The Effect of pH on the Formation of Volatile Compounds Produced by Heating a Model System Containing 5 ’-Imp and Cysteine

A identificação de compostos voláteis formados a partir de reações entre Inosina-5’-Monofosfato (5’-IMP) e cisteína a três diferentes pH (3,0; 4,5; 6,0) e 140 °C foi realizada através da análise de ‘‘headspace’’ dinâmico. Os resultados mostraram que mais de 90 compostos voláteis foram produzidos, principalmente compostos heterocíclicos, incluindo furanos sulfurados, tiofenos, tiazoles, furanos, sulfitos alquilados, compostos bicíclicos e sulfitos cíclicos. Os estudos demonstraram que os furanos sulfurados, mercaptocetonas e alquil-furanos foram formados principalmente a pH ácido, enquanto que as pirazinas foram completamente inibidas a pH elevado. Estes resultados confirmam observações preliminares de que o pH exerce uma grande influência nos compostos voláteis formados em reações de Maillard.


Introduction
It has been established that the Maillard reaction between pentose sugars and cysteine or hydrogen sulphide is very important for meat flavour 1,2,3 .The main source of pentoses in meat are ribonucleotides and 5'-IMP is the principal ribonucleotide in post-mortem meat.The reaction between pentose and cysteine has been examined in model systems 4,5,6 .It was also observed that pH influenced such reactions 7,8 and that meat flavour formation also seems to be pH dependent 9 .Work has been done on the thermal stability of nucleotides in aqueous solutions and the thermal reaction of 5'-IMP with amino acids at pH 2.3 has been studied 10 .However the influence of different pHs on the volatile products from reactions of nucleotides with amino acids has not been studied.To better elucidate the participation of 5'-IMP and cysteine, as well as pH, in the formation of some important meat flavour compounds, heated model systems were studied using the reactants, 5'-IMP and cysteine, at three different pHs, i.e., 6.0, 4.5 and 3.0.

Preparation of reaction mixtures
Reactions between 5'-IMP and cysteine were carried out at three different pHs, i.e., 3.0, 4.5 and 6.0.Cysteine (0.121 g) and 5'-IMP (0.348 g) were dissolved in 10 mL of an appropriate pyrophosphate buffer (pH 3.0; 4.5, 6.0).One millilitre (1.0 mL) aliquots of each solution were transferred to the pyrex ampoules, nitrogen was blown over each sample for 3 min, and the ampoules flame-sealed.The final concentrations in each reaction were 12.1 mg/mL and 34.8 mg/mL of cysteine and 5'-IMP respectively.These concentrations were related to the amount of these compounds in meat muscle 11 .The reactions were carried out in triplicate.Ampoules containing the reaction mixtures were placed in a horizontal position in a CERTOclav autoclave (Kelomat, Traun, Austria) and heated for 1 h at 140 °C under a pressure of 0.28 MPa (2.7 bar).After the reaction, the mixtures were left to cool and then submitted to headspace analysis.A pH measurement was carried out before and after each heat treatment.In general the pH of reaction mixtures decreased by less than 0.2 pH unit after heating.

Headspace Collection of volatiles
For the collection of the volatile compounds produced by heat treatment, a dynamic headspace technique was used.It was similar to that described by Madruga and Mottram 12 ,and Madruga 13 .The cooled ampoules were broken manually and the samples were immediately transferred to a 250 mL conical flask fitted with 30mm screw joints to take a sliding joint with PTFE seal (SVL fittings; J. Bibby Science Products, Stone) and Dreschel head.Pyrophosphate buffer (20 mL) at the appropriate pHs were added to the reaction mixtures and aimed to dilute the mixtures.The volatiles were collected in a glass-lined stainless steel trap (155 mm long x 0.75 mm id) packed with Tenax-GC (SGE Ltd).During the collection of the volatile components, the conical flask was maintained at 60 °C, in a water-bath with constant agitation.The volatiles were swept on to the adsorbent in the trap using a flow of oxygen-free nitrogen (40 mL/min) and collected during 1 h.The oxygen-free nitrogen supply was purified by passing through granular charcoal to remove traces of organic compounds.The flow was adjusted via a pressure regulator and a mass flow controller.At the end of the volatile collection, the flask was removed and the trap was connected directly to the nitrogen supply for 5min, to remove moisture.Usually no less than three headspace collections were performed for each experiment.

Gas chromatography with FID detection and odour assessment
After collection, the volatiles were thermally desorbed, using a modified injector port, directly on to the front of a DB-5 (30 m x 0.32 mm id x 1.0 µm film, J & W Scientific Inc) or a CP-Wax-52CB 52CB (50 m x 0.32 mm id x 0.21 µm film, Chromapak UK Ltd) fused silica capillary column, in the oven of a Hewlett-Packard HP5890 Gas Chromatograph.The oven was held initially at 0 °C for 5 min while the the volatiles were desorbed from the Tenax trap (held at 250 °C in the modified injector).After the removal of the coolant, the column temperature was rapidly increased to 60 °C and maintained at this temperature for 5 min.Then it was ramped at 4 °C/min to the final temperature, where it was held for a further 20 min.For the DB-5 column the final temperature was 250 °C, but a lower temperature of 220 °C was used with the less-stable CP-Wax column.At the end of the column, the effluent was split 1:1, into two deactivated fused silica capillaries (0.4 mm od; 0.2 mm id) of equal length, using a 1/16 in.stainless steel union with a two-hole ferrule carrying the two fused silica capillaries (SGE Ltd).
One of the ends was connected to the FID, and the other was pushed through a length of glass-lined stainless steel tubing (1/16 in id; 0.7 mm id) (SGE Ltd).This tubing passed through the GC detector heater block and into a glass nose connection, which was used as the ''sniffing port'' mounted above in the GC.Another line was connected to the sniffing port, carrying air, which was moistened by bubbling it through distilled water.For each analysis the aroma of the effluents was assessed by four individuals, with experience in flavours, who marked the chromatogram and noted a description for each aroma detected.A clean nose-cone was fitted for each assessor.Helium at 2 mL/min was used as carrier gas.

Determination of linear retention indices (LRI)
A standard mixture of n-alkane (C6 -C20) in ethanol was analyzed each day before GC runs to allow a check of the instrument performance and the calculation of retention indices of each component in the samples.The standard (1.0 µl) was injected to the trap and the solvent was removed by purging with oxygen-free nitrogen (40 mL min -1 ) for 5 min.These alkanes were used as external standard references in Linear Retention Indice (LRI) calculations.LRI of each compound was calculated from the standard alkane retention time and the peak retention time using the Eq. 1.
where: LRI = Linear Retention Index RTx = retention time of compound RTn = retention time of n-alkane before peak RTn+1 = retention time of n-alkane after peak n = carbon number of n-alkane before peak Gas chromatography-mass spectrometry (GC-MS) Analyses were performed on a Hewlett Packard 5988A Mass Spectrometer with a HP5890 Gas Chromatograph, linked to a HP 59970 GC/MS Workstation.The GC columns and conditions used were the same as those described above.The following conditions were used for the mass spectrometer; source temperature = 200 °C, ionising voltage = 70 eV); scan range from m/z 29-290, with 1.44 scans per second

Results and Discussion
The major headspace components of the model system involving 5'-IMP + cysteine at three different pHs (3.0, 4.5, 6.0) are listed in Table 1 in order of elution.The 98 compounds presented are those which gave significant peaks in gas chromatograms, together with minor components of possible odour significance, e.g.compounds which were described as having a meat-like aroma or some similar description.The identity of 56 of these have been established by comparison of mass spectral data and Linear Retention Indices (LRI) with those of authentic materials.Thirty-nine identities have been suggested for compounds whose mass spectra agree with literature spectra, but with-out reference to LRI, and three other compounds (sulphur containing structures) were suggested by the interpretation of mass spectra and comparison with those of related compounds.Eight compounds (in italics) have been synthesised 13 .The gas Chromatogram of headspace volatiles collected from 5'-IMP/cysteine model system heated at pH 3.0, in a DB-5 column is presented in Fig. 1.
The volatiles identified in these three reaction mixtures were dominated by sulphur-containing compounds, especially disulphides and thiophenes.Also thiazoles, alkyl sulphides, cyclic sulphides, bicyclic compounds, furans and ketones were found.The total quantity of volatile compounds increased significantly as the pH decreased.On both columns, a large number of heterocyclic disulphides were identified from the reaction of 5'-IMP with cysteine at pH 4.5 and 3.0, but at pH 6.0 only one was detected.
The presence of thiol substituted furans and thiophenes, such as compounds (19, 29, 36, 37, 50, 54), gave rise to a number of important disulphides in the reaction mixtures.Those identified were: In the reaction mixture at pH 3.0, all of these 12 compounds were formed.Nevertheless four were not detected at pH 4.5 and none were found at pH 6.0.
Dithianes, trithianes and tetrathianes, which are heterocyclic compounds with two, three and four sulphur atoms in six-membered rings, were also formed in these systems.Formation of dithianones and dithianes was favoured by acid pH; however more trithianes /tetrathianes were found at high pH.Trithianes can be formed from the reaction between saturated aldehydes, such as acetaldehyde, and hydrogen sulphide 27 .In the 5'-IMP+cysteine model systems the thermal degradation of cysteine to give acetaldehyde and hydrogen sulphide could be a route for their formation 28 .
Except for thiazoles, there were no other nitrogen-containing volatile compounds identified from the 5'-IMP/cysteine at any of the three pHs.Absence of pyrazines at low pH was probably because the carbonyl-amino interaction is not favourable under acidic pH.Lack of pyrazine was noted by Zhang & Ho 10 in a 5'-IMP/cysteine mixture at pH 2.3 and Whitfield et al. 41 found that concentrations of nitrogen-containing heterocyclic compounds were reduced in systems containing cysteine and ribose, compared with other amino acids.Five thiazoles, including a series of acylthiazoles [16, 42, 44, 53] and a benzothiazole [75], were generated in the 5'-IMP/cysteine mixtures.Their formation does not seem to be highly influenced by changes in pH.The acylthiazoles have been reported in a model system containing cysteine and ribose 4,5 .Compound 75 was not reported in the volatiles of the ribose/cysteine system, however it has been found among the volatiles of roasted, fried and pressure cooked beef 42 .According to Vernin & Parkanyi 27 , the thermal degradation of cysteine and cystine, either alone or in the presence of reducing sugars such as ribose, is a source of thiazoles, also they can be formed by heat-degradation of thiamine.
The breakdown of 5'-IMP appears to be the main source of furans, furfurals and furanones found in the reaction mixtures.Four alkylfurans containing chains of C1-C5 [18, 24, 41, 45], together with two furylketones [34, 51] and two acylfurans [27, 39] were formed.Another group of furans identified were furfural [15] and its derivatives [30, 32, 48, 64] and 4-hydroxy-5-methyl-3(2H)-furanone [21].These compounds were highly influenced by pH changes.There was a clear tendency for many more furans being formed at low pH.Only 2-butylfuran [24] and 2-furfural [15] were detected in the reaction mixture at pH 6.0.One exception was found for 2,4-dimethylfuran [18] which was not detected at pH 4.5 and 3.0, although this compound may not have been fully captured during the headspace analysis due to its low boiling point and consequently to its high volatility.4-Hydroxy-5-methyl-3(2H)-furanone [21] was found only in the reaction mixture at pH 4.5.It may have been formed at pH 3.0 but, in the presence of H2S, this compound could have been converted into other volatiles.According to Mottram & Leseigneur 8 , concentrations of furans/furfural/ furanones dropped markedly as the pH increased in model systems containing ribose and aminoacids.
The presence of ketones in the 5'-IMP/cysteine system can be explained by Maillard-type reactions and Strecker degradation of cysteine, which can lead to the formation of short chain ketones [3, 6, 7, 8, 9, 11, 17].However the formation of 2-hexanone [12], 2-heptanone [23] and 3-heptanone [25] cannot be explained by these routes/mecha-nisms; they may have been formed by the condensation reactions between other carbonyl products.Ketones were more abundant in 5'-IMP/cysteine system at pH 6.0, which could result from the fact that enolisation of Amadori products in the Maillard reaction is pH dependent and dicarbonyl formation is more pronounced at alkaline conditions.Alternatively, at lower pH they may be lost due to further reaction with cysteine or hydrogen sulphide.In general, dicarbonyls like 2,3-pentanedione [8] and 2,3-butanedione are important intermediates in the formation of other volatile products, as has been discussed in this text.They can react with hydrogen sulphide leading to the formation of mercapto ketones which can produce important meat-like volatiles.2-Methylcyclopentanone [17] was identified in the volatiles of fresh cooked ground beef and freeze-dried defatted rehydrated beef.It was also reported to contribute to increase meatiness in some flavour isolates 18 .

Conclusion
The results have shown that the reaction between inosine-5'-monophosphate (5'-IMP) and cysteine led mainly to the formation of heterocyclic compounds (seventy-six volatiles).Many of these were sulphur-containing volatiles, such as disulphides and thiophenes, other compounds identified were thiazoles, furans, mercaptoketones, bicyclic and cyclic sulphur-compounds.A clear tendency was observed for some classes of compounds to be formed more at higher or lower pH, for instance, sulphur-containing compounds, such as sulphur-substituted furans and mercaptoketones, as well as alkylfurans, were more readily formed at lower pH, while pyrazines were inhibited by acidic conditions.Other heterocyclic compounds like thiazoles and trithiolanes were not affected by pH changes.

Figure 1 .
Figure 1.Gas Chromatogram of headspace volatiles collected from 5'-IMP/cysteine model system heated at pH 3.0, showing a summary of the aromas detected in the polar column effluent.Peak numbers related to compounds in Table1.

Table 1 .
1.Compounds identified in the volatiles from the reaction between 5'-IMP and Cysteine, at different pHs, using DB-5 and CP-Wax columns.