Juice limonin levels

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. 

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. 

Limonin Levels of Citrus Juices. A compilation of limonin values in various citrus juices is given in (3). Limonin has been found in essentially all varieties of citrus juices examined. Although there is a considerable range of limonin values reported, certain trends are apparent. 

In the food industry, high-performance liquid chromatography (HPLC) is the most widely used technique for the determination of limonin levels in citrus juices, since it is accurate and reliable. Most of the HPLC methods developed are for the analysis of limonin in citrus juices. These methods may not work for other bitter limonoid aglycones and/or tissues. 

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). 

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. 

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). 

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. 

Limonoid glucosides and deoxylimonin were found to cross-react with the antibodies. The relatively low levels of deoxylimonin in citrus juices should not contribute to the limonin values significantly. However, the high concentration of limonoid glucosides, which can be over 400 ppm, can give artificially high limonin values. 

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