The
Anatomy When is biological senseware like noses and tongues at least as important to your work as conventional labware like gas chromatographs and mass spectrometers? When you're a flavor chemist. Flavor, food, and agriculture scientists are using both categories of instruments to create better flavors, breed and raise tastier animals and plants, and optimize food processing while preserving flavor. Some scientists say that flavor chemistry could provide the means for feeding the ever-increasing global population. For anyone equipped with a tongue and a nose, a food's flavor is primarily a blend of its taste and odor. Taste buds detect primarily sweet, bitter, salty, and sour compounds. Olfactory recep-
FOCUS tors in the nasal cavities can detect a huge variety of volatile aroma chemicals at sensitivities that beat out most modern instrumental methods of detection. For chemists equipped with instruments like gas chromatographs and mass spectrometers, flavors can be depicted as sets of numbers, or, more conveniently and routinely, as sets of peaks. By correlating instrumental data with a food's sensory and physical features and with agricultural practices, food scientists and farmers are gaining control over the flavor of food products. They also are learning how flavors change as foods are processed and packaged and as consumers prepare and eat them. History of flavor analysis techniques Until recently, noses and tongues were the only tools available for analyzing flavors. Today, these facial instruments continue to provide the last
words even on compounds that flavor researchers isolate and characterize using the mighty analytical arsenal available to them. To date, flavor analysts have identified roughly 4600 flavor compounds in foods, a number that may be only half of the total cast. In the 1950s, 500 flavor compounds were known. Although tactics may have changed since then, the strategy of flavor scientists hasn't. "Even with the advent of modern instrumentation, we must still separate and isolate fractions, use some means of detecting materials separated, and identify and characterize chemical structure and biological activity," Roy Teranishi said at a recent meeting of the American Oil Chemists Society, whose members work with animal and vegetable oils. Teranishi is a flavor scientist working at the U.S. Department of Agriculture's (USDA) Western Regional Research Center (WRRC) in Berkeley, CA. Before modern instruments became available, workers typically used fractional distillations to isolate and concentrate some of the flavor chemicals in a food. They tried to make derivatives that they then could separate by paper or column chromatography. If successful, the researchers then crystallized the isolated materials and identified them by their melting points. Unfortunately, derivatization often was achieved at the expense of the compound's odoriferous feature or was accompanied by isomerizations. In addition, the poor sensitivity of these techniques limited researchers to discovering only the most abundant components. During the 1960s and early 1970s, flavor analysis mushroomed. The routine availability of gas chromatographs and infrared, NMR, and mass spectrometers enabled researchers to achieve isolations and characterizations at unprecedented resolutions and sensitivities. These instruments were to classical techniques what an electron
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microscope was to light microscopy. During this period, food companies hoped that flavor analysis would solve most of their problems, according to Ron Buttery, a flavor chemist with the WRRC, one of the USDA's four regional research centers. Also, government money was readily available for basic flavor research, says Gary Reineccius, professor of food science at the University of Minnesota in St. Paul. But the flavor research boom ended in the late 1970s. U.S. funding agencies began refocusing support from basic food studies to nutrition and toxicology studies. This shift in dollars forced many flavor scientists to shift their research foci to these areas, Reineccius says. Now U.S. flavor research is making a
of Flavor modest comeback, according to But tery, Reineccius, and others. Gas chro matography (GC) and mass spectrome try (MS) remain the workhorses, and their capabilities routinely are extend ed with purge-and-trap devices that can preconcentrate trace volatile flavor chemicals. Many U.S. researchers— most in government, some in industry, and a few still surviving in academic laboratories—continue to learn more about what makes a food such as cat fish taste like catfish instead of like pineapple. Tastes of today's flavor analysis landscape "The most important characteristic of flavor is aroma," notes Reineccius. It is mostly the profile of a food's volatile
aroma chemicals that enables a baker to distinguish cookies made with margerine from those made with butter. Many foods contain hundreds of these volatile compounds. Coffee, for exam ple, has more than 500 of them. Toma toes have nearly 400. Cooked beef may have more than 1000. "Capillary gas chromatography to gether with mass spectrometry allows you to identify a lot more compounds than you ever could before," remarks Cynthia Mussinan, director of research analytical services for the New Jersey research facility of International Fla vors and Fragrances (IFF). When these instruments became routinely available, flavor discoveries were guaranteed for anyone studying one of the scads of previously unana-
lyzed foods. "But the fun days are over," says Thomas Parliment, a senior scientist with the General Foods Cor poration in Tarrytown, NY. "You can't do the first paper on blueberries." As far as simply finding out what fla vor chemicals are present in food, fla vor chemists have reached the point at which the law of diminishing returns takes over—no strain, no gain. The challenge now is to develop methods for detecting flavor chemicals that are present at extremely low levels, are too labile to survive gas chromatography, or are difficult to chromatograph be cause of their water solubility, says USDA's Buttery. "A 'peak' that you don't even see in the gas chromatogram may be the most important flavor molecule," he says. Buttery and his col leagues recently detected in tomatoes a potent flavor compound called β-demascenone, which they found to be present at the 2-ppb level. The same chemical plays a big part in the aroma and taste of Concord grape products. Another challenge is to quantify what has been determined to be present with qualitative analyses. "No one is saying how much of these flavor compounds are there," Buttery says. At food companies, professional flavor ists and sensory panels determine the appropriate relative amounts of the flavor compounds that the company chemists have found to be present. Here, art presides over science. Quantification in flavor science is difficult for several reasons, Buttery says. In foods, a variety of enzymes change the relative concentrations of flavor compounds. These enzymes are active at different times during anallyses, processing, growth, and matura tion of the food source. The measure ments that a flavor researcher makes during a GC/MS analysis of garlic probably do not agree with the actual profile of flavor chemicals present half an hour after a chef slices the garlic's own enzymes into action. Also, it is dif ficult to precisely quantify materials 925 A
FOCUS such as 0-demascenone, which are present in extreme trace amounts. Pyrazine, the chemical that gives green pepper its characteristic smell, is another example. "It is so powerful [and present in such low concentrations] that you would never detect it in a packed column, and you even have to preconcentrate it to detect it in a capillary column," says IFF's Mussinan. This adds steps and theoretical inferences that weaken the confidence of trace quantification calculations. Despite these problems, researchers are learning more about the flavor dynamics of meats, fruits, vegetables, grains, herbs, and spices, as well as processing, cooking, and packaging. Take Catharina Ang. She is a research food technologist with the USDA Poultry Meat Quality and Safety Research Unit in Athens, GA, who has been comparing chromatograms of fresh-cooked chicken with those of reheated chicken. She and others such as Arthur Spanier, who is doing similar studies with beef, are attempting to discover which chemicals are responsible for the "warmed-over flavor." They are out to end the leftover blahs. Ang and her colleagues have found that if they cook the chicken and then chromatograph extracts after 1, 2, 3, and 5 days of refrigerated storage, the chemical profiles change. The size of some peaks changes, and some appear or disappear. Interpreting these changes is proving more difficult than detecting them. One of the major peaks in all of these chromatograms is hexanal, and its size increases with storage time. But sensory studies by Ang's colleague reveal that hexanal "does not seem to be the 'warmed-over-flavor' chemical," Ang says. Other USDA scientists also are hard at work. About 20 years ago, Harold Dupuy and others at the Southern Regional Research Center (SRRC) in New Orleans, LA, built a simple device that allowed them to analyze the volatiles of just about any food. Now patented and sold by a Louisiana company, the external closed inlet device, or ECID, consists of a finger-sized cartridge that contains food samples. As the cartridge is heated, purging gas shunts the evolving volatile components into the GC column. "As chemists, we're attempting to set [objective] baselines for the stimulusresponse effect between the flavor compounds in foods and the actual sensation that the subject perceives when he's eating these foods," explains John Vercellotti, research leader of the Food Flavor Quality Research Unit at the SRRC. This basic information is neces-
USDA researchers are studying the chemical differences between freshly cooked and warmed-over chicken flavor.
puy, now retired from the USDA but working at SRRC labs on cooperative research projects with Virginia Polytechnic Institute and State University in Blacksburg, tells this story. A few years ago, an East Coast company was having problems with repulsive off-flavors in their processed rice products. Together with the USDA, they had spent a dozen years breeding a rice strain that had all the qualities you would want—good yield, good nutrition, and good processing characteristics. One problem: The rice tasted awful. " T h e breeders thought they had bred something bad into the rice," recalls Dupuy. "We told them what the problem was nearly overnight." Dupuy and his colleagues learned that the offflavors stemmed from a reaction between a fumigant the rice company was using during shipping and a protein in the rice. There was nothing wrong with the strain of rice, after all. Flavor in the Ivory Tower
sary, he says, to maintain acceptable flavor during food production, preservation, and processing. "At the United States Department of Agriculture we are trying to improve our commodities and world market position," Vercellotti says. "And flavor quality is the bottom line on anything you sell." Such research today might save the $45 billion/year beef business, the $7 billion/year peanut business, and other food industries from making extremely costly errors in the future. Other spinoffs from the research include salvaging food that otherwise would be lost. For instance, ugly strawberries that are unfit for the fresh market might be fine as raw stock for flavor chemicals. A quick screen with the ECID-equipped gas chromatograph could help an inspector decide whether to process or dump multi-ton lot of strawberries. An example of fruit flavor research appeared in the May/June 1988 issue of the ACS Journal of Agricultural and Food Chemistry. Teranishi, Buttery, Karl-Heinz Engel of the Technical University in West Berlin, and others described experiments on nectarine flavor. Using liquid-liquid extraction with ether to isolate the components, and GC and GC/MS to detect and identify them, the scientists learned that a cast of four lactones probably is sufficient to characterize the aroma and flavor of the nectarine. Grains are another area in which flavor analysis is doing some good. Du-
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Only a few academic flavor research strongholds survived the funding drought of the 1970s. One of them is the lab of Robert Lindsay in the Department of Food Science at the University of Wisconsin—Madison. Among other projects, Lindsay is working on fish and seafood flavor chemistry with the goal of learning how fresh and rotten fish flavors develop and which chemicals are responsible for them. "With our sophisticated tools, we are finding that a lot more of the flavor is caused by compounds that are present in the low ppb range, as opposed to those that emerge earlier during the analyses in the ppm range," he observes. In one of his projects involving salmon (which spend part of their time in marine waters and part in fresh waters), Lindsay is making discoveries that could help salmon aquaculturists. In marine environments, salmon eat foods such as shrimp, which contain carotenoids. These compounds bestow the salmon's meat both with its color and much of its flavor. In fresh water, the salmon are deprived of carotenoidcontaining food sources. The fish are less colored and less flavorful as a result. Aquaculturists could benefit from such information, Lindsay says, by adding carotenoid sources to the feed of salmon they raise in fresh water. He also sees biotechnological and genetic interventions in the future when more of the biochemical details of salmon flavor are uncovered. Another of the success stories in aca-
FOCUS demie flavor research is the University of Minnesota's Reineccius. "We are trying to take liquid flavors (which they virtually all are) and turn them into a nice, stable, dry, free-flowing powder," he explains. His tactic in this project is to immobilize droplets of flavor in a starch matrix to make flavor powder that has the consistency of dry milk. The advantages of dry flavor powder over liquid flavor, Reineccius says, are that it is better suited for flavoring dry foods, it is easier to ship because you don't need glass bottles, and it has a longer shelf life. One of the 10 graduate students working under Reineccius's tutelage is developing an on-line GC surveillance system for milk production. What is needed for these sorts of systems is baseline information about the chemical signature of the food product during different processing phases. Reineccius says this already is possible with some items such as tequila and apples. But as a rule, he says, it is not. "The tools exist, but the background data is lacking." Terry Acree, a Cornell biochemistry professor whose lab is at the New York Agricultural Experiment Station in Geneva, has been using one of the more sensational hyphenated pieces of equipment: the GC-nose. Flavor researchers have been using their tongues and noses for decades, mostly for making qualitative, informal confirmations (e.g., methyl anthranilate really smells like grape Kool-Aid). But Acree has developed a more formal, quantitative, and reproducible technique called "charm" analysis. Acree injects extracts from grape skin, orange juice, or other foods into the gas chromatograph, which he has modified with a sniff port so that a person can smell what the instrument separates and detects. Trained sniffers push a button when they smell something and push it again when they no longer smell anything. Successive dilutions of the flavor or aroma chemical help Acree to determine a compound's odor threshold, or its minimal humanly detectable concentration. This results in an aromagram, or a charm vs. retention index plot, that correlates with the chromatogram. The technique is good for determining how people respond to individual compounds of a fragrant mixture, Acree says. But an odor or flavor is more than simply the sum of its parts. Acree admits that he cannot predict human responses to a new mixture of flavor chemicals by simply looking at a catalog of their charm responses. The problem is compounded by non-
rosemary differ markedly from the chemical profiles of their oils, which are obtained from the dead and harvested plants. His company intends to use this information to make "living spice oils." Others at the meeting spoke on topics such as the development of natural flavors using biotechnological processing and techniques for studying the biochemical processes underlying grape and wine flavor. The future
Building flavor-producing genes into plants and vegetables is in the offing, researchers suggest.
chemical factors important in flavor perception. These include what is technically known as a food's "mouthfeel," or how the food feels in the mouth; cultural and social biases; and the way a food sounds in your mouth as you chew it. If a potato chip doesn't crunch, chances are it won't taste right, even though a GC analysis would depict it as identical to a crunchy chip. A smorgasbord of research Many other flavor scientists are working on projects ranging from the change in chemical profiles of fresh fruits as they ripen to the relationship between chirality and aroma; from identifying the flavor chemical precursors in papaya to determining how packaging polymers change a food's flavor by absorbing important volatile flavor chemicals; and from developing automated techniques for analyzing volatile compounds on line to developing alternative fish sources into highly acceptable substitutes for premium seafoods like crabs and shrimp. At the recent Third Chemical Congress in Toronto, Teranishi described how various constituents of pineapple volatiles contribute to the pineapple flavor. Braja Mookherjee, a research director and vice-president at IFF, described his headspace analysis technique in which he can analyze the volatiles of living herbs and plants. He discovered that the chemical profiles of living plants such as peppermint and
As researchers learn more about flavor chemistry, there will be more opportunities for monitoring and optimizing flavor quality throughout all phases of food production. "Once you know how an animal and its raising conditions relate to the flavor of its meat, then you can start doing something about your feeding regimens or your genetic selection," remarks University of Wisconsin's Lindsay. The same goes for fruits and vegetables, says USDA's Buttery. Now that a fair amount is known of the chemistry of tomato flavor, tomato breeders can conduct more rational breeding programs. Building flavor-producing genes into plants and vegetables is in the offing, researchers suggest. "When there are twice as many people in the world, we're going to have to use biotechnology and some other tricks to feed them," says USDA's Vercellotti. "Knowing as much as possible about flavor chemistry, we will be able to build flavor into biosynthesized foods," he predicts. As more and more food companies emphasize natural over artificial flavor additives, surveillance will become more important, says IFF's Mussinan. Consumers will want to know if they actually are getting the natural flavors that they may be digging deep into their pockets to pay for. She predicts that NMR instruments equipped with deuterium probes will become more common in the future for discerning what she calls the "naturalness" of certain chemicals. For instance, the celebrity flavor chemical vanillin is found in vanilla beans or can be made from scratch in flavor chemists' labs. "The deuterium probe may say if it's natural," she says. But no matter how food and flavor analysis evolves, researchers unanimously agree that noses and tongues will continue to be a couple of their most important instruments—ones that are not in danger of being replaced by newer models. They say this also will help keep capital expenses in check. Ivan Amato
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