COVER STORY
MICHAEL KELLETT PHOTOGRAPHY
TEXAS FRUIT Citrus fruits contain limonoids, which may help prevent cancer. Researchers are trying to maximize Texas grapefruits’ high level of limonoids.
DEVISING HEALTHIER FOODS
Enhancing the good components and removing the unhealthy ones may PREVENT DISEASE RACHEL PETKEWICH, C&EN WEST COAST NEWS BUREAU
LEONARD M. PIKE set out to kick up some school spirit and ended up boosting the nutritional content of a carrot. As a member of Texas A&M University’s faculty, now professor emeritus of horticulture, Pike knew the pride that surrounded maroon, one of the school’s colors. He thought cultivating a maroon carrot would be fun for the Texas Aggies. But in the process of breeding a vegetable with novel aesthetics, the focus changed to building a healthier carrot. More than a decade later, Pike’s maroon carrots are known to grocery store patrons as BetaSweets. They also sell in the form of presliced carrot “coins” called
BetaBites, which display their orange core and maroon-purple edge. The carrots’ unusual coloring stems from high levels of anthocyanins, a group of antioxidants that give various fruits and flowers a purple hue and are touted as being able to inhibit the growth of cancer cells. β-Carotene, a fat-soluble terpene and antioxidant, gives regular carrots their characteristic orange color and converts to vision-improving vitamin A in the human body. Human trials investigating the effect of maroon carrot juice on breast cancer are scheduled to begin soon at the medical schools of Texas A&M and the University of Arizona.
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Pike’s carrots reflect the recognition among both researchers and the public that a healthy diet starts in the field, and momentum for nutritional improvement of several food crops is building. The number of published papers exploring the properties of bioactive constituents of foods has quintupled since 2003, showing that researchers are interested in parsing out exactly what cocktails of bioactive compounds are good for us, says James Seiber, director of the Department of Agriculture’s Western Regional Research Center (WRRC) in Albany, Calif., and editor of the Journal of Agricultural & Food Chemis-
oids’ structure-activity relationships show that limonoids may reduce the risk of try, published by the American Chemical and learn how to maximize levels of the several cancers, including oral, stomach, Society. The public is interested because bioactive compounds in Texas-grown and colon cancers. they have heard results of epidemiologigrapefruits, says Bhimanagouda S. Patil, Limonoids are colorless, highly oxygenatcal studies, for example, that say drinking director of the Vegetable & Fruit Imed triterpenoid compounds that are unique cranberry juice or eating blueberries could provement Center at Texas A&M. He to citrus fruits. Researchers have speculated help prevent certain diseases, he adds. notes that growers think the improved that the compounds, which contain a furan Creating healthier foods that appeal to fruits would give them a niche in the consumers is a collaborative effort that marketplace. combines the talents of chemists, nutriO Patil explains that there are two ways tionists, food scientists, horticulturists, to enhance bioactive compounds such as physiologists, medical experts, and plant limonoids: by boosting their biosynthesis breeders. The teams are using analytiOH O Glucose via traditional breeding techniques or by cal chemistry techniques and traditional CO2H isolating the precursor compounds, makbreeding methods rather than the biotechHO2C ing structural modifications, and adding nology and genetic engineering that would O the bioactive compounds to the final juice be needed to, for example, grow a banana HO O or processed product. Some bioactive that could serve as an edible vaccine. compounds could be synthesized from Nutritionists usually first discover that Deacetylnomilinic acid glucoside scratch, but a more efficient strategy, Patil a whole fruit provides a particular health says, might be to pinpoint, usbenefit in clinical studies. Cheming analytical techniques, the ists then determine the responSOLUBLE FIBER Fortefiber (generic structure shown) time during the growing season sible component. Next, plant is made by modifying cellulose from trees. that the compounds are most breeders increase the amount of abundant in the natural fruit. For that component in the offspring OCH3 instance, the Texas grapefruit plants. Chemists, food scientists, CH3O OH O HO growing season is from October and nutritionists work together HO O OH O to May. Patil’s group noticed that again in human trials to test the HO HO O O levels of certain limonoids, such nutritional efficacy of the isolated O as deacetylnomilinic acid glucocomponent. O OCH n-2 3 side, are very high earlier in the Pike had applied traditional OH OCH3 season. This means that clinical breeding on several different researchers doing studies with crops before using it to increase whole fruit should note the point during amounts of antioxidants in the maroon ring, may prevent cancer by activating enthe growth cycle when the subject eats the carrots. Another group of researchers is zymes called glutathione S-transferases. fruit, Patil says. breeding out negative factors such as cerThese enzymes can transform harmful subIn addition, researchers have found tain fatty acids from corn to make healthier stances into more water-soluble forms that that bioactive compounds can degrade oils. Some researchers are hoping a modican be excreted from the body. during storage. Therefore, fruit that is harfied soluble fiber that lowers cholesterol Chemists want to understand limoneven more than traditional bulk fiber may soon find its way into ready-to-eat products such as tortillas, pasta, and cakes. Yet other researchers are investigating the effect of harvest time on food chemistry by, for example, pinpointing when grapefruit Cost effective & easy to use high precision should be picked to maximize the levels of metering pumps for chemical research certain anticancer compounds in the fruit. GRAPEFRUITS GROW in warm climates,
including California and Florida, but the reddest and sweetest varieties come from Texas. These highly prized Texas grapefruits have piqued the interest of food chemists because they contain high levels of several bioactive compounds, including limonoids. Animal and cell culture studies
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People have historically resisted dramatic changes in diet unless the shift is toward something that really tastes good.
COVER STORY
IN STRUM E NTAT I ON
Understanding Healthier Foods With Analytical Chemistry and crushed. Hexane is used to extract oil from the germ. The fatty acids in the oil are then converted to methyl esters for two reasons: to pluck them from a glycerol backbone and to make them volatile enough for the gas chromatograph. Extracting limonoids—triterpenoid compounds that are unique to citrus fruit and that may help prevent cancer— from grapefruits originally took 10 steps. So when food scientists in Bhimanagouda S. Patil’s group at MICHAEL KELLETT PHOTOGRAPHY (BOTH)
Figuring out exactly what’s in the food you eat calls for chemists and their analytical instrumentation. Advances in analytical instrumentation have allowed food scientists to answer increasingly intricate questions about food and health. In developing healthier foods, researchers use the techniques to find bioactive compounds and standardize how much of those compounds are fed to the test subjects in clinical trials. The researchers also rely on these tools to analyze what happens to compounds in the frying pan or after the body digests them. All of this means that food scientists have a huge number of samples to analyze. The analytical workhorses among modern food science labs are mass spectrometers and gas or liquid chromatographs. Whatever the instrument is, the method has to be fast because hundreds of samples are run on a regular basis, says Linda M. Pollak, a corn breeder at the Department of Agriculture’s Agricultural Research Service at Iowa State University who uses several analytical techniques for a project on modifying the fatty acid content in corn oils. For many food research labs, attaching autosamplers to the instrumentation is a must, she adds. Gas chromatographs analyze volatile samples, so they are the best instrument for analyzing fatty acid content in corn that has been processed into oil, says Pamela White, a food chemist at Iowa State who collaborates with Pollak. Freshly processed oils are tested for composition. Researchers also examine oils for stability after they have been stored in a sealed container on a shelf or have been used, for example, to fry a basket of coconut shrimp. Preparing samples for food studies is time-intensive. To procure corn oil for her studies, White explains, a corn kernel is first soaked. Then the germ is removed
ANALYSIS
Researchers frequently use highperformance liquid chromatography to analyze nonvolatile food samples.
Texas A&M University figured out how to cut out six steps, they patented the process. Now only four steps are needed to obtain crystallized limonoids for analysis or for dosing in clinical trials. In the process, the researchers first use wet chemistry to extract limonoids from the fruit’s pulp. The compounds are further purified with flash chromatography techniques. Patil and colleagues use liquid chromatography to profile limonoids because, like the β-carotene and anthocyanins found in maroon carrots studied at the same university, they are not volatile enough for gas chromatography. The researchers determine the structure and purity with a nuclear magnetic resonance spectrometer. Animal studies and human clinical trials create numerous biological samples that must be prepared for analytical
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instrumentation. Techniques and methods vary depending on the analytes and sample matrix; Wallace H. Yokoyama can attest to that. Yokoyama is a research chemist at USDA’s Western Regional Research Center in Albany, Calif., collaborating with Dow Wolff Cellulosics, which developed Fortefiber, a cellulose material functionalized to reduce blood glucose and cholesterol levels in those who eat it. He studies the effects of Fortefiber and other kinds of fiber intake on metabolic disease in hamsters. He created analytical methods suitable for blood and fecal samples from the dosed hamsters. To understand how the animals digest fat, for example, he uses a liquid chromatograph and mass spectrometer to quantify triglycerides and sterols—components of cholesterol—that have been centrifuged from blood samples or extracted from feces (J. Ag. Food Chem. 2007, 55, 9750). Although the types of instrumentation may be fairly consistent in labs examining food, the methodologies can vary considerably. These disparities can draw into question what the results really mean for the eating public, write food scientists Edwin N. Frankel of the University of California, Davis, and John W. Finley of Louisiana State University in the Journal of Agricultural & Food Chemistry (DOI: 10.1021/jf800336p). They are particularly concerned about antioxidants because different labs have used multiple protocols to evaluate the activity of natural antioxidants. These techniques employ a wide range of free-radical-generating systems, as well as different methods of inducing oxidation and measuring the endpoint of oxidation. To help make these studies more comparable, the authors recommend that each antioxidant evaluation be carried out under various conditions of oxidation, using several methods to measure different products of oxidation related to real food quality. Advances in analytical chemistry have allowed food scientists to answer many questions, but other questions, including the debate on how best to analyze antioxidants, may have to wait for more sensitive detection techniques to emerge.
fatty acids found in edible vested and stored adds a level of fats and oils, the levels of complexity that may not be adwhich have various effects dressed in many health studies, on health and taste. Patil suggests. The five major fatty acids Previous laboratory studies R in edible fats and oils are show that feeding grapefruit pulp OH grouped according to their to rats with cancer improves the HUE’S WHO β-Carotene + carbon-carbon bonds. Palrats’ recovery rate. Trials with O HO (top) makes carrots orange, mitic and stearic acids have citrus products in patients recovand anthocyanins (such as only single bonds. Oleic acid ering from breast cancer treatOH the example shown here, has one double bond. Linolements are scheduled to run at the right) add a purple color. OH ic acid has two double bonds, medical schools of Texas A&M and linolenic acid has three and the University of Arizona. that every culture likes to fry some sort of double bonds. To simplify food labels, nuAs consumers hear and read more about food in oil because the process adds crisptritionists group palmitic and stearic acids the perils of high cholesterol and obesity, ness, flavor, and unique texture. under saturated fats, oleic acid as monothe pros and cons of edible oils continue to unsaturated fat, and linoleic and linolenic splash across health-related headlines. For under polyunsaturated fat. example, numerous stories based on cliniTO THAT END, food researchers attempt to Finding the right balance of fatty acids cal studies indicate that people who subappease consumers’ taste for oil while prethat meets criteria for taste, health, and sist on traditionally Mediterranean diets, serving their health. For their part, White function is tricky, White says. To taste which include a lot of olive oil, have healthy and other researchers are focusing on altergood, oils have to have certain amounts of weights and cholesterol levels. ing the chemical composition of corn oil particular fatty acids. But oils must have Although everyone seems to grasp that using breeding and analytical techniques to other fatty acids to maintain a shelf-life too much, or the wrong kind, of oil can be make healthful salad oil or frying oil. and not degrade in a fryer. And the corn hazardous to their health, people aren’t That project is not as easy as it may plant itself needs certain fatty acids to imabout to give it up. Pamela J. White, a food seem. The researchers’ tinkering is fopart strength to cell structure so the plant chemist at Iowa State University, explains cused on complex combinations of five
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COVER STORY
intensive because it can take years to breed the plants, grow the crops, harvest the corn, analyze hundreds of samples, process them into oils, and test their performance. White, Duvick, and Pollak are now cooperating with EnerGenetics International, a private Iowa-based agricultural research company, to test the oils in food applications, improve the agronomic traits of the corn so it will be more profitable for farmers to grow, and provide samples to food companies. MICHAEL KELLETT PHOTOG RA PHY
can stand up in the field and provide good crop yields. Linoleic acid contributes to good taste, but is not very stable in frying and storage. Oleic acid has been shown in clinical trials to reduce low-density lipoprotein, or “bad” cholesterol. But a corn oil made with 70% oleic acid would impart a bland, waxy taste that is not very desirable, White says. White works with Linda M. Pollak, a corn breeder, and Susan A. Duvick, a plant biologist, both with USDA’s Agricultural Research Service at Iowa State. Pollak breeds
MAROON CARROTS the plants and exAnthocyanins— tracts samples of corn, and Duvick ana- antioxidant compounds that lyzes the corn’s fatty turn flowers, fruits, acid content with a and the edges of these carrots gas chromatography violet—may help method. The trio prevent certain takes the varieties cancers. with the most promising fatty acid profile to a pilot plant on campus to be processed into oils, which are tested for taste and performance in storage or for applications, most notably frying. White and Pollak have been working to modify the fatty acid composition of corn oil for nearly two decades. Even with fast analytical methods, the research is time-
FIBER ALSO frequently grabs news cover-
age. Studies have shown that fiber in foods and bulk fiber supplements can lower cholesterol, reduce the risk of colon cancer, and generally help people lose weight and control diabetes by maintaining lower levels of blood glucose. Fiber-filled foods and supplements, with their bland taste and rough texture, however, are not consumer favorites. For the same reasons, many people don’t like to eat whole-grain breads or skins of fruits and vegetables. Psyllium powder or other bulk fiber supplements add unappetizing black flecks or an unpleasant thick consistency to drinks. But what about a fiber product that is designed to target a specific health-related purpose such as lowering cholesterol and is disguised in yogurts and desserts, soft breads and tortillas, and snack foods? Dow Wolff Cellulosics hopes its new food additive Fortefiber will fit the bill. A few years ago, Wallace H. Yokoyama, a research chemist at USDA’s WRRC, was studying how consuming modified cellulose reduces metabolic diseases in hamsters. He found that the soluble fiber helps keep blood sugar from spiking too high, a problem in diabetes. So Dow Wolff Cellulosics asked Yokoyama to collaborate on studies of Fortefiber, a water-soluble material created from the microcrystalline cellulosic pulp of a tree, the species of which the company declined to disclose. The additive is commercially available as a thickening agent, and the polymer lengths can be custom-designed to achieve specific food textures or to accommodate different formulations, ranging from liquids to snack bars. The proprietary, colorless semisynthetic compound is based on hydroxypropyl methylcellulose (HPMC), a compound that has been modified in hundreds of ways since Dow developed it 60 years ago as an alternative to animal gelatin in capsules,
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for example. Removing excess hydrogen bonds from cellulose makes HPMC-based molecules soluble. Clinical trials demonstrate that Fortefiber can reduce high blood glucose levels. It’s the same for low-density lipoprotein. The product is not found in stores yet, but Dow Wolff Cellulosics hopes to blend Fortefiber into ready-to-eat foods or to market it in supplement form as a powder or tablet, says Maciej Turowski, an analytical-chemist-turned-technical-marketing manager for the company. Although these new food research avenues show promise, the transition from lab to table isn’t guaranteed to suddenly boost public health. People have historically resisted dramatic changes in diet unless the shift is toward something that really tastes good. Clinical studies indicate that antioxidant compounds called polyphenols, which are found in grapes and other berries, boost cardiovascular health. But “it doesn’t taste good to dump old red wine in potatoes,” says John W. Finley, chair of the food science department at Louisiana State University. THE GLUT of nutritional advice flooding the public could make it difficult for fortified or functionalized foods like Fortefiber and BetaSweets to get a hold in the food market, Finley worries. He cautions that the myriad of products available in health stores with outrageous and possibly dangerous claims could bias people against these science-based innovations. For instance, consumers see many products “fortified” with ginseng or green tea, but the levels of these components are either undeclared or so low that they provide little or no benefit, he says. Also, scientists still have questions to answer. Adding various nutrients to a number of foods and juices may be too much of a good thing, says Jeffrey B. Blumberg, director of the USDA Antioxidants Research Laboratory and professor of nutrition science and policy at Tufts University. For example, it remains to be seen whether extra fatty acids, limonoids, or anthocyanins could be toxic if consumed in the increased amounts. In addition, he points to data showing that antioxidant activity in the lab may not translate to bioavailability in the human body. In time, science might in fact help identify an ideal diet. For now, the old standards of eating many different foods and in moderation are still the best guides. ■
