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Enzymatic oxidation of hexanal by oat
Journal of Cereal Science 38 (2003) 199–203 www.elsevier.com/locate/jnlabr/yjcrs
Enzymatic oxidation of hexanal by oat S. Lehto, S. Laakso*, P. Lehtinen Helsinki University of Technology, Laboratory of Biochemistry and Microbiology, P.O. Box 6100, Helsinki 02015 HUT, Finland Received 28 October 2002; revised 14 February 2003; accepted 24 February 2003
Abstract The fate of hexanal added to flours of five different grain varieties was studied. All the flours appeared to be capable in decreasing hexanal concentration, but the abilities of oat flours were clearly the best. Depending on the oat flour used, the rate of hexanal decrease was 2.45 –2.73 mg hexanal min21 g21 flour when 100 mg of hexanal was added to 1 g flour. This ability was highest amongst oat flour fractions rich in protein and fiber, which originated from the aleurone layers. The time course of hexanal decrease followed typical first order kinetics and the reaction was susceptible to heat inactivation. Hexanoic acid was identified as the predominant product correlating with hexanal decrease. These observations suggested the presence of aldehyde dehydrogenase type activity in oat. Hexanal was also absorbed to oat matrix to some extent, but it became saturated relatively quickly. It was concluded that the aldehyde dehydrogenase type activity present in oat efficiently prevents the accumulation of short chain aldehydes that lead to off odours in oat products. q 2003 Elsevier Ltd. All rights reserved. Keywords: Oat; Hexanal; Oxidation; Hexanoic acid
1. Introduction Oxidation of unsaturated food lipids initiates a complex series of free radical reactions yielding a family of secondary lipid oxidation products (Frankel, 1998). In multi-component foods, the interaction of secondary oxidation products with proteins and other components has a significant impact on oxidative and flavor stability and texture during food processing and storage. Flavor release depends on the composition of food matrix and interactions of different food components during processing and storage (Lubbers et al., 1998). Several different binding mechanisms for the secondary oxidation products have been identified including covalent bonds, hydrogen bonds, hydrophobic bonds, and formation of inclusion complexes (Le Thanh et al., 1992). The composition of secondary lipid oxidation products varies but almost invariably includes the short chain aldehyde, hexanal, derived from the carbon skeleton of unsaturated fatty acids. Hexanal has a very low aroma threshold and has therefore, been used as an indicator of lipid rancidity of foods (Heinio¨ et al., 2002; Sjo¨vall et al., * Corresponding author. Tel.: þ 358-9-462-373. E-mail address: [email protected] (S. Laakso). 0733-5210/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0733-5210(03)00028-6
2000). Several studies have been done to prevent the release of hexanal and other short chain aldehydes. Soy protein has been examined for its ability to bind several volatiles, especially aldehydes (Aspelund and Wilson, 1983; O’Keefe et al., 1991; Chung and Villota, 1989). Maier and Hartmann (1977) studied the absorption of volatile carbonyl compounds by 22 different amino acids and found great differences between them. Lysine absorbed best, and often the reaction was irreversible. Other good adsorbers were arginine, histidine, phenylalanine, tryptophan, proline, and valine. However, less information exists about enzymatic conversion of short chain aldehydes to less volatile compounds. Oxidation of hexanal to hexanoic acid has been reported by liver of mammals (Poole and Halestrap, 1989; Eckey et al., 1988), and reduction of hexanal to hexanol has been carried out by using alcohol dehydrogenase from Saccharomyces cerevisiae (Willoughby et al., 1999). Lipid oxidation and development of rancidity are phenomena typically associated with cereal processing. Therefore, a study was undertaken to characterise the interaction of hexanal with different cereal flours. From the cereals studied, oat appeared to have, in addition to the capacity of absorbing hexanal, a significant activity oxidising hexanal to hexanoic acid.
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2. Experimental 2.1. Treatment of the grains The oat varieties Veli and hulless Lisbeth, rye variety Akusti, barley variety Saana, and wheat variety Mahti were all 2000 harvest and were purchaced from Finland States Grain Storage. The grains had been dried in farm driers to moisture content of 11%. The grains were ground and sieved to particle size smaller than 0.5 mm using a laboratory grinder (Fritsch Pulverisette, Germany). The flours were used within a day. Oat was fractionated into fiber, starch and protein fractions according to a wet fractionation method described by Liukkonen et al. (1992). Oat flour made as described above was mixed in water (15 8C) to give a 25% (w/w) slurry. The slurry was mixed for 15 min and homogenized with Ultra Turrax for 1 min. The fiber was separated from the starch –protein mixture in a continuous centrifuge (AEG ESF 102) and washed with water. The starch – protein mixture was sieved (mesh size 88 mm) to remove residual fiber and protein separated from starch by centrifuging at 16,000 g for 20 min. Heat-treated and non-heated oat fiber and oat flour were obtained from whole meal flour by milling and air classification (Avena Oy, Finland). The heat treatment included steaming for 20 min at 100 8C to final moisture content of 17%. The samples were placed in headspace vials (0.5 g sample per vial) and the vials were stored for 2 weeks at 35 8C and exposed to light. They were then analysed by a GC/MS method described below. 2.2. Preparation of hexanal – flour mixtures Flours or flour fractions were mixed in water at a ratio of 12:1 into air-tight headspace vials. Depending on the experiment, the aqueous suspensions were subjected to different treatments prior to addition of hexanal from a stock solution. Heat treatments were done by incubating the vials containing either the aqueous suspensions or dry flours in water baths of different temperatures for 30 min. Appropriate amounts of hexanal –water stock solution were added into the vials so that, depending on the experiment, the total concentration of hexanal in the flour became 100 or 300 mg hexanal g21 flour. If nothing else is mentioned, the used quantities in results section always refer to mg of hexanal per g of flour. The vials were incubated at room temperature for 0.5– 4 h. Aqueous hexanal stock solutions were prepared by homogenisation using Ultra Turrax (T 25, Janke & Kunkel GmbH & co&kg, Germany). Hexanal (0.1 ml) was homogenised with 99.9 ml of distilled water to give a solution of 1000 mg g21. Other stock solutions required were prepared from this specific solution. Hexanal stock solution was also used as an external standard together with 1-hexanol of which the stock solutions were prepared the same way as
hexanal solutions. Hexanal standard (purity 100%) was received from Sigma Chemical Co, Switzerland and 1-hexanol standard (purity $ 99%) was received from Sigma-Aldrich Chemie GmbH, Switzerland. 2.3. Analyses Hexanal and 1-hexanol were analysed straight in the incubation vials with static Headspace Sampler (HP 7694) connected to mass spectrometer (HP 5971A)—gas chromatograph (HP 5890 Series II). The column used was HP5MS, a crosslinked 5% PHME Siloxane 19091S-133 (Hewlett Packard, USA). The sample was injected at a column temperature of 40 8C. The temperature program maintained this temperature for 4 min followed by a rise of 20 8C min21 up to 200 8C where it was held for 20 min. External hexanal and 1-hexanol standards were used as appropriate stock solutions and analysed in the headspace vials. The amounts of other short chain aldehydes originating from stored flour were determined by the similar GC/MS technique (Heinio¨ et al., 2002). To compensate for the variation in the performance of the GC/MS technique during the 2 week storage, all detector responses of duplicate samples were standardised to the response of an external isobutanol standard (purity 100%, received from J.T. Baker Chemical Co., Phillipsburg). The amounts of hexanal and 1-hexanol are reported as quantitative units and the other volatiles are reported as arbitrary detector responses (Heinio¨ et al., 2002). All the results are the means of two measurements (standard deviation , 6%). Hexanoic acid (caproic acid) was methylated, identified and quantified by gas chromatography with a flame ionisation detector (Suutari et al., 1990). Identification was done by using a commercial hexanoic acid standard, and an internal capric acid (C10:0) standard was used to ensure proper quantification. Caproic acid (purity 99– 100%) and capric acid (purity 99– 100%) standards were received from Sigma Chemical Co, USA. The results are the means of two measurements (standard deviation , 8%).
3. Results 3.1. Decrease in hexanal by different cereal flours All the hexanal supplemented water– flour mixtures decreased the amount of headspace hexanal. Fig. 1 shows, however, that flours from both hulled and the hulless oat varieties were the most efficient. With 100 mg g21 of hexanal, the reaction rates of reduction were 2.73 (Veli) and 2.45 mg g21 min21 (Lisbeth). The efficiency of rye flour was also quite noticeable (2.13 mg g21 min21), but the reaction rates by barley and wheat flours were only 0.83 and 1.24 mg g21 min21, respectively. Compared with the odour threshold of 0.005 mg g21 of hexanal in water (Buttery et al., 1999), the reaction rates were high. It can be assumed that
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Table 1 Development of volatile rancidity in headspace of heat treated and nonheated oat products, expressed in relative units. The samples were stored for 2 weeks at 35 8C and were exposed to light Aldehyde
Relative headspace response Non-heated Heat-treated Non-heated Heat-treated fiber fiber flour flour
Fig. 1. The rates of hexanal decrease by different water– flour mixtures. The initial amount of hexanal added was 100 mg g21. Headspace stabilisation time was 25 min at 60 8C.
especially oat flour can very rapidly remove hexanal amounts, which lie far above this odour threshold. The fact that oat flour was able to eliminate all of the added 100 mg g21 of hexanal from the headspace in 1.5 h suggested that the flours had either exceptional capacity for binding hexanal or a capacity to convert hexanal to other compounds. Evidence supporting the latter possibility came from the observation that no headspace hexanal emerged back into the mixture even though the incubation was continued for additional 12 h after complete removal of hexanal. It was, therefore, concluded that at room temperature, the removal of hexanal from the oat water – flour mixture was a relatively fast and irreversible process. 3.2. Effect of heat treatment Some of the oat water –flour mixtures were heat treated for 30 min at temperatures between 60 and 100 8C prior to addition of hexanal (300 mg g21). As shown in Fig. 2, the ability to reduce hexanal was conserved when the mixture was heated for 30 min at temperatures between 60 and 70 8C, and it diminished slightly between 70 and 80 8C after which it collapsed rapidly. Yet, some hexanal
Fig. 2. The effect of heating of water–flour mixtures before hexanal addition on the rate of hexanal decrease. Heating time was 30 min, hexanal addition was 300 mg g21 and incubation time was 1 h at room temperature. Headspace stabilisation time was 5 min at 60 8C.
Isobutyraldehyde Butyraldehyde Isovaleraldehyde Pentanal Hexanal Heptanal Nonanal
0.50 0.29 0.39 0.14 0.13 0.14 0.14
0.82 0.57 0.76 0.33 0.36 0.63 0.66
1.00a 0.73 0.73 0.78 0.52 0.66 0.64
0.98 1.00 1.00 1.00 1.00 1.00 1.00
a Values presented as arbitrary detector response with the highest value taken as 1.00.
was eliminated even after heating of water –flour mixture at 100 8C for 30 min, suggesting that two separate phenomena may be responsible for the decrease of hexanal. The heat-labile activity also probably plays a role in the decrease of endogenously formed hexanal and other short chain aldehydes. Those aldehydes found to be formed abundantly upon prolonged storage of heat treated oat products were present at much lower levels in the corresponding non-heated products after the same storage period under similar conditions (Table 1). 3.3. Decrease in hexanal by flour fractions In order to identify the kernel material primarily responsible for the elimination of hexanal, the whole oat flour was fractionated into fiber, starch and protein fractions. The protein fraction was clearly the most efficient in decreasing hexanal (Fig. 3). This activity was also present in fiber fraction whereas starch fraction practically lacked this ability. The fiber fraction also contained protein (Liukkonen et al., 1992) suggesting that the capability to eliminate hexanal by oat flour was associated with protein rich kernel material probably originating from kernel surface.
Fig. 3. The rates of decrease of hexanal by starch, fiber and protein fractions. The hexanal concentration was 300 mg g21. Headspace stabilisation time was 5 min at 60 8C.
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Fig. 4. The decrease of hexanal and the formation of hexanoic acid at room temperature. Hexanal concentration was 300 mg g21. Headspace stabilization time was 5 min at 60 8C.
3.4. Formation of hexanoic acid and 1-hexanol The facts that the capability of the oat flour to decrease hexanal was higher than anticipated by simple absorption, this phenomenon was temperature sensitive and concentrated onto a protein rich fraction, led to a search for a possible conversion product of hexanal. As shown in Fig. 4, the decrease in hexanal content coincided with accumulation of hexanoic acid. The amount of 1-hexanol produced in 3 h was only 1.2 mg g21 which was relatively small compared with the amount of hexanoic acid formed. There was only 6 mg g21 of headspace hexanal remaining after 3 h and simultaneously 240 mg g21 of hexanoic acid (Fig. 4) and 1 mg g21 of 1-hexanol had been formed. The fate of the rest of the hexanal removed, which was approximately 20% of that originally added, remains unknown. It may have been either adsorbed or otherwise reacted with flour material to make the headspace hexanal undetectable. Approximately 80% of added hexanal was transformed to hexanoic acid under the experimental conditions.
interconnected (Lehtinen et al., 2003; Ekstrand et al., 1993). Several explanations may exist. Unsaturated fatty acids may be oxidized and degraded into volatile products without a prior hydrolysis and cause the rancidity occurring especially during storage of oat products. Alternatively, the small amounts of free fatty acids found already in intact noninactivated kernels are basically sufficient to provide volatile oxidation products in excess of their sensory threshold. In either case, the excessive formation of volatile compounds, such as hexanal and pentanal, is linked to the perception of rancid sensory attributes in oat material (Heinio¨ et al., 2002). Consequently, the inactivation of lipase does not grant long shelf life of oat products, as it does not prevent the oxidative rancidity. The present paper brings an additional aspect regarding the relationship between lipid hydrolysis and development of rancidity. It is demonstrated that short chain aldehydes do not accumulate in non-inactivated oat material but are efficiently converted to less volatile products. This means that against the sensory symptoms of rancidity, oat has an intrinsic mechanism of resistance based on aldehyde dehydrogenase type activity. At present, it is customary to use heat for the inactivation of lipid hydrolysing activities in oat to improve storage stability. This requires temperatures that exceed the temperature tolerance of the aldehyde dehydrogenase type activity. This may explain, at least in part, the earlier observation that in lipase inactivated oat, the accumulation of hexanal exceeds significantly that of lipase active oat (Lehtinen et al., 2003). Therefore, heat inactivation of grains or grain products may not represent the best routines to achieve optimum keeping quality. This applies especially to oat where the amount of both free and acylated unsaturated fatty acids is invariably higher than that needed to cause rancid perception by their secondary oxidation products.
References 4. Discussion An increment in the content of free fatty acids amongst the lipids of processed oat has generally been considered as an early indicator of a forthcoming oxidative rancidity. Therefore, much attention is being paid to prevent lipid hydrolysis during processing and storage of oat containing products. In light of recent studies, the view of lipid hydrolysis as an initiator of rancidity process is probably too straightforward. In processed oat, volatile lipid oxidation products such as hexanal have shown to be formed abundantly without observable hydrolysis of acyl lipids (Kiilia¨inen et al., 2001). On the other hand, slight variations in process conditions can give fractions where lipid hydrolysis progresses to a great extent but the formation of volatile oxidation products is minimal. It seems as if hydrolysis and oxidation of oat lipids were only loosely or not at all
Aspelund, T.G., Wilson, L.A., 1983. Adsorption of off-flavor compounds onto soy protein: a thermodynamic study. J. Agric. Food Chem. 31, 539 –545. Buttery, R.G., Orts, W.J., Takeoka, G.R., Nam, Y., 1999. Volatile flavor components of rice cakes. J. Agric. Food Chem. 47, 4353–4356. Chung, S., Villota, R., 1989. Binding of alcohols by soy protein in aqueous solutions. J. Food Sci. 54, 1604–1606. Eckey, R., Agarwal, D.P., Volkens, T., Goedde, H.W., 1988. A simple and rapid method for the puridication of aldehyde dehydrogenase isozymes form human liver. Adv. Biosci. 71, 171–175. ˚ kesson, G., Sto¨llman, U., Lingnert, H., Dahl, S., Ekstrand, B., Gangby, I., A 1993. Lipase activity and development of rancidity in oats and oat products related to heat treatment during processing. J. Cereal Sci. 17, 247 –254. Frankel, E.N., 1998. Lipid Oxidation, The Oily Press, Dundee, UK, pp. 13–41, 55–77. Heinio¨, R.-L., Lehtinen, P., Oksman-Caldentey, K.-M., Poutanen, K., 2002. Difference between sensory profiles and development of rancidity during long-term storage of native and processed oat. Cereal Chem. 79, 367 –385.
S. Lehto et al. / Journal of Cereal Science 38 (2003) 199–203 Kiilia¨inen, K., Virtanen, E., Lehtinen, P., Laakso, S., 2001. Lipid deterioration in lipase-inactivated. Third European Conference on Functional Properties in OATS Uppsala, Sweden, May 17–18. Lehtinen, P., Kiilia¨inen, K., Lehtomaki, I., Laakso, S., 2003. Effect of heat treatment on lipid stability in processed oat. J. Cereal Sci. 37, 215–221. Le Thanh, M., Thibeaudeau, P., Thibaut, M.A., Voilley, A., 1992. Interactions between volatile and non-volatile compounds in the presence of water. Food Chem. 43, 129– 135. Liukkonen, K.H., Montfoort, A., Laakso, S., 1992. Water-induced lipid changes in oat processing. J. Argic. Food Chem. 40, 126–130. Lubbers, S., Landy, P., Voilley, A., 1998. Retention and release of aroma compounds in foods containing proteins. Food Technol. 52, 68see also pages 70, 72, 74, 208, 210, 212, 214. Maier, H.G., Hartmann, R.U., 1977. The adsorption of volatile aroma constituents by foods. VIII. Adsorption of volatile carbonyl compounds by amino acids. Z. Lebensm.-Unters. Forsch 163, 251– 254.
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O’Keefe, S.F., Resurreccion, A.P., Wilson, L.A., Murphy, P.A., 1991. Temperature effect on binding of volatile compounds to soy protein in aqueous model systems. J. Food Sci. 56, 802–806. Poole, R.C., Halestrap, A.P., 1989. Purification of aldehyde dehydrogenase from rat liver mitochondria by a-cyanocinnamate affinity chromatography. Biochem. J. 259, 105 –110. Sjo¨vall, O., Virtalaine, T., Lapvetela¨inen, A., Kallio, H., 2000. Development of rancidity in wheat germ analyzed by headspace gas chromatography and sensory analysis. J. Agric. Food Chem. 48, 3522–3527. Suutari, M., Liukkonen, K., Laakso, S., 1990. Temperature adaptation in yeast. The role of fatty acids. J. Gen. Microbiol. 136, 1469–1474. Willoughby, N.A., Kirschner, T., Smith, M.P., Hjorth, R., TitchenerHooker, N.J., 1999. Immobilized metal ion affinity chromatography purification of alcohol dehydrogenase from baker’s yeast using an expanded bed adsorption system. J. Chromatogr., A 840, 195 –204.
Enzymatic oxidation of hexanal by oat S. Lehto, S. Laakso*, P. Lehtinen Helsinki University of Technology, Laboratory of Biochemistry and Microbiology, P.O. Box 6100, Helsinki 02015 HUT, Finland Received 28 October 2002; revised 14 February 2003; accepted 24 February 2003
Abstract The fate of hexanal added to flours of five different grain varieties was studied. All the flours appeared to be capable in decreasing hexanal concentration, but the abilities of oat flours were clearly the best. Depending on the oat flour used, the rate of hexanal decrease was 2.45 –2.73 mg hexanal min21 g21 flour when 100 mg of hexanal was added to 1 g flour. This ability was highest amongst oat flour fractions rich in protein and fiber, which originated from the aleurone layers. The time course of hexanal decrease followed typical first order kinetics and the reaction was susceptible to heat inactivation. Hexanoic acid was identified as the predominant product correlating with hexanal decrease. These observations suggested the presence of aldehyde dehydrogenase type activity in oat. Hexanal was also absorbed to oat matrix to some extent, but it became saturated relatively quickly. It was concluded that the aldehyde dehydrogenase type activity present in oat efficiently prevents the accumulation of short chain aldehydes that lead to off odours in oat products. q 2003 Elsevier Ltd. All rights reserved. Keywords: Oat; Hexanal; Oxidation; Hexanoic acid
1. Introduction Oxidation of unsaturated food lipids initiates a complex series of free radical reactions yielding a family of secondary lipid oxidation products (Frankel, 1998). In multi-component foods, the interaction of secondary oxidation products with proteins and other components has a significant impact on oxidative and flavor stability and texture during food processing and storage. Flavor release depends on the composition of food matrix and interactions of different food components during processing and storage (Lubbers et al., 1998). Several different binding mechanisms for the secondary oxidation products have been identified including covalent bonds, hydrogen bonds, hydrophobic bonds, and formation of inclusion complexes (Le Thanh et al., 1992). The composition of secondary lipid oxidation products varies but almost invariably includes the short chain aldehyde, hexanal, derived from the carbon skeleton of unsaturated fatty acids. Hexanal has a very low aroma threshold and has therefore, been used as an indicator of lipid rancidity of foods (Heinio¨ et al., 2002; Sjo¨vall et al., * Corresponding author. Tel.: þ 358-9-462-373. E-mail address: [email protected] (S. Laakso). 0733-5210/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0733-5210(03)00028-6
2000). Several studies have been done to prevent the release of hexanal and other short chain aldehydes. Soy protein has been examined for its ability to bind several volatiles, especially aldehydes (Aspelund and Wilson, 1983; O’Keefe et al., 1991; Chung and Villota, 1989). Maier and Hartmann (1977) studied the absorption of volatile carbonyl compounds by 22 different amino acids and found great differences between them. Lysine absorbed best, and often the reaction was irreversible. Other good adsorbers were arginine, histidine, phenylalanine, tryptophan, proline, and valine. However, less information exists about enzymatic conversion of short chain aldehydes to less volatile compounds. Oxidation of hexanal to hexanoic acid has been reported by liver of mammals (Poole and Halestrap, 1989; Eckey et al., 1988), and reduction of hexanal to hexanol has been carried out by using alcohol dehydrogenase from Saccharomyces cerevisiae (Willoughby et al., 1999). Lipid oxidation and development of rancidity are phenomena typically associated with cereal processing. Therefore, a study was undertaken to characterise the interaction of hexanal with different cereal flours. From the cereals studied, oat appeared to have, in addition to the capacity of absorbing hexanal, a significant activity oxidising hexanal to hexanoic acid.
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2. Experimental 2.1. Treatment of the grains The oat varieties Veli and hulless Lisbeth, rye variety Akusti, barley variety Saana, and wheat variety Mahti were all 2000 harvest and were purchaced from Finland States Grain Storage. The grains had been dried in farm driers to moisture content of 11%. The grains were ground and sieved to particle size smaller than 0.5 mm using a laboratory grinder (Fritsch Pulverisette, Germany). The flours were used within a day. Oat was fractionated into fiber, starch and protein fractions according to a wet fractionation method described by Liukkonen et al. (1992). Oat flour made as described above was mixed in water (15 8C) to give a 25% (w/w) slurry. The slurry was mixed for 15 min and homogenized with Ultra Turrax for 1 min. The fiber was separated from the starch –protein mixture in a continuous centrifuge (AEG ESF 102) and washed with water. The starch – protein mixture was sieved (mesh size 88 mm) to remove residual fiber and protein separated from starch by centrifuging at 16,000 g for 20 min. Heat-treated and non-heated oat fiber and oat flour were obtained from whole meal flour by milling and air classification (Avena Oy, Finland). The heat treatment included steaming for 20 min at 100 8C to final moisture content of 17%. The samples were placed in headspace vials (0.5 g sample per vial) and the vials were stored for 2 weeks at 35 8C and exposed to light. They were then analysed by a GC/MS method described below. 2.2. Preparation of hexanal – flour mixtures Flours or flour fractions were mixed in water at a ratio of 12:1 into air-tight headspace vials. Depending on the experiment, the aqueous suspensions were subjected to different treatments prior to addition of hexanal from a stock solution. Heat treatments were done by incubating the vials containing either the aqueous suspensions or dry flours in water baths of different temperatures for 30 min. Appropriate amounts of hexanal –water stock solution were added into the vials so that, depending on the experiment, the total concentration of hexanal in the flour became 100 or 300 mg hexanal g21 flour. If nothing else is mentioned, the used quantities in results section always refer to mg of hexanal per g of flour. The vials were incubated at room temperature for 0.5– 4 h. Aqueous hexanal stock solutions were prepared by homogenisation using Ultra Turrax (T 25, Janke & Kunkel GmbH & co&kg, Germany). Hexanal (0.1 ml) was homogenised with 99.9 ml of distilled water to give a solution of 1000 mg g21. Other stock solutions required were prepared from this specific solution. Hexanal stock solution was also used as an external standard together with 1-hexanol of which the stock solutions were prepared the same way as
hexanal solutions. Hexanal standard (purity 100%) was received from Sigma Chemical Co, Switzerland and 1-hexanol standard (purity $ 99%) was received from Sigma-Aldrich Chemie GmbH, Switzerland. 2.3. Analyses Hexanal and 1-hexanol were analysed straight in the incubation vials with static Headspace Sampler (HP 7694) connected to mass spectrometer (HP 5971A)—gas chromatograph (HP 5890 Series II). The column used was HP5MS, a crosslinked 5% PHME Siloxane 19091S-133 (Hewlett Packard, USA). The sample was injected at a column temperature of 40 8C. The temperature program maintained this temperature for 4 min followed by a rise of 20 8C min21 up to 200 8C where it was held for 20 min. External hexanal and 1-hexanol standards were used as appropriate stock solutions and analysed in the headspace vials. The amounts of other short chain aldehydes originating from stored flour were determined by the similar GC/MS technique (Heinio¨ et al., 2002). To compensate for the variation in the performance of the GC/MS technique during the 2 week storage, all detector responses of duplicate samples were standardised to the response of an external isobutanol standard (purity 100%, received from J.T. Baker Chemical Co., Phillipsburg). The amounts of hexanal and 1-hexanol are reported as quantitative units and the other volatiles are reported as arbitrary detector responses (Heinio¨ et al., 2002). All the results are the means of two measurements (standard deviation , 6%). Hexanoic acid (caproic acid) was methylated, identified and quantified by gas chromatography with a flame ionisation detector (Suutari et al., 1990). Identification was done by using a commercial hexanoic acid standard, and an internal capric acid (C10:0) standard was used to ensure proper quantification. Caproic acid (purity 99– 100%) and capric acid (purity 99– 100%) standards were received from Sigma Chemical Co, USA. The results are the means of two measurements (standard deviation , 8%).
3. Results 3.1. Decrease in hexanal by different cereal flours All the hexanal supplemented water– flour mixtures decreased the amount of headspace hexanal. Fig. 1 shows, however, that flours from both hulled and the hulless oat varieties were the most efficient. With 100 mg g21 of hexanal, the reaction rates of reduction were 2.73 (Veli) and 2.45 mg g21 min21 (Lisbeth). The efficiency of rye flour was also quite noticeable (2.13 mg g21 min21), but the reaction rates by barley and wheat flours were only 0.83 and 1.24 mg g21 min21, respectively. Compared with the odour threshold of 0.005 mg g21 of hexanal in water (Buttery et al., 1999), the reaction rates were high. It can be assumed that
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Table 1 Development of volatile rancidity in headspace of heat treated and nonheated oat products, expressed in relative units. The samples were stored for 2 weeks at 35 8C and were exposed to light Aldehyde
Relative headspace response Non-heated Heat-treated Non-heated Heat-treated fiber fiber flour flour
Fig. 1. The rates of hexanal decrease by different water– flour mixtures. The initial amount of hexanal added was 100 mg g21. Headspace stabilisation time was 25 min at 60 8C.
especially oat flour can very rapidly remove hexanal amounts, which lie far above this odour threshold. The fact that oat flour was able to eliminate all of the added 100 mg g21 of hexanal from the headspace in 1.5 h suggested that the flours had either exceptional capacity for binding hexanal or a capacity to convert hexanal to other compounds. Evidence supporting the latter possibility came from the observation that no headspace hexanal emerged back into the mixture even though the incubation was continued for additional 12 h after complete removal of hexanal. It was, therefore, concluded that at room temperature, the removal of hexanal from the oat water – flour mixture was a relatively fast and irreversible process. 3.2. Effect of heat treatment Some of the oat water –flour mixtures were heat treated for 30 min at temperatures between 60 and 100 8C prior to addition of hexanal (300 mg g21). As shown in Fig. 2, the ability to reduce hexanal was conserved when the mixture was heated for 30 min at temperatures between 60 and 70 8C, and it diminished slightly between 70 and 80 8C after which it collapsed rapidly. Yet, some hexanal
Fig. 2. The effect of heating of water–flour mixtures before hexanal addition on the rate of hexanal decrease. Heating time was 30 min, hexanal addition was 300 mg g21 and incubation time was 1 h at room temperature. Headspace stabilisation time was 5 min at 60 8C.
Isobutyraldehyde Butyraldehyde Isovaleraldehyde Pentanal Hexanal Heptanal Nonanal
0.50 0.29 0.39 0.14 0.13 0.14 0.14
0.82 0.57 0.76 0.33 0.36 0.63 0.66
1.00a 0.73 0.73 0.78 0.52 0.66 0.64
0.98 1.00 1.00 1.00 1.00 1.00 1.00
a Values presented as arbitrary detector response with the highest value taken as 1.00.
was eliminated even after heating of water –flour mixture at 100 8C for 30 min, suggesting that two separate phenomena may be responsible for the decrease of hexanal. The heat-labile activity also probably plays a role in the decrease of endogenously formed hexanal and other short chain aldehydes. Those aldehydes found to be formed abundantly upon prolonged storage of heat treated oat products were present at much lower levels in the corresponding non-heated products after the same storage period under similar conditions (Table 1). 3.3. Decrease in hexanal by flour fractions In order to identify the kernel material primarily responsible for the elimination of hexanal, the whole oat flour was fractionated into fiber, starch and protein fractions. The protein fraction was clearly the most efficient in decreasing hexanal (Fig. 3). This activity was also present in fiber fraction whereas starch fraction practically lacked this ability. The fiber fraction also contained protein (Liukkonen et al., 1992) suggesting that the capability to eliminate hexanal by oat flour was associated with protein rich kernel material probably originating from kernel surface.
Fig. 3. The rates of decrease of hexanal by starch, fiber and protein fractions. The hexanal concentration was 300 mg g21. Headspace stabilisation time was 5 min at 60 8C.
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Fig. 4. The decrease of hexanal and the formation of hexanoic acid at room temperature. Hexanal concentration was 300 mg g21. Headspace stabilization time was 5 min at 60 8C.
3.4. Formation of hexanoic acid and 1-hexanol The facts that the capability of the oat flour to decrease hexanal was higher than anticipated by simple absorption, this phenomenon was temperature sensitive and concentrated onto a protein rich fraction, led to a search for a possible conversion product of hexanal. As shown in Fig. 4, the decrease in hexanal content coincided with accumulation of hexanoic acid. The amount of 1-hexanol produced in 3 h was only 1.2 mg g21 which was relatively small compared with the amount of hexanoic acid formed. There was only 6 mg g21 of headspace hexanal remaining after 3 h and simultaneously 240 mg g21 of hexanoic acid (Fig. 4) and 1 mg g21 of 1-hexanol had been formed. The fate of the rest of the hexanal removed, which was approximately 20% of that originally added, remains unknown. It may have been either adsorbed or otherwise reacted with flour material to make the headspace hexanal undetectable. Approximately 80% of added hexanal was transformed to hexanoic acid under the experimental conditions.
interconnected (Lehtinen et al., 2003; Ekstrand et al., 1993). Several explanations may exist. Unsaturated fatty acids may be oxidized and degraded into volatile products without a prior hydrolysis and cause the rancidity occurring especially during storage of oat products. Alternatively, the small amounts of free fatty acids found already in intact noninactivated kernels are basically sufficient to provide volatile oxidation products in excess of their sensory threshold. In either case, the excessive formation of volatile compounds, such as hexanal and pentanal, is linked to the perception of rancid sensory attributes in oat material (Heinio¨ et al., 2002). Consequently, the inactivation of lipase does not grant long shelf life of oat products, as it does not prevent the oxidative rancidity. The present paper brings an additional aspect regarding the relationship between lipid hydrolysis and development of rancidity. It is demonstrated that short chain aldehydes do not accumulate in non-inactivated oat material but are efficiently converted to less volatile products. This means that against the sensory symptoms of rancidity, oat has an intrinsic mechanism of resistance based on aldehyde dehydrogenase type activity. At present, it is customary to use heat for the inactivation of lipid hydrolysing activities in oat to improve storage stability. This requires temperatures that exceed the temperature tolerance of the aldehyde dehydrogenase type activity. This may explain, at least in part, the earlier observation that in lipase inactivated oat, the accumulation of hexanal exceeds significantly that of lipase active oat (Lehtinen et al., 2003). Therefore, heat inactivation of grains or grain products may not represent the best routines to achieve optimum keeping quality. This applies especially to oat where the amount of both free and acylated unsaturated fatty acids is invariably higher than that needed to cause rancid perception by their secondary oxidation products.
References 4. Discussion An increment in the content of free fatty acids amongst the lipids of processed oat has generally been considered as an early indicator of a forthcoming oxidative rancidity. Therefore, much attention is being paid to prevent lipid hydrolysis during processing and storage of oat containing products. In light of recent studies, the view of lipid hydrolysis as an initiator of rancidity process is probably too straightforward. In processed oat, volatile lipid oxidation products such as hexanal have shown to be formed abundantly without observable hydrolysis of acyl lipids (Kiilia¨inen et al., 2001). On the other hand, slight variations in process conditions can give fractions where lipid hydrolysis progresses to a great extent but the formation of volatile oxidation products is minimal. It seems as if hydrolysis and oxidation of oat lipids were only loosely or not at all
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