TECHNOLOGY
New Methods Remove Citrus Bitterness Immobilized bacterial cells, adsorbent resin, and inclusion compounds used to remove limonoids, flavonoids in citrus juices
MIAMI DEACH One of the major problems of the citrus industry worldwide is bitter ness in citrus juices and products caused by limonoids (predominantly limonin) and flavonoids (primarily naringin). Certain citrus fruits are unsuitable for processing because their juice becomes bitter soon after it is extracted—including certain va rieties of oranges, grapefruits, lem ons, mandarins, and minor citrus fruits such as Natsudaidai (Japanese summer orange), Iyokan, and Ponkan. California alone loses an esti
mated $8 million yearly, for example, owing to limonoid-caused "delayed bitterness" in citrus, primarily na vel oranges. There is no acceptable commer cial method for reducing levels of bitter citrus components, however. The usual method is just to blend excessively bitter juice with less-bit ter juice. Several new approaches to remov ing the bitterness from citrus were presented at a symposium on isola tion, identification, and control of bitter compounds in foods, orga nized by the flavor subdivision of the Division of Agricultural & Food Chemistry. The methods include use of an adsorbent resin, immobilized bacterial cells, and cyclodextrin poly mers to form inclusion compounds. And one participant outlined possi ble use of byproduct bitter compo nents from citrus for insect control. Twenty-nine limonoids, chemi cally related triterpene derivatives, have been isolated from citrus and citrus hybrids. Four of them are bit ter: limonin, nomilin, ichangin, and nomilinic acid. Limonin is the ma jor cause of juice bitterness. The
"bitterness threshold" for detec tion of its bitterness is 6 ppm. Intact fruits do not normally con tain limonin, note Shin Hasegawa and Vincent P. Maier of the Depart ment of Agriculture's Fruit & Vege table Chemistry Laboratory in Pas adena, Calif. However, they con tain a nonbitter precursor, limonoate Α-ring lactone, which differs from limonin only in having a free carboxy 1 group at carbon-16 and a free hydroxyl group at carbon-17. When the juice is extracted, this precursor lactonizes to the intensely bitter dilactone, limonin, upon standing for a few hours under the juice's acidic conditions (pH 3.5 to 4.5) or upon heating. The conversion is ac celerated by the enzyme limonin D-ring lactone hydrolase present in citrus. Thus, processes of juice extrac tion, heat treatment, and storage of juice or concentrate result in limonin-induced bitterness, especially in early-season orange, navel orange, and lemon juices. Early season or anges and tangerines have an espe cially high level of precursor. No milin bitterness is a relatively mi-
Limonin, nomilin, and naringin are causes of bitterness in citrus juices
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Limonin
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May 27, 1985 C&EN
Nomilin
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Naringin
nor problem and occurs mainly in grapefruit juices. Several approaches have been used to try to remove limonininduced juice bitterness—including preharvest treatment of citrus trees, postharvest fruit treatment, and treatment of the extracted juice. For example, Hasegawa and his coworkers found that preharvest treatment of citrus trees with triethylamine derivatives reduced formation of the limonin precursor. In preliminary tests, they are now finding that treating the trees with auxins, plant growth regulators, also significantly reduces precursor levels. Methods used until now have had severe economic and technical limitations, however. In addition, they have often impaired the juice's stability and quality by affecting other components besides limonin. Flavonoid bitterness is generally due to the presence of naringin (^S^-trihydroxyflavanone-Z rhamnoglucoside). It is the main bitter compound in juices of grapefruit and Natsudaidai, and is present in the intact fruit. The Florida state government sets a limit of 600 ppm on naringin in grapefruit juice. Several methods have been tried for removal of naringin in citrus juices, based on enzymic conversion to nonbitter naringenin by the enzyme naringinase. However, commercial naringinase preparations contain as impurities other enzymes that cause deterioration of the flavor and color of treated juice. And the added cost of purifying naringinase discourages its use by industry. Use of immobilized naringinase columns seems to reduce impurity and cost problems, and they have been used to debitter grapefruit juice and Natsudaidai juice. However, notes Ajai Puri of Coca-Cola Foods' citrus R&D laboratory in Plymouth, Fla., the immobilized enzyme techniques are limited in application. Puri instead has developed a unique method of removing both naringin and limonin at the same time. He does this by treating citrus juices with a polystyrene-divinylbenzene crosslinked copolymeric adsorbent resin. The bitter compounds are adsorbed onto the insoluble resin. His firm was issued a patent on the method last year (U.S.
| nutrients intact and does not introfc duce off-flavors. Columns can readily be regenerated by washing with dilute lye, which is used anyway in the citrus industry. An industrial firm is now planning pilot-plant tests. And patent protection is being sought. Meanwhile, Hasegawa is developing a biological process to reduce limonin and nomilin levels of citrus juices to below the bitterness threshold. He and his coworkers isolated from soil five bacterial species that can convert limonoids to nonbitter metabolites. They have established five metabolic pathways of limonoids in bacteria, and isolated a dozen limonoid-metabolizing enzymes. Use of the enzymes reduced limonin content below the bitterness Hasegawa: bacteria remove bitterness threshold in navel orange juice, but it requires large amounts of en4,439,458), but is not yet ready to zymes. Hasegawa therefore has tried discuss plans for commercialization. whole bacterial cells, immobilized Puri has used the resin to debitter in acrylamide gel and packed in a grapefruit, orange, lemon, tanger- column. He finds them to be very ine, and other citrus juices, with no effective. For example, a column significant effect on total soluble packed with 2.8 g of immobilized solids or ascorbic acid levels. A ma- cells reduces 20 ppm or higher levjor advantage is that he uses a els of limonin to below 5 ppm. The commercially available resin, Duo- column can be used 15 to 20 times lite S-861, from Diamond Shamrock. before losing effectiveness and reThe resin has a high capacity and quiring regeneration. Hasegawa can process a large quantity of juice finds no significant adverse effects on other juice components. A pribefore needing regeneration. vate firm, California Citrus ProducAnother method of removing both limonin and naringin is being ers of Lindsay, Calif., is planning developed by Philip E. Shaw and pilot-plant tests of the process on Charles W. Wilson III of USDA's navel orange juice. citrus and subtropical products lab At present, the biological funcin Winter Haven, Fla. They poly- tions of citrus limonoids are unmerize water-soluble cyclodextrins known. However, in lab tests, the with epichlorohydrin and other limonoids—limonin, nomilin, and crosslinking agents to form insolu- obacunone—show antifeeding activble polymers. Some of these poly- ity against some insects, including mers retain the cyclodextrin mono- the fall armyworm and cotton bollmer's ability to form inclusion com- worm, says Isao Kubo of the Depounds with molecules that have partment of Entomology and Parathe right shape and size to fit in the sitology at University of California, cavity of the doughnut-shaped Berkeley. His group has not yet done cyclodextrin—in particular, limonin field tests. However, he notes, limonand naringin. oids are natural, environmentally The researchers find that /3-cyclo- compatible products and probably dextrin polymers can selectively re- would be available as inexpensive move limonin from navel orange by-products from citrus juice, peels, juice, and limonin and naringin and seeds. For example, he estimates, from grapefruit juice. They have more than 300 metric tons of limonstudied the optimum conditions for oids would be available annually both batch and continuous-column just from grapefruit seeds. This could treatment of clear and pulpy juices. make citrus limonoids promising D Shaw says the method leaves juice candidates for pest control. May 27, 1985 C&EN
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