Authentication and quality assessment of food products

Conference Report Ever since the origins of food science (e.g. in the fonn of dairy science) a century or so ago, many resources have been dedicated to detecting economic fraud in the fonn of the adulteration of food products. Although it is still necessary to screen for unsophisticated fonns of adulteration (e.g. the detection of added water in milk, the detection of added ash, and the detection of added pulp wash in orange juice), the methods employed by adulterators have become increasingly elaborate in recent years. A range of sophisticated new techniques has been developed to tackle the problem, with the result that the secondary literature and other documents on the authentication of food products and food ingredients are rapidly becoming out of date. Thus, 'Authentication and quality assessment of food products' was convened at an appropriate time to review progress and difficulties in the area. Jointly organized by the Laboratoires Eurofins, I'Universite de Nantes and the Centre Euro¢en d' Analyse Isotopique Specifique (CEAIS), the meeting took place over five days. Four main themes were covered, in addition to an outline of general principles: fruit juices, fruit products and honey; wines, spirits and vinegars; practical applications of isotope analysis; and flavours and essential oils. The presentations focused mainly on isotopic analysis using two methods: stable isotope ratio analysis by mass spectrometry (SIRA-MS) and site-specific natural isotope fractionation by nuclear magnetic resonance spectroscopy (SNIF-NMR). SNIF-NMR, which was developed by Gerard J. Martin and colleagues at l'Universite de Nantes, France, is one of the most sophisticated and most specific methods available for the determination of authenticity. Given the many new applications of NMR in food analysis, it seems appropriate that Richard Ernst received his Nobel prize for the development of Fourier transfonn NMR during the week following the conference.

Authentication and quality assessment of food products* John O'Brien pathways that the molecules encounter. The difference between C 3 and C4 plants (see Box 1) is a good example of the contribution of metabolic pathways (in the fonn of kinetic isotope effects) to the isotopic composition of plant sugars. Such differences fonn the basis of the application of 13C measurement for determining the origin of a sugar. Thus, sugars from C 3 plants are lower in 13C than those from C4 plants, making it possible to differentiate between sugars from high-fructose com syrup and orange or grape. Similarly, as pointed out in another presentation at the meeting, by Landis W. Doner (US Department of Agriculture), all the significant floral sources of honey are C 3 plants and, surprisingly, honeys from all parts of the world have a narrow range of 13e values, which enables the detection of added com syrups. However, the determination of 13C content alone is not sufficient to detect added syrups prepared from beet sugar, for example, in a citrus product, because both citrus plants and sugar beet are C 3 plants. Gilles G. Martin (Laboratoires Eurofins, Nantes, France) described how multidimensional data analysis techniques could overcome this problem - for example, by plotting 8(13C1 12C) values (see below) versus 8(2HPH) values. The higher 2H content of citrus sugars, compared with beet sugars, assists in differentiating between the two.

Natural isotope distribution The rationale for and the principles of measuring natural isotope distributions were explained by Maryvonne L. Martin and Gerard J. Martin (l'Universite de Nantes). Knowledge of the natural abundance of 14C has been exploited regularly to differentiate between purely synthetic and natural molecules I. However, the method cannot discriminate between semisynthetic and authentic natural molecules. In addition, samples can be artificially enriched with 14C to mislead the analyst. Although the natural abundances of isotopes such as 13C or 2H are low (Table 1), their distribution among and within molecules in nature varies enough to be of value in determining the origin of such molecules. Factors influencing isotopic contents and distributions in molecules from plants and animals include climate, the isotopic distribution in the nutrients absorbed, and the metabolic • Held in Nantes, France, 7-11 October 1991

Stable isotope ratio analysis The total isotope ratios (e.g. 180Jl60, 13CJl2C or 2H/ 1H) of molecules are detennined using isotope ratio mass

Table 1. Average natural abundance levels {)f isotopes of hydrogen, carbon and {)xygen Natural abundance Element

Isotope

(%)

Hydrogen

IH 2H 3H

99.985 0.015 <10-16

Carbon

12C BC 14C

98.892 1.108

Oxygen

160 170 180

99.763 0.037 0.2

trace

lohn O'Brien is the editor of Trends in Food Science & Technology.

Trends in Food Science & Technology January 1992 [Vol. 3]

©1992, Elsevier Science Publishers Ltd, (UK)

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Box 1. The dark reactions of photosynthesis - C3 and C4 plants The dark reactions of photosynthesis are independent of light and use the ATP and NADPH produced in the light reactions to reduce carbon dioxide. In plants, such reactions take place in chloroplasts and there are two possible metabolic pathways. In C) plants, the first product of CO 2 reduction is 3-phosphoglycerate, a three-carbon (C)) compound. Two molecules of 3-phosphoglycerate are formed when carbon dioxide combines with ribulose l ,S-bisphosphate, catalysed by ribulose-bisphosphate carboxylase (EC 4.1.1.39). The 3-phosphoglycerate is usually reduced by NADPH and ATP to form glyceraldehyde 3-phosphate, which feeds a complex series of reactions (the Calvin cycle or carbonreduction cycle) from which hexoses such as glucose and fructose are formed. In C4 plants, the first products of CO 2 reduction are four-carbon (C 4) dicarboxylic acids, the first of which is oxaloacetate. Oxaloacetate is the product of the combination of phosphoenolpyruvate and COl! catalysed by the enzyme phosExamples of C3 and C4 plants phoenolpyruvate carboxylase (EC 4.1.1.31). Oxaloacetate is C3 plants C4 plants converted to malate and aspar(sugars lower in 13C) (sugars higher in 13C) tate, which are then transported to specia lized chloroplasts in the Maize Sugar beet Orange so-ca lled bundle sheath cells of Sugar cane Grape Potato the plants. In the bundle sheath Barley Apple Sorghum cells, CO 2 is released, which is Wheat then a substrate for the Calvin cycle as in C) plants. Thus, the key metabol ic difference between C) and C4 plants is that C4 plants possess a 'pump' that pumps CO 2 from mesophyll cells into the specialized bundle sheath cells. The result is high CO 2 concentrations in the bundle sheath cells; this inhibits photorespiration and, therefore, increases the photosynthetic efficiency of C4 plants. C4 plants also use less water per unit increase in dry weight than C) plants, and sugars formed by C4 plants have higher DC contents than sugars synthesized by C) plants.

spectrometry (IRMS). The standard used for determining the 180/ 160 and 2HJlH ratios is 'standard mean ocean water' (SMOW), whereas the standard used for the determination of the I3C/I2C ratio is the PDB carbonate standard (carbonate from the Pee Dee belemnite formation in South Carolina, USA). Ratios are normally expressed in terms of their deviation from the standards in the form of values (%0):

o

oeHl1H)

=[eH/1H)samPle -1] x 1000 CZH/1H)SMOW

O(I3C/ 12C) = [(I3CJl 2C )samPle 1 ] x 1000 (I3CJl2C)PDB K. Habfast (Finnigan MAT GmbH, FRG) described an extension of the IRMS technique that may prove to be very useful in authenticity testing: GC-IRMS. This is a relatively new technique and is in use in fewer than 40 laboratories worldwide. However, the method is already being used successfully by oil companies to characterize different crude oils.

SNIF-NMR The SNIF-NMR method was described in a number of presentations throughout the meeting by Gerard 1. Martin and Maryvonne L. Martin. The specificity of the method depends on the determination of the 2H concentrations at specific sites on the test molecule 2 (Fig. 1). In the case of ethanol, there are four different monodeuterated forms of the molecule (CH2DCH 20H, CH3CHDOH, CH3CDHOH and CH3CH 20D). Of the possible sites, the 2H on the methyl and methylene carbons are most useful

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because the hydrogen on the hydroxyl group is exchangeable. Thus, determinations of the 2H concentrations of the methyl and methylene groups of ethanol can be used as a 'fingerprint' of the origin of the molecule. The 2H1IH ratios of the methyl [eHl1Hb] and methylene [(ZHl1H)C1] sites of ethanol are closely related to those of the starting products. The value of CZHl1H)C2 is very sensitive to the 2H content of the non-exchangeable sites of the glucose, whereas the (ZHl1H)c, value is closely related to the 2H content of the fermentation water. Such relationships can be exploited to determine the origin of a wine or spirit alcohol and whether sugar was added before the fermentation. A combination of determination of the 2H concentrations on the C 1 and C 2 groups together with measurement of the overall I3C content is a powerful way of discriminating ethanols from different sources (e.g. barley, beet, cane and whey)3. Such combinations of parameters can be analysed statistically using a variety of multidimensional methods. Ethanol derived from whey is interesting because it is easily distinguishable from other ethanols and because its isotopic composition has been shown to depend strongly on the feed of the cow from which the milk was obtained.

Wine authentication Not surprisingly, a major focus of the meeting was the authentication of wines. The SNIF-NMR method was officially adopted in 1987 by the International Office of Vine and Wine and by the Commission of the European Communities as a means of detecting the chaptalization (adding extra sugar before fermentation) of wine with beet sugar. Chaptalization is permitted only for certain wines in France and the FRG, to increase the natural alcoholic strength in areas where the natural sugar content of the must is too low. Elsewhere it is illegal. R. Passelorgue (Ministry of Finance, France) described how the old method for detecting chaptalization - determination of the alcohol extract ratio - has now been replaced by the NMR method. Five EC countries have already purchased a total of 15 NMR spectrometers for the application of the SNIF-NMR method in their government laboratories. A large project is now upderway to increase the usefulness of SNIF-NMR by analysing reference samples from different parts of France. Part of the contribution of the French Ministry of Finance is to prepare reference wines on a laboratory scale from grape samples without added sugar. Such analyses are not restricted to France. H. Spiesecke (Environment Institute of the Commission of the European Communities, Ispra, Italy) described progress to date in the creation of the European Isotopic Databank of reference wines from EC member states. The databank, which is due to be completed by March 1993, will contain analytical data Trends in Food Science & Technology January 1992 [Vol. 3[

on a minimum of 400 samples from each of France and Italy, 200 from the FRG, 100 from Spain, with smaller numbers of samples from Luxembourg, Portugal and the UK. The sensitivity and reproducibility of the SNIF-NMR method for the detection of chaptalization was the subject of some debate. However, it was reported by Gilles G. Martin that it is possible to detect ethanol derived from beet sugar in a wine that contains 12% (v/v) alcohol with a precision of 0.3% (v/v) alcohol. Maryvonne L. Martin gave a comprehensive presentation on the origin authentication of wine. She highlighted the potential sources of error in the isotopic analysis of wines. For example, the preliminary distillation step can result in isotopic fractionation and, therefore, must be carefully standardized. Strict quality control procedures also need to be applied during NMR or MS measurements. Professor Martin also outlined the difficulties in determining the vintage of a wine. The determination of the 14C content of a wine has been applied for the determination of vintage. However, a shortcoming of such measurements is that, while there was a steady increase in atmospheric 14C during the 1950s and 1960s as a result of nuclear arms testing, levels have been decreasing for several years. Thus, it is conceivable that a young and an old vintage could contain similar 14C levels. The eHlIHb value of the ethanol is clearly correlated with the environmental conditions of the vintage year. However, reliable environmental data are required on the site where the grapes were grown in order to use eHlIH)cI values effectively. E. Humpfer (Bruker Analytische Messtechnik GmbH, FRG) gave an interesting presentation that examined the application of l3C NMR in wine authentication. From a purely analytical point of view, the measurement of l3C levels has an advantage in that 13C is considerably more abundant than 2H, and is also slightly more sensitive in NMR spectroscopy experiments. However, a disadvantage of using the l3C nucleus is the long relaxation times encountered, which can extend analysis times considerably. The work described by Humpfer, which was conducted in conjunction with workers at the Bundesforschungsanstalt fUr Rebenziichtung Geilweilerhof, FRG, was concerned with the direct determination of methanol, diethylene glycol, sugars, acids and amino acids in wine4. The advantage of the method is that qualitative and quantitative measurements may be conducted rapidly without a previous separation. Additional compounds may be detected in wine extract, and the l3C NMR method has also been applied to fruit juice analysis.

Fruit juice authentication The adulteration of fruit juices, particularly orange juices, has attracted considerable publicity in recent years. Such publicity is not surprising considering the extent of the problem. A report published by the UK Ministry of Agriculture, Fisheries and Food in 1990 revealed that most fruit juices on the UK market at that time had been adulterated in some ways. G.E. Gouin (International Federation of Fruit Juice Producers, Paris, France) reviewed the methods Trends in Food Science & Technology January 1992 [Vol. 3]

H '

.•IIIIIID

A H3C

OH

Fig. 1 The monodeuterated forms ('isotopomers') of ethanol.

available for detecting fruit juice adulteration. These include the determination of organic acids such as citric acid or malic acid. However, it was pointed out that, while the determination of D-malic acid was once a useful method of detecting the addition of synthetic malate, synthetic L-malate produced by biotechnology is now available commercially. The characterization of fruit juices by MS was described by J. Bricout (Centre de Recherches PernodRicard, Creteil, France). He reminded the audience that the 2H content of oranges reflects the climate in which they are grown and tends to be higher in warm climates. In addition, it was pointed out that sugar beet can be grown in a warm climate such as in Texas or Morocco with the result that its 2H content is higher. This may present difficulties for the analyst attempting to detect beet sugar in other products. Allan R. Brause (Analytical Chemical Services of Columbia, Inc., MD, USA) gave an interesting perspective on the detection of fruit juice adulteration in the USA. Brause suggested that adulteration by a manufacturer can sometimes be detected simply by conducting an audit on a company's books to determine if, for example, sugar was being purchased for addition to a fruit juice. The 'matrix method' for the detection of fruit juice adulteration was described in detail. For orange juice, the matrix method involves, inter alia, the determination of fructose, glucose, sucrose, total sugars, sodium, potassium, 180, naringin, sodium benzoate, l3C and carotenoids. Brause set the stage for the next speaker (Gilles G. Martin) by suggesting that SNIF-NMR is the only useful method for the detection of beet sugar adulteration of apple juice. Martin focused on the methodology of determining the authenticity of fruit juices in more detail, with particular emphasis on SNIF-NMR and SIRA. SNIF-NMR of fruit juice is conducted by fermenting the juice and recovering the resulting alcohol by distillation. Determination of the 2H and 180 contents of juice water can help to determine if a juice had been made from a concentrate. However, Martin also cautioned that 180-enriched water can be obtained cheaply and added to adulterated products in order to mimic the 180 content of authentic orange juice or concentrate.

Authentication of flavourings The importance of the trend towards natural and nature-identical flavourings was emphasized by M. Hariel (Neoflaveur, Paris, France), who reported that 21

2

HO

1

CHO

presentation focused largely on GC methods using chiral stationary phases such as Lipodex C (a ~-cyclodextrin) and Lipodex E (a y-cyclodextrin). The choice of stationary phase depends on the enantiomers to be separated.

Conclusions The meeting was not as broad in its scope as might have been expected from the title. Attention was largely focused on two methods: SNIF-NMR and SIRA-MS. In this respect, the meeting was really a new develop1 2 ments seminar; the older traditional techniques of H H authentication received little attention. In addition, perhaps it would have been useful, for the sake of comparison and completeness, if some of the other modern 3 4 6 methods of authenticity testing had been included. Such CH=CH-CH 3 methods include, for example, the application of DNA probes coupled with the polymerase chain reaction to identify plants and animals6 • It has been proposed that Anethole synthetic DNA segments encoding information about products could be added to products and product labels Fig. 2 to provide a means of establishing their authenticity. Hydrogen sites on vanillin and anethole that give NMR signals. Note Similarly, a range of enzyme-linked immunosorbent that one pair of hydrogen sites on vanillin (position 3) and two pairs assays (ELISAs) have been developed in recent years of hydrogen sites on anethole (positions 1 and 2) are equivalent. for the detection and quantification of ingredients such as bovine casein and whey proteins, and skeletal muscle antigens from pork, beef or chicken7•8 • Other methods of 75% of the flavours used by Nestle in 1987 were natu- meat species identification have also been published ral, less than 10% were artificial and -15% were nature- recently9. Research currently devoted to the detection of irradiidentical. Several major frauds involving flavourings were cited throughout the meeting, and impressive new ated foods to and the calculation of the dose of radiation techniques have been developed for the authentication absorbed by a food could also be regarded as a branch of such materials. The application of in vitro biosyn- of authenticity testing. Clearly, a meeting covering all thetic methods for flavour production will present new aspects of authenticity testing would be quite an amopportunities for flavourists (P. Delest, Sanofi Bio- bitious undertaking, and it must be stated that Industries, Paris, France) and, possibly, new problems 'Authentication and quality assessment of food prodfor the analyst. Gilles G. Martin reported that a consid- ucts' was the most comprehensive meeting of its type erable number of flavour molecules can be examined by yet held. Judging by the pace of research in the field, SIRA and SNIF-NMR. For example, the SNIF-NMR there will be plenty of new developments to report when method can be applied to acetic acid, lactic acid, the second conference in this series is held in a few benzaldehyde, geraniol, vanillin, anethole, citral, ethyl years' time. Further information may be obtained from acetate and many other flavour compounds. Molecules the co-organizers Eurofins at Site de la Geraudiere, CP such as vanillin and anethole (Fig. 2) contain many more 4001,44073 Nantes Cedex 03, France. non-equivalent non-exchangeable sites than ethanol and, References thus, the SNIF-NMR becomes more complicated. A Culp, R.A. and Noakes, J.E. (1990) J. Agric. Food Chern. 38, common approach when examining molecules such as 1249-1255 this is to measure the ratios of the 2H contents at certain 2 Guillou, c., Remaud, G. and Martin, G.J. (1991) Trends Food Sci. sites on the molecule to the 2H content at other sites (R jjj Techno/. 2, 85--S9 values). Such an approach provides an internal reference 3 Martin, G.J., Danho, D. and Vallet, C. (1991) J. Sci. Food Agric. 56, and also an additional 'fingerprint' for the characteriz419-434 4 Rapp, A., Markowetz, A. and N~rgall, H. (1991) z. Lebensm. ation of such molecules. Unters. Forsch. 192, 1-6 The application of chirospecific gas chromatography 5 Report on the Quality of Fruit Juices in the UK (1990) Ministry of (GC), on-line GC-IRMS and multidimensional GC to Agriculture, Fisheries and Food, UK flavour analysis was described by Pieter Schreier 6 Jones, J.L. (1991) Trends Food Sci. Technol. 2, 28-32 (University of Wtirzburg, FRG). Schreier emphasized 7 Demeulemester, c., Lajon, A., Abramowski, V., Martin, J-L. and the importance of the enantiomeric configuration in Durand, P. (1991) J. Sci. Food Agric. 56, 325-333 flavour molecules by pointing out the differences 8 Anon. (1990) Trends Food Sci. Technol. 1, 30 between the flavours of (+)- (S)-carvone (caraway) and 9 Sawaya, W.N., Saeed, T., Mameesh, M., EI-Rayes, E., Husain, A., Ali, S. (-)- (S)-carvone (spearmint). Although optical methods and Abdul Rahman, H. (1990) Food Chern. 37, 201-219 and NMR (in conjunction with the use of chiral re- 10 Grootveld, M., Jain, R., Claxson, A.w.D., Naughton, D. and agents) can be used for chiral analysis, Schreier's Blake, D.R. (1990) Trends Food Sci. Techno/. 1,7-14 VaniHin

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Trends in Food Science &Technology January 1992 [Vol. 3]