Juice limonin bitterness

More recently Maier et al. (3) published a comprehensive review of the limonoid constituents of Citrus and the impact of limonin bitterness on juice quality. This paper summarizes the chemical, biochemical and juice quality aspects of limonoids in Citrus (and related genera) and presents relevant advances since previous reviews. 

Limonin has been known to be an intensely bitter substance since it was first isolated in 1841 by Bernay (23). Beginning in 1966 the development of analytical methods for limonin allowed correlations of apparent bitterness with juice limonin content to be undertaken. The various reports of relative bitterness of suprathreshold limonin levels have been reviewed by Maier et al. 

Influence of Sweeteners on Bitterness. In model system studies, natural fruit juice sugars were observed to raise the limonin threshold (24). An expanded study of natural and artificial sweeteners (26) demonstrated that sucrose, neohesperidin dihydro-chalcone (NHD), hesperetin dihydrochalcone glucoside (HDG) and aspartylphenylalanine methyl ester (AP) all raise the limonin threshold. At low sweetness levels HDG was the most effective followed by AP and NHD. Sucrose was without effect up to the 2% level. At sweetness levels equivalent to 1% sucrose, HDG, AP and NHD raised the limonin threshold in water from 1.0 ppm to 3.2, 2.5 and 1.3 ppm, respectively. Because of its high sweetness intensity, the concentration of NHD (16 ppm) was considerably lower than HDG (80 ppm) and AP (90 ppm). At 3-10% sucrose sweetness equivalency, the effectiveness of NHD increased substantially, sucrose moderately and HDG slightly, while that of AP decreased. Therefore, the sweeteners HDG, AP and NHD can effectively suppress limonin bitterness at low concentrations. 

The intact fruits do not contain bitter limonin, but rather a nonbitter precursor, limonoate A-ring lactone (8). When juice is extracted, this nonbitter precursor is gradually converted to limonin under acidic conditions and the conversion is accelerated by the action of limonin D-ring lactone hydrolase, which has been isolated from citrus (9). The bitterness due to nomilin in juices most likely develops in a manner similar to that of limonin bitterness, but this has not yet been directly proven. However, the contribution of nomilin to juices is minor. It occurs mainly In grapefruit juices (4). 

Physical Removal During the past several years, significant progress has been made in the development of selective adsorption processes for removal of two bitter principles, limonin and naringin, from citrus juices. Adsorbents such as cellulose esters (43), cross-linked polystyrenes (44) and 6-cyclodextrins (45, 46) have been shown to reduce effectively the limonin bitterness in the juice. 

Limonoid UDP-D>glucose transferase Limonin bitterness is a problem in juices extracted from early-season to mid-season winter fruit, but not a problem in juice extracted from the late season fruit. As the fruit ripens, the concentration of limonoate A-ring lactone (2) decreases (23,24). This natural limonin debittering process has been known for over a century, but the mechanism of this metabolism was not understood until the recent discovery that limonoid glucosides are present in mature fruit tissues and seeds. We observed that in navel orange the initial... 

Hasegawa, S., M. N. Patel, and R. C. Snyder, Reduction of limonin bitterness in navel orange juice serum with bacterial cells immobilized in acrylamide gel, J. Agric. Food Chem., 30, 509-511 (1982). 

Limonin bitterness is especially acute in juice obtained from short maturation time fruits such as navel oranges. Juices obtained from most of the other citrus crops, such as Valencia oranges, usually do not have the limonin bitterness problem, as the concentration of limonin has decreased sufficiently by the time the processing season starts. Juices from navel oranges, particularly early- to mid-season fruits, can contain over 30 ppm of limonin, which is well above the bitterness detection threshold of 6 ppm. Grapefruit also has significant levels of limonin, which vary from 10 ppm in the early season to 1 ppm or less in the late season. 

Limonoids are a group of chemically related triterpene derivatives found in the Rutaceae and Meliaceae families. There are two groups of limonoids the aglycones and their corresponding glucosides. A total of 36 limonoid aglycones have been isolated from Citrus and its hybrids (Table 1). Five of them are bitter in taste -limonin, nomilin, nomilinic acid, obacunoic acid and ichangin (Fig. 1). In citrus juices, limonin is the major cause of delayed bitterness. 

Hasegawa S., Maier V.P. Solutions to the limonin bitterness problem of citrus juices. Food Technology, 73-75 (1983). 

A number of other processes have become commonplace in the manufacture of fruit juices. For example, if oranges of the varieties Navel or Navellina are processed the juice becomes unpleasantly bitter because of the biochemical development of a glycoside, limonin. This substance can be partially or totally removed by the use of appropriate ion-exchange resins to yield a juice of acceptable taste. 

Fruit juices can be deacidified with a weak base anion-exchange resin. Removal of compounds which cause a bitter taste is a more popular application (26,27). It is accomplished with resins that have no ion-exchange fimctionality. In essence, they are similar to the copolymer intermediates used by resin manufacturers in the production of macroporous cation and anion exchangers. These products are called polymeric adsorbents. They are excellent for removal of limonin [1180-71-8] and naringin [1023647-2], the principal compounds responsible for bitterness in orange, lemon, and grapefruit juices. The adsorbents are regenerated with steam or alcohol. Decaffeination of coffee (qv) and tea (qv) is practiced with the same polymeric adsorbents (28). 

Limonoids are a group of chemically related triterpene derivatives found in the Rutaceae and Meliaceae. Limonin, a bitter member of the group, occurs widely in citrus juices. It has commercial significance because bitterness (excessive bitterness in the case of grapefruit) reduces juice quality. Dreyer (1) and Connolly et al. (2) have reviewed the chemistry and biochemistry of limonoids. 

The general conclusions reached in these tests were that limonin levels less than about 6 ppm were generally nonbitter and that the bitterness perceived at higher limonin levels varied with the sweetness, acidity and oil levels of the juice. 

Another important interaction is that of limonin with the bitter flavanone glycoside naringin. Both of these bitter substances are present in grapefruit juice and Guadagni et al. (25) found that they interact at subthreshold levels in an additive way. Less than threshold amounts of limonin or naringin contribute to the bitterness of a mixture of the two compounds. The bitterness of the mixture can be predicted by adding the taste-unit contribution of each component (taste unit = concentration/ threshold). 

While most of the limonoids of Citrus have been isolated from seeds, several occur in detectable amounts in other parts of the fruit. Minor amounts of deacetylnomilin, nomilin, obacunone, de-acetylnomilinic acid and nomilinic acid were identified in extracts of navel orange peel (37). 17-Dehydrolimonoate A-ring lactone was isolated from peel and juice of navel oranges (38) and nomilin has been reported to occur in grapefruit juice and juice vesicles (39). The only Citrus limonoids known to be bitter are limonin, VI, XII, obacunoic acid and IX (3). 

Control of Juice Bitterness. A number of advances have been reported in this field since it was last reviewed (3). A commercial application of the cellulose acetate adsorption technique for the removal of limonin from citrus juices was undertaken (49). New sorbent gel forms of cellulose esters for adsorption of limonin were developed (50). Knowledge was gained that limonoids are biosynthesized in citrus leaves and translocated to the fruit (12) and that specific bioregulators can inhibit accumulation of XIV in citrus leaves (15). Additional studies were carried out on the use of neodiosmin to suppress limonin and other types of bitterness (30,51). The influence of extractor and finisher pressures on the level of limonin and naringin in grapefruit juice was reported (34). Also, further studies were conducted on the microbial sources and properties of limonoate dehydrogenase (52), the enzyme that converts XIV to XV and can be used to prevent limonin from forming in freshly expressed citrus juices (53). 

The quality of extracted citrus juices depends on enzyme reactions that occur not only in the fruit during the development period, but also in the juice during processing. When juice is extracted from citrus fruit, enzymes are released from their normal restraint in the cell. Several of these enzymes catalyze reactions that adversely affect taste and appearance of the juice. Unless the reactions are controlled, the juice products will not meet the standards of quality set up by the USDA Food Safety and Quality Service. The two reactions of commercial importance are the hydrolysis of pectin to pectic acid, which clarifies juice, and the lactonization of limonoic acid A-ring lactone to the bitter compound, limonin. Research efforts to identify and characterize the reactions, to isolate and purify the enzymes, and to develop methods to control the reactions are described in this review. 

Maier, V. P. Beverly, G. D. Limonin monolactone, the nonbitter precursor responsible for delayed bitterness in certain citrus juices. J. Food Sci., 1968, J3, A88-A92. 

Several postharvest treatments to citrus fruits have been tested in an effort to improve the quality of the extracted juice. Bruemmer and Roe subjected citrus fruits to anaerobic conditions for periods of 20 to 32 hours at 32.2 to 43°C (228, 229). This treatment reduced the titratable acidity and increased the Brix-acid ratio by about 10%. The decrease in acidity was accompanied, however, by a 20-fold increase in ethanol (229). Since the soluble solids-acid ratio is a major criterion of citrus juice quality, this procedure, if perfected, could allow earlier harvesting of fruit and a more consistent supply of fruit during the processing season. Bitterness of products from navel oranges, lemons, and grapefruit is related to limonin content. A 3-hour treatment of fruit with 20 ul ethylene/1 of air lowered the limonin content, reduced bitterness, and the juice was judged more palatable than juice from untreated fruit (230). 

Limonin and naringen, which can give citrus juices an undesirable bitter flavor, can be removed selectively by passing juice through a crosslinked polymer of (3-cyclodextrin.87,88 Only the bitter components are removed. The polymer can be regenerated for reuse by washing the polymer with a 1-2% solution of sodium hydroxide. 

V. P. Maier S. Hasegawa R. D. Bennet L. C. Echols, Limonin and Limonoids-Chemistry, Biochemistry and Juice Bitterness. In Citrus Nutrition and Quality, S. Nagy, J. A. Attaway, Eds. ACS Symposium Series No. 143 American Chemical Society Washington, DC, 1980 pp 63-82. 

Several limonoids are known to be bitter principles of citrus (Rutaceae). A typical example is limonin. Although fresh juice does not elicit a bitter taste, sometimes it becomes bitter after heating or storage. This is explained by the formation of bitter-tasting limonin by deglycosylation and further cyclization from limonin glucoside, which is present in citrus fruit tissue and seeds and does not exhibit bitterness.146 Recently, it was reported that limonin had antitumor activity.147 Besides limonin, nomilin and obakunone, which are considered to be... 

Not only analytical or preparative separations can be performed on cyclodextrin polymer columns, but also undesired components can be removed from aqueous solutions, bitter tasting substances (narin-gin, limonin) can be removed or at least their concentration can be strongly reduced after treatment of citrus juice with cyclodextrin polymers in batch or column process (65,66). Phenylalanine can be eliminated from dietetic protein hydrolysates (67), water-soluble organic substances (e.g. polychlorinated biphenyls (68), 2-naph-talenecarboxylate or phenol can be removed from aqueous solutions (e.g. from pharmaceutical wastewater) by polystyrene-cyclodextrin derivatives (69), by 8-cyclodextrin immobilized on cellulose (70) or by 6-cyclodextrin-polyurethane polymer (71). 

Bitter taste remover. Based on an immobilised enzyme, naringinase, in a cellulose acetate film to remove the bitter taste (naringin and limonin ) from grapefmit juice. 

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