Methods for determining the adulteration of citrus juices

Review

Methods for determining result in lost value for the consumer. As a result, standards of identity, grade standards and quality specifications have been developed by government and industry for many juice products. Qualitative and quantitative parameters allow products to be tested for quality and authenticity. In addition, laws have been passed that impose fines and prison sentences for anyone apprehended adulterating a juice product. Over the years, analytical methods have become more accurate, sensitive and capable of separating greater numbers of juice constituents, and adulterators have been forced to become more sophisticated in their practices to avoid detection. As the level of sophistication required rises, there comes a point at which it is no longer economical to adulterate a product. Juice manuThe adulteration of fruit juices and juice-based beverages is a facturers buying bulk product are continually increasing serious economic problem. Juice adulteration has progressed the number of sophisticated tests performed for quality from simple dilution with water and the substitution of cheap control, either by in-house testing or through the use of service laboratories. ingredients to highly sophisticated manipulations designed to The evolution and use of analytical methods to detect mask the adulteration process. Major adulteration problems and determine the degree of adulteration has been the currently encountered by regulatory agencies include un- subject of several review articles and books. This paper declared addition of sugar and pulp wash, the admixture of provides information on the more newly developed juices, and the addition of colorants, amino acids and organic methods. For information on more established methods, see Ref. 1. acids. Also of regulatory concern is ascertaining the geoIt is beyond the scope of this article to discuss all graphical origin of a juice. While numerous methods have facets of citrus juice adulteration. Thus, we discuss here been developed to detect juice adulteration, only a limited only the major areas that we perceive to be especially number have proved workable in deterring sophisticated troublesome, critically evaluating the most sophisticated tests available to combat illegal manipulations of citrus adulteration. Methodologies critically evaluated in this report juices. include isotopic analyses, SNIF-NMR, HPLC, tracer addition,

the adulteration of citrus juices

Wilbur W. Widmer, Paul F. Cancalon and Steven Nagy

UVN/S spectrophotometry, ICP-AES, electrochemical detec-

Water addition

tion and pattern recognition.

The major atoms found in living tissue (carbon, oxygen and hydrogen) are present in nature in combination with traces of heavier stable isotopes (Table I). Numerous studies have shown that the distribution of these isotopes in living tissues is not random, but can vary according to factors such as plant botanical origin and geographical distribution. These differences have been extensively used to detect adulteration (for reviews, see Refs 2-4).

The adulteration of fruit juices and juice-based beverages has been an economic problem faced by the consumer and by the food industry for many years. Fortunately, adulterated products comprise a relatively small portion of the products on the US market. Adulterants can be added by unscrupulous producers to increase profits by the addition of inexpensive ingredients to make an inferior product appear of higher quality or to extend supplies to meet consumer demand; product adulteration may also result from a malfunction or poor control of processing parameters. Juice concentrate prices are determined on the basis of the level of total soluble solids (primarily sugars and acids), whereas single-strength product prices are based on volume. Adulteration can be accomplished by simple dilution, or by addition of inexpensive sugars or acids or of complex mixtures meant to simulate the natural product and, thus, avoid detection. All such forms of adulteration Wilbur W. Widmer, Paul F. Cancalon and Sleven Nagy are with the Scientific Research Department of the Florida Department of Citrus stationed at CREC, 700 Experimental Station Road, Lake Alfred, FL 33850, USA.

278

([)1992, ElseVier SCience

Puhlishers Ltd, (UK)

Oxygen isotope ratios The distribution of oxygen isotopes III water can be affected by various factors: climatic factors (the proportion of 180 increases with temperature but decreases with precipitation), geographical factors (the proportion of 180 decreases with increases in latitude and with increases III the distance from the sea and in the elevation),

I Table 1. Relative abundances in I nature of the main isotopes of ~ carbon, oxygen and hYdrOge~ I

Isotope

I Carbon 12e

lJe

Relative abundance

I

98.916% 1.084%

I

I

I Oxygen 16

0

17

I

0 1110

99.758% 0.038% 0.204%

Hydrogen IH 2H

I

99.983% 0.014%

L-

Trends in Food Science & Technology November 1992 IVol. 31

I

I I

I

I

biological factors (the plant becomes enriched in the heavier isotopes during transpiration) and technical factors (juice concentration results in IKO enrichment). The IKO/IOO ratio (R) is usually measured with an isotope ratio mass spectrometer, and is usually determined in '8' units, in terms of the IKO enrichment of the sample with respect to a standard: 8 1R O

- RSMOW) x 101 RSMOW

= (R""ll Pk

(I)

where 'SMOW' is the reference standard: 'standard mean ocean water'. The water fraction of juice has been studied in numerous fruits 5!>. Fruit water, particularly that of citrus juices, is significantly enriched in 18 0 as compared with ground water. Therefore, a reconstituted juice will be poorer in the heavier isotope than a fresh juice, allowing differentiation between natural and reconstituted juices. Furthermore, the residual water in concentrated orange juice is further enriched in the heavier isotope; 8 18 0 is positive (8 = 10-15) for juices with a sugar content of 65° Brix 7 ; any addition of water will decrease the 18 0 concentration.

Hydrogen and deuterium ratios The factors influencing variations in IKO content also affect the distribution of "H in water. However, a major complication arises in the analysis of 2H/I H ratios, since hydrogen bound to oxygen or nitrogen is, in most cases, very labile, and can be exchanged readily with that of water. In mass spectroscopic methods to determine "H/IH ratios (R). non-exchangeable sample hydrogens are transformed into water by combustion, reduced to hydrogen, and the ratio measured. Deuterium enrichment is expressed as 8 2H, as described above for the determination of IKO enrichment:

from Pee Dee belemnite (PDB) - a limestone fossil from the Pee Dee range, SC, USA. In plants, the carbon isotopic distribution is determined during the dark phase of photosynthesis. The fixation of carbon dioxide leading to glucose synthesis can proceed through different pathways in different plants. For example, plants such as citrus, beet, maple, potato, apple and grape produce glucose via 3-phosphoglyceric acid, in a pathway known as the C 1 or Calvin cycle. These plants preferentially accumulate the low molecular weight isotope and, consequently, have a very negative 8: -30 < 8 < -24. Other plants (e.g. sugar cane and corn) fix CO 2 via phosphoenolpyruvate, in the HatchSlack (C~) cycle. This pathway induces very little carbon isotope fractionation and, thus, in these plants the carbon isotope distribution is similar to that of the atmosphere: -12 < 8 < -9. A few plants (e.g. pineapple) known as CAM CCrassulacian acid metabolism') plants can use both pathways depending on lighting conditions. Other factors, such as the differential diffusion rates of the different isotopic forms of CO 2 , also affect the distribution of the two isotopes within plant tissues. Determination of the 8 11 C value can be used to assess whether a product from one type of plant has been adulterated with a product from another. For example, adulteration with corn or cane sugar can easily be detected in citrus juices, which have 8 l1 C values of -25.5, as the average value for high-fructose corn syrups is -9.7. However, 8 l1 C values cannot be used to determine whether products from plants using the same CO 2 fixation pathway have been mixed, as the values for the different products will be similar. For example, beet sugar, with an average 8 l1 c value of -26, cannot be differentiated from citrus products.

Oxygen isotope ratios

Since heavier isotopes accumulate in fruit juice water, determination 8 2H values has been used to determine whether a juice is fresh or reconstituted, as seen for 8 18 0 values 5.x.

The drop in the positive IKO value of 65° Brix concentrated juices that occurs on dilution with water or adulterants has been used for indirect assessment of the addition of beet medium invert sugar, which cannot be detected using determination of the carbon isotope ratio. However, this method is limited to juices with a sugar content of ~65° Brix.

Sugar addition

Hydrogen/deuterium ratios

Perhaps the simplest form of citrus juice adulteration is by the undeclared addition of cane and/or beet sugars to citrus juices (primarily to juice concentrates) to extend supplies. These additions are primarily detected by isotopic and chemical methods.

Deuterium concentrations determined by mass spectrometry have also been used to quantify sugar levels, particularly levels of beet sugar added to orange juice IO- 12 . Orange sugar is much richer in 2H than beet sugar, making the detection of beet medium invert sugar possible. However, the exchangeable oxygen-bound hydrogen atoms have first to be removed, through the formation of nitrate esters, before the level of nonexchangeable carbon-bound 2H can be determined. This makes the method rather complex and prone to explosions. A modified method has been proposed I1 whereby the sample is first oxidized into formic acid before being reduced into hydrogen. Because of natural variations in the isotope ratio, this method does not allow the detection of small amounts of added beet MIS.

8"H = (R,arnPlc - RSMOW) R SMDW

x

101

(2)

Carbon isotope ratios Stable carbon isotope ratios can be measured by mass spectrometry. The sample is combusted to CO 2 and the I1C/ 12C ratio (R) determined in terms of 8 11 e, the I1C enrichment of the sample with respect to a standard 9 .

8 11 C = (R,arnPk - R"andard) x 101

(3)

R . . tandard

The standard most often used being calcium carbonate

Trends in Food Science & Technology November 1992 [Vol. 31

27'

SNIF-NMR method More recently, a method has been developed that takes into account the deuterium distribution within the molecule. It has been shown l4 that deuterium is not randomly distributed within organic molecules. The differences in distribution are particularly significant in ethanol, and a method called site-specific natural isotope fractionation - nuclear magnetic resonance (SNIF-NMR) has been developed to detect chaptalization and to determine wine origin l5 . It has been shown that the 2H/IH ratio of the methyl site of ethanol (H/IH)I is related to the ratio in the glucose molecule from which it is derived, whereas that of the methylene group of ethanol (H/ IH)11 and that of the fermentation water eH/IH)~ are dependent on that of the ratio in the must water. The method has subsequently been applied to the detection of beet sugar in citrus juices. It has been shown that the methyl site eH/IH)1 ratio is significantly lower in ethanol derived from beet sugar than in ethanol originating from grapes or citrus fruits. It has been reported that the contribution of I % beet ethanol induces a eHlIH)1 shift of 0.88 ppm. This methodology for the detection of beet sugar is complex and demands very expensive equipment. It requires a relatively large amount of juice (at least 500 ml), which must first be fermented under controlled conditions. The resulting ethanol is then extracted by a very precise quantitative distillation, using an automated Cadiot column, in order to avoid isotopic fractionation. The isotope analysis is subsequently performed with a 400 MHz NMR spectrometer (AM 400 Bruker) fitted with a specific probe tuned to the deuterium frequency (61.4 MHz) and run in proton decoupling mode. Ten spectra of each sample are recorded for statistical analysis. The plant isotopic content varies greatly with climatic and geographic factors 16. Therefore, an accurate determination of beet sugar adulteration requires knowledge of the origin of the juice as well as an extensive database to determine how the unknown sample compares with pure juices from the same area. The method provides accurate detection and quantification of the level of added beet medium invert sugar when the origin of the juice is known and can be compared to background data 17. However, this is difficult with some commercial juices, which may be poorly characterized blends of products from several countries. With such unknown juices, the sensitivity of the method decreases significantly. The addition of sugars of various origins may also affect the quantification process. Nevertheless, the SNIF-NMR method seems to provide one of the most reliable techniques for the quantification of added beet sugar.

Beet sugar detection using 01 igosaccharides Medium invert sugar (MIS) has been extensively used as an adulterant, since its sugar composition mimics that of citrus juices. However, differentiating between endogenous and exogenous saccharides to detect sugar 280

addition has remained a major challenge. As discussed above, several methods have been developed that use measurement of the level of stable isotopes, but these techniques are complex and expensive. Another approach to the detection of adulterants is monitoring minor contaminants present with the main compound. MIS is manufactured by hydrolysing crystallized beet or cane sucrose to produce an equimolar solution of fructose, glucose and sucrose. Low and Swallow IX and Swallow et al. 19 developed a method to detect beet sugar adulteration by monitoring four oligosaccharides specific to medium invert sugar. However, the origin of these oligosaccharides remained unclear. Low and Swallowl~ postulated that oligosaccharides are produced by hydrolytic enzymes specific to beets (in the case of beet MIS oligosaccharides) or citrus (in the case of naturally occurring orange oligosaccharides). The authors also raised the possibility that the beet-specific oligosaccharides arose from the action of HCl on beet sucrose. The oligosaccharides specific to beet sugar were examined using a modification 20 of the method of Swallow et al. 19 Samples were analysed with a Waters (Milford, MA, USA) high-performance liquid chromatography (HPLC) system with two Carbo Pac anion exchange columns. A valve between the two columns allowed the elution to waste of the monosaccharides and sucrose eluted within the first 10 minutes, at which time the flow valve was switched to elute the oligosaccharides onto the second column and the electrochemical detector. This modification, developed by White et al. 20, represents a major improvement: as oligosaccharides are present only as traces in MIS, the large quantities of simple sugars overload the detector and the resulting chromatogram using previous methods. Chromatography of beet MIS revealed two series of peaks. The first group, present in citrus juices as well as in beet MIS, elutes between 15 and 23 minutes. It contains a mixture of di- and trisaccharides whose composition is extremely variable, but appears to be greatly affected by the level of microbial activity. The second group, which elutes after 23 minutes, appears to correspond mostly to trisaccharides, and has been characterized as the 'beet-MIS-specific oligosaccharide'. However, analysis of commercial sucrose from both cane and beet origin did not detect significant amounts of oligosaccharides intrinsic to purified sucrose 21 . To assess the specificity to beets of MIS oligomers, acid-hydrolysed beet or cane sucrose samples were examined (prepared either in our laboratory or obtained from industrial sources). Similar peaks were found in both samples, independent of the origin of the product. Identical peaks could also be produced from citrus sugars21. It appeared, therefore, that the MIS oligosaccharides were by-products of the sucrose inversion process, not related to the botanical origin of the sugar. The type and amount of oligosaccharides generated were shown to depend on the composition of the simple sugar solution from which they were produced. Since, during inversion, the sugar composition changes constantly from pure sucrose to a mixture of fructose and Trends in Food Science & Technology November 1992 [Vol. 31

glucose, a specific oligosaccharide pattern will be determined by the relative proportions of these three sugars when the hydrolysis is stopped. Conversely, any oligosaccharide pattern can be reproduced by acid incubation of sugars in the appropriate ratios. This oligomerization process, called reversion, has been extensively examined (for reviews, see Refs 22 and 23), and it has been shown that, in aqueous acidic solutions, monosaccharides can condense with each other to produce various oligomers. Thus, the generation of oligosaccharides is not due to inversion per se, but to the presence of simple sugars in acidic media. It should be added that microorganisms, both yeasts and bacteria, generate many types of oligosaccharides that, in nature and/or composition, are different from the acid-produced oligosaccharides. The presence of enzymatically generated oligosaccharides explains that the 'MIS-specific oligosaccharides' are masked in juices such as apple juice, in which starch is hydrolysed with enzymatic mixtures. These 'enzymatic oligosaccharides' are also characteristic of MIS produced with invertase rather than by acid treatment2 4 . The oligomerization of saccharides follows the mass action law until an equilibrium is reached between the levels of monomers and polymers22. In acidic medium, monosaccharides become ionized, and can interact preferentially with water (at low concentration) or with another sugar molecule (at high concentration). As a result, the amount of oligosaccharide generated increases exponentially with saccharide concentration. This explains why only very limited amounts of oligosaccharides are produced during juice concentration, since excessive heat is applied only to juice at or below 16° Brix. Diluted sucrose solutions do not produce significant amounts of oligosaccharides during inversion, and even favor hydrolysis of the saccharides present. A similar process occurs in single-strength commercial juices that have been severely heated, such as aseptically packed juices or juices kept at moderate temperature for extended periods of time: such juices contain almost no oligosaccharides, and the level of sucrose can be reduced to about one third of its normal value. The opposite situation occurs in concentrated juice solutions, in which heating shifts the equilibrium towards the formation of oligomers. The equimolar solution of glucose, fructose and sucrose that constitutes citrus juices produces, in a very short time, the same pattern of oligosaccharides as a partially (50%) hydrolysed sucrose solution or a prepared equimolar solution of the three sugars. This reaction occurs in juices that are heated after concentration, such as aseptically packed juice concentrates. Slow formation of oligo saccharides also occurs in concentrated juices kept at room temperature. The oligosaccharides generated in such juices will mask the presence of any oligomers of extraneous origin, making detection of any added sugars more difficult. In summary, an acidic concentrated solution of sugars of any origin will produce oligosaccharides when heated. These results demonstrate the importance of examining the oligosaccharide profile of a suspect product. Heated, concentrated juices generate an oligosaccharide profile Trends in Food Science & Technology November 1992 IVai. 31

identical to that of half-hydrolysed sucrose, but not to that of fully inverted sugar. Thus, detection of acid-induced oligosaccharides generated in concentrated solutions of simple sugars can be used to infer the presence of added beet sugar in fruit juices. However, results should be carefully interpreted, since these oligosaccharides are not specific to beet sugar or to hydrolysis per se, and may be produced or removed from sugar solutions depending on their sugar composition. Overall, despite some problems related to determining the origin of the oligosaccharides, this method offers a relatively rapid and inexpensive way of monitoring large numbers of samples for the presence of added medium invert sugar of any origin.

Addition of pulp wash The pulp expelled from citrus juice finishers (,finisher pulp') contains about 80% juice. These soluble juice solids can be recovered by countercurrent washing and refining. Pulp wash has been defined by the Florida Department of Citrus, USA, as 'water-extracted soluble fruit solids recovered in the presence of water from unfermented excess fruit pulp removed during the production of citrus juice products'. The State of Florida currently prohibits the addition of pulp wash to 'frozen concentrated orange juice' and 'concentrated orange juice for manufacturing' produced in Florida 25 • The addition of pulp wash to citrus juice is also forbidden in many European countries. However, US federal regulations 26 do allow for the inline addition of pulp wash to freshly extracted juice. There is currently considerable controversy in promulgating uniform regulation on pulp wash addition for both the State of Florida and the rest of the USA. Pulp wash is not a chemically characterized commodity, but its composition may be determined by numerous physical parameters. Furthermore, juice composition can vary significantly according to geographical and botanical origin. Methods developed to detect pulp wash in juice include the following.

Tracer studies In Florida, in order to facilitate the detection of pulp wash addition, sodium benzoate tracer must be added to pulp wash to a concentration equivalent to 50-100 ppm when reconstituted to single-strength level. This tracer does not have to be added when pulp wash is exported to some countries. The detection of added pulp wash containing tracer can easily be accomplished using the method of Lee et al. 27 A polystyrene divinylbenzene column with a methanol: water mobile phase is used for analysis after sample cleanup by solid-phase extraction on C-18 packing. A detection limit of 0.2 ppm for benzoic acid in orange juice enables this method to detect less than 5% added pulp wash with tracer. This method will also detect the addition of sorbic acid, methyl paraben or propyl paraben preservatives. An earlier method developed by Fisher28 , using a reversed-phase C-18 column, did not separate benzoate from sorbate. Added pulp 28

wash without tracer is more difficult to detect. The State of Florida also requires that drums of pulp wash be clearly labeled and encircled by a five-inch-wide yellow band.

The Petrus method This method 29 , the most widely used at present, can be divided into two parts, both performed on juice diluted in ethanol (90% final concentration). The samples are scanned from 600 nm to 200 nm with an ultraviolet/visible (UV!VIS) spectrophotometer. Various components of the juice (carotenoids, polyphenols, flavonoids and ascorbic acid) absorb at different wavelengths, producing peaks whose area is proportional to the concentration. Since pulp wash is poorer in color than juices, but richer in flavonoids and polyphenols, differences can be established for juice and pulp wash. Further qualitative information can be obtained by examining the juice fluorometric scans at various wavelengths. The method had been developed for use with Florida products, for which an extensive database has been established. It should also be pointed out that, since flavonoids are more soluble in methanol than in ethanol, differences induced by the addition of pulp wash are significantly enhanced when methanol solutions are examined (Cancalon, P.F., unpublished). A major problem in the use of this method has come from juice produced from freeze-damaged oranges. This product exhibits chemical characteristics very similar to that of pulp wash. If damaged oranges are processed within 24 hours of the freeze, the product is still similar to juice, and can be differentiated from pulp wash by fluorometry, particularly with methanol solutions. However, if the fruits are pressed at a later time, the juice becomes very similar to pulp wash both chemically and organoleptically (Cancalon, P.F., unpublished). Another difficulty has been introduced by the debittering of pulp wash, which decreases the amount of flavonoids and lessens the efficiency of the Petrus method. The application of the Petrus method to juice blends from various geographical locations has unfortunately met with difficulties. Nevertheless, the method offers the possibility of rapidly screening large numbers of juices.

Pectin levels Pulp wash contains about twice as much watersoluble pectin as expressed juice, but less oxalate and alkali-soluble pectins. These differences can be used to provide an indication of the presence of pulp wash in juice10 .11 . However, very high extraction pressures will produce a juice with a high pectin content, or pectinmodifying enzymes might be used to lower the juice's pectin content, as is done with apple juice12 • In most citrus juice products, pectin-modifying enzymes (e.g. pectinesterase; EC 3.1.1.11) are destroyed by thermal processing (pasteurization); therefore, free galacturonic acid should be present in low amounts. High levels of free galacturonic acid indicate the use of partly spoiled fruit, the use of products that have been allowed to

282

ferment, or the use of products that have been treated enzymically to reduce pectin levels. Galacturonic acid can also be monitored spectrophotometrically by producing chromogens with m-hydroxydiphenyl and carbazole 11 . Preliminary results indicate that galacturonic acid can be quantified by HPLC with pulse amperometric detection, eluting after most oligosaccharides (Cancalon, P.F., unpublished).

Mineral profiles Pulp wash is richer in most minerals than pure juice. Levels of calcium, silicon and sodium increase the most. An analysis method has been developed by NikdeP", using inductively coupled plasma - atomic emission spectrometry (ICP-AES) coupled with a pattern-recognition program (Artificial Neural Network, Wards Systems Group, Inc., Frederick, MD, USA), that can provide information on the presence of pulp wash in JUices.

HPLC, electrochemical detection and pattern recogn ition One limitation of the Petrus method is the use of equipment that analyses only an entire class of chemicals. An indication of the addition of pulp wash to orange juice by determination of the narirutin to hesperidin ratio has also been reported 15 ·16 . Pulp wash contains much higher levels of both narirutin and hesperidin than juice does. The actual ratio of narirutin to total hesperidin differs very little between pulp wash and orange juice from the same fruit type; levels depend on extractor type, pressure settings, fruit variety and the integrity of the fruit. However, because of solubility differences, much of the hesperidin in juice is lost during sample preparation, and the measured ratio reflects the narirutin to soluble hesperidin ratio. The amount of solubilized hesperidin in juice is very dependent on time elapsed after extraction, processing history and storage conditions. In fruit, hesperidin occurs as the chalcone (open-ring) form. During storage, the ring closes to form a molecule that is much less soluble and forms a precipitate in the juice. Juice concentration accelerates the process, and precipitated hesperidin is primarily responsible for evaporator fouling. Juice from Navel oranges or from freeze-damaged fruit also contains higher narirutin levels than juice from other varieties, such as Hamlin, Pineapple or Valencia oranges; this will affect the ratio, as will sample filtration efficiency during sample preparation. All of these factors must be considered when evaluating the narirutin to hesperidin ratio as a criterion for determining the addition of pulp wash. A method has been developed by Gamache 37 that simultaneously examines the levels of several hundred chemicals present in juices. Juice compounds are separated on a reversed-phase C-IS column and detected with an oxidative array of 16 coulometric sensors, each set at a higher potential than the previous set. Compounds that are easily oxidized can be observed with the low-potential electrodes, whereas measurement

Trends in Food Science & Technology November 1992 [Vol. 31

of more stable compounds is achieved with the highpotential electrodes. Consequently, chemicals that are not separated on the high-performance liquid chromatography (HPLC) column may be detected by successive electrodes. Up to 300 peak clusters were revealed by this method, corresponding to the oxidation products of phenolic compounds, flavonoids and amino acids. Pattern-recognition analysis of these peaks provides a means of identifying and quantifying pulp wash addition as well as juice dilution. A similar method has been reported by Nolan and Koski 1x , who monitored polyphenol profiles using gradient HPLC combined with electrochemical, UV and fluorescence detection. Kirksey et al. 19 have developed an HPLC procedure specifically for determining added pulp wash by using fluorescence detection. An HPLC method coupled with pattern recognition has been proposed by Perfetti et at. 40 using UVNIS detectors. Various types of instruments were tested by Page et at. 41 using pattern-recognition techniques; of these, HPLC gave the best results in differentiating between pure and adulterated juices. Preliminary experiments using capillary electrophoresis of numerous juice components have shown promising differences between juice and pulp wash using both UV and visible detectors 4 ". Efforts are being made to quantify these variations using patternrecognition procedures. It has been recommended that a matrix of several analyses should be used to monitor the presence of pulp wash in juices 41 . It is hoped that new techniques using statistical analysis of a large number of parameters will be able to circumvent the inherent difficulty in determining pulp wash addition due to the variability of the material examined.

The undeclared admixture of juices Whenever the price of one type of citrus juice (e.g. grapefruit juice) is much lower than the price of orange juice, the temptation to admix orange and grapefruit solids becomes inevitable. The most benign scenario is inadvertent mixing. The two fruit types are frequently processed with partial sharing of equipment and, unless processing practices are carefully controlled, unintentional blends may result. Orange juice may be distinguished from orange-grapefruit juice blends by determination of the flavanone glycoside profile. Juice from sweet orange (Citrus sinensis) is known to contain the flavanone glycosides hesperidin and narirutin but lack naringin and neohesperidin, while grapefruit (Citrus paradisi) contains all four glycosides, with naringin being predominant. Mixtures of orange and grapefruit juices can be distinguished from pure orange juice by the presence and absence, respectively, of naringin. However, US Department of Agriculture regulations allow orange juice to contain up to 5% sour orange (Citrus aurantium) and 10% tangerine (Citrus reticulata) and tangerine hybrids. Both sour orange and the 'K-Early' tangelo grown in Florida contain naringin. Fortunately, the ratio of naringin to neohesperidin differs markedly among grapefruit, sour orange and

Trends in Food Science & Technology November 1992 [Vol. 3J

K-Early tangelo, and fruit origin in juice mixtures may be easily determined 44 • Juices with a naringin to neohesperidin ratio greater than 10 contain juice from grapefruit; a ratio of 1-4 indicates sour orange; and ratios less than I contain juice from K-Early. Both isocratic 45 .46 and gradient 14 .40.47 reversed-phase HPLC using a C-IS stationary phase may be employed with acetonitrile: water: acetic acid or acidified phosphate buffer. Greiner and Walbrauch 45 report small interferences in some samples containing less than 5 ppm naringin unless juice samples are subjected to a polyamide 'cleanup' prior to injection. Rouseff and Martin 46 also reported small peaks eluting near naringin that could be interpreted as naringin if the chromatography is not optimized. Depending on the solvent strength and chemistry of the C-IS stationary phase employed, potassium sorbate and sodium benzoate, if present, may also interfere with naringin or neohesperidin quantification. These interfering compounds are eliminated by use of a solvent with 30 mM phosphate buffer at pH 6.2-6.3. Tailing peaks are avoided under these conditions by using a C-IS stationary phase with thorough coverage of the silica surface and very low surface activit y 4x. Another potential problem is the use of sample filter membranes made of polysulfone and nylon, which adsorb naringin and neohesperidin, particularly when small volumes are filtered. Anotop inorganic membrane (Whatman Inc., Clifton, NJ, USA) and cellulose acetate filters with 0.2 /lm pore ratings did not adsorb flavanone glycoside components, even with filtrate volumes of 0.3 ml (Ref. 4S). Utilizing a gradient HPLC method, Kirksey et a1. 15 .19 analysed and reported the differences found in the phenolic composition of citrus fruits. Juice from several orange varieties (grapefruit, lemon and lime) were analysed along with juice from grape and apple. Their HPLC method was able to distinguish juice type and juice blends from pure juices.

Minor undeclared additives Over the years many methods have been developed to detect undeclared additives in citrus juices. Most methods are non-specific and measure a general loss of compounds, whereas others are highly sophisticated and detect a particular targeted compound.

Amino acids Amino acids are an important group of compounds that have received considerable attention. The formol index method was perhaps the first used to detect adulteration; it is still used today 49. Amino acid profiles are used extensively in Europe but have received little attention in the USA. Both commercial amino acid analysers and HPLC methods are available, with methods most often employing precolumn or post-column derivatization to facilitate detection. Ion-exchange chromatography followed by postcolumn derivatization with the ninhydrin reaction and detection at visible wavelengths is common and has

283

been used for orange juice analysis 15 . Pre-column derivatization techniques employ reversed-phase chromatographic separation; amino acid derivatives separated by reversed-phase HPLC methods include o-phthaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) with UV or fluorescence detection 50 , phenylisothiocyanate (PITC or PTC) with UV detection 51 , or dimethylamino azobenzene sulfonyl chloride (DABS) with visible detection 52 .

Organic acids Organic acids are important components of citrus juices, as they impart tartness. If the method of adulteration is by dilution and/or the addition of sugar, an acid, particularly citric acid, must be added, to balance the taste. Whereas commercial citric acid cannot be readily differentiated from authentic juice citric acid, the isocitric acid levels in juice have become more meaningful in assessing adulteration. Commercial isocitric acid is expensive and, therefore, is not readily added. The citric to isocitric acid ratio can indicate the addition of acid to orange juice. Isocitric acid is measured using isocitrate dehydrogenase (EC l.l.l.41 )51,54. L-malic acid occurs naturally in citrus juice, whereas D-malic acid is not a natural constituent. D-malic acid has been detected in commercial orange juices and is probably the result of the addition of synthetic malic acid, which contains both the D and L forms. Levels of added synthetically produced DL-malic acid can be calculated from the differences between the total malic acid content determined chemically and the L-malic acid content determined enzymatically (the difference represents the D-malic acid content)55. More recently, Eisele and Heuser 56 reported an HPLC method for the rapid separation and quantification of D- and L-malic acids in fruit juices, using a styrene-divinylbenzene column and a chiral mobile phase consisting of L-valine and copper acetate in water. Although the procedure has not been applied to orange juice, its potential application appears promising. Colorants Many methods have been developed or adapted to determine natural and/or added synthetic colorants to citrus juices 57 . Colorants are often added to improve the color score of a citrus product and, thereby, improve the juice'S final grade level. As an example, material may be added to juice from fruit low in pigment content to improve color. This can be accomplished by the addition of tangerine juice, which has a high level of carotenoids. In the USA, tangerine juice may be legally added at up to 10% the juice content; however, addition of tangerine juice to orange juice is illegal in Europe. Ting and Rouseff49 have described a qualitative method utilizing column chromatography to detect the addition of tangerine juice. More recently, Phillip et al. 58 described an HPLC method to detect the addition of tangerine juice based on the quantification of several carotenoid esters. Addition of annatto or turmeric food coloring is less 284

expensive than adding tangerine JUice. Turmeric can easily be detected by visible spectroscopy using the method developed by Petrus et al. 59 Ting and Rouseff49 have also described an HPLC method for detecting turmeric or annatto in orange juice, utilizing solid-phase extraction of juice to isolate pigments. Detection of curcumin, demethoxycurcumin, and bisdemethoxycurcumin indicates addition of turmeric. Detection of bixin and norbixin indicates adulteration with annatto. Other methods of determining carotenoids and colorants in citrus juices have been reviewed by Stewart60 and by Petrus and Nagl l .

Determination of geographical origin In the USA, geographical origin of an orange juice product is covered by both federal and state laws. A statement of the country of origin on the labelling of imported foods is not required by the Federal Food, Drug and Cosmetic Act. However, country of origin is required by the US Customs Service62 , which has determined that fruit juice concentrate that is imported and used in the production of concentrated or reconstituted fruit juices is not substantially transformed in processing and blending with other batches of concentrate and, therefore, that the country of origin marking is required on single fruit juices and concentrates containing the imported concentrate 61 . Florida law as related to the 'Florida sunshine tree' symbol states that the symbol should be restricted to use in advertising, promotion, merchandising and packaging of citrus products processed or manufactured from citrus fruit grown only in the State of Florida 25 • Methods to determine country of origin and to distinguish citrus products from different geographical areas of the same country (e.g. California versus Florida in the USA) have been developed. A large database has been acquired over many years to verify the applicability of the methods.

Mineral analysis Elemental analysis of orange juice samples by inductively coupled plasma - atomic emission spectroscopy (ICP-AES) can aid determination of the geographical origin of a juice. Each geographical region possesses a unique mineral composition, and these differences are reflected in fruit juice due to mineral adsorption and translocation from the root zone to the growing fruit. The high sensitivity of ICP-AES instrumentation can accurately quantify minerals at the ppb or ppt level. Applying computerized pattern-recognition techniques to 15 elements, Nikdel et al. 64 found that juice processed from fruit grown in Florida was readily distinguished from juice produced in Brazil, Mexico or Belize. Products from California and Arizona could also be distinguished. Barium, rubidium, calcium and copper were the most important elements in geographical differentiation. More recently, using refined pattern-recognition techniques available through the Ein*Sight software package (Informetrix, Seattle, DC, USA), Nikdel 65 found that juice blends from Florida and Brazil could be Trends in Food Science & Technology November 1992 [Vol, 3[

differentiated from non-blended juices. His method differs from previously published ICP-AES work comparing Florida and Brazilian orange juices 66 by utilizing acid digestion of samples in enclosed teflon containers combined with microwave radiation instead of dry ashing. Dry ashing was found to cause significant losses of rubidium, which is important in distinguishing geographical regions 67 . In addition, foaming problems associated with conventional acid digestion were eliminated, digestion times were shortened and less acid per sample was required.

9 10 11 12

13 14 15 16

Statistical evaluation of juice quality Many sophisticated instruments are at the disposal of food chemists wishing to acquire accurate and precise data on a juice sample. However, the acquisition of data is but the first step in the process of evaluating the authenticity of a juice. Evaluation of the acquired data must take into consideration important parameters related to fruit cultivar, growing conditions, geographical origin and processing variables. Nevertheless, the composition of citrus juice can be defined in a reasonable manner. It is highly doubtful that the determination of a single natural juice component can provide sufficient information to define the authenticity of a sample. However, if the juice contains a foreign substance the analyst can be reasonably certain that the juice has been adulterated. Most analysts agree that measurements of several components yield better estimates of juice content than a single parameter. Some of the first effective methods to combat adulteration employed multiple regression equations 68 - 7o • Other statistical adaptations have also been utilized: the chi-square tese l , groups of constituent ratios 72 , hierarchical classification73 and the directional tese 4 • Some of the newer methods that show considerable promise include the chemical matrix 43 , canonical variates analysis - analytical matrix 75 , discriminant analysis or pattern recognition 76 and neural networks n . Statistical programs to define the authenticity of a juice work best when a large database of information about authentic juices, such as that developed by the Florida Department of Citrus, is available.

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18 19 20 21 22

23 24 25

26 27 28 29 30 31 32 33 34 35

36 37

References 2 3 4 5 6 7 8

Nagy, S., Attaway, IA and Rhodes, MA, eds (1988) Adulteration of Fruit Juice Beverages, Marcel Dekker Krueger, D.A. (1988) in Adulteration of Fruit Juice Beverages (Nagy, S., Attaway, IA and Rhodes, M.A., eds), pp. 109-124, Marcel Dekker Doner, L,W. (1988) in Adulteration of Fruit Juice Beverages (Nagy, S., Attaway, IA and Rhodes, M.A., edsi, pp. 125-138, Marcel Dekker Martin, G.I., Danko, D. and Vallet, C (1991) J. Sci. Food Agrie. 56, 419-434 Bricout, I., Fontes, I.C and Merlivat, L. (1972) C. R. Acad. Sci. Paris Ser. D 274,1803-1806 Bricout, I. (1973) Ann. Falsif Expert. Chim. 66, 195-202 Brause, A.R., Raterman, I.M., Petrus, D.R. and Doner, L,W. (1984) }. Assoe. Off Anal. Chem. 67, 535-539 Dunbar, I. and Wilson, A.T. (1983) Plant Physiol. 72, 725-727

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Lee, H.S., Rousef!, R.L. and Fisher, IJ. (1986) j. Food Sci. 51, 568-570 Fisher, IJ. (1983) J. Agrie. Food Chem. 31, 66-68 Petrus, D.R. and Attaway, I.A. (1980) j. Assoe. Off. Anal. Chem. 63, 1317-1331 Walbrauch, S. (1986) Fluess. Obst. 53, 25-27 Rouse, A.H., Atkins, CD. and Moore, E.L. (1959) Proe. Fla. State Hortie. Soe. 72, 227-233 Bartolini, M.E. and len, I.J. (1990) j. Food Sci. 55, 564-565 Robertson, G.L. (1981) J. Food Biochem. 5, 139-143 Nikdel, S. (1991) in 42nd Annual Citrus Processors' Meeting, p. 23, Citrus Research and Education Center, Lake Alfred, FL, USA Kirksey, S., Schwartz, 1.0., Hutfilz, I.A. and Gudat, M.A. (1990) in 20ath National Meeting of the American Chemical Society, Division of Agricultural and Food Chemistry, Abstr. 134, American Chemical Society Rousef!, R.L. and Marcy, I.E. (1984) in 35th Annual Citrus Processors' Meeting, p. 29, Citrus Research and Education Center, Lake Alfred, FL, USA Gamache, P.H. (1989) ESA Application Notes 10-1244, ESA Inc., Bedford, MA, USA Nolan, S.M. and Koski, P.G. (1992) in 1992 1FT Annual Meeting Abstracts, p. 131, Institute of Food Technologists, USA Kirksey, S.T., Schwartz, 1.0. and Wade, R.L. (1992) in 1992 Annual Meeting Abstracts, p. 131, Institute of Food Technologists, USA Perfetti, G.A., loe, F.L., Fazio, T. and Page, S'w. (1988) j. Assoe. Off. Anal. Chem. 71,469-473 Page, S'w., loe, F.L. and Dusold, L.R. (1988) in Adulteration of Fruit Juice Beverages (Nagy, S., Attaway, I.A. and Rhodes, M.E., edsi, pp. 269-278, Marcel Dekker Cancalon, PJ. and Bryan, CR. (1992) in 43rd Annual Citrus Processors'Meeting, p. 6, Citrus Research and Education Center, Lake Alfred, FL, USA Brause, A. (1992) Int. Food Ingred. 1, 4-11 Rousef!, R.L., Martin, SJ. and Youtsey, CD. (1987)]. Agrie. Food Chem.35,1027-1030

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Richard, J,p, (1988) in Adulteration of Fruit juice Beverages (Nagy, S" Attaway, JA and Rhodes, M,E" edsL pp, 235-268, Marcel Dekker Brown, M,B" Katz, B,P, and Cohen, E, (1988) in Adulteration of Fruit juice Beverages (Nagy, S" Attaway, JA and Rhodes, M,E" edsL pp, 215-234, Marcel Dekker Fry, J, (1990) in Production and Packaging of Non-carbonated Fruit juices and Fruit Beverages (Hicks, D" ed.), pp, 68-113, Van Nostrand Reinhold Page, SW" Joe, F,L, and Dusold, L,R, (1988) in Adulteration of Fruit juice Beverages (Nagy, S" Attaway, JA and Rhodes, M,E" edsl, pp, 269-278, Marcel Dekker

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Nikdel, S, (1992) in 1992 1FT Annual Meeting Abstracts, p, 132, Institute of Food Technologists, USA

Review

Biosurfactants: moving counterparts. So far, the use of biosurfactants has been

towards industrial

limited to a few industrial applications, because biosurfactants have been economically uncompetitive. There is a need to gain a greater understanding of the physiology,

application

genetics and biochemistry of biosurfactant-producing strains, and to improve process technology to reduce production costs.

Armin Fiechter Chemically synthesized surface-active compounds are widely used in the pharmaceutical, cosmetic, petroleum and food industries. However, with the advantages of biodegradability, and production on renewable-resource substrates, biosurfactants may eventually replace their chemically synthesized Armin Fiechter is at the Institut fur Biotechnologie, ETH-Hbnggerberg, CH-8093 Zurich, Switzerland,

286

\01992, cbp\ ler SCierH e Puhll~h{-'r~ Ltd, (UK)

Surfactants and emulsifiers are integral to many industrial, agricultural and food processes. Most of the compounds are chemically synthesized, and it is only in the past few decades that surface-active molecules of biological origin have been described. Their surfactant and emulsification properties result from the presence of both hydrophilic and hydrophobic regions on the same molecule; aggregates form and accumulate at surface boundaries, thus separating the two phases. The industrial demand for surfactants is high: the market value for soaps and detergents reached Trends in Food Science & Techno[ogy November 1992 [Vol. 31