Biochemistry and Biological Removal of Limonoid Bitterness in Citrus

Chapter 8

Biochemistry and Biological Removal of Limonoid Bitterness in Citrus Juices

Quality Factors of Fruits and Vegetables Downloaded from pubs.acs.org by CORNELL UNIV on 08/30/16. For personal use only.

Shin Hasegawa Fruit and Vegetable Chemistry Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 263 South Chester Avenue, Pasadena, CA 91106 This review paper summarizes the biochemical and b i o l o g i c a l aspects of limonoids i n c i t r u s and presents possible preharvest approaches to reduce b i t t e r limonoids i n c i t r u s f r u i t s .

Bitterness due to limonoids i n a variety of c i t r u s juices i s a major problem of the c i t r u s industry worldwide and has s i g n i f i c a n t negative economic impact. Recently, Maier, et a l . (1) published a comprehensive review of limonoid chemistry. More recently, Dekker (2) reviewed the impact of the limonoid bitterness on juice quality and postharvest approaches to the problem. Hasegawa (3) reviewed the metabolism of limonoids i n microorganisms and the development of a reactor, that uses immobilized b a c t e r i a l c e l l s f o r reduction of b i t t e r limonoids i n citrus juices. Limonoids are a group of chemically related triterpene derivatives found i n the plant f a m i l i e s , Rutaceae and Meliaceae. Among 37 limonoids reported to occur i n c i t r u s and hybrids, four are known to be b i t t e r . They are limonin, nomilin, ichangin and nomilinic a c i d . Limonin i s the major cause of c i t r u s juice bitterness and i s widely d i s t r i b u t e d . Nomilin i s also known to contribute to limonoid bitterness i n grapefruit juice (4). Recently, limonoids were shown to occur as glucoside derivatives (Hasegawa, S., et a l . Phytochemistry i n press). Ten limonoid glucosides have been isolated from grapefruit seeds and orange j u i c e s . Each consists of a limonoid linked with one glucose molecule at C-17 by a 3-glucosidic linkage. Limonin 17-0-3-D-glucopyranoside appears to have an astrigent and s l i g h t l y b i t t e r taste (Koski, P., Tropicana, FL. personal communication) as compared to the intense bitterness of i t s aglycone. Since c i t r u s juices contain r e l a t i v e l y high concentrations of limonoid glucosides, these compounds may contribute to the c i t r u s juice flavors. The monolactones such as limonoate Α-ring lactone are the This chapter not subject to U.S. copyright Published 1989 American Chemical Society

8. HASEGAWA

Removal ofLimonoid Bitterness in Citrus Juices

85

predominant limonoid aglycones present n a t u r a l l y i n c i t r u s leaves and f r u i t tisues, whereas the dilactones such as limonin are the predominant limonoid aglycones present i n c i t r u s seeds. In t h i s a r t i c l e the author w i l l not make a d i s t i n c t i o n between monolactones and dilactones.


Delayed Bitterness Limonin and nomilin are two b i t t e r limonoids present i n c i t r u s juices. Systematic organoleptic tests showed that the bitterness threshold i s 6 ppm (5) f o r limonin, and 6 ppm (6) or 3 ppm (7) f o r nomilin. The bitterness due to limonin develops gradually i n juices after extraction from c e r t a i n v a r i e t i e s of oranges, grapefruit, lemon, Natsudaidai, mandarin and some other minor c i t r u s such as Iyokan and Ponkan. This phenomenon i s generally referred to as "delayed bitterness". The intact f r u i t s do not contain b i t t e r limonin, but rather a nonbitter precursor, limonoate Α-ring lactone (8). When juice i s extracted, t h i s nonbitter precursor i s gradually converted to limonin under a c i d i c conditions and the conversion i s accelerated by the action of limonin D-ring lactone hydrolase, which has been isolated from c i t r u s (9). The bitterness due to nomilin i n juices most l i k e l y develops i n a manner similar to that of limonin bitterness, but this has not yet been d i r e c t l y proven. However, the contribution of nomilin to juices i s minor. I t occurs mainly i n grapefruit juices (4). Biosynthesis of Limonoids i n Citrus During the past several years, s i g n i f i c a n t progress has been made i n studies on the biosynthesis of c i t r u s limonoids. Based on recent radioactive tracer work, the biosynthetic pathways of the major limonoids i n common c i t r u s are well established ( F i g . 1). Preparations of Radioactive Substrates. Young c i t r u s seedlings are excellent tools for the preparation of 14-C nomilin from labeled acetate. When fed to stems of lemon seedlings, up to 5% of the labeled acetate was incorporated into nomilin (10). Incorporation of acetate was the best followed by mevalonate and farnesyl pyrophosphate. This high value of incorporation supports the fact that nomilin i s one of the major compounds which accumulate i n seedlings. Labeled obacunone was prepared enzymatically from labeled nomilin with nomilin acetyl-lyase which was isolated from Corynebacterium fascians (11). Labeled obacunoate was prepared enzymatically from labeled obacunone with obacunone Α-ring lactone hydrolase obtained from c e l l - f r e e extracts of £. fascians (12). Citrus seedlings were not capable of accumulating deacetylnomilin or deacetylnomilinate. Therefore, labeled deacetylnomilin was synthesized from labeled acetate i n young seedlings of calamondin (Citrus r e t i c u l a t a cv. Austera χ Fortunella sp.). Labeled deacetylnomilin was chemically converted to labeled deacetylnomilinate (13). Labeled 6-keto-73-nomilol was biosynthesized from labeled acetate i n calamondin seedlings (14).


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QUALITY FACTORS OF FRUITS AND

VEGETABLES

Biosynthesis of Major Limonoids. Radioactive tracer work showed that 14-C labeled acetate, mevalonate and farnesyl pyrophosphate are incorporated into nomilin i n young lemon seedlings (10). Nomilin did not appear to be further metbolized i n the seedlings. On the other hand, i n older c i t r u s , nomilin was further converted to obacunone, obacunoate and limonin (11, 15). The conversion of nomilin to obacunone appears to be catalyzed by the action of nomilin acetyl-lyase and the conversion of obacunone to obacunoate by obacunone Α-ring lactone hydrolase. Both enzymes have been isolated from c e l l - f r e e extracts of _C. fascians (16), but they have not yet been isolated from c i t r u s . The major difference i n the limonoid biosynthetic enzymes of young seedlings as compared with older trees i s that nomilin acetyl-lyase a c t i v i t y i s missing i n young seedlings and, therefore, nomilin accumulates as the predominant limonoid (10). However, when labeled obacunone was fed to a young shoot of either young seedlings or older lemon trees, both tissues converted obacunone to obacunoate and to limonin (11). In young seedlings, although there i s no detectable nomilin acetyl-lyase a c t i v i t y , the enzyme systems leading from obacunone to limonin are present just as i n older c i t r u s . Thus, nomilin acetyl-lyase appears to play a key role i n the regulatory system which controls the biosynthesis and accumulation of limonoids i n c i t r u s .

from stems

Deacetylnomilinate b > Nomilin

->

Deacetylnomilin

->

Nomilinate

4

Obacunone i

-> 7a Obacunol 7a-0bacunyl acetate

Obacunoate (Ichangin) Limonin

Limonol Limonyl acetate Deoxylimonin Deoxylimonol 17-Dehydrolimonoate Α-ring lactone

Limonoids -> Limonoid glucosides (Late stages of f r u i t growth and maturation) Figure 1.

Biosynthetic pathways of limonoids i n c i t r u s .

Limonin appears to be the l a s t major limonoid to be biosynthesized. There are several possible precursors present between obacunoate and limonin, including isoobacunoate and


8. HASEGAWA


87

ichangin, a l l of which have been isolated from c i t r u s . Since labeled isoobacunoate was not converted to limonin i n tissues of either seedlings or mature trees i n several t r i a l s (Hasegawa, S., USDA, ARS, Pasadena, CA, unpublished data), i t i s most l i k e l y that ichangin i s the direct precursor of limonin. We have not been able to biosynthesize labeled ichangin for use as a substrate. Recently, deacetylnomilinate was found to be the i n i t i a l precursor of a l l the limonoids known to be present i n c i t r u s (13). When 14-C labeled deacetylnomilinate was fed to the stem of a lemon seedling, i t was converted mainly to nomilin. According to the previously proposed pathways (17), nomilin and obacunone were thought to be biosynthesized from deacetylnomilinate v i a nomilinate and/or deacetylnomilin. However, recent work has c l e a r l y demonstrated that deacetylnomilinate i s the i n i t i a l precursor of a l l the known c i t r u s limonoids. This finding well established the biosynthetic pathways of the major limonoids i n c i t r u s . Biosynthesis of Minor Citrus Limonoids The conversion of limonin to 17-dehydrolimonoate Α-ring lactone (18) and to deoxylimonin (19) have been shown i n c i t r u s . Deoxylimonate i s most l i k e l y formed from deoxylimonin as seen i n bacteria. We recently observed the conversion of labeled nomilin to nomilinate i n young lemon seedlings (15), showing that nomilinate i s a metabolite of nomilin. Most l i k e l y , deacetylnomilin i s biosynthesized from deacetylnomilinate (11). There i s no evidence of the direct conversion of nomilin to deacetylnomilin i n common c i t r u s . The other minor limonoids such as deoxylimonol, limonol and limonyl acetate are considered to be metabolites of limonin. 7a-0bacunol and 7a-obacunyl acetate are most l i k e l y metabolites of obaccunone Biosynthesis of Limonoids i n Citrus ichangensis Citrus ichangensis possesses an unusual d i s t r i b u t i o n of limonoids (20). Unlike other species of c i t r u s , f r u i t tissues and seeds of C_. ichangensis contain very high concentrations of nonbitter ichangensin and very low concentrations of b i t t e r limonin. I t also contains r e l a t i v e l y high concentration of nonbitter deacetylnomilin. Quantitative analysis showed that a r a t i o of ichangensin to limonin i n the f r u i t tissue i s approximately 50 to 1. When labeled nomilin was fed to £. ichangensis f r u i t tissues, i t was metabolized to deacetylnomilin and ichangensin (Hasegawa, S. et a l . , USDA, ARS, Pasadena, CA, unpublished data). Therefore, ichangensin i s most l i k e l y biosynthesized from nomilin v i a deacetylnomin as shown i n F i g 2. The conversion of nomilin to deacetylnomilin i s catalyzed by nomilin acetyl esterase. The presence of this enzyme a c t i v i t y i n C_. ichangensis i s unique to this species. In other c i t r u s or microorganisms, nomilin i s mainly converted to obacunone by nomilin acetyl-lyase (15). The formation of ichangensin i s the predominant biosynthetic pathway of limonoids i n iC. ichangensis. Biosynthesis of Calamin Group Limonoids i n Calamondin In addition to the above, there i s another group of limonoids present i n a c i t r u s hybrid, calamondin (Citrus r e t i c u l a t a cv.

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QUALITY FACTORS OF FRUITS AND VEGETABLES

Nomilin

> Deacetylnomilin

ι

1

(See F i g . 1)

Ichangensin

1 Limonin


Figure 2.

Biosynthetic pathways of limonoids i n Citrus ichangensis

Austera χ Fortunella sp.) and kumquat (Fortunella magarita). This group includes calamin, retrocalamin, cyclocalamin, isocyclocalamin, methyl isoobacunoate diosphenol, 6-keto-7$-nomilol, 6-keto-73 -deacetylnomilol (21-4). Based on radioactive tracer work, we have proposed the biosynthetic pathways of this group of limonoids as shown i n F i g . 3.

Deacetylnomilinate 6-keto-73-Nomilol 6-keto-7$-Deacetylnomilol Calamin

> Nomilin (See F i g . 1) Limonin > Retrocalamin

Cyclocalamin Isocyclocalamin Figure 3.

Possible biosynthetic pathways of limonoids i n calamondin.

Biosynthesis of Limonoid Glucosides Limonoids are also present i n c i t r u s as glucoside d e r i v a t i v e s . Recently, we reported the findidng of 17-0-3-D-glucopyranosides of the major limonoids such as limonin, nomilin, deacetylnomilin, obacunone, nomilinic acid, deacetylnomilinic acid, isoobacunoic acid, epiisoobacunoic acid, obacunoic acid and trans-obacunoic acid i n grapefruit seeds (Hasegawa, S. et a l . , Phytochemistry i n press). These glucosides are present i n high concentrations r e l a t i v e to the aglycones. For example, commercial orange, grapefruit and lemon juices contain an average 320 ppm, 200 ppm and 90 ppm of t o t a l glucosides, respectively (25). They may be of significance i n the area of taste of c i t r u s products, limonoid debittering and human nutrition. In further research on the limonoid glucosides, we found that they appear to be present only i n f r u i t tissues and seeds. Radioactive tracer work showed that the f r u i t tissue i t s e l f i s

8. HASEGAWA

Removal of Limonoid Bitterness in Citrus Juices

89

capable of biosynthesizing the glucosides. They are biosynthesized during late stages of f r u i t growth and maturation i n navel and Valencia oranges, and lemon ( F i g . 1). In navel oranges, they appeared i n the edible portion of f r u i t before they appeared i n the peel, and their concentrations increased as maturity of the f r u i t progressed.


Sites of Limonoid Biosynthesis i n Citrus Stems are the major s i t e of nomilin biosynthesis from acetate i n c i t r u s (26). Analysis of the phloem, the cortex and the inner core regions of the stem showed that the phloem region i s the s i t e of nomilin biosynthesis from acetate (27). Root tissues also have this capacity. Leaves, f r u i t s and seeds are either incapable of biosynthesizing limonoids from acetate or have a very low capacity. However, these tissues are capable of biosynthesizing limonoids from nomilin. Nomilin i s translocating from the stem to other locations, where i t i s further biosynthesized to other limonoids (26). B i o l o g i c a l A c t i v i t y of Limonoids Certain c i t r u s limonoids induce glutathione S-transferase, a detoxifying enzyme, i n the l i v e r and small i n t e s t i n a l mucosa of mice, and i n h i b i t a benzo(a)pyrene-induced neoplasia i n the forestomch (28). This work i s at a preliminary stage and further work i s needed to f u l l y determine the anti-cancer a c t i v i t y of limonoids, and, i n p a r t i c u l a r , their glucosides. Since we have found a number of species of bacteria capable of hydrolyzing limonoid glucosides, these compounds may be hydrolyzed by the i n t e s t i n a l f l o r a to l i b e r a t e limonoid aglycones. Therefore, c i t r u s f r u i t and juices, as a source of these natural products, may provide chemopreventative benefits against cancer. Citrus seeds are also an excellent source of these compounds. Certain limonoids also possess an antifeedant a c t i v i t y against insect pests such as f a l l armyworm, cotton bollworm and spruce budworm (29, 30). Limonoids do not d i r e c t l y k i l l the pests, but gradually lower their growth rates. Preharvest Approaches to the Limonoid Bitterness Problem Three possible preharvest approaches have been proposed and on these subjects i s underway at the Pasadena laboratory.

research

Inhibition of Limonoid Biosynthesis by Auxins. Auxins are potent i n h i b i t o r s of nomilin biosynthesis i n c i t r u s seedlings (31). For instance, up to 91% i n h i b i t i o n was observed when 10 ppm of indoleacetic acid was fed to the stem of a lemeon seedling two days prior to and two days following feeding of 25 pCi of 14-C acetate (Table 1). Other auxins tested include 1-naphthaleneacetic acid (NAA), indolepropionic a c i d , indolebutyric a c i d , 3 -indole a c e t o n i t r i l e , ethyl indole-3-acetate, 3-indoleacrylic a c i d , 3-(2-hydroxyethyl)indole, indole-2-carboxylic acid and 2,3,4-trichlorophenoxyacetic acid. They were a l l very e f f e c t i v e .

90


Radioactive tracer work also demonstrated that NAA i n h i b i t s the accumulation of limonoids i n f r u i t tissues of lemon trees (32). When 3-cm-long stems with small lemon f r u i t s attached were fed with 14-C labeled acetate after having already been treated with 20 ppm

Table 1.

Inhibition of Nomilin Biosynthesis by Indoleacetic Acid i n Young Lemon Seedlings


IA (ppm) 0 10 25 50

Nomilin (cpm) 1,127.900 100,400 125,000 95,000

Inhibition (%) 0 91 89 92

Source: Reprinted with permission from r e f . 31. Copyright 1986. of NAA for three days, there was an 80-100% i n h i b i t i o n of nomilin accumulation i n the f r u i t when compared to the controls. These results show that auxins may have potential as bioregulators for reduction of b i t t e r limonoids i n commercially grown c i t r u s f r u i t s . F i e l d experiments with NAA on orchard navel orange trees are underway. A l t e r a t i o n of Biosynthetic Pathways of Limonoids. As pointed out previously, unlike other c i t r u s , Citrus ichangensis accumulates nonbitter ichangensin i n the f r u i t tissue as the predominant limonoid instead of b i t t e r limonin (20). Ichangensin i s biosynthesized from nomilin v i a deacetylnomilin, which i s the major biosynthetic pathway of limonoids i n this species. I f the genes for the pathway from nomilin to ichangensin or even from nomilin to deacetynomilin (nomilin acetyl esterase) could be transferred by genetic engineering or breeding techniques to other c i t r u s species which have the limonoid bitterness problem, nonbitter ichangensin or nonbitter deacetylnomilin might accumulate as the major limonoid i n the f r u i t tissue rather than b i t t e r limonin. This presents another approach to the limonoid bitterness problem. Stimulation of Limonoid Glucoside Formation. Limonoids are converted to their glucoside derivatives during late stages of f r u i t growth and maturation. The conversion appears to be catalyzed by UDP-D-G glucosyltransferase. Since the glucosides appear to be p r a c t i c a l l y nonbitter as compared to the intense bitterness of the parent limonoids, the formation of the glucosides represents a debittering pathway that can be explored i n future research dealing with pathway regulation. Postharvest Approaches to the limonoid Bitterness Problem Since this subject was reviewed i n 1988 by Johnson and Chandler (33), Dekker (2), and Hasegawa (3), no s i g n i f i c a n t progress has

8. HASEGAWA

Removal of Limonoid Bitterness in Citrus Juices

been made i n this f i e l d . approaches.

91

Following i s a b r i e f description of the


B i o l o g i c a l Removal Six species of bacteria, each capable of metabolizing limonoids, have been isolated from s o i l by enrichment using limonoids as carbon sources. They are Arthrobacter globiformis (34), Pseudomonas 321-18 (35), Arthrobacter globiformis II (36), Bacterium 342-152-1, Corynebacterium fascians (37) and Acinetobacter sp. (38). Based on the metabolites and enzymes produced by these species of bacteria, f i v e metabolic pathways of limonoids have been established i n bacteria (3)(Fig. 4).

Limonol t Limonoate 4 Limonin > Deoxylimonin 4 4 4 trans-19-Hydroxy Limonoate Α-ring lactone Deoxylimonate obacunoate ^ 17-Dehydrolimonoate Α-ring lactone Figure 4.

Metabolic pathways of limonin i n bacteria.

In addition, nomilin was also metabolized v i a obacunone i n immobilized c e l l s of £. fascians (39). The 17-dehydrolimonoid path way i s th major metabolic pathway of limonoids i n bacteria, and four limonoate dehydrogenases, which catalyze the conversion of limonoate Α-ring lactone to 17-dehydrolimonoate Α-ring lactone, have been isolated from d i f f e r e n t species of bacteria (34. 35, 37). Several other enzymes including limonin D-ring lactone hydrolase, nomilin acetyl-lyase, deoxylimonin hydrolase and obacunone Α-ring lactone hydrolase have been isolated and characterized (16, 40, 41). Among limonoid-metabolizing enzymes, limonoate dehydrogenase isolated from A. globiformis was the f i r s t enzyme used to demonstrate the preven tion of the limonin bitterness i n navel orange juices (42). A bioreactor, which uses immobilized b a c t e r i a l c e l l s , has been developed at the Pasadena laboratory for removal of b i t t e r limonoids i n c i t r u s juices (3). When the juice was passed through a column packed with immobilized b a c t e r i a l c e l l s , limonin i n the juice was converted to either 17-dehydrolimonoate Α-ring lactone by A. globiformis or limonol by A. globiformis II and C. fascians. Nomi l i n i n grapefruit juice was also metabolized s i m i l a r l y , except that i t was also metabolized to obacunone i n immobilized c e l l s of _C. fascians. Debittering of c i t r u s juice by b a c t e r i a l c e l l s entrapped i n a d i a l y s i s sac was also demonstrated by Vaks and L i f s h i t z (38). The technical f e a s i b i l i t y of an immobilized c e l l system was demonstrated on a laboratory scale for use i n reducing limonoid bitterness of c i t r u s j u i c e s . The treatment caused no s i g n i f i c a n t adverse effect on the other juice constituents as judged by analysis of ascorbic a c i d , c i t r i c a c i d , malic acid, sucrose, glucose and fructone.


92


Physical Removal During the past several years, s i g n i f i c a n t progress has been made i n the development of selective adsorption processes for removal of two b i t t e r p r i n c i p l e s , limonin and naringin, from c i t r u s j u i c e s . Adsorbents such as c e l l u l o s e esters (43), cross-linked polystyrenes (44) and $-cyclodextrins (45, 46) have been shown to reduce e f f e c t i v e l y the limonin bitterness i n the juice. Cellulose acetate j e l s and beads were used to e f f e c t i v e l y remove limonin below the bitterness threshold from c i t r u s juies (43). The resins can be regenerated readily by washing with a small volume of warm water. The process has been used commercially i n A u s t r a l i a i n debittering b i t t e r orange juices (47). Polystyrene-divinylbenzene cross-linked copolymer adsorbent resins removed e f f e c t i v e l y both limonin and naringin from c i t r u s juices (44). Over 85% of limonin was adsorbed from about 50 bed volume juices of grapefruit, navel orange, lemon and tangerine. Up to 70% of naringin was also removed from grapefruit juice. 3-Cyclodextrins form i n c l u s i o n compounds with limonin and naringin. Reduction of limonin and naringin i n c i t r u s juices has been demonstrated using β-cylodextrin monomers (45) and polymers (46). A continuous flow process with polymers, for example, reduced both limonin and naringin contents an average of 50% or more i n 16 bed volumes of c l a r i f i e d grapefruit juice (48). The column can be r e a d i l y regenerated with 2% NaOH solution and the same column can be used many times without losing i t s effectiveness.

General Remarks Biochemical studies carried out at the Pasadena laboratory have shown how, where and when limonoids are biosynthesized and accumulate i n c i t r u s . The recent discovery of limonoid glucosides has cleared an unanswered question of how limonoid aglycones disappear at late stages of f r u i t growth and maturation. Certain limonoids have been shown to possess antifeedant e f f e c t s on some insects and anticarcinogenesis a c t i v i t y i n mice. Although further research i s needed, limonoids appear to have potential as i n s e c t i c i d e s or chemopreventative agents against cancer. Citrus f r u i t and seeds are an excellent source of these compounds. At present there are no t o t a l l y s a t i s f a c t o r y methods of commercially removing limonoid bitterness. Several new approaches are being developed which may lead to a more complete solution to the problem. These include s p e c i f i c adsorbents of limonoids, a b i o l o g i c a l reactor that uses immobilized b a c t e r i a l c e l l s , and the preharvest approaches which are presented i n the text. To help v i s u a l i z e the compounds i n this chapter, I have included two pages of structures (pp 93 and 94).


8. HASEGAWA


Limonin

Obacunone

Calamin

Nomilin

93

Limonoate Α - r i n g lactone

Obacunoate

Retrocalamin

Deacetylnomilin

Cyclocalamin


94 QUALITY FACTORS OF FRUITS AND VEGETABLES

Nomilin 17-D-fi-D-glucopyranoside

8. HASEGAWA


95

Acknowledgments The author thanks Dr. V. P. Maier f o r h i s support and guidance of our limonoid research program, and f o r e d i t o r i a l help i n the preparation of t h i s manuscript. Literature Cited


1.

Maier, V. P.; Hasegawa, S.; Bennett, R. D.; Echols, L. C. In Citrus Nutrition and Quality; Nagy, S.; Attaway, J . Α., Ed.; American Chemical Society: Washington, D C, 1980, p 63. 2. Dekker, R. F. H. Aus. J . Biotechnol. 1988, 2, 65. 3. Hasegawa, S. Food Biotechnol. 1987, 1, 249. 4. Rouseff, R. L. J . Agric. Food Chem. 1982, 30, 504. 5. Guadagni, D. G.; Maier, V. P.; Turnbaugh, J . C. J . Food S c i . 1973, 24, 1277. 6. Rouseff, R. L.; Matthew, R. F. J . Food S c i . 1984, 49, 777. 7. Hashinaga, F.; Eijima, H.; Nagahama, Η.; Itoo, S. B u l l . Fac. Agric. Kagoshima Univ. 1977, 27, 171. 8. Maier, V. P.; Beverely, G. D. J . Food S c i . 1968, 33, 488. 9. Maier, V. P.; Hasegawa, S.; Hera, E. Phytochemistry 1969, 8, 405. 10. Hasegawa, S.; Bennett, R. D.; Maier, V. P. Phytochemistry 1984, 23, 1601. 11. Herman, Z.; Hasegawa, S. Phytochemistry 1985, 24, 2911. 12. Herman, Z.; Hasegawa, S.; Ou, P. J . Food S c i . 1985, 50, 118. 13. Hasegawa, S.; Herman, Z. Phytochemistry 1986, 25, 2523. 14. Herman, Z.; Bennett, R. D.; Ou, P.; Fong, C. H.; Hasegawa, S. Phytochemistry 1987, 26, 2247. 15. Hasegawa, S.; Herman, Z. Phytochemistry 1985, 24, 1973. 16. Herman, Z.; Hasegawa, S.; Ou, P. J . Food S c i . 1985, 50, 118. 17. Maier, V. P.; Bennett, R. D.; Hasegawa, S. In Citrus Science and Technology Nagy, S.; Shaw, P. E.; Veldhuis, M. K. Ed.; Avi Publishing: CT., 1977; V o l . 1, p 355. 18. Hasegawa, S.; Maier, V. P.; Bennett, R. D. Phytochemistry 1974, 13,103. 19. Hasegawa, S.; Bennett, R. D.; Verdon, C. P. Phytochemistry 1980, 19, 1445 20. Bennett, R. D.; Herman, Z.; Hasegawa, S. Phytochemistry 1988, 27, 1543. 21. Hasegawa, S.; Bennett, R. D.; Verdon, C. P. J . A r i c . Food Chem. 1980, 28, 922. 22. Bennett, R. D.; Hasegawa, S. Tetrahedron 1981, 37, 17. 23. Hasegawa, S.; Herman, Z.; Ou, P.; Fong, C. H. Phytochemistry 1988, 27, 1349. 24. Herman, Z.; Bennett, R. D.; Ou, P.; Fong, C. H.; Hasegawa, S. Phytochemistry 1987, 26, 2247. 25. Hasegawa, S.; Fong, C H.; Herman, Z.; Ou, P. 196th ACS National Meeting, Los Angeles, CA. AGFD 90. 26. Hasegawa, S.; Herman, Z.; Orme, E. D.; Ou, P. Phytochemistry 1986, 25, 1984. 27. Ou, P.; Hasegawa, S.; Herman, Z.; Fong, C. H. Phytochemistry 1988, 27, 115. 28. Lam, L. K. T.; Hasegawa, S. Nur. Cancer. 1989, 12, 43. 29. A l f o r d , A. R.; Bentley, M. D. J . Econ. Entomol. 1986, 79, 35.

96 30. 31. 32. 33. 34. 35.


36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

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May 22, 1989

Biochemistry and Biological Removal of Limonoid Bitterness in Citrus

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