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Changes in Maillard reaction products in ghee during storage
Food Chemistry 135 (2012) 921–928
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Changes in Maillard reaction products in ghee during storage P. Andrewes ⇑ Fonterra Research Centre, Fonterra Cooperative Group Limited, Private Bag 11029, Dairy Farm Road, Palmerston North, New Zealand
a r t i c l e
i n f o
Article history: Received 10 April 2012 Received in revised form 10 May 2012 Accepted 8 June 2012 Available online 16 June 2012 Keywords: Ghee Maillard reaction Milkfat Oxidation
a b s t r a c t Ghee (milkfat from heat clarification) was made using direct cream (DC), cream butter (CB) or pre-stratification (PS) methods and stored at 60 °C, in air, for at least two weeks. Milkfat degradation, particularly oxidation, occurred in all types of ghee, resulting in increases in aldehydes and free fatty acids. However, there was little difference in fat degradation rates in each type of ghee. DC and CB ghee contained volatile Maillard reaction products, whereas PS ghee did not. The concentrations of 3,4-dihydroxy-3-hexen-2,5dione (DHHD) and 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (pyranone), Maillard reaction products found in DC and CB ghee, rapidly decreased during storage, associated with increases in acetic acid. This work suggests hydration of both DHHD and pyranone, during storage, could form reactive 1-deoxy-D-erythro-hexo-2,3-diulose (1-deoxyglucosone) that degrades mostly to acetic acid. Thus, the Maillard reaction cascade appears to continue in food, in the absence of proteins and sugars, long after cooking has ceased. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The isolation of milkfat by ‘boiling-off’ water from milk, cream or butter is an ancient process dating back as far as 1500 BC (Sserunjogi, Abrahamsen, & Narvhus, 1998). Usually, after the bulk of the water is removed, heating continues until the non-fat solids are deeply coloured. Non-fat solids are then often removed by either filtration or decanting the oil (Ganguli & Jain, 1973; Sserunjogi et al., 1998). Isolation of milkfat in this manner was traditionally used (and is still widely done today) because it is a valuable means to both preserve and add flavour to milkfat (Ganguli & Jain, 1973; Sserunjogi et al., 1998). Ghee (from the sanskrit Ghrita) is widely used in India (and South Asia) and is perhaps the most common example of a milkfat isolated by ‘boiling-off’ water. However, there are numerous related indigenous products (Sserunjogi et al., 1998), such as samin (Sudan) and samuli (Uganda). Western chefs often make beurre noisette (brown butter) and beurre noir (black butter) using similar techniques, but the primary objective is usually flavour creation rather than fat preservation. There are four methods commonly used for the manufacture of ghee (Ganguli & Jain, 1973; Sserunjogi et al., 1998): milk butter (or desi method), direct cream (DC), cream butter (CB), and pre-stratification (PS). Heat clarification of cream directly produces DC ghee. Churning cream to butter, discarding the buttermilk, and heat clarification of butter, produces CB ghee. PS ghee is made by churning cream to butter, melting the butter, discarding serum, ⇑ Tel.: +64 6 3504649; fax: +64 6 3561476. E-mail address: [email protected] 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.06.013
and using mild heat to remove residual water. The cream used in manufacture of DC, CB and PS ghee has also often undergone fermentation (Sserunjogi et al., 1998). Desi ghee was not investigated in this work but is produced via churning fermented milk to butter, and heating. High temperatures used in the manufacture of desi, DC and CB ghee causes non-fat milk solids to ‘caramelise’ (sugar degradation) and also participate in the Maillard reaction (Ganguli & Jain, 1973; Sserunjogi et al., 1998; Wadhwa & Jain, 1990; Wadodkar, Punjrath, & Shah, 2002). The Maillard reaction is used to describe a cascade of reactions initiated by a reaction between reducing sugars and amino groups (from free amino acids, peptides, or proteins) to form Amadori compounds (Baltes, 1982; Belitz, Grosch, & Schieberle, 2004; Hodge, Mills, & Fisher, 1972). Subsequent reactions of the Amadori compounds produce complex mixtures of reaction products, including numerous compounds that can also be derived from sugar degradation in the absence of any intermediate Amadori compounds (Belitz et al., 2004). In this paper, for simplicity, Maillard reaction products (MRP) are defined as any compounds produced by heating of non-fat solids, regardless of formation via Amadori compounds or sugar degradation. The MRP include insoluble melanoidins (removed from ghee by filtration), pigments (many also removed by filtration but residual pigments can add colour to ghee), and volatile compounds, some of which are potent flavours (Baltes, 1982; Belitz et al., 2004; Hodge et al., 1972). Maillard reactions in dairy products produce many compounds that are derived from lactose (van Boekel, 1998). Most of these compounds are believed to be formed via highly reactive deoxyosone intermediates (a-dicarbonyl compounds). Typically lactose
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either forms 3-deoxyosones (at acidic pH via 1,2 enolization), or 1-deoxyosones (at neutral to high pH via 2,3 enolization). Further reactions of 3-deoxyosones can produce the well-known hydroxymethylfurfural (van Boekel, 1998). Reactions of 1-deoxyosones are usually more important in dairy products due to the neutral pH of dairy products (van Boekel, 1998). Some features of the 1-deoxyosone pathway for glucose are illustrated in Fig. 1 (the 1-deoxyosone derived from glucose is 1-deoxy-D-erythro-hexo-2,3-diulose or 1-deoxyglucosone). The disaccharide lactose undergoes analogous reactions to glucose but the galactosyl group adds additional complexity (van Boekel, 1998). Either galactose or water can be eliminated from lactose at different points in these pathways. If galactose is eliminated the same products form as for glucose. However, if the galactosyl group is not eliminated, analogues to the products formed from glucose, such as galactosyl isomaltol and galactosyl beta-pyranone, are found (van Boekel, 1998). Additionally, the galactosyl group of lactose plays an important role in directing maltol formation from the 1-deoxyosone (Yaylayan & Mandeville, 1994). Maltol is an important flavour compound in heated dairy products (Patton, 1950) and was part of this research. However, galactosyl MRP were not considered further because they were not detectable using the methods described. The reaction of 1-deoxyosones can take either of two paths (Fig. 1) to produce 5 and 6-membered ring compounds, such as acetylformoin (right branch of Fig. 1) and 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (pyranone), respectively (Kim & Baltes, 1996; Voigt & Glomb, 2009; Voigt, Smuda, Pfahler, & Glomb, 2010). Acetylformoin is notable in that it has a linear tautomer (3,4-dihydroxy-3-hexen-2,5-dione, DHHD, Fig. 1) that is the only known open-chain compound with a caramel odour
(Engel, Hofmann, & Schieberle, 2001). Its other tautomers do not appear to have any caramel odour. Also, in the presence of water, the caramel odour of DHHD disappears and Engel et al. (2001) claim this is due to tautomerisation of DHHD. Both acetylformoin and pyranone retain the original ‘carbonbackbone’ of glucose minus two moles of water for diagnostic masses of 144 (Kim & Baltes, 1996). Their formation (and formation of related compounds), from 1-deoxyglucosone, is explained by appropriate combinations of keto-enol tautomerisation, cyclization, and dehydration. Such reactions (that do not break the ‘carbon-backbone’) are often reversible. In particular, hydration of pyranone can reform 1-deoxyglucosone (Kim & Baltes, 1996). In practice, this means compounds like DHHD, pyranone and 1-deoxyglucosone often have the same chemistry. For example, pyranone (Kim & Baltes, 1996), acetylformoin (Hofmann, 1998), and 1-deoxyglucosone (Davidek, Robert, Devaud, Vera, & Blank, 2006; Voigt & Glomb, 2009; Voigt et al., 2010) have all been shown to produce the same numerous end products of glucose degradation. Furthermore, pyranone and DHHD formed compounds (e.g., acetic acid, formic acid, lactones) that are best explained by conversion to deoxyglucosone followed by hydrolytic and oxidative fragmentation. As already noted, galactose elimination allows lactose to access all the pathways illustrated in Fig. 1, producing all of the MRP (including intermediates) produced by glucose. Thus, the model studies above for glucose can explain the presence of numerous compounds found in heated dairy products and ghee (van Boekel, 1998; Wadodkar et al., 2002). In the course of investigating Maillard reaction products in ghee, it was observed the two related compounds, pyranone and DHHD, were both rapidly lost from samples during storage. The objective
O
OH
O
O
HO
Formicacid Aceticacid Acetol Glycericacid Lacticacid Aldehydes Hydroxyacids Lactones ...
OH OH
O OH
OH
-H 2O
+H 2O
1-Deoxy-D-erythro-hexo-2,3-diulose (1-deoxyglucosone) +H 2O
O O OH
HO
-H 2O OH
OH
O
O O
2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (pyranone)
Maltol Isomaltol Furans Furanones ...
HO
O
2,4-dihydroxy-2,5-dimethyl-3(2H)-furanone
OH
3,4-dihydroxy-3-hexen-2,5-dione (DHHD)
Maltol Isomaltol Furans Furanones ...
Fig. 1. Possible reaction pathways for the reactive glucose derivative, 1-deoxyglucosone (taken from Hodge et al., 1972; Voigt et al., 2010).
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of this work was to further investigate the behaviour of these two compounds in ghee during storage and determine if there were any implications for the chemistry of ghee. The storage conditions selected (60 °C, air, in the dark) would usually not be encountered during typical use of ghee. In this work it was assumed these accelerated storage conditions will predict the chemistry that will occur, albeit more slowly, in more moderate conditions. 2. Materials and methods 2.1. Materials Iron(III) chloride hexahydrate was from BDH (Poole, England). Iron chloride reagent was made by dissolving iron(III) chloride (5 g/L) in hydrochloric acid (0.5 M). Maltol and hexanal were from Aldrich (St. Louis, MO, USA). All reagents were analytical grade or better. DHHD was provided by GlycoSyn, Industrial Research Limited (Lower Hutt, New Zealand). The DHHD had an estimated 70% purity (by 1H and 13C NMR) and was synthesised by condensation of 2-oxopropanal using a previously described procedure (Goto, Miyagi, & Inokawa, 1963). Fresh cream (40%, w/w, fat) was purchased from a local supermarket (Palmerston North, New Zealand) each day that ghee was made. 2.2. Gas chromatography–mass spectrometry Two gas chromatography mass spectrometry (GC–MS) methods were used to analyse the samples. Both methods used common conditions wherever possible. However, each method used a different GC column (and corresponding temperature programme). In this section, the common conditions for each method are given first followed by the specifics applicable to each method. The analysis was performed using a Shimadzu QP2010 Plus GC–MS (Shimadzu, Kyoto, Japan) The GC–MS was equipped with an AOC5000 (CTC Combipal, CTC Analytics, Zwingen, Switzerland) autosampler configured for solid phase microextraction (SPME). Samples were extracted and introduced into the GC–MS using headspace SPME using a 1 cm long fibre (Supelco, Bellefonte, PA, USA) that was coated with a 65 lm layer of polydimethylsiloxane/divinylbenzene and housed in a 23 gauge needle. The SPME fibre was exposed to the headspace of a sample of ghee (3 g) in a 20 ml Chromacol (Welwyn Garden City, Herts, UK) volatile organic compound (VOC) tube sealed with Teflon-backed septa (Chromacol). The headspace was extracted for 30 min at 60 °C with shaking at 250 rpm. Analytes were desorbed from the SPME fibre in the GC injection port, which contained a silanised SPME liner (Restek) and was held at 220 °C. The split injection technique was used to inject sample with a 5:1 split ratio. The column flow (Helium carrier gas) was 1 ml min 1, with a purge flow of 3 ml min 1. The MS interface was held at 230 °C and the ion source at 200 °C. The MS was operated in EI mode at 70 eV with an interval of 1 s. MS acquisition used the FASST program (allowing simultaneous scan and SIM) with a microscan of 0.1 amu. The scan acquisition used an m/z range of 45–350. DHHD, and the related pyranone, were analysed using a Rtx-5 column (Restek, Bellefonte, PA, USA), simultaneous scan and SIM (m/z = 144), and an appropriate temperature programme (GC oven was held at 50 °C for 2 min, increased to 80 °C at 3 °C min 1, increased to 120 °C at 5 °C min 1, and then increased to 320 °C at 20 °C min 1 and held at this final temperature for 2 min). For all other compounds, a second method using a polar column (EC-Wax; Alltech Associates, Deefield, IL, USA) was used as it was more appropriate for the polar analytes and matrix. The GC oven
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temperature programme was slightly modified (the oven was held at 50 °C for 2 min, increased to 80 °C at 3 °C min 1, increased to 120 °C at 5 °C min 1, and then increased to 220 °C at 20 °C min 1 and held at this final temperature for 6 min). Standards of hexanal, maltol, acetic acid, and DHHD were spiked into samples to identify and where necessary quantify (using standard additions) compounds. 2.3. Colourimetric measurement of maltol Ghee (1.0 g) was dissolved in heptane (10 ml) in a 30 ml Teflon Oak Ridge centrifuge tube (Nalgene, Rochester, NY, USA). Iron chloride reagent (5.00 ml) was added to the tube, and agitated thoroughly to extract maltol into the aqueous phase as a coloured complex with iron(III). An aliquot (1 ml) of the aqueous phase was carefully removed from the Oak Ridge tube, using a pipette, and the absorbance of the iron(III) complex was measured at 525 nm. Samples of milkfat, spiked with maltol, were treated in the same manner and used to construct a calibration curve. 2.4. Ghee preparation All types of ghee were prepared from the same batch of fresh cream that was divided into two portions. One portion was used to make DC ghee and the other used to make butter for the CB and PS ghee. Two equivalent trials were done on different days. The procedures described below to prepare ghee are broadly representative of documented procedures (Ganguli & Jain, 1973; Singh & Ram, 1978; Sserunjogi et al., 1998), commercial experience (Illingworth, Janssen, Cant, & Stephens, 2009), and observations in domestic or traditional settings. DC ghee was prepared by heating cream (300–600 g) in a Teflon-coated wok over a moderate heat, stirring regularly. The temperature of the cream was monitored during the cooking. Until the majority of the water had evaporated from the cream the temperature did not exceed 110 °C. After the water had evaporated, the slurry of milk solids in liquid fat was heated to 135 ± 5 °C and held at that temperature for 10 min. The slurry was cooled to below 50 °C and filtered through a paper towel. To make CB and PS ghee, cream (1 L at room temperature) was whipped with an egg beater until phase inversion. The granules were gently worked with a fork to separate the butter and buttermilk. Buttermilk was decanted from the butter, and discarded. On average the discarded buttermilk represented 50 ± 5% (w/w) of the original cream. The butter was divided into two portions. One portion was used to make PS and the other CB ghee. To make CB ghee, the butter was heated in a wok over a moderate heat, stirring regularly. The temperature of the butter was monitored during the cooking. Until the majority of the water had evaporated from the butter the temperature did not exceed 110 °C. After the water had evaporated, the slurry of milk solids in liquid fat was heated to 135 ± 5° C and held at that temperature for 10 min. The slurry was cooled to below 50° C and filtered through a paper towel. To make PS ghee, butter was melted and placed in a separating funnel in an incubator at 50 °C and left to settle into two layers. The serum layer was discarded. The oil layer was collected while filtering through a paper towel. The oil was then heated to 110 °C and immediately cooled back to 50 °C. Minimal heat was desired to achieve milkfat with low moisture and low amounts of MRP. 2.5. Ghee storage Equal amounts of each type of ghee were placed in the same type of sample container. Samples were wrapped in aluminium foil
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to exclude light and stored together in an incubator (Contherm, Upper Hutt, New Zealand) at 60 °C. Samples were removed from the incubator periodically for analysis. Samples were analysed for maltol using a colourimetric assay (Section 2.3) and for other volatile compounds using gas chromatography mass spectrometry (Section 2.2). All samples were handled in exactly the same manner. Two independent storage trials were performed with freshly made DC, CB and PS ghee, made on the same day, from the same batch of cream (Section 2.4). Variation from analysis (10% or less for repeat analyses of the same sample) was always much less than the variation due to ghee preparation and analysis (40% or less). For practical reasons, manufacture and storage was only done twice for each type of ghee and results are an average from the two trials unless otherwise indicated. Statistical analysis was not done due to the small sample size and the qualitative nature of this investigation.
3. Results and discussion 3.1. Analytical methods In preliminary experiments, DHHD was often not detected in fresh ghee when using a highly polar GC stationary phase (ECWax), whereas pyranone always gave a large peak. This did not appear to be an issue peculiar to an individual column because low response of DHHD, relative to pyranone, was also observed with
an EC-1000 column and two other EC-Wax columns (not shown). In contrast, the DHHD peak area was at least 100-fold greater when using a mildly polar stationary phase (Rtx-5), all else being equal (Fig. 2). Peak area of the pyranone (Fig. 2) was about the same on both types of columns (confirming the equivalence of the methodology); but, by only changing the column, it was possible to greatly increase DHHD peak area. On the Rtx-5 column DHHD elutes at a much lower temperature. It is possible that in a polar stationary phase at higher temperatures there is significant DHHD degradation. DHHD was identified using a standard. However, a standard for pyranone was not available. The peak, assumed to be pyranone, had a mass spectrum (Kim & Baltes, 1996) and retention indices (Osada & Shibamoto, 2006; Yu & Ho, 1995) that agreed with literature data. Furthermore, the 144 mass chromatograms of the ghee samples (Fig. 2a) contained only 2 significant peaks: DHHD identified from the synthetic standard, and the second larger peak, assumed to be pyranone. Pyranone and DHHD are two of only a few compounds, formed via heating, that produce the diagnostic 144 ion (and related ions) in abundance in GCMS. Pyranone is commonly found in heated foods that are rich in glucose or lactose (Kim & Baltes, 1996). Thus, pyranone is commonly identified in MRP without using an authentic standard (Yaylayan & Mandeville, 1994), and such an approach was assumed as adequate for this work. The formation of an intense purple coloured complex when maltol reacts with iron(III) has long been used to characterise maltol in dairy products (Patton, 1950). This approach was applicable
Fig. 2. Comparison of the 144 mass chromatograms of DC ghee obtained on (a) a polar (EC-Wax) and (b) a non polar (Rtx-5) GC column. All aspects of the GC methodology were equivalent except for the column. Peak 1 is DHHD and peak 2 is pyranone.
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to ghee and the colourimetric maltol assay gave results comparable with GCMS (not shown). 3.2. Qualitative comparisons of ghee The fresh samples of DC, CB and PS ghee all had similar appearance (i.e., the molten fat was clear and golden-yellow). The DC and CB ghee had stronger aromas (caramel, cooked, and burnt) than the PS ghee. A qualitative comparison of DC, CB and PS ghee is shown in Fig. 3, based upon GCMS peak areas for a selection of compounds found in both fresh and stored (2 weeks) samples. Compounds that have typically (Sarrazin, Frerot, Bagnoud, Aeberhardt, & Rubin, 2011; Wadodkar et al., 2002) been found in milkfat (associated with fat degradation via oxidation and lipolysis) are shown on the left side of Fig. 3. All three types of ghee contained similar amounts of these compounds immediately after production. All three samples were made from the same cream. Thus, all three traditional methods of fat isolation have similar impact on fat degradation (which did not appear to be any greater than in commercial anhydrous milkfat – results not shown). Compounds on the right side of Fig. 3 are all MRP. The identity of maltol and acetic acid was confirmed with authentic standards. The identity of the other MRP is only tentative based upon library searching. Nevertheless, for fresh samples, it is clear that DC ghee
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contained the most MRP, and CB ghee contained less MRP, while PS ghee contained little, or undetectable, amounts of MRP. This finding was consistent with prior literature (Wadodkar et al., 2002) and consistent with the relative amounts of non-fat solids in cream compared to butter. DC ghee is often said to be preferred over CB ghee due to its richer and stronger flavour (Wadhwa & Jain, 1990), which could be due, in part, to greater amounts of MRP in DC ghee. After the ghee samples had been stored for two weeks (at 60 °C), the DC samples had distinctly less golden-yellow colour than the CB and PS samples (all molten fat samples were still clear liquids). Numerous changes in the volatile profiles of all three ghee types were apparent after storage. Some select examples are given in Fig. 3. Very few of the MRP in CB and DC ghee appeared to be greatly affected by storage. For example, peak areas for furfural, acetylfuran, HMF and maltol were similar after storage. In stark contrast, both pyranone and DHHD could no longer be detected in DC and CB ghee after two weeks storage. The disappearance of pyranone and DHHD was most striking in the 144 mass chromatogram of DC ghee; the chromatogram contained only two large peaks in fresh samples (Fig. 2) that disappeared on storage. The only other MRP that appeared to change in the CB and DC ghee was acetic acid that doubled in peak area over the storage period. More detail is given in the next section.
Fig. 3. Qualitative comparison of peak areas of some of the compounds found in (a) PS, (b) CB, and (c) DC ghee when analysed using SPME GC–MS with an EC-Wax column. Samples were analysed immediately after making the ghee (open bars) and again after two weeks storage at 60 °C (solid bars). The results given represent the average of two different ghee samples. To the left of the vertical line are compounds associated with fat degradation, and to the right are MRP.
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The other qualitative difference seen in all three types of ghee over storage was a large increase in the peak areas of compounds typically associated with fat degradation (compounds on the left side of Fig. 3). Hexanal, heptanal, and nonenal are all associated with (milk) fat oxidation (Kochhar, 1996). In all three types of ghee the concentrations of these compounds greatly increased over the storage period. The concentration of hexanal increased more in the PS ghee than in the CB and DC ghee, but the concentration of heptanal and nonenal increased more in the CB and DC ghee. Thus, fat oxidation appears to have different consequences in the different types of the ghee. However, there is no clear evidence, from these data, that oxidation was occurring significantly faster, or slower, in any of the types of ghee. It has been reported, based on sensory evaluations, that DC and CB ghee have better keeping quality than PS ghee (Ganguli & Jain, 1973; Singh & Ram, 1978; Sserunjogi et al., 1998). However, this could be due to caramel notes in DC and CB ghee masking oxidised flavours, rather than slower fat oxidation rates per se. Butyric acid is also due to fat degradation but it could be from either hydrolysis (Deeth & Fitz-Gerald, 1994) or oxidation (Kochhar, 1996). In all three types of ghee the butyric acid increased during storage (Fig. 3), slightly more so in the CB and DC ghee. Overall (considering hexanal, heptanal, nonenal and butyric acid), there does not appear to be much difference in total fat degradation in the types of ghee. But the specifics of fat degradation are different. It could be assumed, based on fat degradation, all three types of ghee will have a similar (sensory-based) shelf-life, but a sensory investigation of ghee shelf-life versus fat degradation would be confounded by the (caramel) flavours of the MRP. Linking the chemical data to sensory properties was not within the scope of this paper and is not considered further.
Table 1 Concentrations of maltol (ppm) in two samples (A and B) of both CB and DC ghee, as determined by colourimetry, during storage at 60 °C.
3.3. Time-course of MRP in ghee
Fig. 4. Time-course showing the loss of pyranone (j) and DHHD (N) in DC ghee during storage at 60 °C (represented as a peak area on right axis). As these compounds were lost, acetic acid () formed (concentration given in ppm on left axis). The results given represent the average of two different ghee samples.
Samples of ghee were periodically analysed using GCMS during storage (at least 2 weeks), and it was found that many MRP, such as maltol, change little in concentration during storage of the CB and DC ghee. Maltol is one of the most abundant MRP found in ghee (Wadodkar et al., 2002) and is readily quantified using colourimetry (Patton, 1950). Therefore, to confirm the GCMS results, analysis of maltol by colourimetry was done. The results are shown in Table 1. DC ghee contained an average of 210 ppm maltol, while the CB ghee contained 40 ppm maltol. In both types of ghee the maltol concentration changed little during storage. In contrast to the high stability of maltol, both DHHD and pyranone rapidly diminished in both the CB and DC ghee. A timecourse, showing the loss of DHHD and pyranone from DC ghee is shown in Fig. 4. Similar behaviour was seen in CB ghee (not shown) but further attention was placed only on the DC ghee due to it containing much greater amounts of MRP that facilitated analysis. The loss of DHHD in ghee during storage may have been due to tautomerisation to 2,4-dihydroxy-2,5-dimethyl-3(2H)-furanone (Fig. 1) in the presence of water (Engel et al., 2001). However, the furanone was not detected. In the DC ghee, in parallel to DHHD and pyranone decreasing in concentration, acetic acid increased in concentration from 45 ppm to 75 ppm. The acetic acid was not coming from milkfat degradation because acetic acid did not form in the PS ghee (Fig. 3). Acetic acid stopped increasing in concentration after DHHD and pyranone were no longer detectable (Fig. 4). This suggested that the acetic acid could be derived from pyranone and DHHD. A standard for pyranone was not available and the DHHD standard was not 100% pure, so accurate quantification of these compounds was not possible. However, by assuming the DHHD standard to be pure, and assuming that the pyranone had the same TIC response factor as maltol, it was estimated that these compounds were present in concentrations between 10 and
Storage time (days)
0 3 6 9 12 14
CB ghee
DC ghee
Sample A
Sample B
Sample A
Sample B
44 53 47 44 44 48
28 36 37 30 34 41
200 220 190 210 200 200
230 240 240 230 220 230
100 ppm in DC ghee. Pyranone typically occurs in ppm concentrations in heated foods (Kim & Baltes, 1996). Therefore, the amount of pyranone and DHHD in DC ghee does appear sufficient to account for the amount of acetic acid formed (30 ppm). The DHHD standard contained large amounts of acetic acid as a significant impurity (determined by H and 13C NMR, and GCMS – not shown). When this DHHD standard was placed in milkfat and stored, it degraded and additional acetic acid was formed (not shown). It has been proposed that water addition to pyranone (Fig. 1) regenerates the highly reactive 1-deoxyglucosone (Kim & Baltes, 1996; Voigt et al., 2010). The reactive 1-deoxyglucosone, depending on the conditions, can then form numerous compounds including acetic acid (Voigt & Glomb, 2009; Voigt et al., 2010). It is likely that water addition to DHHD can also regenerate 1-deoxyglucosone (and again, this could then proceed to acetic acid). However, the DHHD probably first rapidly tautomerises, losing its caramel odour, as shown by Engel et al. (2001). It is hypothesised that in DC and CB ghee during storage, pyranone and DHHD form acetic acid via 1-deoxyglucosone (Fig. 1). If this hypothesis is true, it should be possible to isolate 1-dexoxygluosone (e.g., as a quinoxaline-derivative) from ghee, during storage, and it should also be possible to detect other products associated with 1-deoxyglucosone fragmentation (e.g., glyceric acid and threosones). Various proportions of glucose degradation products could form depending on ghee storage conditions (Novotny, Cejpek, & Velisek, 2008). 3.4. Time-course of hexanal in different types of ghee during storage Pyranone and DHHD can possibly act as antioxidants. Various antioxidant mechanisms are available for both of these com-
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Fig. 5. Time course showing the changes in hexanal concentration in PS (), CB (j) and DC (N) ghee during storage at 60 °C. The results given represent the average of two different ghee samples.
pounds. For example, pyranone could abstract hydroxyl radicals (Osada & Shibamoto, 2006), and DHHD has a reductone structure analogous to that of vitamin C (Belitz et al., 2004). This suggested a hypothesis that decreases in DHHD and pyranone in CB and DC ghee during storage are related to these compounds acting as antioxidants. To test this hypothesis the concentration of hexanal was measured in all three types of ghee (PS, CB and DC) during storage (Fig. 5). Hexanal was followed because it is a popular indicator for fat oxidation (Kochhar, 1996). In all three types of ghee, the change in hexanal concentration, during storage was slow to begin with, then greatly accelerated after nine days (Fig. 5). Such an increase in oxidation rate after the DHHD and pyranone were depleted might support our hypothesis. But this behaviour was seen in the PS ghee (no MRP present) and such non-linear kinetics is routinely observed when fats oxidise (Kochhar, 1996). Based upon hexanal measurement alone, the PS ghee appeared to oxidise at the fastest rate; both CB and DC ghee oxidised at a slower rate than PS ghee. Based only on hexanal measurement, the CB ghee appeared to be the most stable ghee with respect to oxidation. Therefore, these data did not support the hypothesis; i.e., if pyranone and DHHD were acting as antioxidants the CB ghee should have behaved more like the PS ghee since the CB ghee contained substantially less MRP than the DC ghee. Based on hexanal, the CB and DC ghee appear more stable than the PS ghee. However the stability did not appear correlated to the amount of MRP in these samples. It is likely that the apparently higher (based on hexanal) stability of CB and DC ghee is due to some other factor like phospholipid content, as suggested by others (Singh & Ram, 1978). However, if other compounds are used as a measure of fat oxidation (e.g., nonenal, Fig. 3) the trend is reversed. So, overall, there is no compelling chemical evidence that any of these traditional methods of fat isolation yield a more stable fat. From a sensory perspective DC ghee appears to have slightly better keeping quality (Ganguli & Jain, 1973), but that could just as easily be from cooked flavours of MRP masking other flavours.
4. Conclusions Changes in the concentration of volatile compounds in PS, CB and DC ghee were monitored over storage. The most significant observations were increases in compounds from fat degradation, little change in the majority of MRP, and complete loss of the related compounds, pyranone and DHHD. While it is likely that both pyranone (Osada & Shibamoto, 2006) and DHHD have antioxidant properties, no evidence of these
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compounds being responsible for any large improvement in the stability of milkfat in ghee could be found. The loss of pyranone and DHHD correlated to an increase in acetic acid. It is suggested that in ghee DHHD and pyranone are degrading to acetic acid. This is based on an observation that a DHHD standard degrades to acetic acid, and literature reports on the reactions of pyranone (Kim & Baltes, 1996). An alternative explanation is that DHHD and pyranone are forming compounds not detected, while another unknown (also undetected) is degrading to acetic acid at a similar rate. Thus there is a need to do further experiments to link loss of DHHD and pyranone to acetic acid formation. Furthermore, based on these observations, we propose that DHHD and pyranone are degrading via 1-deoxyglucosone. If this pathway is proven, finding it in ghee is notable because ghee contains only traces of proteins or sugars. The implication is that the Maillard reaction cascade could have an impact on foods well after cooking has ceased. Obviously, it is well known that Maillard reactions occur in food during storage (Baltes, 1982). However, it is remarkable that the Maillard reaction may continue to play a role in food that contains minimal protein or sugar. It was not possible to determine what impact the loss during storage of DHHD, and pyranone, or acetic acid formation, has on the shelf-life or flavour of ghee. It is possible that it will affect flavour (e.g., loss of some DHHD’s caramel notes as proposed by Engel et al., 2001). This might be most notable with moderate storage (allowing changes in MRP, without fat becoming excessively oxidised). Further work will be needed to determine if the chemistry observed in these accelerated storage trials occurs at lower temperatures and if it is related to known changes in ghee flavour that occur during storage (Singh & Ram, 1978). Acknowledgements Jeremy Jones and Graham Caygill (of GlycoSyn, Industrial Research Limited) are thanked for the synthesis and characterisation of DHHD. Tim Coolbear and Euan Cant are thanked for reviewing the manuscript. References Baltes, W. (1982). Chemical changes in food by the Maillard reaction. Food Chemistry, 9, 59–73. Belitz, H. D., Grosch, W., & Schieberle, P. (2004). Food chemistry (3rd ed.). Berlin: Springer-Verlag [Chapter 4]. Davidek, T., Robert, F., Devaud, S., Vera, F. A., & Blank, I. (2006). Sugar fragmentation in the Maillard reaction cascade: Formation of short-chain carboxylic acids by a new oxidative a-dicarbonyl cleavage pathway. Journal of Agricultural and Food Chemistry, 54, 6677–6684. Deeth, H. C., & Fitz-Gerald, C. H. (1994). Lipolytic enzymes and hydrolytic rancidity in milk and milk products. In P. F. Fox (Ed.), Advanced dairy chemistry. Lipids (Vol. 2, pp. 247–308). London: Chapman & Hall. Engel, W., Hofmann, T., & Schieberle, P. (2001). Characterization of 3,4-dihydroxy-3hexen-2,5-dione as the first open-chain caramel-like smelling flavour compound. European Food Research and Technology, 213, 104–106. Ganguli, N. C., & Jain, M. K. (1973). Ghee: Its chemistry processing and technology. Journal of Dairy Science, 56, 19–25. Goto, R., Miyagi, Y., & Inokawa, H. (1963). Syntheses and structures of acetylformoin and its related compounds. I. Bulletin of the Chemical Society of Japan, 36, 147–151. Hodge, J. E., Mills, F. D., & Fisher, B. E. (1972). Compounds of browned flavour derived from sugar-amine reactions. Cereal Science Today, 17, 34–40. Hofmann, T. (1998). Acetylformoin – a chemical switch in the formation of coloured Maillard reaction products from hexoses and primary and secondary amino acids. Journal of Agricultural and Food Chemistry, 46, 3918–3928. Illingworth, D., Janssen, P. W. M., Cant, P. A. E., & Stephens, G. R. (2009). Dairy Product and Process. Patent WO2009/011598. Kim, M., & Baltes, W. (1996). On the role of 2,3-dihydro-3,5-dihydroy-6-methyl4(H)-pyran-4-one in the Maillard reaction. Journal of Agricultural and Food Chemistry, 44, 282–289. Kochhar, S. P. (1996). Oxidative pathways to the formation of off-flavours. In M. J. Saxby (Ed.), Food taints and off-flavours (pp. 168–218). Glasgow: Blackie Academic and Professional.
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Novotny, O., Cejpek, K., & Velisek, J. (2008). Formation of carboxylic acids during degradation of monosaccharides. Czech Journal of Food Science, 26, 117–131. Osada, Y., & Shibamoto, T. (2006). Antioxidative activity of volatile extracts from Maillard model systems. Food Chemistry, 98, 522–528. Patton, S. (1950). The isolation of maltol from heated skim milk. Journal of Dairy Science, 33, 102–106. Sarrazin, E., Frerot, E., Bagnoud, A., Aeberhardt, K., & Rubin, M. (2011). Discovery of new lactones in sweet cream butter oil. Journal of Agricultural and Food Chemistry, 59, 6657–6666. Singh, S., & Ram, B. P. (1978). Effect of ripening of cream manufacturing temperature and packaging materials and flavour and keeping quality of ghee (butter fat). Journal of Food Science and Technology, 15, 142–145. Sserunjogi, M. L., Abrahamsen, R. K., & Narvhus, J. (1998). A review paper: Current knowledge of ghee and related products. International Dairy Journal, 8, 677–688. Van Boekel, M. A. J. S. (1998). Effect of heating on Maillard reactions in milk. Food Chemistry, 62, 403–414.
Voigt, M., & Glomb, M. A. (2009). Reactivity of 1-deoxy-erythro-hexo-2,3-diulose: A key intermediate in the Maillard chemistry of hexoses. Journal of Agricultural and Food Chemistry, 57, 4765–4770. Voigt, M., Smuda, M., Pfahler, C., & Glomb, M. A. (2010). Oxygen-dependent fragmentation reactions during degradation of 1-deoxy-erythro-hexo-2,3diulose. Journal of Agricultural and Food Chemistry, 58, 5685–5691. Wadhwa, B. K., & Jain, M. K. (1990). Chemistry of ghee flavour – A review. Indian Journal of Dairy Science, 43, 601–607. Wadodkar, U. R., Punjrath, J. S., & Shah, A. C. (2002). Evaluation of volatile compounds in different types of ghee using direct injection with gas chromatography–mass spectrometry. Journal of Dairy Research, 69, 163–171. Yaylayan, V. A., & Mandeville, S. (1994). Stereochemical control of maltol formation in Maillard reaction. Journal of Agricultural and Food Chemistry, 42, 771–775. Yu, T.-H., & Ho, C.-T. (1995). Volatile compounds generated from thermal reaction of methionine and methionine sulfoxide with or without glucose. Journal of Agricultural and Food Chemistry, 43, 1641–1646.
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Changes in Maillard reaction products in ghee during storage P. Andrewes ⇑ Fonterra Research Centre, Fonterra Cooperative Group Limited, Private Bag 11029, Dairy Farm Road, Palmerston North, New Zealand
a r t i c l e
i n f o
Article history: Received 10 April 2012 Received in revised form 10 May 2012 Accepted 8 June 2012 Available online 16 June 2012 Keywords: Ghee Maillard reaction Milkfat Oxidation
a b s t r a c t Ghee (milkfat from heat clarification) was made using direct cream (DC), cream butter (CB) or pre-stratification (PS) methods and stored at 60 °C, in air, for at least two weeks. Milkfat degradation, particularly oxidation, occurred in all types of ghee, resulting in increases in aldehydes and free fatty acids. However, there was little difference in fat degradation rates in each type of ghee. DC and CB ghee contained volatile Maillard reaction products, whereas PS ghee did not. The concentrations of 3,4-dihydroxy-3-hexen-2,5dione (DHHD) and 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (pyranone), Maillard reaction products found in DC and CB ghee, rapidly decreased during storage, associated with increases in acetic acid. This work suggests hydration of both DHHD and pyranone, during storage, could form reactive 1-deoxy-D-erythro-hexo-2,3-diulose (1-deoxyglucosone) that degrades mostly to acetic acid. Thus, the Maillard reaction cascade appears to continue in food, in the absence of proteins and sugars, long after cooking has ceased. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The isolation of milkfat by ‘boiling-off’ water from milk, cream or butter is an ancient process dating back as far as 1500 BC (Sserunjogi, Abrahamsen, & Narvhus, 1998). Usually, after the bulk of the water is removed, heating continues until the non-fat solids are deeply coloured. Non-fat solids are then often removed by either filtration or decanting the oil (Ganguli & Jain, 1973; Sserunjogi et al., 1998). Isolation of milkfat in this manner was traditionally used (and is still widely done today) because it is a valuable means to both preserve and add flavour to milkfat (Ganguli & Jain, 1973; Sserunjogi et al., 1998). Ghee (from the sanskrit Ghrita) is widely used in India (and South Asia) and is perhaps the most common example of a milkfat isolated by ‘boiling-off’ water. However, there are numerous related indigenous products (Sserunjogi et al., 1998), such as samin (Sudan) and samuli (Uganda). Western chefs often make beurre noisette (brown butter) and beurre noir (black butter) using similar techniques, but the primary objective is usually flavour creation rather than fat preservation. There are four methods commonly used for the manufacture of ghee (Ganguli & Jain, 1973; Sserunjogi et al., 1998): milk butter (or desi method), direct cream (DC), cream butter (CB), and pre-stratification (PS). Heat clarification of cream directly produces DC ghee. Churning cream to butter, discarding the buttermilk, and heat clarification of butter, produces CB ghee. PS ghee is made by churning cream to butter, melting the butter, discarding serum, ⇑ Tel.: +64 6 3504649; fax: +64 6 3561476. E-mail address: [email protected] 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.06.013
and using mild heat to remove residual water. The cream used in manufacture of DC, CB and PS ghee has also often undergone fermentation (Sserunjogi et al., 1998). Desi ghee was not investigated in this work but is produced via churning fermented milk to butter, and heating. High temperatures used in the manufacture of desi, DC and CB ghee causes non-fat milk solids to ‘caramelise’ (sugar degradation) and also participate in the Maillard reaction (Ganguli & Jain, 1973; Sserunjogi et al., 1998; Wadhwa & Jain, 1990; Wadodkar, Punjrath, & Shah, 2002). The Maillard reaction is used to describe a cascade of reactions initiated by a reaction between reducing sugars and amino groups (from free amino acids, peptides, or proteins) to form Amadori compounds (Baltes, 1982; Belitz, Grosch, & Schieberle, 2004; Hodge, Mills, & Fisher, 1972). Subsequent reactions of the Amadori compounds produce complex mixtures of reaction products, including numerous compounds that can also be derived from sugar degradation in the absence of any intermediate Amadori compounds (Belitz et al., 2004). In this paper, for simplicity, Maillard reaction products (MRP) are defined as any compounds produced by heating of non-fat solids, regardless of formation via Amadori compounds or sugar degradation. The MRP include insoluble melanoidins (removed from ghee by filtration), pigments (many also removed by filtration but residual pigments can add colour to ghee), and volatile compounds, some of which are potent flavours (Baltes, 1982; Belitz et al., 2004; Hodge et al., 1972). Maillard reactions in dairy products produce many compounds that are derived from lactose (van Boekel, 1998). Most of these compounds are believed to be formed via highly reactive deoxyosone intermediates (a-dicarbonyl compounds). Typically lactose
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either forms 3-deoxyosones (at acidic pH via 1,2 enolization), or 1-deoxyosones (at neutral to high pH via 2,3 enolization). Further reactions of 3-deoxyosones can produce the well-known hydroxymethylfurfural (van Boekel, 1998). Reactions of 1-deoxyosones are usually more important in dairy products due to the neutral pH of dairy products (van Boekel, 1998). Some features of the 1-deoxyosone pathway for glucose are illustrated in Fig. 1 (the 1-deoxyosone derived from glucose is 1-deoxy-D-erythro-hexo-2,3-diulose or 1-deoxyglucosone). The disaccharide lactose undergoes analogous reactions to glucose but the galactosyl group adds additional complexity (van Boekel, 1998). Either galactose or water can be eliminated from lactose at different points in these pathways. If galactose is eliminated the same products form as for glucose. However, if the galactosyl group is not eliminated, analogues to the products formed from glucose, such as galactosyl isomaltol and galactosyl beta-pyranone, are found (van Boekel, 1998). Additionally, the galactosyl group of lactose plays an important role in directing maltol formation from the 1-deoxyosone (Yaylayan & Mandeville, 1994). Maltol is an important flavour compound in heated dairy products (Patton, 1950) and was part of this research. However, galactosyl MRP were not considered further because they were not detectable using the methods described. The reaction of 1-deoxyosones can take either of two paths (Fig. 1) to produce 5 and 6-membered ring compounds, such as acetylformoin (right branch of Fig. 1) and 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (pyranone), respectively (Kim & Baltes, 1996; Voigt & Glomb, 2009; Voigt, Smuda, Pfahler, & Glomb, 2010). Acetylformoin is notable in that it has a linear tautomer (3,4-dihydroxy-3-hexen-2,5-dione, DHHD, Fig. 1) that is the only known open-chain compound with a caramel odour
(Engel, Hofmann, & Schieberle, 2001). Its other tautomers do not appear to have any caramel odour. Also, in the presence of water, the caramel odour of DHHD disappears and Engel et al. (2001) claim this is due to tautomerisation of DHHD. Both acetylformoin and pyranone retain the original ‘carbonbackbone’ of glucose minus two moles of water for diagnostic masses of 144 (Kim & Baltes, 1996). Their formation (and formation of related compounds), from 1-deoxyglucosone, is explained by appropriate combinations of keto-enol tautomerisation, cyclization, and dehydration. Such reactions (that do not break the ‘carbon-backbone’) are often reversible. In particular, hydration of pyranone can reform 1-deoxyglucosone (Kim & Baltes, 1996). In practice, this means compounds like DHHD, pyranone and 1-deoxyglucosone often have the same chemistry. For example, pyranone (Kim & Baltes, 1996), acetylformoin (Hofmann, 1998), and 1-deoxyglucosone (Davidek, Robert, Devaud, Vera, & Blank, 2006; Voigt & Glomb, 2009; Voigt et al., 2010) have all been shown to produce the same numerous end products of glucose degradation. Furthermore, pyranone and DHHD formed compounds (e.g., acetic acid, formic acid, lactones) that are best explained by conversion to deoxyglucosone followed by hydrolytic and oxidative fragmentation. As already noted, galactose elimination allows lactose to access all the pathways illustrated in Fig. 1, producing all of the MRP (including intermediates) produced by glucose. Thus, the model studies above for glucose can explain the presence of numerous compounds found in heated dairy products and ghee (van Boekel, 1998; Wadodkar et al., 2002). In the course of investigating Maillard reaction products in ghee, it was observed the two related compounds, pyranone and DHHD, were both rapidly lost from samples during storage. The objective
O
OH
O
O
HO
Formicacid Aceticacid Acetol Glycericacid Lacticacid Aldehydes Hydroxyacids Lactones ...
OH OH
O OH
OH
-H 2O
+H 2O
1-Deoxy-D-erythro-hexo-2,3-diulose (1-deoxyglucosone) +H 2O
O O OH
HO
-H 2O OH
OH
O
O O
2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (pyranone)
Maltol Isomaltol Furans Furanones ...
HO
O
2,4-dihydroxy-2,5-dimethyl-3(2H)-furanone
OH
3,4-dihydroxy-3-hexen-2,5-dione (DHHD)
Maltol Isomaltol Furans Furanones ...
Fig. 1. Possible reaction pathways for the reactive glucose derivative, 1-deoxyglucosone (taken from Hodge et al., 1972; Voigt et al., 2010).
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of this work was to further investigate the behaviour of these two compounds in ghee during storage and determine if there were any implications for the chemistry of ghee. The storage conditions selected (60 °C, air, in the dark) would usually not be encountered during typical use of ghee. In this work it was assumed these accelerated storage conditions will predict the chemistry that will occur, albeit more slowly, in more moderate conditions. 2. Materials and methods 2.1. Materials Iron(III) chloride hexahydrate was from BDH (Poole, England). Iron chloride reagent was made by dissolving iron(III) chloride (5 g/L) in hydrochloric acid (0.5 M). Maltol and hexanal were from Aldrich (St. Louis, MO, USA). All reagents were analytical grade or better. DHHD was provided by GlycoSyn, Industrial Research Limited (Lower Hutt, New Zealand). The DHHD had an estimated 70% purity (by 1H and 13C NMR) and was synthesised by condensation of 2-oxopropanal using a previously described procedure (Goto, Miyagi, & Inokawa, 1963). Fresh cream (40%, w/w, fat) was purchased from a local supermarket (Palmerston North, New Zealand) each day that ghee was made. 2.2. Gas chromatography–mass spectrometry Two gas chromatography mass spectrometry (GC–MS) methods were used to analyse the samples. Both methods used common conditions wherever possible. However, each method used a different GC column (and corresponding temperature programme). In this section, the common conditions for each method are given first followed by the specifics applicable to each method. The analysis was performed using a Shimadzu QP2010 Plus GC–MS (Shimadzu, Kyoto, Japan) The GC–MS was equipped with an AOC5000 (CTC Combipal, CTC Analytics, Zwingen, Switzerland) autosampler configured for solid phase microextraction (SPME). Samples were extracted and introduced into the GC–MS using headspace SPME using a 1 cm long fibre (Supelco, Bellefonte, PA, USA) that was coated with a 65 lm layer of polydimethylsiloxane/divinylbenzene and housed in a 23 gauge needle. The SPME fibre was exposed to the headspace of a sample of ghee (3 g) in a 20 ml Chromacol (Welwyn Garden City, Herts, UK) volatile organic compound (VOC) tube sealed with Teflon-backed septa (Chromacol). The headspace was extracted for 30 min at 60 °C with shaking at 250 rpm. Analytes were desorbed from the SPME fibre in the GC injection port, which contained a silanised SPME liner (Restek) and was held at 220 °C. The split injection technique was used to inject sample with a 5:1 split ratio. The column flow (Helium carrier gas) was 1 ml min 1, with a purge flow of 3 ml min 1. The MS interface was held at 230 °C and the ion source at 200 °C. The MS was operated in EI mode at 70 eV with an interval of 1 s. MS acquisition used the FASST program (allowing simultaneous scan and SIM) with a microscan of 0.1 amu. The scan acquisition used an m/z range of 45–350. DHHD, and the related pyranone, were analysed using a Rtx-5 column (Restek, Bellefonte, PA, USA), simultaneous scan and SIM (m/z = 144), and an appropriate temperature programme (GC oven was held at 50 °C for 2 min, increased to 80 °C at 3 °C min 1, increased to 120 °C at 5 °C min 1, and then increased to 320 °C at 20 °C min 1 and held at this final temperature for 2 min). For all other compounds, a second method using a polar column (EC-Wax; Alltech Associates, Deefield, IL, USA) was used as it was more appropriate for the polar analytes and matrix. The GC oven
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temperature programme was slightly modified (the oven was held at 50 °C for 2 min, increased to 80 °C at 3 °C min 1, increased to 120 °C at 5 °C min 1, and then increased to 220 °C at 20 °C min 1 and held at this final temperature for 6 min). Standards of hexanal, maltol, acetic acid, and DHHD were spiked into samples to identify and where necessary quantify (using standard additions) compounds. 2.3. Colourimetric measurement of maltol Ghee (1.0 g) was dissolved in heptane (10 ml) in a 30 ml Teflon Oak Ridge centrifuge tube (Nalgene, Rochester, NY, USA). Iron chloride reagent (5.00 ml) was added to the tube, and agitated thoroughly to extract maltol into the aqueous phase as a coloured complex with iron(III). An aliquot (1 ml) of the aqueous phase was carefully removed from the Oak Ridge tube, using a pipette, and the absorbance of the iron(III) complex was measured at 525 nm. Samples of milkfat, spiked with maltol, were treated in the same manner and used to construct a calibration curve. 2.4. Ghee preparation All types of ghee were prepared from the same batch of fresh cream that was divided into two portions. One portion was used to make DC ghee and the other used to make butter for the CB and PS ghee. Two equivalent trials were done on different days. The procedures described below to prepare ghee are broadly representative of documented procedures (Ganguli & Jain, 1973; Singh & Ram, 1978; Sserunjogi et al., 1998), commercial experience (Illingworth, Janssen, Cant, & Stephens, 2009), and observations in domestic or traditional settings. DC ghee was prepared by heating cream (300–600 g) in a Teflon-coated wok over a moderate heat, stirring regularly. The temperature of the cream was monitored during the cooking. Until the majority of the water had evaporated from the cream the temperature did not exceed 110 °C. After the water had evaporated, the slurry of milk solids in liquid fat was heated to 135 ± 5 °C and held at that temperature for 10 min. The slurry was cooled to below 50 °C and filtered through a paper towel. To make CB and PS ghee, cream (1 L at room temperature) was whipped with an egg beater until phase inversion. The granules were gently worked with a fork to separate the butter and buttermilk. Buttermilk was decanted from the butter, and discarded. On average the discarded buttermilk represented 50 ± 5% (w/w) of the original cream. The butter was divided into two portions. One portion was used to make PS and the other CB ghee. To make CB ghee, the butter was heated in a wok over a moderate heat, stirring regularly. The temperature of the butter was monitored during the cooking. Until the majority of the water had evaporated from the butter the temperature did not exceed 110 °C. After the water had evaporated, the slurry of milk solids in liquid fat was heated to 135 ± 5° C and held at that temperature for 10 min. The slurry was cooled to below 50° C and filtered through a paper towel. To make PS ghee, butter was melted and placed in a separating funnel in an incubator at 50 °C and left to settle into two layers. The serum layer was discarded. The oil layer was collected while filtering through a paper towel. The oil was then heated to 110 °C and immediately cooled back to 50 °C. Minimal heat was desired to achieve milkfat with low moisture and low amounts of MRP. 2.5. Ghee storage Equal amounts of each type of ghee were placed in the same type of sample container. Samples were wrapped in aluminium foil
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to exclude light and stored together in an incubator (Contherm, Upper Hutt, New Zealand) at 60 °C. Samples were removed from the incubator periodically for analysis. Samples were analysed for maltol using a colourimetric assay (Section 2.3) and for other volatile compounds using gas chromatography mass spectrometry (Section 2.2). All samples were handled in exactly the same manner. Two independent storage trials were performed with freshly made DC, CB and PS ghee, made on the same day, from the same batch of cream (Section 2.4). Variation from analysis (10% or less for repeat analyses of the same sample) was always much less than the variation due to ghee preparation and analysis (40% or less). For practical reasons, manufacture and storage was only done twice for each type of ghee and results are an average from the two trials unless otherwise indicated. Statistical analysis was not done due to the small sample size and the qualitative nature of this investigation.
3. Results and discussion 3.1. Analytical methods In preliminary experiments, DHHD was often not detected in fresh ghee when using a highly polar GC stationary phase (ECWax), whereas pyranone always gave a large peak. This did not appear to be an issue peculiar to an individual column because low response of DHHD, relative to pyranone, was also observed with
an EC-1000 column and two other EC-Wax columns (not shown). In contrast, the DHHD peak area was at least 100-fold greater when using a mildly polar stationary phase (Rtx-5), all else being equal (Fig. 2). Peak area of the pyranone (Fig. 2) was about the same on both types of columns (confirming the equivalence of the methodology); but, by only changing the column, it was possible to greatly increase DHHD peak area. On the Rtx-5 column DHHD elutes at a much lower temperature. It is possible that in a polar stationary phase at higher temperatures there is significant DHHD degradation. DHHD was identified using a standard. However, a standard for pyranone was not available. The peak, assumed to be pyranone, had a mass spectrum (Kim & Baltes, 1996) and retention indices (Osada & Shibamoto, 2006; Yu & Ho, 1995) that agreed with literature data. Furthermore, the 144 mass chromatograms of the ghee samples (Fig. 2a) contained only 2 significant peaks: DHHD identified from the synthetic standard, and the second larger peak, assumed to be pyranone. Pyranone and DHHD are two of only a few compounds, formed via heating, that produce the diagnostic 144 ion (and related ions) in abundance in GCMS. Pyranone is commonly found in heated foods that are rich in glucose or lactose (Kim & Baltes, 1996). Thus, pyranone is commonly identified in MRP without using an authentic standard (Yaylayan & Mandeville, 1994), and such an approach was assumed as adequate for this work. The formation of an intense purple coloured complex when maltol reacts with iron(III) has long been used to characterise maltol in dairy products (Patton, 1950). This approach was applicable
Fig. 2. Comparison of the 144 mass chromatograms of DC ghee obtained on (a) a polar (EC-Wax) and (b) a non polar (Rtx-5) GC column. All aspects of the GC methodology were equivalent except for the column. Peak 1 is DHHD and peak 2 is pyranone.
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to ghee and the colourimetric maltol assay gave results comparable with GCMS (not shown). 3.2. Qualitative comparisons of ghee The fresh samples of DC, CB and PS ghee all had similar appearance (i.e., the molten fat was clear and golden-yellow). The DC and CB ghee had stronger aromas (caramel, cooked, and burnt) than the PS ghee. A qualitative comparison of DC, CB and PS ghee is shown in Fig. 3, based upon GCMS peak areas for a selection of compounds found in both fresh and stored (2 weeks) samples. Compounds that have typically (Sarrazin, Frerot, Bagnoud, Aeberhardt, & Rubin, 2011; Wadodkar et al., 2002) been found in milkfat (associated with fat degradation via oxidation and lipolysis) are shown on the left side of Fig. 3. All three types of ghee contained similar amounts of these compounds immediately after production. All three samples were made from the same cream. Thus, all three traditional methods of fat isolation have similar impact on fat degradation (which did not appear to be any greater than in commercial anhydrous milkfat – results not shown). Compounds on the right side of Fig. 3 are all MRP. The identity of maltol and acetic acid was confirmed with authentic standards. The identity of the other MRP is only tentative based upon library searching. Nevertheless, for fresh samples, it is clear that DC ghee
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contained the most MRP, and CB ghee contained less MRP, while PS ghee contained little, or undetectable, amounts of MRP. This finding was consistent with prior literature (Wadodkar et al., 2002) and consistent with the relative amounts of non-fat solids in cream compared to butter. DC ghee is often said to be preferred over CB ghee due to its richer and stronger flavour (Wadhwa & Jain, 1990), which could be due, in part, to greater amounts of MRP in DC ghee. After the ghee samples had been stored for two weeks (at 60 °C), the DC samples had distinctly less golden-yellow colour than the CB and PS samples (all molten fat samples were still clear liquids). Numerous changes in the volatile profiles of all three ghee types were apparent after storage. Some select examples are given in Fig. 3. Very few of the MRP in CB and DC ghee appeared to be greatly affected by storage. For example, peak areas for furfural, acetylfuran, HMF and maltol were similar after storage. In stark contrast, both pyranone and DHHD could no longer be detected in DC and CB ghee after two weeks storage. The disappearance of pyranone and DHHD was most striking in the 144 mass chromatogram of DC ghee; the chromatogram contained only two large peaks in fresh samples (Fig. 2) that disappeared on storage. The only other MRP that appeared to change in the CB and DC ghee was acetic acid that doubled in peak area over the storage period. More detail is given in the next section.
Fig. 3. Qualitative comparison of peak areas of some of the compounds found in (a) PS, (b) CB, and (c) DC ghee when analysed using SPME GC–MS with an EC-Wax column. Samples were analysed immediately after making the ghee (open bars) and again after two weeks storage at 60 °C (solid bars). The results given represent the average of two different ghee samples. To the left of the vertical line are compounds associated with fat degradation, and to the right are MRP.
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The other qualitative difference seen in all three types of ghee over storage was a large increase in the peak areas of compounds typically associated with fat degradation (compounds on the left side of Fig. 3). Hexanal, heptanal, and nonenal are all associated with (milk) fat oxidation (Kochhar, 1996). In all three types of ghee the concentrations of these compounds greatly increased over the storage period. The concentration of hexanal increased more in the PS ghee than in the CB and DC ghee, but the concentration of heptanal and nonenal increased more in the CB and DC ghee. Thus, fat oxidation appears to have different consequences in the different types of the ghee. However, there is no clear evidence, from these data, that oxidation was occurring significantly faster, or slower, in any of the types of ghee. It has been reported, based on sensory evaluations, that DC and CB ghee have better keeping quality than PS ghee (Ganguli & Jain, 1973; Singh & Ram, 1978; Sserunjogi et al., 1998). However, this could be due to caramel notes in DC and CB ghee masking oxidised flavours, rather than slower fat oxidation rates per se. Butyric acid is also due to fat degradation but it could be from either hydrolysis (Deeth & Fitz-Gerald, 1994) or oxidation (Kochhar, 1996). In all three types of ghee the butyric acid increased during storage (Fig. 3), slightly more so in the CB and DC ghee. Overall (considering hexanal, heptanal, nonenal and butyric acid), there does not appear to be much difference in total fat degradation in the types of ghee. But the specifics of fat degradation are different. It could be assumed, based on fat degradation, all three types of ghee will have a similar (sensory-based) shelf-life, but a sensory investigation of ghee shelf-life versus fat degradation would be confounded by the (caramel) flavours of the MRP. Linking the chemical data to sensory properties was not within the scope of this paper and is not considered further.
Table 1 Concentrations of maltol (ppm) in two samples (A and B) of both CB and DC ghee, as determined by colourimetry, during storage at 60 °C.
3.3. Time-course of MRP in ghee
Fig. 4. Time-course showing the loss of pyranone (j) and DHHD (N) in DC ghee during storage at 60 °C (represented as a peak area on right axis). As these compounds were lost, acetic acid () formed (concentration given in ppm on left axis). The results given represent the average of two different ghee samples.
Samples of ghee were periodically analysed using GCMS during storage (at least 2 weeks), and it was found that many MRP, such as maltol, change little in concentration during storage of the CB and DC ghee. Maltol is one of the most abundant MRP found in ghee (Wadodkar et al., 2002) and is readily quantified using colourimetry (Patton, 1950). Therefore, to confirm the GCMS results, analysis of maltol by colourimetry was done. The results are shown in Table 1. DC ghee contained an average of 210 ppm maltol, while the CB ghee contained 40 ppm maltol. In both types of ghee the maltol concentration changed little during storage. In contrast to the high stability of maltol, both DHHD and pyranone rapidly diminished in both the CB and DC ghee. A timecourse, showing the loss of DHHD and pyranone from DC ghee is shown in Fig. 4. Similar behaviour was seen in CB ghee (not shown) but further attention was placed only on the DC ghee due to it containing much greater amounts of MRP that facilitated analysis. The loss of DHHD in ghee during storage may have been due to tautomerisation to 2,4-dihydroxy-2,5-dimethyl-3(2H)-furanone (Fig. 1) in the presence of water (Engel et al., 2001). However, the furanone was not detected. In the DC ghee, in parallel to DHHD and pyranone decreasing in concentration, acetic acid increased in concentration from 45 ppm to 75 ppm. The acetic acid was not coming from milkfat degradation because acetic acid did not form in the PS ghee (Fig. 3). Acetic acid stopped increasing in concentration after DHHD and pyranone were no longer detectable (Fig. 4). This suggested that the acetic acid could be derived from pyranone and DHHD. A standard for pyranone was not available and the DHHD standard was not 100% pure, so accurate quantification of these compounds was not possible. However, by assuming the DHHD standard to be pure, and assuming that the pyranone had the same TIC response factor as maltol, it was estimated that these compounds were present in concentrations between 10 and
Storage time (days)
0 3 6 9 12 14
CB ghee
DC ghee
Sample A
Sample B
Sample A
Sample B
44 53 47 44 44 48
28 36 37 30 34 41
200 220 190 210 200 200
230 240 240 230 220 230
100 ppm in DC ghee. Pyranone typically occurs in ppm concentrations in heated foods (Kim & Baltes, 1996). Therefore, the amount of pyranone and DHHD in DC ghee does appear sufficient to account for the amount of acetic acid formed (30 ppm). The DHHD standard contained large amounts of acetic acid as a significant impurity (determined by H and 13C NMR, and GCMS – not shown). When this DHHD standard was placed in milkfat and stored, it degraded and additional acetic acid was formed (not shown). It has been proposed that water addition to pyranone (Fig. 1) regenerates the highly reactive 1-deoxyglucosone (Kim & Baltes, 1996; Voigt et al., 2010). The reactive 1-deoxyglucosone, depending on the conditions, can then form numerous compounds including acetic acid (Voigt & Glomb, 2009; Voigt et al., 2010). It is likely that water addition to DHHD can also regenerate 1-deoxyglucosone (and again, this could then proceed to acetic acid). However, the DHHD probably first rapidly tautomerises, losing its caramel odour, as shown by Engel et al. (2001). It is hypothesised that in DC and CB ghee during storage, pyranone and DHHD form acetic acid via 1-deoxyglucosone (Fig. 1). If this hypothesis is true, it should be possible to isolate 1-dexoxygluosone (e.g., as a quinoxaline-derivative) from ghee, during storage, and it should also be possible to detect other products associated with 1-deoxyglucosone fragmentation (e.g., glyceric acid and threosones). Various proportions of glucose degradation products could form depending on ghee storage conditions (Novotny, Cejpek, & Velisek, 2008). 3.4. Time-course of hexanal in different types of ghee during storage Pyranone and DHHD can possibly act as antioxidants. Various antioxidant mechanisms are available for both of these com-
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Fig. 5. Time course showing the changes in hexanal concentration in PS (), CB (j) and DC (N) ghee during storage at 60 °C. The results given represent the average of two different ghee samples.
pounds. For example, pyranone could abstract hydroxyl radicals (Osada & Shibamoto, 2006), and DHHD has a reductone structure analogous to that of vitamin C (Belitz et al., 2004). This suggested a hypothesis that decreases in DHHD and pyranone in CB and DC ghee during storage are related to these compounds acting as antioxidants. To test this hypothesis the concentration of hexanal was measured in all three types of ghee (PS, CB and DC) during storage (Fig. 5). Hexanal was followed because it is a popular indicator for fat oxidation (Kochhar, 1996). In all three types of ghee, the change in hexanal concentration, during storage was slow to begin with, then greatly accelerated after nine days (Fig. 5). Such an increase in oxidation rate after the DHHD and pyranone were depleted might support our hypothesis. But this behaviour was seen in the PS ghee (no MRP present) and such non-linear kinetics is routinely observed when fats oxidise (Kochhar, 1996). Based upon hexanal measurement alone, the PS ghee appeared to oxidise at the fastest rate; both CB and DC ghee oxidised at a slower rate than PS ghee. Based only on hexanal measurement, the CB ghee appeared to be the most stable ghee with respect to oxidation. Therefore, these data did not support the hypothesis; i.e., if pyranone and DHHD were acting as antioxidants the CB ghee should have behaved more like the PS ghee since the CB ghee contained substantially less MRP than the DC ghee. Based on hexanal, the CB and DC ghee appear more stable than the PS ghee. However the stability did not appear correlated to the amount of MRP in these samples. It is likely that the apparently higher (based on hexanal) stability of CB and DC ghee is due to some other factor like phospholipid content, as suggested by others (Singh & Ram, 1978). However, if other compounds are used as a measure of fat oxidation (e.g., nonenal, Fig. 3) the trend is reversed. So, overall, there is no compelling chemical evidence that any of these traditional methods of fat isolation yield a more stable fat. From a sensory perspective DC ghee appears to have slightly better keeping quality (Ganguli & Jain, 1973), but that could just as easily be from cooked flavours of MRP masking other flavours.
4. Conclusions Changes in the concentration of volatile compounds in PS, CB and DC ghee were monitored over storage. The most significant observations were increases in compounds from fat degradation, little change in the majority of MRP, and complete loss of the related compounds, pyranone and DHHD. While it is likely that both pyranone (Osada & Shibamoto, 2006) and DHHD have antioxidant properties, no evidence of these
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compounds being responsible for any large improvement in the stability of milkfat in ghee could be found. The loss of pyranone and DHHD correlated to an increase in acetic acid. It is suggested that in ghee DHHD and pyranone are degrading to acetic acid. This is based on an observation that a DHHD standard degrades to acetic acid, and literature reports on the reactions of pyranone (Kim & Baltes, 1996). An alternative explanation is that DHHD and pyranone are forming compounds not detected, while another unknown (also undetected) is degrading to acetic acid at a similar rate. Thus there is a need to do further experiments to link loss of DHHD and pyranone to acetic acid formation. Furthermore, based on these observations, we propose that DHHD and pyranone are degrading via 1-deoxyglucosone. If this pathway is proven, finding it in ghee is notable because ghee contains only traces of proteins or sugars. The implication is that the Maillard reaction cascade could have an impact on foods well after cooking has ceased. Obviously, it is well known that Maillard reactions occur in food during storage (Baltes, 1982). However, it is remarkable that the Maillard reaction may continue to play a role in food that contains minimal protein or sugar. It was not possible to determine what impact the loss during storage of DHHD, and pyranone, or acetic acid formation, has on the shelf-life or flavour of ghee. It is possible that it will affect flavour (e.g., loss of some DHHD’s caramel notes as proposed by Engel et al., 2001). This might be most notable with moderate storage (allowing changes in MRP, without fat becoming excessively oxidised). Further work will be needed to determine if the chemistry observed in these accelerated storage trials occurs at lower temperatures and if it is related to known changes in ghee flavour that occur during storage (Singh & Ram, 1978). Acknowledgements Jeremy Jones and Graham Caygill (of GlycoSyn, Industrial Research Limited) are thanked for the synthesis and characterisation of DHHD. Tim Coolbear and Euan Cant are thanked for reviewing the manuscript. References Baltes, W. (1982). Chemical changes in food by the Maillard reaction. Food Chemistry, 9, 59–73. Belitz, H. D., Grosch, W., & Schieberle, P. (2004). Food chemistry (3rd ed.). Berlin: Springer-Verlag [Chapter 4]. Davidek, T., Robert, F., Devaud, S., Vera, F. A., & Blank, I. (2006). Sugar fragmentation in the Maillard reaction cascade: Formation of short-chain carboxylic acids by a new oxidative a-dicarbonyl cleavage pathway. Journal of Agricultural and Food Chemistry, 54, 6677–6684. Deeth, H. C., & Fitz-Gerald, C. H. (1994). Lipolytic enzymes and hydrolytic rancidity in milk and milk products. In P. F. Fox (Ed.), Advanced dairy chemistry. Lipids (Vol. 2, pp. 247–308). London: Chapman & Hall. Engel, W., Hofmann, T., & Schieberle, P. (2001). Characterization of 3,4-dihydroxy-3hexen-2,5-dione as the first open-chain caramel-like smelling flavour compound. European Food Research and Technology, 213, 104–106. Ganguli, N. C., & Jain, M. K. (1973). Ghee: Its chemistry processing and technology. Journal of Dairy Science, 56, 19–25. Goto, R., Miyagi, Y., & Inokawa, H. (1963). Syntheses and structures of acetylformoin and its related compounds. I. Bulletin of the Chemical Society of Japan, 36, 147–151. Hodge, J. E., Mills, F. D., & Fisher, B. E. (1972). Compounds of browned flavour derived from sugar-amine reactions. Cereal Science Today, 17, 34–40. Hofmann, T. (1998). Acetylformoin – a chemical switch in the formation of coloured Maillard reaction products from hexoses and primary and secondary amino acids. Journal of Agricultural and Food Chemistry, 46, 3918–3928. Illingworth, D., Janssen, P. W. M., Cant, P. A. E., & Stephens, G. R. (2009). Dairy Product and Process. Patent WO2009/011598. Kim, M., & Baltes, W. (1996). On the role of 2,3-dihydro-3,5-dihydroy-6-methyl4(H)-pyran-4-one in the Maillard reaction. Journal of Agricultural and Food Chemistry, 44, 282–289. Kochhar, S. P. (1996). Oxidative pathways to the formation of off-flavours. In M. J. Saxby (Ed.), Food taints and off-flavours (pp. 168–218). Glasgow: Blackie Academic and Professional.
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