters. Moreover, through Lipozyme TL IM-mediated transesterification, valuable methionol-derived esters were synthesised from the readily available natural material butter oil as the fatty acid source. The conversion of methionol and yield of each methionyl ester were also elucidated by GC–MS-FID. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Esters are important flavour compounds because of their occurrence in a wide range of natural sources, various odour characteristics and their wide range of uses in flavourings. They are naturally present in fruits, mostly at concentrations between 1 and 100 ppm. They are employed in fruit-flavoured products (i.e., beverages, candies, jellies, and jams), baked goods, wines, and dairy products (i.e., cultured butter, sour cream, yogurt, and cheese) (Janssens, De Pooter, Schamp, & Vandamme, 1992). The sensory notes of esters are related to the organic acid and alcohol from which they are derived. Moreover, the odour intensity of esters decreases with the increase of molecular weight (Belitz, Grosch, & Schieberle, 2009); however, the flavour industry needs esters with strong odours and high molecular weight to prolong the duration of the odour of flavourings. Esters containing sulfur can be synthesised to yield flavour substances with strong odour and high molecular weight, as flavour compounds containing sulfur exhibit very low sensory threshold levels and very good odour characteristics (Liu & Crow, 2010). The sulfur-containing esters can be divided into two classes; one in which the sulfur atom belongs to the organic acids, such as methyl 3-(methylthio)propionate, and the other in which the sulfur atom is from an alcohol or mercaptan (AOM), such as methionol (Boustany, 1966).

⇑ Corresponding authors. Tel.: +65 6516 2687 (S.Q. Liu). Tel.: +65 6516 8821; fax: +65 6775 7895 (D. Huang). E-mail addresses: [email protected] (S.Q. Liu), [email protected] (D. Huang). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.08.124

Methionol is an alcohol with low odour threshold ranging from about 1 to 3 ppm and imparts a powerful odour, described as souplike, meaty, boiled potato-like, vegetable-like, savoury or toasted cheese-like. Although methionol has been regarded as an off-flavour compound in beer and wine (Hill & Smith, 2000), it is considered as an important constituent of the overall aroma profiles in cheeses, particularly premium quality Cheddar and Camembert cheese (Landaud, Helinck, & Bonnarme, 2008). Among the methionyl esters, methyl and ethyl 3-(methylthio)propanoate dominated in the vacuum headspace extract (VHS) of yellow passion fruits. Both compounds have previously been found in pineapple juice. 3-(Methylthio)propyl acetate (methionyl acetate) possesses herbaceous odour impressions and a typical vegetable-like character, as described in the literature in numerous flavour systems. In addition, methionyl butanoate (sulfury, cheese-like, mushroom-like) and methionyl hexanoate (tropical fruit note, methional-like, canned pineapple), which have threshold concentrations of 10–20 ppb and 500 ppb respectively, were also identified in the VHS extract obtained from the juice of the yellow variety (Nijssen, Visscher, Maarse, Willemsens, & Boelens, 1996). Werkhoff, Güntert, Krammer, Sommer, and Kaulen (1998) revealed the presence of 3-(methylthio) esters of propanoic acid in yellow passion fruit. The aroma properties of the 3-(methylthio) esters of propanoic acid are not very interesting, with the exception of the hexyl derivative. In general, the 3-(methylthio)propanoic acid esters have a sulfury, vegetable-like odour, and only hexyl 3-(methylthio)propanoate with its fruity and geranium-like odour note may contribute to the overall olfactory impression of the passion fruits. Overall, these research results

C. Li et al. / Food Chemistry 145 (2014) 796–801

indicated that methionol derived organic compounds usually have a basic meaty flavour. This presumption can be used to direct the development of new meaty aromas and promote research and development of meaty flavour. There are two methods to synthesise esters by esterification. Fatty acid or butter oil and methionol were used as the starting materials in both methods. In the chemical method, 4-dimethylaminopyridine (DMAP) was applied as the chemical catalyst and N,N0 -dicyclohexylcarbodiimide (DCC) was used as an esterification agent by removing water and driving the reaction to completion (Farshori, Banday, Zahoor, & Rauf, 2010). The advantage of the DMAP/DCC system is that it does not require a toxic metal catalyst. The second method is the enzymatic method, in which lipase TL IM was employed as the biocatalyst (Soumanou & Bornscheuer, 2003a, 2003b; Wang, Wu, & Zong, 2008). The biocatalytic conversion of a structurally related precursor molecule is often a superior strategy, which allows the accumulation of a desired flavour product to be significantly enhanced. As a prerequisite for this strategy, the precursor must be present in nature, and its isolation in sufficient amounts from the natural source must be easily feasible in an economically viable fashion (Dhake, Thakare, & Bhanage, 2013). Inexpensive, readily available, and renewable natural precursors, such as fatty acids which were used as the starting materials in our study, can be converted to more highly valued flavours (Sun et al., 2012). The use of enzymes for synthesis of flavour compounds is also of great importance, due to the characteristics of enzymatic reactions such as high substrate specificity, high reaction specificity, mild reaction conditions, and reduction of waste product formation. The transesterification of butter oil with methionol in a solvent-free system is particularly attractive as it meets all these requirements plus it is a food grade processing method. In the food industry, the solvent-free system is preferred because of safety concerns when solvent is used (Sun, Yu, Curran, & Liu, 2012). The increase in consumer demand for nutritious and flavourful food supply has led to an increased demand for flavouring materials that may be considered natural. The use of specific enzymes in biosynthetic processes presents great potential to meet this demand. The objectives of the present study were to synthesise new methionol-derived flavours by chemical-catalysed and enzymecatalysed esterification of fatty acid with methionol for the first time, since there is no report on the enzymatic esterification of fatty acid with methionol. Moreover, the two methods were compared qualitatively and quantitatively. The sensory description and threshold of each ester were also measured and it was seen that fatty acid carbon chain length has an effect on the flavour attributes of methionyl esters. In addition, we aimed to convert butter oil and methionol to valuable methionol-derived esters through Lipozyme TL IM-mediated solvent-free transesterification.

2. Materials and methods 2.1. Materials and reagents Hydrogen chloride solution (2 M) in diethyl ether, 4-dimethylaminopyridine (DMAP), N,N0 -dicyclohexylcarbodiimide (DCC), and methionol were purchased from Sigma–Aldrich Chemical Company (Singapore) and used directly. Fatty acids (C4, C6, C8, C10, C12 and C14) were obtained from Firmenich Asia Private Ltd (Singapore). The solvents were all of analytical grade and used as received. Butter oil which contained no free fatty acids (analysed by SPME/GC–MS method) was purchased from PGEO Edible Oils Sdn. Bhd, (Pasir Gudang, Malaysia). Butter oil was supplied by

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Firmenich Asia Private Ltd (Singapore). The major fatty acid composition of butter oil by weight is as follows: butyric (C4) 10%; caproic (C6) 5%, caprylic (C8) 2.6%; capric (C10) 5%; lauric (C12) 5%; myristic (C14) 12%; palmitic (C16) 27%; stearic (C18) 10%; and oleic (C18:1) 23% (Lubary, ter Horst, Hofland, & Jansens, 2009). The molecular weight of butter oil used was estimated to be 863 g/mol according to its fatty acid composition and confirmed further with its average saponification value (Riel, 1962). Lipozyme TL IM is a lipase immobilised on silica gel from Novozymes (Bagsvaerd, Denmark); it is a food-grade lipase from Thermomyces lanuginosus with sn-1,3-specific selectivity. Internal standard, methyl pentadecanoate (>98%) used for determining the composition of fatty acid esters was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 2.2. Analytical method Gas chromatography was performed with a QP2010 GC–MS system (Shimadzu, Kyoto, Japan) equipped with a DB-5 ms capillary column (30 m  0.25 mm  0.25 lm; Supelco, Bellefonte, PA), and flame ionisation detector (FID). The injector and detector temperatures were set at 230 °C. The initial oven temperature was set at 50 °C for 5 min, ramped to 230 °C at 5 °C /min, and then held for 30 min. Injection of 1.0 lL was performed and helium was used as the carrier gas with a flow rate of 1.2 mL/min. Split mode was applied with a split ratio of 10:1. Proton NMR spectra were recorded on a 300 MHz spectrophotometer with chemical shift values reported in d units (parts per million) relative to tetramethylsilane. Thin layer chromatography was done on pre-coated 0.2-mm Merck silica gel 60 F254 plates. High-resolution mass spectra were obtained using microTOF LC-MS (Bruker Biospin, Rheinstetten, Germany). 2.3. Esterification of methionol with fatty acid through chemical method Fatty acids (C4, C6, C8, C10, C12 and C14 fatty acids) (3.0 mmol), DCC (619.0 mg, 3.0 mmol), and methionol (265.5 mg, 2.5 mmol) were dissolved in dichloromethane (20 mL). To the solution, a catalytic amount of DMAP (55.0 mg, 0.45 mmol) was added and stirred at room temperature for 24 h. The N,N-dicyclohexylurea solid formed was filtered off and the filtrate was transferred to a 50.00 mL volumetric flask and made to volume with dichloromethane. An aliquot of the resulting solution (100 lL) was taken and spiked with 75 lL of internal standard solution of methyl pentadecanoate (2.2 mg/mL) and diluted to 1.0 mL with dichloromethane. The resulting solution was stored at room temperature for GC-FID analysis. The remaining filtrate was washed with hydrochloric acid (1 M, 3  20 mL), saturated sodium bicarbonate (3  20 mL) again with water (3  20 mL) and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to give the esters, which were chromatographed over silica gel column using n-hexane–ethyl acetate (25:1, v/v) as an eluent. All newly synthesised compounds were characterised by 1H NMR, and high resolution mass spectrometry. 2.4. Lipase-catalysed esterification of methionol with fatty acid Methionol (265.5 mg, 2.5 mmol), fatty acid (3.0 mmol), Lipozyme TL IM (5% (w/w) of reactants) and n-hexane (20 mL) were mixed a 100-mL screw-capped glass bottle, followed by incubation at 40 °C in a water bath at 150 rpm shaking speed for 24 h. The reaction was stopped by separating the enzyme from the reaction solution through centrifugation. The supernatant was transferred to a 50.00-mL volumetric flask and topped up with n-hexane. An aliquot of the solution (100 lL) was pipetted into a GC vial and

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spiked with internal standard methyl pentadecanoate solution (2.2 mg/mL, 75 lL) and diluted to 1.0 mL with n-hexane for GCFID analysis. The solvent of the remaining solution was removed under reduced pressure to give the crude esters, which were chromatographed over a column of silica gel, using n-hexane–ethyl acetate (25:1, v/v) as an eluent to obtain pure esters, the identities of which were confirmed by 1H NMR and high resolution electron impact mass spectrometry. 2.5. 1H NMR and HRMS results 3-(Methylthio)propyl butanoate, liquid, Rf = 0.5 (n-hexane: EA = 16:1 v/v), 1H NMR (300 MHz, CDCl3): d 4.13 (t, J = 6.0 Hz, 2H, CH2O), 2.52 (t, J = 6.0 Hz, 2H, CH2S), 2.25 (t, J = 7.5 Hz, 2H), 2.00 (s, 3H, CH3S), 1.91–1.84 (m, 2H), 1.68–1.55 (m, 2H), 0.91 (t, J = 7.5 Hz, 3H, CH3CH2). HRMS calculated for C8H16O2S m/z 176.0871 (M+), found 176.0871. 3-(Methylthio)propyl hexanoate, liquid, Rf = 0.5 (n-hexane:EA = 16:1 v/v), 1H NMR (300 MHz, CDCl3) d 4.14 (t, J = 6.0 Hz, 2H, CH2O), 2.53 (t, J = 6.0 Hz, 2H, CH2S), 2.27 (t, J = 7.5 Hz, 2H, CH2CO), 2.01 (s, 3H, CH3S), 1.92–1.85 (m, 2H), 1.62–1.57 (m, 2H), 1.30– 1.27 (m, 4H), 0.87 (t, J = 7.5 Hz, 3H, CH3CH2). HRMS calculated for C10H20O2S m/z 204.1184 (M+), found 204.1182. 3-(Methylthio)propyl octanoate, liquid, Rf = 0.5 (n-hexane:EA = 16:1 v/v), 1H NMR (300 MHz, CDCl3) d 4.14 (t, J = 6.0 Hz, 2H, CH2O), 2.54 (t, J = 6.0 Hz, 2H, CH2S), 2.25 (t, J = 7.5 Hz, 2H, CH2CO), 1.92 (s, 3H, CH3S), 1.90–1.88 (m, 2H), 1.62–1.57 (m, 2H), 1.28–1.26 (m, 8H), 0.86 (t, J = 7.5 Hz, 3H, CH3CH2). HRMS calculated for C12H24O2S m/z 232.1497 (M+), found 232.1490. 3-(Methylthio)propyl decanoate, liquid, Rf = 0.5 (n-hexane:EA = 16:1 v/v), 1H NMR (300 MHz, CDCl3) d 4.16 (t, J = 6.3 Hz, 2H, CH2O), 2.55 (t, J = 6.0 Hz, 2H, CH2S), 2.29 (t, J = 7.5 Hz, 2H, CH2CO), 2.01 (s, 3H, SCH3), 1.91–1.87 (m, 2H), 1.63–1.60 (m, 2H), 1.26– 1.24 (m, 12H), 0.87 (t, J = 7.5 Hz, 3H, CH3CH2). HRMS calculated for C14H28O2S m/z 260.1810 (M+), found 260.1820. 3-(Methylthio)propyl dodecanoate, liquid, Rf = 0.5 (n-hexane:EA = 16:1 v/v), 1H NMR (300 MHz, CDCl3) d 4.16 (t, J = 6.0 Hz, 2H, CH2O), 2.55 (t, J = 6.0 Hz, 2H, CH2S), 2.29 (t, J = 7.5 Hz, 2H, CH2CO), 2.00 (s, 3H, CH3S), 1.93–1.87 (m, 2H), 1.62–1.56 (m, 2H), 1.26–1.24 (m,16H), 0.87 (t, J = 7.5 Hz, 3H, CH3CH2). HRMS calculated for C16H32O2S m/z 288.2123 (M+), found 288.2135. 3-(Methylthio)propyl tetradecanoate, liquid, Rf = 0.5 (n-hexane:EA = 16:1 v/v), 1H NMR (300 MHz, CDCl3) d 4.16 (t, J = 6.0 Hz, 2H, CH2O), 2.55 (t, J = 6.0 Hz, 2H, CH2S), 2.29 (t, J = 7.5 Hz, 2H, CH2CO), 2.10 (s, 3H, CH3S), 1.94–1.90 (m, 2H), 1.61–1.58 (m, 2H), 1.27–1.24 (m, 20H), 0.88 (t, J = 7.5 Hz, 3H, CH3CH2). HRMS calculated for C18H36O2S m/z 316.2436 (M+), found 316.2446.

2.7. Standard solutions for calibration curve The purified esters were used as standards. A stock solution was prepared by dissolving six esters at 2000 ppm each in n-hexane (10 mL). These esters include 3-(methylthio)propyl butanoate, 3(methylthio)propyl hexanoate, 3-(methylthio)propyl octanoate, 3-(methylthio)propyl decanoate, 3-(methylthio)propyl dodecanoate, and 3-(methylthio)propyl tetradecanoate. The internal standard stock solution (220 ppm) was prepared by accurately measuring methyl pentadecanoate (2.2 mg) and dissolving in nhexane (10.00 mL). The internal standard solution (75 lL) was added into each sample vial for analysis with a final concentration of 165 ppm. The final concentrations of the mixed ester standards were 150, 200, 400, 600, 800, and 1000 ppm. These standard solutions were analysed by GC-FID test to calculate a standard curve. 2.8. Qualitative and quantitative analysis of methionol fatty acid esters Methionol-derived fatty acid esters (3-(methylthio)propyl butanoate, hexanoate, octanoate, decanoate, dodecanoate and tetradecanoate) were analysed by GC–MS and GC-FID. The synthesised compounds found in esterified samples or transesterified oil samples were identified through mass spectral analysis and comparing their mass spectra with Wiley database. 2.9. Stability evaluation of fatty acid methionyl esters in acidic media and heat treatment 2.9.1. Acid stability test Esters (10 mg) and hydrogen chloride in diethyl ether (9 mL, with concentration of 10, 0.1, and 0.001 mM respectively) and de-ionised water (10 mL) were added into 100 mL screw-capped glass bottles and kept at room temperature for 24 h. The mixtures (100 lL) were mixed with internal standard solution (75 lL methyl pentadecanoate, 2.2 mg/mL) and diluted to 1.0 mL with n-hexane. The resulting solutions were analysed by GC–MS and GC-FID. 2.9.2. Thermal stability test Ester (10 mg) and toluene (10 mL) were added into a 50-mL three-necked flask and stirred at 200 rpm at a stipulated temperature for 2 h at ambient atmosphere or under N2 protection. The temperatures tested were 75, 80, 85, 90, 95, 100 and 105 °C. Samples were collected every two hours for GC–MS and GC-FID analysis; 100 lL of reaction samples obtained were spiked with 75 lL of internal standard solution (methyl pentadecanoate, 2.2 mg/mL) and diluted to 1.0 mL with toluene. The resultant solution was stored at room temperature until GC–MS and FID analysis. 2.10. Odour descriptions and threshold testing of methionyl esters

2.6. Transesterification of butter oil with methionol by lipase Butter oil (4.000 g, 4.6 mmol, acid-free as checked by GC), methionol (1.4653 g, 13.8 mmol) and Lipozyme TL IM (0.2750 g, 5% (w/w) of reactants) were added into a 100-mL screw-capped glass bottle and the mixture was incubated at 40 °C in a water bath with stirring at 130 rpm. Samples were collected at different times for GC-FID analysis, to monitor the reaction progress. The reaction was stopped by separating the enzyme from the reaction solutions through centrifugation. Samples (20 lL) obtained were spiked with internal standard solution (methyl pentadecanoate, 75 lL, 2.2 mg/ mL) and diluted to 1.0 mL with n-hexane. The resulting solution was stored in a fridge at 0 °C overnight to precipitate undesirable products, such as glycerol, mono-, di- and triacylglycerols. The top layer was collected for GC–MS and GC-FID analysis. All the reactions were conducted in triplicate and responses presented for GC–MS-FID were the average of three determined values.

In the odour description test, the flavour attributes of all the six compounds were described by a sensory panel consisting of five experienced flavourists from Firmenich Asia Private Ltd (Singapore). Before sensory analysis all the ester samples were diluted with 5% ethanol in deionised water and kept at a room temperature for 24 h. Blank samples containing 5% ethanol in deionised water were also provided for each tested compound. The detection thresholds of methionyl esters in water were determined using the forced-choice ascending concentration series method of limits (ASTM, 1988). Compounds were diluted in absolute ethanol (for the water threshold) before addition to the deionised water. The compound concentrations were serially diluted for five concentrations for the threshold test. Blank samples in each set were adjusted with the same concentration of ethanol to eliminate any bias due to the solvent used. Each 20-mL volume screwcapped test tube was filled up to 8 mL and was allowed to

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C. Li et al. / Food Chemistry 145 (2014) 796–801 Table 1 Comparison of yields of methionol-derived fatty acid esters synthesised by chemical and enzymatic methods. Methionol-derived fatty acid esters

3-(methylthio)propyl 3-(methylthio)propyl 3-(methylthio)propyl 3-(methylthio)propyl 3-(methylthio)propyl 3-(methylthio)propyl tetradecanoate a

butanoate hexanoate octanoate decanoate dodecanoate

Yield (%) Chemical method

Enzymatic methoda

67.24 90.41 87.18 82.61 78.18 85.99

90.66 84.65 77.67 81.95 86.51 84.69

Table 2 The yield of methionol-derived fatty acid esters synthesised by lipase-catalysed transesterification of butter oil and methionol.a Methionol-derived fatty acid esters

Yield (mg) per gram of butter oil

3-(methylthio)propyl 3-(methylthio)propyl 3-(methylthio)propyl 3-(methylthio)propyl 3-(methylthio)propyl 3-(methylthio)propyl

13.2 10.6 6.9 10.4 9.4 20.6

butanoate hexanoate octanoate decanoate dodecanoate tetradecanoate

a Lipase TL IM was applied as the catalyst and the reaction was incubated for 48 h.

In the enzymatic method, Lipase TL IM was used as the catalyst.

equilibrate for 24 h before testing. Each concentration in the series was presented to the panels with the blank in a randomised order. The threshold of each compound was calculated as the geometric mean of the estimated thresholds of each individual flavourist. 3. Results and discussion 3.1. Qualitative and quantitative analysis of methionol fatty acid esters Fatty acid esters can be synthesised with high yields via many chemical methods with low costs and can also be prepared with lipase as catalyst. Here we compared both methods for generating methionyl esters and determined the conversion of the reaction using GC-FID (Table 1). The data showed that the conversions of the enzymatic reaction were comparable to that of the chemical method. Typically chemical methods require less expensive catalysts. However, when we take into consideration other factors, the enzymatic method is preferred. Firstly, chemically catalysed reaction requires purification steps that compromise the final yields of the product and add to the processing costs. In addition, the use of synthetic and toxic chemicals is strongly discouraged for industrial applications because of pollution concerns. In comparison, enzymes are costly but have great advantages, such as high substrate specificity, high reaction specificity, mild reaction conditions, and reduction of waste product formation. In the food industry, the use of environmentally friendly lipases is preferred because there is no need to purify the products as the reaction mixture is food grade and can be directly used as flavouring materials. This alleviates complex downstream processes and thus can lead to reduction of overall operation costs. Moreover, immobilised lipases are especially suitable for food industry applications, due to several advantages, such as lack of contamination of products with enzymes, enzyme reusability, and easy separation of immobilised

enzymes from the reaction mixture or easy retention of enzymes in batch reactors. The most important advantage is the improvement of enzyme activity and stability against extreme conditions, such as high temperatures, pH and adverse effects from the organic solvent medium (Klibanov, 2001). These advantages overcome the drawback of high cost, if the conversion of the enzymatic reaction is satisfactory as it is the case here. To illustrate the utility of enzymatic reaction in food system, we applied the same enzyme to convert butter oil and methionol to methionyl esters via transesterification. The major volatile compounds identified in the transesterified butter oil are shown in the GC–MS chromatogram (Fig. 1). The newly synthesised compounds were characterised by 1H NMR and high resolution mass spectrometry. In addition to the known synthesised compounds (peaks 2–4 and 6–8), there are three more peaks with higher retention time (peaks 9, 10, and 11). From the MS data, we can deduce their potential structures as methionyl esters of fatty acids with longer chain lengths. Peak 9 shows m/z of 344 (M+) for palmitic methionyl ester. In addition, characteristic fragments were detected for CH3SCH2CH = CH2 (m/z 88), methionol cation (m/z 106), and 239 ([C15H31CO]+). Peak 10 is methionyl ester of oleic acid, with characteristic peaks at m/z 265 ([C17H33CO]+), 106, and 88. Peak 11 is methionyl ester of stearic acid with peaks at m/z 372 (M+), 267 ([C17H35CO]+), 106 and 88. These compounds are expected, as the butter oil contains the respective fatty acids (palmitic (27%), oleic (23%), and stearic (10%)). These high molecular weight esters are not of interest as flavouring compounds; therefore, we did not attempt to prepare them from their respective acids and methionol. The overall product profile shown in Fig. 1 is consistent with the chemical compositions of the butter oil. Through the solvent-free transesterification, about 13.2 mg of 3-(methylthio)propyl butanoate, 10.6 mg of 3-(methylthio)propyl hexanoate, 6.9 mg of 3-(methylthio)propyl octanoate, 10.4 mg of 3-(methylthio)propyl decanoate, 9.4 mg of 3-(methylthio)propyl dodecanoate and 20.6 mg of 3-(methylthio)propyl tetradecanoate

mVolts 9/39.18

70 5/26.13

60

8/35.22

50 40 30 20 10 0 -3

2/14.07 1/5.56

3/19.05

10

10/44.53

6/27.83 7/31.67

11/.45.54

4/23.63

20

30

40

Time (min) Fig. 1. GC-FID chromatogram of the reaction products of methionol and butter oil. Peak 1, methionol; peak 2, 3-(methylthio)propyl butanoate; peak 3, 3-(methylthio)propyl hexanoate; peak 4, 3-(methylthio)propyl octanoate, peak 5, internal standard; peak 6, 3-(methylthio)propyl decanoate; peak 7, 3-(methylthio)propyl dodecanoate; peak 8, 3(methylthio)propyl tetradecanoate; peak 9, 3-(methylthio)propyl hexadecanoate; peak 10, 3-(methylthio)propyl oleate; peak 11, 3-(methylthio)propyl octadecanoate.

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C. Li et al. / Food Chemistry 145 (2014) 796–801

Yield of C4 to C14 methionyl esters and conversion of methionol (mmol/per mol of butter oil)

Table 3 Comparison of sensory descriptors and thresholds of methionyl esters.

3-(methylthio)propyl butanoate 3-(methylthio)propyl hextanoate 3-(methylthio)propyl octanoate 3-methylthio-propyl decanoate 3-methylthio-propyl dodecanoate 3-methylthio-propyl tetradecanoate Methionol

90.0

80.0

70.0

60.0

50.0

40.0

Compounds

Odour assessment

Threshold

3-(methylthio)propyl butanoate 3-(methylthio)propyl hexanoate 3-(methylthio)propyl octanoate 3-(methylthio)propyl decanoate

Sulfury, cheese, mushroom-like

10– 20 ppb 500 ppb

3-(methylthio)propyl dodecanoate 3-(methylthio)propyl tetradecanoate

30.0

20.0

10.0

Tropical fruit-like, methional-like, canned pineapple-like Green, fruity, slightly metallic, 1 ppm sulfury 50 ppm Canned pineapple-like, potato-like, metallic, methional-like, soft, passion fruit like Metallic, ripe papaya, pineapple-like 50 ppm 50 ppm Fermented, juicy, pineapple-like, metallic, green, seedy, heavy, honey dew

0.0 0

5

10

15

20

25

30

35

40

45

Incubation time (h)

Fig. 2. Time-course production of methionyl fatty acid esters during lipasecatalysed transesterification of butter oil with methionol. A reaction mixture containing 4.0 g of coconut oil, 1.5 g methionol and 0.275 g of Lipozyme TL IM was incubated at 40 °C.

were produced per gram of butter oil. The conversion of methionol obtained was satisfactory at 81.3% (Table 2 and Fig. 2). Using butter oil as a starting material, we can thus obtain a host of methionyl esters. Enzymatic transesterification of butter oil with methionol is generally deemed as a direct alcoholysis reaction and/or a two-step process of simultaneous hydrolysis and esterification. Since methionol contains a certain amount of water, hydrolysis and esterification may also occur during the lipase-catalysed transesterification in the solvent-free environment, but a further study is needed to verify the mechanism. In the reaction between butter oil and methionol, the conversion of methionol cannot reach 100%. Our results show that the reaction reached equilibrium with conversion of 81.3% after 48 h. This may be due to the fact that glycerol formed cannot be removed from the reaction mixture. 3.2. Acid and thermal stability assay of methionol derived fatty acid esters One potential concern of flavour active esters is their stability towards weakly acidic food matrices and against heat during food thermal processing. To evaluate the resistance of the methionyl esters against acids, we treated the esters with different concentrations of hydrochloric acid (10, 0.1, 0.001 mM) at room temperature for 24 h and analysed the mixture by GC. There were no new peaks detected compared to untreated samples. Therefore, we concluded that the esters can resist the acidic environment fairly well. Moreover, heating these esters in toluene at 105 °C for 2 h did not result in any noticeable decomposition. Therefore, the esters are acid and thermally stable. It should be pointed out that the food matrix is typically complicated, as it contains many types of molecules including proteins, lipids, and carbohydrates. The stability of the esters when used as flavouring agents in real food would need to be evaluated. 3.3. Odour descriptions and threshold testing of methionyl esters The aromatic characterisation of the six methionyl esters and their thresholds were compared in Table 3. Our results are in agreement with the sensory description of C4 and C6 methionyl esters (Nijssen, 1996). The sensory properties of the other four

methionyl esters (C8, C10, C12, and C14) were unreported. From our sensory test results, these methionol-derived esters possess unique flavour notes. The threshold increased from 20 ppb to 50 ppm from C4 to C10 esters and remains at 50 ppm for C10, C12 and C14 esters. The other noticeable trend is the fruity notes of these esters (except C4) in general but their difference in terms of specific type of fruit. Therefore, it is of potential for these esters to be used for creating unique flavouring materials by taking advantage of these differences. Finally, the longer-chain fatty acid methionyl esters with high threshold can prolong the duration of their flavouring. In summary, we reported that both chemical-catalysed and enzyme-catalysed esterification of fatty acid with methionol can effectively generate methionol-derived flavours but the enzymatic method is preferred due to its high conversion and generation of food grade flavouring materials. Fatty acid carbon chain length has an effect on the flavour attributes of the methionyl esters, with all methionyl esters having unique sensory notes. Moreover, through Lipozyme TL IM-mediated transesterification, 3-(methylthio)propyl esters were generated by using a low cost natural material, butter oil, as the source of carboxylic acids. The high acid and heat stability of our synthesised methionyl esters makes them suitable for food use. In particular, the long fatty acid methionyl esters with high odour thresholds can provide long-lasting flavouring in food products. Our results provided valuable information for the food and flavour industry on the Lipozyme TL IM-mediated transesterification of butter oil with methionol. Transesterification of butter oil could also provide other useful flavouring esters, such as those derived from phenethyl alcohol. Acknowledgment The sensory evaluation was supported by flavourists from Firmenich Asia Private Ltd (Singapore). References ASTM. (1988). Standard practice for determination of odour and taste thresholds by a forced-choice ascending concentration series of limits. In ASTM Standards on Sensory Evaluation of Materials and Products, ASTM (pp. 77). Philadelphia. Belitz, H., Grosch, W., & Schieberle, P. (2009). Food Chemistry, 4th revised and extended edn. Heidelberg: Springer. Boustany, K. (1966). Addition of methanethiol to some a, bunsaturated aldehydes and study of the reduction and acetalisation of the addition products. Journal of Chemistry of the United Arab Republic, 9(3), 317–322. Dhake, K. P., Thakare, D. D., & Bhanage, B. M. (2013). Lipase: A potential biocatalyst for the synthesis of valuable flavour and fragrance ester compounds. Flavour and Fragrance Journal, 28(2), 71–83. Farshori, N. N., Banday, M. R., Zahoor, Z., & Rauf, A. (2010). DCC/DMAP mediated esterification of hydroxy and non-hydroxy olefinic fatty acids with b-sitosterol: In vitro antimicrobial activity. Chinese Chemical Letters, 21(6), 646–650.

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Synthesis and evaluation of odour-active methionyl esters of fatty acids via esterification and transesterification of butter oil.

Methionol-derived fatty acid esters were synthesised by both chemical and lipase catalysed esterification between fatty acids and methionol. Beneficia...
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