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Separation and Characterization of Flavor Components from Vegetables RICHARD A. BERNHARD
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Department of Food Science and Technology, University of California, Davis, Calif.
Aliphatic disulfides appear to be the principal compounds responsible for the aroma and flavor of onions. Gold has demonstrated that alkylidene phthalides and dihydrophthalides are intimately concerned with the flavor of celery. While little is known about the flavor compounds of lettuce, certain off-flavors of carrots are due to the violet -like aromas of the ionones. Aliphatic acids produce the cheeselike flavor of sauerkraut, while the odor of rutabaga is caused by mercaptans and isothiocyanates. Forss found that cucumber flavor arises from a series of aliphatic aldehydes, as does the flavor of potatoes, tomatoes, and beans. Cooked vegetable flavors arise from a variety of simple substance produced during heating. Methods of isolation, separation, and characterization of vegetable flavor are critically discussed.
Comparatively few studies have been made on the flavor of vegetables ^* vs. fruits and essential oils. It is interesting to speculate on w h y this is so. Examination of agricultural statistics shows that total production i n the United States of 18 of the most common fruits is i n the neighborhood of 18 million tons a year, and that of a l l common vegetables is about 19 million tons. Thus on the basis of sheer bulk, one could expect the two groups to be of about equal importance. F r o m an economic stand point, the total value of a l l fruits i n the United States for 1960 was ap proximately $1.2 billion and that of a l l vegetables about $1.15 billion (24): not much to choose from here either. It appears that the two groups are of equal importance in both volume and value. W h y then is research on vegetable flavor chemistry lagging so far behind that of fruit flavor chemistry? It is this reviewers opinion that
13 1 Hornstein; Flavor Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1969.
F L A V O R CHEMISTRY
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this lag results from the difficulty of working with vegetable flavor. The flavors and odors of many vegetables are difficult to describe and worse to differentiate from each other. Potatoes and peas have little characteristic aroma and flavor, but such close relatives as oranges and lemons have quite different and easily recognizable flavor and aroma differences. One may raise the objection, what about onions, cabbage, turnips, and even rutabaga? D o not these have distinctive aromas and flavors? In deed they do, and as a consequence, the preponderance of investigations conducted i n vegetable flavor chemistry has been directed toward some of these plants. Apparently the strong, characteristic aromas and flavors of these vegetables have influenced many experimenters' choice of study area. This cannot be the sole reason for selection of a research program since economic factors and many issues influence this decision too. Some of these are brought out in the following discussion. W i t h the increasing sophistication in analytical techniques and in strumentation has come a greater knowledge of food flavor composition. There has been a flood of research papers on the volatile compounds in foods, and most of these studies are reported i n the name of "flavor" chemistry. There seems little doubt that many important constituents contributing to the aroma of foods have been isolated and identified, but what has not been done, to any appreciable extent, is to establish which of these components is responsible for what sensory properties. Stewart (34) has stated that many of the compounds isolated probably have little or no sensory effect. H e suggests greater emphasis on what he refers to as the significance of these compounds of flavor. Reliable tools are available for this purpose, and others should be developed. Flavor profile, odor recognition, dilution index, and difference tests can all prove useful for studying this problem. Appropriate tests with isolated fractions and compounds w i l l help answer such questions as: Does the compound have sensory properties? If so, what is their nature? What combination of components results in the sensory properties typical of the original product from which they were derived? Only after such studies have been made, w i l l we know the significance of the compounds isolated.
Isolation and Concentration Procedures Vegetables by their very nature are difficult to examine chemically. Flavor substances frequently occur i n concentrations of a few parts per million, surrounded by large amounts of other organic materials and huge quantities of water. This poses a real problem. The vegetable is usually comminuted, chopped, or otherwise re duced to a pulpy or powdery mass and then subjected to an isolation procedure. Five major methods are commonly employed to obtain flavor
Hornstein; Flavor Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1969.
Downloaded by CORNELL UNIV on May 17, 2017 | http://pubs.acs.org Publication Date: January 1, 1969 | doi: 10.1021/ba-1966-0056.ch008
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fractions from plant materials. Traditionally steam distillation has been used for years to isolate volatile organic substances from plants. This is still the preferred method of many researchers although it has many obvious disadvantages. The plant material is subjected to considerable heat and hydrolytic activity of a high order for long periods of time. Dis tillation under reduced pressure is another common procedure for iso lating flavor fractions. A variant of this technique is the use of shortpath distillation, frequently called molecular distillation. Using this procedure, decomposition of high-boiling, high molecular weight com pounds is often materially retarded. Another way to isolate organic material is by liquid-solid or liquidliquid extraction. Generally the product is exposed to prolonged heating and the less volatile components are subjected to pot heats that can materially alter many labile organic constituents. A l l this is further complicated b y the introduction of large volumes of solvent and the problems of its subsequent removal or concentration. Three newer methods use gas chromatographic techniques. One method places the sample i n a closed system and allows an inert gas to sweep across it and carry away entrained volatiles (21). These gases are condensed i n a series of cold-finger traps and the gas is recirculated over the food material. The cycle is usually repeated a number of times until sufficient volatile material is collected i n traps for subsequent analysis. There are other variations of this gas sweeping procedure, i n which single passes are made i n open systems (33). Sampling of headspace vapors with subsequent introduction of the sample into the chromatograph has been the choice of a number of workers i n recent months ( I , 2, 3, 5, 6, 22, 26, 30, 31). This has the great advantage of offering a rapid, convenient technique for analysis. Using flame ioniza tion detectors, Buttery and Teranishi (6) have studied direct injection of aqueous vapors from a number of foods. Fortunately, flame ionization detectors show little or no response to moderate amounts of water vapor, and thus this procedure can be applied most successfully. There is an obvious advantage to the latter three svstems over the former four procedures when working with volatile flavor substances. The conditions for removal of the flavor segment are m i l d , usually room temperature, and contact period is brief. This minimizes sample handling with any attendant possibility of artifact production. Its major dis advantages are that only the most volatile components are usually de termined, since only substances with appreciable vapor pressures would be expected to be i n the vapor phase. Additionally, compounds present in minute concentration may remain dissolved or entrapped i n the main mass of material. Thus it is not always possible to obtain a representative sample of the substances desired for study using gas sweeping or headspace techniques. Here the concentration of volatiles w i l l vary also
Hornstein; Flavor Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1969.
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with sample temperature, and the relative concentrations collected w i l l not necessarily be those of the same materials normally present i n the vegetable. One frequently reasons that the samples garnered by these two methods are fairly representative of the odor components of the products examined, but this has yet to be experimentally verified to any extent. Of course, there is the fervent hope that better isolation tech niques w i l l come i n the coming years. Certainly there is a pressing need for them.
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Separation and Characterization Once the flavor fraction has been isolated from the vegetable, atten tion must be directed to separation and identification of the individual components responsible for its make-up. T o date the preferred means of separating these constituents has been by gas-liquid chromatography ( G L C ) . Other procedures run a poor second to the versatility, speed, and resolution of G L C . Separation by additional chromatographic techniques has been used to some extent too—e.g., solid-liquid, liquid-liquid, thin-layer, paper partition, and ion exchange (13, 14, 18, 19). Some have resorted to fractional distillation, crystallization, and sublimation procedures (7,11,12,13). A l l , of course, can be most useful, and one is often the procedure of choice i n a given situation; but for universality, G L C is hard to surpass. Despite its unquestioned advantages as a tool for separation, G L C does not offer the best means for identifying substances. True, retention volumes (and its imprecise variations—e.g., retention time) are most helpful i n a rough qualitative sense, but one can never be certain as to identities based solely on retention data, no matter how many stationary liquid phases have been employed. This leaves the problem of char acterization to other physical and chemical means. Preparation of solid chemical derivatives—e.g., hydrazones of carbonyl compounds—is a useful and reliable means of identification. In flavor work one is often prohibited from using this time-honored method for lack of sufficient material. Frequently, one may trap effluent substances from a chromatograph i n a suitable reagent and form these derivatives, but this method suffers from a number of disadvantages, most of which are associated with sample size. A crystalline derivative, whose melting point agrees with accepted literature values (and is not depressed by admixture with an authentic sample ) w i l l usually provide accepted identification. If this method gives no satisfactory agreement with any known derivatives, one must resort to combustion analyses, molecular weight determinations, and lastly, to degradation studies. It takes a skilled analyst to get by on much less than 20 mg. of a compound that
Hornstein; Flavor Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1969.
Downloaded by CORNELL UNIV on May 17, 2017 | http://pubs.acs.org Publication Date: January 1, 1969 | doi: 10.1021/ba-1966-0056.ch008
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has been described i n the literature; a good deal more may be necessary for a compound that has not been described. If less than 0.1 mg. is available, the investigator is forced to seek refuge i n physical methods of identification that require only minute amounts of material for subsequent examination. I n this instance spectral methods of analysis are most common. Infrared and ultraviolet spectra can be determined i n solution using microcells. The solvent can be removed and a mass spectrum obtained. There is an increasing trend to wards the use of mass spectrographs for identification. I n the hands of a skilled analyst, this provides an elegant way i n which to identify effluent fractions directly from a gas chromatograph. T o date the major deterrent to the use of such instrumentation has been the high cost of the equip ment. Infrared spectrometry has provided the major means of identifying flavor components during the current decade.
Allium, Onions The largest number of investigations of vegetable flavor chemistry has been carried out with members of the genus Allium, onions. Their distinctive aromas have no doubt been largely responsible for this i n terest. Although investigation of Allium flavor has been going on since the late nineteenth century (32), much remains to be learned. I n 1961 Carson and W o n g (7) conducted an extensive study of the volatile components of a particularly pungent variety of onion called sunspice. This onion was the result of breeding programs designed to produce an onion of unusual pungency suitable for dehydration. After drying, the onion has a flavor potency similar to that of common fresh onions. Thus they selected a strain that is a rich source of volatile ma terials. T w o separate batches of ca. 142 pounds of onions were chopped, mixed with 25 gallons of water, and distilled under vacuum. The distilling vapors were led through two parallel columns containing 400 grams of activated charcoal. Distillation was continued for 40 hours at a pot temperature of 25°. The carbon columns were dried under vacuum and extracted i n a Soxhlet extractor with peroxide-free ether for 40 hours. This extraction was performed i n the dark to minimize possible lightinduced decomposition or isomerization. The combined ether extracts (3 liters ) were dried, and the solvent was removed b y distillation to yield 115 m l . of concentrate. Further concentration yielded 14 grams of a pale yellow oil, which was further distilled under vacuum to give 8.7 grams of an o i l boiling between 40° a n d 60° at 1 m m . of H g . This represents an oil concentration of about 75 p.p.m. based o n fresh weight of onion. Another procedure was tried i n which the juice from 142 pounds of onions was steam distilled, and extracted with isopentane. The two
Hornstein; Flavor Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1969.
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Downloaded by CORNELL UNIV on May 17, 2017 | http://pubs.acs.org Publication Date: January 1, 1969 | doi: 10.1021/ba-1966-0056.ch008
methods of isolation yielded oils giving different chromatographic pat terns, but the same disulfides and trisulfides were found i n each case. Analysis of the concentrates was made using G L C , and effluent frac tions were identified by derivatization and infrared spectral analyses. The compounds that Carson and W o n g (7) isolated and identified are recorded in Table I. Their most important new findings were that allyl 1-propyl disulfide is not present i n onions i n significant amounts; methyl as well as 1-propyl derivatives occur; and substantial quantities of trisulfides corresponding to the disulfides are present. Indeed, this work is be lieved to represent the first unequivocal isolation of pure, well-defined aliphatic trisulfides from plant sources. Table I. Volatile Components F o u n d in Onions by Carson and W o n g (7) Hydrogen sulfide 1-Propanethiol Methyl disulfide Methyl 1-propyl disulfide 1 -Propyl disulfide Methyl trisulfide Methyl 1 -propyl trisulfide 1-Propyl trisulfide
Ethanol 1- Propanol 2- Propanol Methanal Propanal 1-Butanal Acetone Methyl ethyl ketone
Carson and W o n g (7) presented no experimental evidence that any of the compounds isolated from onions has a role i n the flavor or aroma of onions. In reality the association of these onion components with onion flavor is intuitive. This is not leveled as a criticism of Carson and W o n g s work but really applies to many so-called flavor studies. Members of the genus Allium—onions, garlic, leek, chives, etc. (16)—possess strong, characteristic flavors and aromas not found i n other vegetables. Another remarkable attribute is that most members of the genus have no odor unless there is damage to the plant tissue. These characteristic volatiles are absent from intact tissue, and are produced enzymatically when injury occurs (35). Substrates for the production of these volatiles are known as alliins, and are derivatives of the amino acid cysteine. These derivatives give rise, through several reactions, to the sulfur-containing volatiles. S-AUyl cysteine sulfoxide of garlic, the first alliin to be identified (35), when treated with a preparation of garlic containing the enzyme alliinase (28), gives rise to allicin (diallyl thiosulfinate), pyruvic acid, and ammonia (26,35): Ο Î 2CH
