Not So Radical Antioxidants

Today’s consumers are continually looking for new sources of assistance in their quest for health and longevity. With growing awareness of how many diseases result from cellular damage, growing numbers of health-hunters are seeking foods rich in antioxidants, the components that guard against cell-ravaging free radicals.

Back to school 

First, a quick refresher in molecular chemistry. Each atom on a molecule has a nucleus that contains positively charged protons, while negatively charged electrons orbit the nucleus in one or more shells. Electrons will fill the shells in order: the first shell can hold two electrons; the second, eight; the third, 18; the fourth, 32; the fifth, 50; and the sixth, 72. The number of electrons in the outermost, or valence, shell determines the atom’s ability to participate in reactions with other atoms. If the outermost shell is full, the atom is less likely to participate in reactions and is referred to as “inert.”

Typically, atoms “match up” in a way that leaves no unpaired electrons. Oxygen can interact with certain compounds, though, leaving an unpaired electron behind. These highly unstable atoms are called “radicals” or “free radicals.” Seeking stability, free radicals will attack stable molecules and steal an electron, creating a new radical.

The body sometimes initiates the creation of free radicals to defend itself against viruses or bacteria. Chemical oxidation can, however, attack cells’ mitochondria, membranes, enzymes and DNA. Left unchecked, the cascade effect continues degenerating the structures, causing the cells to malfunction or reproduce improperly.

The heart of the matter 

Case in point: Many researchers believe that oxidation, and subsequent attack by free radicals, of the innermost layer of the artery (endothelium) is the initial cause of atherosclerosis, a form of arteriosclerosis that describes the building up of “plaque” (cholesterol, fatty material, calcium, fibrin and cellular waste products), which can lead to a heart attack or stroke. Another theory is that oxidized low-density lipoprotein (LDL) is preferentially taken up by macrophage cells that, when filled, become “foam cells.” The foam cells build up into fatty streaks—early indicators of atherosclerosis.

In 1975, Bruce N. Ames, professor and researcher, biochemistry and molecular biology, University of California, Berkeley, developed a method for predicting DNA damage that would lead to mutation, the initial steps in the development of cancer. He went on to indicate dietary links to degenerative diseases, suggesting that these disorders are oxidative in origin.

In addition to cancer and cardiovascular disease, effects of oxidative reactions have been implicated in numerous afflictions, such as premature aging, inflammatory diseases, Alzheimer’s disease, immune dysfunction, macular degeneration, Parkinson’s, periodontal disease and cataracts.

To help fight these diseases, Mother Nature has given us numerous substances that can halt the damaging chain reaction started by free radicals. In nutrition, “antioxidants” are nutrients that can donate an electron to unstable free radicals without becoming free radicals themselves. In doing so, they terminate the oxidative chain reaction and the progression of damage to the body. While our bodies cannot manufacture these materials for themselves, Mother Nature provides us with numerous sources for these crucial dietary elements. And while there may be debate over the exact mechanisms of antioxidants in our bodies and the exact amounts needed, there can be little doubt that we’re better off with them than without.

The antioxidant content of a food or ingredient is usually expressed as ORAC (oxygen radical absorbance capacity). The ORAC assay measures a specific reaction of a free radical reactive species and uses the data to plot an area under the curve to quantify the amount of antioxidants. (See “Determining ORAC” in the Dec. 2004 issue of Food Product Design for more information.) Trolox equivalent antioxidant capacity (TEAC) is another assay used and measures antioxidants in Trolox equivalents (TE).

Seeing red, orange and yellow 

Red, orange and yellow tones in fruits and vegetables come, in part, from a family of pigments known as carotenoids. This roughly 700-member family of fat-soluble nutrients is split into two groups. All carotenoids are polyunsaturated; they possess double bonds between carbon atoms. The number of double bonds and their position determine the color. Carotenoid chains that end with a cyclic hydrocarbon group are carotenes. Adding an oxygen-containing functional group yields xanthophylls.

Beta carotene, probably the most well-known carotenoid, is a precursor to vitamin A. Although most often associated with carrots, we can also obtain this powerful antioxidant from red palm oil, apricots, cantaloupe, kiwi and papaya. Research has linked it to preventing certain types of cancers and strengthening of the immune system. Oil- and water-miscible beta carotene ingredients are available for a variety of food and beverage applications.

Lycopene, another well-known carotene, has been the subject of much recent study, and has been linked to reduced risk of cancers of the prostate, colon, breast, lung and digestive tract. Lycopene can be obtained from such fruits as watermelon, pink grapefruit, peaches and apricots, but “tomatoes and tomato products, both today and in the past, were the main source of lycopene,” says Zohar Nir, Ph.D., vice president, new product development and scientific affairs, LycoRed Ltd., Beer Sheva, Israel. “The Mediterranean diet, widely viewed as a ‘healthy’ diet, made extensive use of tomatoes. The high content of lycopene in this type of diet contributes to its beneficial effect on health. This effect can also be achieved today by fortification with lycopene.”

Absorption rate of lycopene from tomatoes increases when cooked, and when eaten together with a healthy oil. Natural lycopene extracts for fortification are produced from tomatoes bred for high lycopene content. Because the tomato derivatives initially used for lycopene fortification could impart color and flavor to a product, applications have typically been limited to tomato-based products. Advances in extraction technology have, however, opened new areas for developers to explore. “Recently, a Japanese company has broken new ground with a lycopene-fortified tomato liquor,” Nir says.

“Each serving of this bright-red wine provides 10 mg of lycopene. And in Korea, a company recently launched a yogurt drink fortified with lycopene and other natural antioxidants.”

Xanthophylls are found in egg yolks, yellow fruits and some green vegetables, although the green chlorophyll often obscures the yellow color in the latter. Two xanthophylls, lutein and zeaxanthin, create the color of a yellow spot on the retina known as the macula lutea, and scientists believe they also protect the eye by filtering out harmful blue light and neutralizing free radicals. As the eye ages, oxidized proteins and debris build up and break down the light-sensitive cells within the macula, causing age-related macular degeneration (AMD). According to a 2003 report by USDA’s Agricultural Research Service, over 1.6 million Americans over the age of 55 were suffering from advanced AMD, while as many as 7 million more were at some intermediate stage. While AMD is irreversible, consumption of lutein and zeaxanthin might reduce risk of developing the disease.

Carotenoids are available in various forms—dry, beadlets, liquid, fat-based, water-based— to meet a variety of application needs. Additional flexibility may be gained by utilizing an encapsulated form of the product, or by combining the carotenoids with other antioxidants to inhibit premature interaction of the fortificant with oxygen from the environment or the food system.

Pick your fla-vorite 

Deeply colored fruits, teas and some greens produce a large family of compounds known as flavonoids. Studies associate these compounds’ antioxidant activity with numerous health benefits. The group is divided into classes by variations in chemical structure.

Anthocyanidins include the dietary flavonoids cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin. They are commonly found in red, blue and purple berries; red and purple grapes; and red wine. They might bolster cellular antioxidant defenses and contribute to maintenance of brain function.

Flavanols are grouped by unit size. Monomers are referred to as catechins, and include catechin and epicatechin. These single units can bind with gallic acid to form catechin or epicatechin gallates (ECG). Green and white teas provide rich supplies of catechins, of which epigallocatechin gallate (EGCG) provides the most antioxidant activity. Various studies have found that the EGCG concentration in teas ranges from 127 to 550 mg/L, but the actual content varies with processing, preparation and other factors. Chocolate, grapes, berries and apples also provide catechins.

Flavanol monomers may also bind together to form dimers, trimers, etc. Polymeric forms include theaflavins, thearubigans (in black and oolong teas) and proanthocyanidins (in berries, red grape seeds and skins, apples, and chocolate). The latter group is also referred to as procyanidins, oligomeric proanthocyanidins (OPCs) and procyanidolic oligomers (PCOs). OPCs can scavenge positively and negatively charged free radicals, making them more-effective antioxidants than vitamins C or E.

A less-familiar group, flavanones are found in citrus fruits and juices, such as orange, grapefruit and lemon. Hesperetin, naringenin, tangeritin and eriodictyol are strong antioxidants that neutralize cell-damaging free radicals and enhance cellular antioxidant defenses.

Flavonols differ from flavanols (note the “o” instead of the “a”) in that they possess a double-bonded oxygen (similar to flavones). Widely distributed among yellow onions, scallions, kale, broccoli, apples, berries, tea and cocoa, flavonols include the most-abundant flavonoid, quercetin. In addition to improvement of cardiovascular health and reducing risk of cancer, quercetin’s strong antioxidant action provides anti-inflammatory and anti-allergic effects. Like many of its flavonoid cousins, quercetin also prevents LDL oxidation. Keampferol, another flavonol, provides a strong antioxidant effect, and is stronger when working synergistically with quercetin.

Flavones and isoflavones round out the flavonoid family. Similar to flavonols, flavones are missing an alcohol group. Found in parsley, celery, thyme and hot peppers, examples include apigenin and luteolin. Isoflavones differ from flavones in the position of its third carbon ring. Isoflavones genistein and daidzein can be obtained from red clover and alfalfa, as well as peas, soy and other legumes. Both have been shown to exhibit anti-inflammatory and cardioprotective traits and act as phytoestrogens.

Smaller functional families 

Red and purple grapes are also good sources of phenolic compounds such as resveratrol. First studied as part of the investigations into the healthful effects of wine, resveratrol might improve coronary health and inhibit proliferation of breast, prostate, stomach, colon, pancreatic and thyroid cancer cells. Many fruits contain ellagic acid, another phenolic compound; the highest levels are found in raspberries, strawberries and pomegranates. Animal studies indicate ellagic acid’s inhibitory effect on tumor growth.

Isothiocyanates are breakdown products created by extraction and subsequent enzymatic hydrolysis of glucosinolates—sulfur-containing compounds found in cruciferous vegetables. Chewing certain leafy greens, like broccoli or kale, releases and converts certain components into powerful antioxidants like sulforophane. Studies have shown isothiocyanates prevent the proliferation of bladder-cancer cells.

Garlic and onions supply a group of antioxidants known as sulfides. Compounds such as diallyl sulfide and allyl methyl trisulfide appear to possess antimutagenic and anticarcinogenic properties, as well as immune and cardiovascular protection. They also appear to inhibit growth and/ or progression of tumors, fungi, parasites, cholesterol and platelet and/or leukocyte adhesion factors.

Don’t forget your vitamins 

Vitamin E is well known for its antioxidant effects on heart disease, and ongoing studies indicate positive effects on certain types of cancer. Vitamin E is actually a combination of fat-soluble molecules that occur naturally in eight different forms: alpha, beta, gamma, and delta tocopherols and tocotrienols. Obtained from whole grains, nuts, seeds, fish and vegetable oils, vitamin E is the most-abundant fat-soluble antioxidant in the body, and our primary defender against lipid oxidation.

Alpha-tocopherol, natural vitamin E, the most stable of the group, will oxidize slowly when exposed to air. Additionally, vitamin E can get “confused” about its role in a food system— oxygen scavenger or fortificant. The latter may require protection from the environment, such as encapsulation, to deliver antioxidant nutrient properties.

Fat-soluble vitamin E can also impart turbidity to clear beverages and gels if not in an appropriate form. Encapsulation is one option; however, the encapsulation material must not interact adversely with components of the product being fortified. Gelatin-based coatings, for example, can interact with polyphenols in a fruit juice, resulting in sedimentation. Vitamin C, ascorbic acid, is our most-abundant water-soluble antioxidant. Working mainly in cellular fluid, vitamin C combats the formation of free radicals that result from exposure to pollutants and smoke.

As with alpha-tocopherol, vitamin C’s sensitivity to heat and oxygen, as well as its affinity for minerals, can cause problems with long-term stability in a food system. Lipid encapsulation can protect the vitamin from its environment or the conditions of processing. A heat-stable coating will, for example, protect vitamin C added to a baked-bar product.

Vitamin A also provides an antioxidant effect. Available from animal sources such as liver and butter, or by converting beta carotene from plant sources, vitamin A brings similar health benefits as beta carotene, but it is less stable than the pro-vitamin A carotenoids. Esterifying the alcohol form of vitamin A, retinol, to create retinyl acetate, palmitate and propionate, provides some protection, but does not eliminate the susceptibility of its double bonds. Developers must balance stability with bio-availbility, as the body’s conversion rate for beta carotene to retinol is approximately 6:1.

The essential mineral selenium functions as a component of several enzymes that participate in the removal of free radicals from the body. The RDA is 55 ìg/day for adult (19+) males and females, 60 ìg/day for pregnant females, and 70 ìg/day for lactating females. Its own antioxidant properties appear to inhibit proliferation of lung-, prostate- and colon-cancer cells. Evidence of selenium’s protective effect on pancreatitis, asthma and the effects of rheumatoid arthritis also exists. Formulating with minerals poses unique challenges, as they can impart a metallic taste. Studies worldwide have, however, yielded effective selenium-enriched milk and bread products.

Less familiar faces 

Red beets have been the subject of research into a new chemical class of antioxidants. These antioxidants, “betalains, specifically betanin, possess a strong freeradical- scavenging activity and stimulatory effect on phase 2 enzyme activity and expression,” says Zbigniew (ZB) Pietrzkowski, Ph.D., vice president of research and development, VDF Future- Ceuticals, Inc., Momence, IL. “Studies have shown favorable bioavailability, stability and pharmacokinetics of betalains, in addition to the unique ability to neutralize the devastating effect of hypochlorous acid, the most powerful oxidant generated by human neutrophiles.”

Pietrzkowski also suggests probiotic cultures may hold antioxidant benefits. “Selected strains of probiotics may enhance the ability of metal ions to be chelated, scavenge reactive oxygen species and provide protection from lipid oxidation,” he says. “Studies showed that certain probiotics may significantly reduce lipid peroxidation in rats treated with 1,2-dimethylhydrazine, which is a known inducer of colon carcinogenesis. Under the reported experimental conditions, DNA damage has been shown to be significantly reduced, as well as the number of preneoplastic lesions.”

Some antioxidants regenerate other antioxidants. Ubiquinone, also known as coenzyme Q10 (CoQ10)—the “Q” referring to quinone chemical group and “10” indicating 10 isoprenyl chemical subunits—acts as a powerful antioxidant and is essential in the reduction of oxidized vitamin E. Lipoic acid is also a strong hydroxy-radical quencher active in tissue fluids and lipids. The dual-medium functionality makes lipoic acid an important protectant for vitamins A and C.

Upping the anti 

Product designers can incorporate antioxidants in foods and beverages in many different ways. Cranberries, for example, are a rich source of proanthocyanidins (PAC). According to the “USDA Database for the Proanthocyanidin Content of Selected Foods” (2004), cranberries provide 418.8 mg PAC per 100 grams, compared to 145.0 mg per 100 grams for strawberries; 81.5 mg per 100 grams for green grapes; and 27.0 mg per 100 grams for blackberries. Using specialty cranberry products, developers can add the health-packed fruits without fear of process-induced loss. “Sweetened, dried cranberries are hardy and process tolerant, offering manufacturing stability that maintains piece identity without suffering water migration or color bleed, and keeping their flavor and texture throughout processing and storage,” says Kristen Girard, principal scientist, food ingredients, Ocean Spray ITG, Middleboro, MA. “They can be incorporated directly into a batter or dough with no need to presoak or pretreat, and no risk of compromising end-product consistency.”

Blueberries also offer developers a versatile ingredient for adding antioxidants. The 3-glucosides and 3-galactosides of delphinidin, malvidin, petunidin, cyanidin and peonidin are the primary anthocyanins that have been identified in blueberries. “One of the most-important benefits of blueberries is the multiple variety of formats available year round, such as fresh, dehydrated, concentrate, juice, purée and canned,” explains Tom Payne, industry specialist, U.S. Highbush Blueberry Council, San Francisco. “For example, the benefits of blueberries in dried form include easy integration into dry goods and intermediate-moisture products, consistent piece size and integrity, ability to use as a coating (for chocolate confectionery, breakfast cereals or bars), crisp flavor notes, and specialty products that are moist and plump, yet do not require refrigeration.”

A study at Texas Tech University, Lubbock, examined the effects of adding 5% and 10% blueberry or dried plum purée to a precooked pork patty. Cooked sausages had an average 36% increase in phenolic content. And, when 10- to 12-year-old children were surveyed on the taste, mouthfeel and appearance of the test patties vs. a regular breakfast sausage patty, no difference was noted. Approximately 90% of the children said they would eat the blueberry sausage patty again, leading to the conclusion that phenolic content of pork sausage patties can be increased by adding fruit purées while maintaining consumer acceptance.

But remember that all antioxidant ingredients are not created equal. For example, researchers (“Procyanidin and catechin content and antioxidant capacity of cocoa and chocolate products,” L. Gu, S. House, X. Wu, B. Ou, R.L. Prior, Journal of Agriculture and Food Chemistry, 2006, 54(11):4057-4061) found natural cocoa powders contained the highest levels of total antioxidants and procyanidins, the dominant antioxidant in chocolates. Procyanidin content was related to the nonfat cocoa solid (NFCS) content. The natural cocoa powders (average 87% of NFCS) contained the highest levels of antioxidants (1100±76 mole TE/ gram) and procyanidins (40.8±8.3 mg/gram). Milk chocolates, with the lowest level of cocoa solids, had the lowest TAC and procyanidin levels. Alkalinization, or “dutching,” significantly reduces cocoa’s antioxidant (531±50 mole TE/gram) and procyanidin content (8.9±2.7 mg/gram).

Antioxidant content of plant-based foods also appears to vary by species and maturity, says Ronald L. Prior, Ph.D., USDA Human Nutrition Research Center on Aging Arkansas Children’s Nutrition Center, Little Rock, AR. He observes that “maturity at harvest had a marked effect on ORAC, total anthocyanins and total phenolics” of Brightwell and Tifblue cultivars of rabbiteye blueberries. Those harvested immediately after turning blue had lower ORAC and total anthocyanins than mature berries harvested 49 days later. “ORAC and total anthocyanins increased 224% and 261% respectively, in the Brightwell cultivar, while in Tifblue they increased 164% and 176% respectively, with increasing maturity,” he says. “Total phenolics increased by 169% and 113% in the Brightwell and Tifblue cultivars, respectively, with increased maturity.”

Beyond healthy 

Many antioxidants offer benefits outside the realm of consumer health. Beta carotene can be used to create a range of yellow, orange or red hues in foods. The fat-soluble vitamin can be processed into different forms to allow for addition to water- or fat-based products.

Growing awareness of their antioxidant potential has spurred growing use of anthocyanins as colorants. Colors imparted are most vibrant when pH is 3.0 to 3.5. Although products colored with anthocyanins are sometimes more susceptible to light-induced fading, they offer developers the ability to blend and create a variety of rich colors with an “all natural” label.

Grape seed extracts have been studied for their ability to inhibit textural and aromatic changes in irradiated chicken breast. Addition of OPCs not only minimized the changes noted, they also contributed to improved juiciness during storage.

And there is, of course, the effect antioxidants have on the foods themselves. Damage-control mechanisms operating within the products can work to maintain the appearance and taste of a food, ensuring that consumers enjoy the product, as well as benefit from the healthful compounds contained therein. 

R. J. Foster is a communications specialist with over 15 years of experience in the food industry in technical service, research & development, quality control, regulatory and technical sales. He can be reached by e-mail at [email protected].