Flavor isolation

Frydman A, Weisshaus O, Bar-Peled M, Huhman D, Summer L, Matin F, Lewinsohn E, Fluhr R, Gressel J, Eyal Y (2004) Citrus fruit bitter flavors isolation and functional characterization of the gene Cml,2RhaT encoding a 1,2rhamnosyltransferase, a key enzyme in the biosynthesis of the bitter flavonoids of Citrus. Plant J 40 88-100... 

Figure 1. Temperature-programmed separation of irradiation flavor isolates on a 20% Carbowax 20M column...

The availability of and improvement in membranes has rekindled some interest in dialysis in aroma research. Benkler and Reineccius (19, 20) initially published studies on the use of Nafion (Dupont) membranes for the separation of fat from flavor isolates. This would permit solvent extraction to be used in the isolation of aroma compounds from fat containing foods. Chang and Reineccius (21) later used a continuous tubular counter current flow system to accomplish this fat/aroma separation more efficiently. These membranes can be obtained commercially and have been improved in terms of membrane thickness and purity. While the aroma isolate obtained using this membrane may not perfectly reproduce the aroma being studied, this is an alternate technique for aroma isolation. 

Solvent extraction has also remained a popular technique for flavor isolation. The most recent developments in this area have involved the use of supercritical CO2 in both flavor extraction and analysis (25-27). 

The pH of the steam distillate was 6.2 in all cases. The total flavor isolate (by SDE) of plain cashews had, on the whole, a strong pungent and green aroma, reminiscent of the cashew nut testa and cashew shell, whereas the flavor isolates from roasted samples had the characteristic mildly nutty aroma also. The flavor fractions obtained by selective extraction method gave some information about the chemical nature of compounds responsible for the characteristic flavor notes. Accordingly, the basic fraction of roasted nuts, (both oven-and oil-roasted), had the typical nutty aroma associated with pyrazine. compounds. The basic fraction of plain cashews did not have any characteristic flavor in particular. 

Preliminary GC analysis revealed that there are more number of peaks in roasted samples compared to plain cashews. Also selective extraction method was found to be slightly superior to the SDE method under the conditions of the experiment adopted in this study. However, the compounds in oven-roasted and oil-roasted samples did not differ much, qualitatively and quantitatively. In total, 26 compounds have been identified in plain cashews and 3 compounds in roasted samples. The identified peaks constituted 70 percent of the total peaks registered in GC analysis of the individual samples. The descriptive flavor profile of the eluting peaks of the plain and roasted samples were studied. Since the flavor isolate from oven roasted cashew nuts contained the flavor components of plain cashews also and since analysis showed that there was not much difference between the flavor constituents of oven-roasted and oil-roasted samples, the aromagram of the oven-roasted cashew nuts (SDE) was taken as representative. Fig.l gives the GC profile of oven roasted cashew nuts and the sensory properties of the numbered peaks are included in Table III. 

Analysis of the neutral fraction of flavor isolates revealed that most of the neutral compounds like esters, lactones and carbonyls present in plain cashews were found in roasted samples also. In addition, three furans have been detected in the roasted... 

Several of the alkylthlazoles identified in french fried potato flavor, such as 2,4-dlmethyl-5-propylthiazole, 2,4-dimethyl-5-pentyl-thlazole, 2-butyl-4-methyl-5-ethylthiazole and 2-butyl-4-propylthia-zole have a strong characteristic sweet, sulfury and green aroma (33). This aroma characteristic is quite distinctive and is present in a large number of the fractions generated from the gas chromatographic fractionation of the french fried potato flavor isolate. It is probably an important part of the total french fried potato flavor. 

Use Perfumes, flavoring, isolates and ionones, source of citral. 

The symposium upon which this book is based was intended to provide participants with an overview on flavor isolation as well as a description of new advances in the enzymatic, fermentative, and molecular biological approaches to the generation of aromatic chemicals. Interaction between participants during the meeting helped spark numerous discussions and deepen friendships (new and old) between American and foreign scientists. We were pleased to be able to provide a number of graduate students the opportunity to make presentations before an international audience. 

A Romano cheese-like aroma was produced from a butter-fat emulsion by treating it with a crude enzyme mixture isolated from Candida rugosa. The emulsion consisted of 20% butterfat and 1.5% Tween 80 in a buffer solution. The treated emulsion was held at 37°C for three hours and then aged at room temperature for three days to develop the cheese-like flavor. The volatile flavor components were isolated from both the enzyme modified butterfat (EMB) and a commercial sample of Romano cheese. The flavor isolates were separated into acidic and nonacidic fractions and analyzed by gas chromatography-mass spectrometry. The results showed good correlation between the acidic fractions of the two samples. The acidic fractions contained similar relative concentrations of eight short-chain fatty acids (C2 - Cj q). Methyl ketones and esters were major components in the nonacidic fraction of the EMB. 

Nonacidic Components. Figure 1 shows the total ion chromatograms obtained from the nonacidic fractions of the flavor isolates. A total of 22 compounds were identified in the EMB and 12 in the Romano cheese. Table II lists the volatile flavor compounds identified in the nonacidic fractions. 

Tonsbeek C.H, Copier H, and Plancken A.J, (1971) Components contributing to beef flavor. Isolation of 2-acetyl-2-thiazoline from beef broth. J. Agric. Food Chem. 19, 1014-16. 

Flavor isolation and analysis are made difficult also by the fact that flavors comprise a large number of chemical classes. If they were comprised of one or just a few classes of compounds, isolation methods could focus on molecular properties characteristic of a given class of compounds. Rather, the chemist must attempt to effectively extract and concentrate alcohols, aldehydes, acids, ketones, amines, carbonyls, heterocyclics, aromatics, gases, nonvolatiles (or nearly so), etc. 

Despite the excellent resolving powers of modem capillary chromatography, there are situations where the analyst may chose to prefractionate the aroma isolate. Some of the more common methods to prefractionate flavor isolates prior to GC analysis include acid/base separations, high pressure liquid chromatography (HPLC), silicic acid column chromatography and preparative GC. 

An HPLC method is attractive for flavor fractionation since it uses a different set of physical properties for separation than GC does. A flavor isolate may be separated by adsorption or reverse/normal phase chromatography. Adsorption chromatography is a good initial choice since it has the greatest column capacity and can handle the widest range of types of compounds [53]. Fractions based on adsorption affinity could then be further fractionated on a normal or reverse phase column [54]. 

A simple inexpensive method for flavor fractionation is via silicic acid [55]. Basically, a flavor concentrate is passed through a column of sihcic acid and then eluted with a solvent gradient. This effectively fractionates the flavor isolate by compound polarity. The major problem with this method is potential artifact formation. However, careful control of the activity of the silicic add and limiting contact time minimizes artifact formation. 

The GC analysis of the flavor, or suitable flavor isolate, should be done using two different polarity GC columns (typically Carbowax and methyl silicone phases). One should also do GC olfactometry (GCO) analysis since this helps to identify compounds, tells one if there are compounds with very low sensory thresholds being used (perhaps no GC peak there), and also if there is a compound coeluting with the solvent. The next step is compound identification (this has been discussed in Chapter 3). 

The flavor isolation methods which most readily lend themselves to quality control applications are static headspace, dynamic headspace, direct injection and solvent extraction techniques. Since there are numerous recent reviews in the literature on these methods, there is little need to present any detail here but only summarize the key points about a given method. The reader can refer to reviews provided by Jennings and Shibamoto (4) Reineccius and Anandaraman (5), Reineccius (6) or Teranishi and Kint (7) for more detail. 

A number of references exist on the topic of flavor isolation, and these provide a different perspective on the topic (4-8). To quote Schreier (9) It must be emphasized that sample preparation is the most critical step in the entire analytical process of the investigation of volatiles. ... 

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