Aroma analysis

In numerous studies the volatile fractions of food have been analysed to identify their components. Due to the progress in instrumental analysis, particularly gas chromatography (GC) and mass spectrometry (MS), about 7600 different volatile substances have been identified in more than 400 food products up to 2004 [1]. 

Among all the volatile compounds, only a limited number are important for aroma. According to a proposal by Rothe and Thomas [2] only those compounds actually contribute to aroma whose concentration in food exceeds their odour thresholds. To estimate the importance of a volatile compound for the aroma of a particular food, the ratio of concentration to its odour threshold was calculated. This value was denoted aroma value [2], odour unit [3] or odour activity value (OAV) [4], In the following the latter term is used. 

The knowledge that not all of the volatiles (e.g. more than 800 in roasted coffee) [5] that occur in a food contribute to its aroma was the rationale for changing the methodology of analysis. Since 1984, when the procedure for charm analysis was published [4], techniques have been developed that focus on the identification of compounds contributing to the aroma with higher OAV. 

It is the objective of this chapter to provide a survey of the state of art in the methodology of aroma analysis (6.2.4.2) and to discuss important results which were obtained by the application of these methods (6.2.4.3). The data listed in 6.2.4.3 will give a first orientation on the composition of important food aromas. 

To detect the odour-active volatiles. Fuller et al. [6] described a system for the sniffing of GC effluents which was improved and applied to food samples by Dravnieks and O Donnell [7]. The new technique, named GC olfactometry (GCO), was the starting point for the development of a systematic approach to the identification of the compounds which cause food aromas. As summarised in Table 6.23 the analytical procedure consists of screening for key odorants by special GCO techniques, quantification and calculation of OAVs as well as aroma-recombination studies. During the last decade these steps have been critically reviewed by Acree [8], Blank [9], Grosch [10, 11 ], Mistry et al. [12] and Schieberle /fi/. 

Thermal treatments canning new raw material disinfection fermentation defects preservation 

Microbial deterioration reactions of food constituents (e. g. oxidation, non-enzymatic browning) transfer of aroma compounds from other food or packaging material 

The aroma substances consist of highly diversified classes of compounds, some of them being highly reactive and are present in food in extremely low concentrations. The difficulties usually encountered in qualitative and quantitative analysis of aroma compounds are based on these features. Other difficulties are associated with identification of aroma compounds, elucidation of their chemical structure and characterization of sensory properties. 

The results of an aroma analysis can serve as an objective guide in food processing for assessing the suitability of individual processing steps, and for assessing the quality of raw material, intermediate- and endproducts. In addition, investigation of food aroma broadens the possibility of food flavoring with substances that are prepared synthetically, but are chemically identical to those found in nature, i. e. the so-called nature identical flavors (cf. 5.5). 

The elucidation of the aroma of any food is carried out stepwise the following instrumental and sensory analyses are conducted  

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A number of solvents have been used to extract volatiles for aroma analysis but the optimum choice depends on a compromise. Table Gl.1.2 lists the most common solvents used to extract odorants from foods. Although pentane and ethyl acetate are flammable, they have a very low toxicity, represent extremes in polarity, and a sequential extraction using these two solvents will remove most of the volatile odorants from aqueous samples (see Basic Protocol 2) however, if the desire is to do a simpler one-step extraction, then a solvent should be chosen with a polarity that will extract the volatiles of interest. For example, maltol is not extracted well with pentane, and 4-hydroxy-2,5-dimethyl-3(2H)-furanone, the smell of strawberry, is almost insoluble therefore, the choice of the optimum solvent depends on the analyte and may require some testing to find. 

Wright, D.W. 1997. Application of multidimensional gas chromatography techniques to aroma analysis. In Techniques for Analyzing Food Aroma (R. Marsili, ed.) pp. 113-141. Marcel Dekker, New York. 

For a compound to contribute to the aroma of a food, the compound must have odor activity and volatilize from the food into the head-space at a concentration above its detection threshold. Since aroma compounds are usually present in a headspace at levels too low to be detected by GC, headspace extraction also requires concentration. SPME headspace extraction lends itself to aroma analysis, since it selectively extracts and concentrates compounds in the headspace. Some other methods used for sample preparation for aroma analysis include purge-and-trap or porous polymer extraction, static headspace extraction, and solvent extraction. A comparison of these methods is summarized in Table Gl.6.2. 

The model mouth was developed as part of the doctoral program of Saskia van Ruth at the Wageningen University in the Netherlands between 1992 and 1995. The hypothesis was that only those volatile compounds released under mouth conditions are relevant for aroma analysis. The volume of the mouth, temperature of the mouth, mastication, and salivation were thought to be critical parameters. Those pa-... 

It is not possible to discuss all the methods available for characterizing foods critically and systematically in a single volume. Methods pertaining to interfaces (food emulsions, foams, and dispersions), fluorescence, ultrasonics, nuclear magnetic resonance, electron spin resonance, Fourier-transform infrared and near infrared spectroscopy, small-angle neutron scattering, dielectrics, microscopy, rheology, sensors, antibodies, flavor and aroma analysis are included. 

As was mentioned earlier, distillation and subsequent solvent extraction remains popular in the aroma research area Q). In this method for aroma analysis, the Likens-Nickerson apparatus has been a standard for over 20 years (17, 18). The primary limitation of the Likens-Nickerson distillation/ extraction procedure has been its operation at reduced pressure. It is desirable to operate the system under vacuum in order to reduce the sample boiling point to minimize the formation of thermally induced artifacts. The fact that the solvent side of the distillation-extraction apparatus is also under vacuum makes it difficult to retain the solvent in the apparatus. Even modifications of the apparatus to include a dry ice/acetone condenser followed by a liquid nitrogen trap do not permit easy operation under vacuum. Problems arise in that the solvent or aqueous vapors reach the cryogenic traps, thereby eventually blocking the exit of the condenser. The need to minimize exposure of the sample to heat has resulted in the more frequent use of two step procedures. Very often, the sample is simply placed in a flash evaporator, a certain volume of distillate collected and the distillate is solvent extracted via either separatory funnel or a continuous extractor. In this manner, the distillation process and solvent choice are not conflicting processes. 

From the point of view of aroma analysis, the ultimate objective of developing so-called "multiply hyphenated" instruments is to produce a device which can automatically determine the identity of all of the constituents of a complex volatile mixture. Integrated GC/IR/MS is a step along that path, but a host of crucial issues remain. 

Sensory examination of the effluent from the column of a gas chromatograph by "nasal appraisal" has been introduced into flavor analysis by Fuller et al. (22). Since that time this technique known as "GC-effluent sniffing" has been used in aroma analysis to locate the positions of odorants in a gas chromatogram. It was first... 

The techniques used for the separation of volatiles have been further improved. HPLC and high resolution gas chromatography (HRGC), the latter in combination with effluent sniffing, have been introduced into aroma analysis of bread (25). 

Sniffing. Aroma analysis of separated compounds from each extract was performed on a Hewlett-Packard Model 5880 Gas Chromatograph modified for sniffing off the end of the column. 

In general, any analytical equipment or procedure used in the field of natural products chemistry and environmental engineering is also helpful in aroma analysis 64,65 The history and principles of such art are described in detail elsewhere and will not be featured here. Gas chromatography (GC), GC-mass spectrometry (MS), and nuclear magnetic resonance (NMR) are the most frequently used techniques along with rather specialized setups such as proton transfer reaction-mass spectrometry66 (PTR-MS) used in retronasal aroma analysis (see Chapters 9.02, 9.06, 9.10-9.11). 

Aroma composition is usually very complex and thus on many occasions coelution of components may take place. Utilizing GC columns with different separation characteristics in a tandem manner may allow resolution of such peaks. In a typical setup, a certain portion of an effluent from the first column is concentrated using a cold trap (named cryofocus) and then sent into the second column for further separation. This analyzing technique combining multiple GC columns is referred to as multidimensional GC (MDGC).73,74 Cryofocus repeated in short intervals in combination with a short second column will furnish a whole two-dimensional chromatogram useful in complex aroma analysis (comprehensive GC x GC). 

Aroma analysis is most often performed utilizing GC-MS. This demands separation of volatile constituents from nonvolatile matrices. Additionally, higher concentrations of analytes are favorable to allow detection of trace key compounds.16,64,65 82 Therefore, various preparation methods derived from aroma extract production were developed. The composition of the concentrate may differ depending on the method used and thus selected to accommodate the aim. 

Figure 4 Aroma analysis of roasted spotted shrimp.

I. Blank, Gas Chromatography-Olfactometry in Food Aroma Analysis. In Flavor, Fragrance, and Odor Analysis, R. Marsili, Ed. Marcel Dekker New York, 2002 pp 297-331. 

The second point is related to the simultaneous presence of odorants at g/L levels and of others that can be active at levels as low as ng/L. This means that although it makes sense to use a general screening procedure for detecting by olfactometry the potentially most relevant aroma molecules, it will not be possible to use a single isolation or preconcentration scheme to identify and further quantify the different aroma molecules. Rather, it will be necessary to have an array of chemical isolation and quantification procedures if a comprehensive aroma analysis is our objective. 

In the next part of this chapter some of these techniques are compared, pointing out possible limitations and advantages in free and bound forms aroma analysis, both in the research and winemakers laboratories. In one paragraph, recent quantification methods for some sulphur compounds groups are discussed. 

Applications of the Kaltron method and GC-MS in wine aroma analysis were reported by Rapp et al. (1996). The study of the data repeatability in the analysis performed using both Kaltron (Rapp et al., 1994) and polystyrene XAD-2 resin (Gunata et al., 1985 Versini et al., 1988 Voirin et al., 1992) extraction, has already been performed in the cited papers. Besides this, a test with eight repetitions of the whole analytical process using XAD-2 resin has been performed both on a non-floral (Pinot blanc) and a floral (Morio-Muskat) varietal wine for the quantification of 25 varietal and fermentation compounds (Carlin, 1998) for each wine sample the mean CV % values ranged from about 7.0 to 7.5 with a standard deviation from about 3 to 4.7 %, respectively, depending on the different level of some compounds, mostly monoter-penes, in each wine type. 

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