This thesis deals with the formation of colour of protein-containing foods, such as fish, as caused by the interaction with wood smoke, more particularly a closer examination of some chemical aspects of the formation of smoke colour. As such, it can be considered an extension of previous work done by Ziemba.
Chapter 1 gives a broad survey of the usual smoking procedures and of the attempts to control the process more efficiently. Starting from the assumption that modernization of the smoke process could result into a modified and therefore possibly less acceptable product, it is necessary to acquire a thorough understanding of the details of the classical process in order to apprehend the requirements necessary for a smoke installation.
Chapter 2 describes the investigations, carried out up till now, aimed at elucidating the underlying problems. The following topics are mentioned: flavour and taste of smoked foods, enhancement of keeping quality as a consequence of the microbiocide properties of smoke, retardation of oxidative deterioration of fat, and formation of colour in smoked foods. It appears that many problems are left unexplained, more specifically the formation of the typical smoke colour; for this reason the attention was focussed upon this aspect.
A preliminary investigation is described in Chapter 3. With the help of a simple model, the influence of the diffusion of smoke constituents into the product and the effect of surface desiccation upon the colour formation could be demonstrated. In agreement with the results of Ziemba it was found that proteins are very important in producing the brown colour, for which the free amino group of the proteins is essential. Guanidino groups do not play a noticeable role.
The contribution of the smoke was investigated as follows: Petri dishes containing water were placed in the smoke house or kiln. Inspection of the liquid after the process revealed the presence of a considerable amount of osazone-forming α-hydroxycarbonyl- and α-dicarbonyl compounds. Comparative experiments proved that several carbonyl compounds of this type show a strong tendency to display browning reactions with amino groups.
In Chapter 4 a quantitative analysis of these α-hydroxycarbonyl and α-dicarbonyl compounds is described. Upon addition of 2,4-dinitrophenyl hydrazine to smoke-treated water at 0°C., the α-dicarbonyls are precipitated quantitatively. α-Hydroxycarbonyl compounds remain in solution and can be precipitated in the form of their osazones, after removal of the first formed osazones, by heating the filtrate. In this way, glycolic aldehyde and glyoxal, and also acetol and methyl glyoxal, could be determined together. The method fails for quantitative determination of acetoin and diacetyl together. Separation of the osazones can be accomplished by chromatography on a zinc carbonate column with benzene/pyridine/ethanol 5:5:2 as an eluens, followed by spectrophotometric determination.
Chapter 4 describes further the total carbonyl assay by gravimetric analysis of the precipitated 2,4-dinitrophenyl hydrazine derivatives. Chromatographic separation of an aliquot of this precipitate enables the determination of α-hydroxy-carbonyl and α-dicarbonyl compounds substituted at both ends. The percentages found were calculated as diacetyl.
The difficulties experienced in the determination of formaldehyde are discussed in detail. Two methods were found feasible and are described: chromatographically, after precipitation of the 2,4-dinitrophenylhydrazones, and spectrophotometrically with chromotropic acid.
It turned out that determination of acetaldehyde in smoke-treated water with piperazine and sodium nitroprusside is impracticable. Acetone and furfural, however, could be determined by simple methods (acetone by colour reaction with salicylic aldehyde, furfural by colour reaction with aniline acetate).
A critical survey is given of the usual methods for determination of phenols in smoke-solutions and -condensates; for this purpose the colour reaction with 4-aminoantipyrine was selected.
In order to obtain quantitative data on the relative amounts of α-hydroxycarbonyl and α-dicarbonyl compounds in smoke-treated water, all the abovementioned analytical methods were carried out with a number of samples, together with the determination of total acid. A browning test was carried out as well.
In all cases the presence of appreciable amounts of glycolic aldehyde, acetol and methyl glyoxal could be ascertained, in proportions comparable with formaldehyde or considerably higher. In the majority of cases the total concentration of these three components nearly equals the total amount of titratable acid (calculated as acetic acid).
Upon doubling the depth of the smoke-absorbing water layer in the Petri dishes, a slight increase of the amount of smoke constituents was observed, particularly of the phenols. Furfural shows a large increase.
The aqueous solutions of smoke constituents as obtained in this manner display a moderate stability at room temperature with the exception of methyl glyoxal, the content of which decreases markedly upon standing.
Finally a comparison was made between our data and those of others.
Due to widely differing conditions, both with respect to smoking technique and methods of interception and analysis, a comparison proved to be very difficult.
Chapter 5 describes the formation of colour in greater detail. The capacity of a number of carbonyl compounds and sugars to block the free amino groups of proteins irreversibly, or to develop a brown colour with glycine, was investigated. A marked activity in both respects was established for glyoxal, methyl glyoxal and glycolic aldehyde. Formaldehyde, although notably capable of blocking amino groups irreversibly, does not cause discolouration with these amino groups. Acetol and diacetyl were almost inactive in this respect.
The concentration of methyl glyoxal, formaldehyde and diacetyl in smoketreated water decreases rapidly upon addition of an amino compound. Glycolic aldehyde shows the same phenomenon, but to a slightly lesser degree.
Next, the browning capacity of various samples of smoke-treated water towards glycine was compared with that of equally concentrated solutions containing methyl glyoxal, glyoxal, glycolic aldehyde and formaldehyde. In all cases the colour intensity produced by the latter was approximately 85 to 90% of the corresponding smoke solutions. In another browning test, with a much smaller excess of glycine, the differences were somewhat larger. In spite of this, the conclusion seems justified that the browning of smoke with amino derivatives can be explained satisfactorily by the presence of the above-mentioned carbonyl compounds in smoke.
It was observed that mixtures of these carbonyl compounds engender a stronger discolouration than the components alone. This is true also if formaldehyde is added, although formaldehyde by itself does not cause browning.
It was shown that many other amino acids and amines exhibit the browning reaction, although the presence of an NH 2
group does not automatically imply a distinct colouration.
Secundary amines, tertiary amines and amides give hardly any or no browning with smoke-treated water. Diaminoethane, on the other hand, shows an exceptionally strong discolouration. A table summarizes the results obtained with these compounds and glycine at a pH of 5.0.
This chapter describes furthermore some experiments on the influence of airoxygen on the formation of colour. In the case of hydroxycarbonyl compounds a small effect was detectable, but in the case of dicarbonyl compounds the effect was either very small or non-existent.
The work was concluded with an investigation of the chemical principles, that govern the browning reaction. The allegedly essential role of pyrrole derivatives in the process was scrutinized and finally rejected.
Mixtures of glycolic aldehyde and glycine in the ratio 2:1 produce optimal browning. This does not, however, justify the conclusion that they participate in the reaction in the same proportion since it is highly probable that this optimal ratio is caused by the fact that glycolic aldehyde is present mainly as a dimer.
Glycolic aldehyde and glycine can react in varying proportions, but a large excess of either of them does not lead to further intensification of the colour.
Several reaction products could be isolated from a concentrated solution of glycolic aldehyde and glycine or ethanolamine by means of gel filtration. One of these reaction products, upon further examination, was identified as the Schiff base of hydroxymethyl furfural and ethanolamine. Presumably therefore the mechanism of the browning reaction should be explained as the formation of a Schiff base of glycolic aldehyde with the amino compound in question, followed by successive aldol condensations with further molecules of glycolic aldehyde. The resulting - and conceivably very reactive - condensation products could react further, either by polycondensation to macromolecules, or by cyclisation to the above-mentioned furan derivative. Since this compound is relatively stable it can be considered as a final product.
By UV-spectrophotometry, the presence of characteristic reaction products could be demonstrated in mixtures of methyl glyoxal and amino compounds as well. In this case, an excess of the carbonyl compound causes a more pronounced deepening of the colour than in the case of glycolic aldehyde. These systems were not investigated in more detail.
Finally some experiments are described with mixtures of glycolic aldehyde, methyl glyoxal and an amino compound.
A diversity of opinions exists as to the role of the disperse phase of smoke in the formation of colour. This subject is broached in chapter 6. It was found impossible, at least under normal conditions, to remove the disperse phase completely from the smoke. Electrostatic filtration was not applied since passage through a strong electric field might induce chemical conversions. In an attempt to investigate the role of the disperse phase, several models were exposed to the action of smoke in such a way that the precipitation of smoke particles on a dry surface and the absorption on a moist protein surface could be traced. Reactions on the smoked surface, which could involve smoke constituents, were suppressed by the addition of sodium metabisulfite.
This part of the work has not reached the stage where pertinent conclusions can be drawn; however, several suggestions for further work are given.