Cold-storage defects in butter and their relation to the autoxidation of unsaturated fatty acids

In this thesis investigations are described of the identification of aroma compounds which are formed as a result of oxidative deterioration of butter during cold storage, producing a typical trainy (fishy) off-flavour. As these flavour defects are caused chiefly by autoxidative breakdown of unsaturated fatty acids, it was also studied which fatty acids may act as precursors in the formation of these off-flavours. For this purpose the volatile odorous compounds formed in the autoxidation of pure unsaturated fatty acids were identified and compared with the compounds formed in butter with cold-storage defects.In the introductory chapter (I), a survey is given of results obtained in previous investigations concerning the aroma compounds in butter with coldstorage defects. Aliphatic carbonyl compounds in particular are considered to be responsible for these flavour defects, but these components were isolated only from washed cream and sweet-cream butter, and not from cold-stored butter made from soured cream. In addition, several classes of compounds such as carbonyl compounds with double bonds not in conjunction with the carbonyl group and cis/trans -isomers have been overlooked in previous investigations, because of inadequate analytical techniques.In Chapter II a survey is given of the mechanisms of autoxidation of fatty acids. The mechanisms are discussed of the radical chain reactions leading to the formation of hydroperoxides, and of the dismutation of these compounds to secondary autoxidation products. The latter reactions are of particular interest, as they result in the formation of aroma compounds which may cause flavour defects.In Table 2 a survey is given of the hydroperoxides and the secondary oxidation products that may be expected in the autoxidation of a number of unsaturated fatty acids, and of those which were actually found in previous investigations.Chapter III deals with flavour defects observed in autoxidized food lipids in general and cold-storage defects of butter in particular.First of all, the organoleptic properties and flavour threshold values are given of many secondary autoxidation products (see Table 3) which have very low flavour threshold values and highly repugnant odours.Secondly, autoxidation experiments are described which indicate the contribution of various unsaturated fatty acids to autoxidation off-flavours.Thirdly, the generation of oxidation defects in lipid-containing foods, and the effect of several factors on this type of deterioration, are discussed.Finally, special attention is paid to the development of cold-storage defects in butter. The main factors which influence the keeping quality of cold-stored butter are: the extent to which milk, cream and butter are contaminated with copper, and the pH of the butter serum.It is known that the oxidation processes in butter, which may cause inter alia a trainy flavour, start at the fat/serum interface. Experiments were carried out which provided further evidence that the oxidative deterioration of coldstored butter is due primarily to oxidation of the phospholipids. Further experiments show, however, that 'artificial butter' made from butter fat and milk serum (without fat globule membrane material or phospholipids) may also develop a trainy flavour, provided that a strong pro-oxidant (copper) is present at the lipid/water interface. It must therefore be concluded that it is uncertain whether the off-flavours are formed by oxidation of the unsaturated fatty acids of the phospholipids, or of the butter fat, or even of both.Chapter IV consists of a description of the techniques which were used for the isolation and identification of volatile flavour compounds in oxidized lipids. The special problems concerning the isolation and separation of volatile flavour compounds are outlined.For the isolation of volatile flavour compounds in butter fat, a semi-continuous high-vacuum distillation apparatus was designed. Aroma compounds were isolated from small samples of oxidized lipids by means of a batch-wise high-vacuum distillation technique.The aroma components were first separated by temperature-programmed gas chromatography, using a type of column which minimized the risk of artefact formation.For further separation and identification, the carbonyl compounds from the fractions obtained by gas chromatography were converted into their DNPH-derivatives, because the latter have favourable properties for liquid-chromatographic and spectrometric analyses. A special conversion reaction was used to avoid isomerization and artefact formation. A method of silver nitrate complex chromatography was used for the further separation of mixtures of DNPH's according to type and degree of unsaturation.After these manipulations, the DNPH's were in general separated into the individual compounds. For their further identification ultraviolet, infrared and mass spectrometry were used. Many reference DNPH's have enabled specific characteristics to be determined (absorption wavelengths in ultraviolet and infrared spectrometry, and m/e -values in mass spectrometry).In a few cases odorous compounds other than carbonyl compounds had to be identified. These compounds were separated by gas chromatography (using a second column, if necessary) and subsequently used for spectrometric analysis.Figure 9 is a diagram which visualizes the combination of techniques used for identification of volatile compounds obtained from oxidized lipids. It could be established that even very labile compounds such as 2 cis -enals, 3 cis -enals, etc. were not modified and could be identified correctly by means of this combination of techniques.In Chapter V the results are presented of investigations on the identification of volatile compounds contributing to the trainy flavour of cold-stored butter. For the sake of comparison, the aroma compounds from fresh butter without flavour defects have also been analysed.In Chapter VI the results are given of the identification of aroma compounds which are formed in the autoxidation of a number of unsaturated fatty acids.Many compounds have been identified, which have not been detected previously in butter with oxidation defects or in autoxidized fatty acids.In Chapter VII the results of the present investigations are discussed and summarized. From the analysis of trainy butter and butter without flavour defects it can be seen that the odorous fraction shows large changes as a result of the oxidative deterioration. Many of the aroma compounds in trainy butter are the same as those found in autoxidized fatty acids.By comparing the composition of the mixture of odorous compounds from trainy butter with that of compounds from autoxidized fatty acids (see Table 25 and Figure 10) it has been possible to determine that the autoxidation of unsaturated fatty acids and particularly the autoxidation of linolenic acid (and fatty acids with the same alkyl-terminal structure) chiefly contributes to the development of a trainy flavour. This conclusion was confirmed experimentally; the addition of autoxidized linolenic acid (with a small quantity of arachidonic acid) to butter without flavour defects caused a distinctly trainy flavour.The analyses of aroma compounds which result from the autoxidation of a number of pure unsaturated fatty acids have established that the aroma compounds formed are well in line with those that can be expected from the mechanisms of autoxidation of unsaturated fatty acids and the dismutation of the hydroperoxides (see Tables 21 to 24).It was established that the changes in flavour in the first stages of oxidative deterioration of cold-stored butter can be ascribed to the rapid oxidation of the highly unsaturated fatty acids, which play the main part in the development of a fatty-metallic off-flavour. The tallowy flavour in the last stage of oxidative deterioration of cold-stored butter can be ascribed to the continued oxidation of several fatty acids.This thesis, although dealing in particular with cold-storage defects in butter, also presents general information on the development of oxidation flavours in lipid-containing food products

Cold-storage defects in butter and their relation to the autoxidation of unsaturated fatty acids

In this thesis investigations are described of the identification of aroma compounds which are formed as a result of oxidative deterioration of butter during cold storage, producing a typical trainy (fishy) off-flavour. As these flavour defects are caused chiefly by autoxidative breakdown of unsaturated fatty acids, it was also studied which fatty acids may act as precursors in the formation of these off-flavours. For this purpose the volatile odorous compounds formed in the autoxidation of pure unsaturated fatty acids were identified and compared with the compounds formed in butter with cold-storage defects.In the introductory chapter (I), a survey is given of results obtained in previous investigations concerning the aroma compounds in butter with coldstorage defects. Aliphatic carbonyl compounds in particular are considered to be responsible for these flavour defects, but these components were isolated only from washed cream and sweet-cream butter, and not from cold-stored butter made from soured cream. In addition, several classes of compounds such as carbonyl compounds with double bonds not in conjunction with the carbonyl group and cis/trans -isomers have been overlooked in previous investigations, because of inadequate analytical techniques.In Chapter II a survey is given of the mechanisms of autoxidation of fatty acids. The mechanisms are discussed of the radical chain reactions leading to the formation of hydroperoxides, and of the dismutation of these compounds to secondary autoxidation products. The latter reactions are of particular interest, as they result in the formation of aroma compounds which may cause flavour defects.In Table 2 a survey is given of the hydroperoxides and the secondary oxidation products that may be expected in the autoxidation of a number of unsaturated fatty acids, and of those which were actually found in previous investigations.Chapter III deals with flavour defects observed in autoxidized food lipids in general and cold-storage defects of butter in particular.First of all, the organoleptic properties and flavour threshold values are given of many secondary autoxidation products (see Table 3) which have very low flavour threshold values and highly repugnant odours.Secondly, autoxidation experiments are described which indicate the contribution of various unsaturated fatty acids to autoxidation off-flavours.Thirdly, the generation of oxidation defects in lipid-containing foods, and the effect of several factors on this type of deterioration, are discussed.Finally, special attention is paid to the development of cold-storage defects in butter. The main factors which influence the keeping quality of cold-stored butter are: the extent to which milk, cream and butter are contaminated with copper, and the pH of the butter serum.It is known that the oxidation processes in butter, which may cause inter alia a trainy flavour, start at the fat/serum interface. Experiments were carried out which provided further evidence that the oxidative deterioration of coldstored butter is due primarily to oxidation of the phospholipids. Further experiments show, however, that 'artificial butter' made from butter fat and milk serum (without fat globule membrane material or phospholipids) may also develop a trainy flavour, provided that a strong pro-oxidant (copper) is present at the lipid/water interface. It must therefore be concluded that it is uncertain whether the off-flavours are formed by oxidation of the unsaturated fatty acids of the phospholipids, or of the butter fat, or even of both.Chapter IV consists of a description of the techniques which were used for the isolation and identification of volatile flavour compounds in oxidized lipids. The special problems concerning the isolation and separation of volatile flavour compounds are outlined.For the isolation of volatile flavour compounds in butter fat, a semi-continuous high-vacuum distillation apparatus was designed. Aroma compounds were isolated from small samples of oxidized lipids by means of a batch-wise high-vacuum distillation technique.The aroma components were first separated by temperature-programmed gas chromatography, using a type of column which minimized the risk of artefact formation.For further separation and identification, the carbonyl compounds from the fractions obtained by gas chromatography were converted into their DNPH-derivatives, because the latter have favourable properties for liquid-chromatographic and spectrometric analyses. A special conversion reaction was used to avoid isomerization and artefact formation. A method of silver nitrate complex chromatography was used for the further separation of mixtures of DNPH's according to type and degree of unsaturation.After these manipulations, the DNPH's were in general separated into the individual compounds. For their further identification ultraviolet, infrared and mass spectrometry were used. Many reference DNPH's have enabled specific characteristics to be determined (absorption wavelengths in ultraviolet and infrared spectrometry, and m/e -values in mass spectrometry).In a few cases odorous compounds other than carbonyl compounds had to be identified. These compounds were separated by gas chromatography (using a second column, if necessary) and subsequently used for spectrometric analysis.Figure 9 is a diagram which visualizes the combination of techniques used for identification of volatile compounds obtained from oxidized lipids. It could be established that even very labile compounds such as 2 cis -enals, 3 cis -enals, etc. were not modified and could be identified correctly by means of this combination of techniques.In Chapter V the results are presented of investigations on the identification of volatile compounds contributing to the trainy flavour of cold-stored butter. For the sake of comparison, the aroma compounds from fresh butter without flavour defects have also been analysed.In Chapter VI the results are given of the identification of aroma compounds which are formed in the autoxidation of a number of unsaturated fatty acids.Many compounds have been identified, which have not been detected previously in butter with oxidation defects or in autoxidized fatty acids.In Chapter VII the results of the present investigations are discussed and summarized. From the analysis of trainy butter and butter without flavour defects it can be seen that the odorous fraction shows large changes as a result of the oxidative deterioration. Many of the aroma compounds in trainy butter are the same as those found in autoxidized fatty acids.By comparing the composition of the mixture of odorous compounds from trainy butter with that of compounds from autoxidized fatty acids (see Table 25 and Figure 10) it has been possible to determine that the autoxidation of unsaturated fatty acids and particularly the autoxidation of linolenic acid (and fatty acids with the same alkyl-terminal structure) chiefly contributes to the development of a trainy flavour. This conclusion was confirmed experimentally; the addition of autoxidized linolenic acid (with a small quantity of arachidonic acid) to butter without flavour defects caused a distinctly trainy flavour.The analyses of aroma compounds which result from the autoxidation of a number of pure unsaturated fatty acids have established that the aroma compounds formed are well in line with those that can be expected from the mechanisms of autoxidation of unsaturated fatty acids and the dismutation of the hydroperoxides (see Tables 21 to 24).It was established that the changes in flavour in the first stages of oxidative deterioration of cold-stored butter can be ascribed to the rapid oxidation of the highly unsaturated fatty acids, which play the main part in the development of a fatty-metallic off-flavour. The tallowy flavour in the last stage of oxidative deterioration of cold-stored butter can be ascribed to the continued oxidation of several fatty acids.This thesis, although dealing in particular with cold-storage defects in butter, also presents general information on the development of oxidation flavours in lipid-containing food products