Cocoa detection

The quantity, quality and purity of the template DNA are important factors in successful PGR amplification. The PGR is an extremely sensitive method capable of detecting trace amounts of DNA in a crop or food sample, so PGR amplification is possible even if a very small quantity of DNA is isolated from the sample. DNA quality can be compromised in highly processed foods such as pastries, breakfast cereals, ready-to-eat meals or food additives owing to the DNA-degrading action of some manufacturing processes. DNA purity is a concern when substances that inhibit the PGR are present in the sample. For example, cocoa-containing foodstuffs contain high levels of plant secondary metabolites, which can lead to irreversible inhibition of the PGR. It is important that these substances are removed prior to PGR amplification. Extraction and purification protocols must be optimized for each type of sample. 

Theobromine was determined by GC in various foods (bitter chocolate, milk chocolate, chocolate cake, cocoa powder, chocolate milk), and results are given in graphs and tables.27 Homogenized samples were boiled in alkaline aqueous media, then fat was extracted with n-hexane. The aqueous layer was acidified with diluted HC1 and NaCl was added. Theobromine was extracted from this treated aqueous solution with dichloromethane and the extract was evaporated to dryness. The residue was redissolved in dichloromethane containing an internal standard. GC analysis was performed on a column packed with 1% cyclohexane dimethanol succinate on Gaschrom Q, with FID. Average recoveries were 99 to 101%, coefficient of variation was less than 3% and the limit of detection for theobromine in foods was about 0.005%. 

HPLC allows a quantitative determination with relatively simple extractions. In many cases, extraction only involves a heating of the commodity with water, followed by filtration and injection onto an HPLC column. In the determination of caffeine, theobromine, and theophylline in cocoa, coffee, or tea, as well as in other foods, there is scarcely a month that passes without a new paper on this assay. Kreiser and Martin provide typical conditions for analysis.28 In their studies, samples were extracted in boiling water and filtered prior to injection onto the HPLC column. The HPLC conditions used a Bondapak reversed phase column and a mobile phase of water methanol acetic acid (74 25 1) with detection at 280 nm. This method is accurate, precise, and conserves time. It has also been adopted by the AOAC as an official method for the determination of theobromine and caffeine in cocoa beans and chocolate products.29... 

Foods derived from cocoa beans have been consumed by humans since at least 460 to 480 AD. The source of cocoa beans, the species Theobroma, contains a variety of biologically active components. These include the purine alkaloids theobromine, caffeine, and theophylline. Structurally, they are methylated xanthines and, thus, are often referred to as methylxanthines. Theobromine (3, 7-dimethylxanthine) is the predominant purine alkaloid in cocoa and chocolate. Caffeine (1, 3, 7-trimethylxanthine), the major purine alkaloid found in coffee and tea, is found in cocoa and chocolate at about one eighth the concentration of theobromine. Only trace amounts of theophylline (1, 3-dimethylxanthine) are detected in cocoa and chocolate products. 

Although low levels of methylxanthines have been detected in the leaves and flowers of T. cacao, the primary storage location is within the seed or bean.16 The cocoa bean is the major natural source of the methylxanthine theobromine, but contains only small amounts of caffeine. Theophylline has been detected in cacao beans, but at such low concentrations that its presence generally is ignored. Together, theobromine and caffeine account for up to 99% of the alkaloid content of T. cacao beans. Alkaloid content is affected by genetic makeup, maturity of beans at harvest, and fermentation process. Analytical methodology also is partially responsible for some of the disparity in methylxanthine values since many early methods were unable to separate theobromine and caffeine. 

Acrolein has been detected in effluent water streams from industrial and municipal sources. Municipal effluents from Dayton, Ohio, for example, contained between 20 and 200 pg acrolein/L in 6 of 11 analyzed samples (USEPA 1980 Beauchamp et al. 1985). Acrolein is also a component of many foods, and processing may increase the acrolein content (USEPA 1980). Acrolein has been identified in raw turkey, potatoes, onions, coffee grounds, raw cocoa beans, alcoholic beverages, hops (USEPA 1980), white bread, sugarcane molasses, souring salted pork, and cooked bluefin tuna (Thunnus thynnus) (Beauchamp et al. 1985). 

Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometric Study of the Flavonoids of Cocoa (Jheobroma cacao) with Negative Ion Detection... 

Oxazoles have been found in relatively few cooked foods, although over 30 have been reported in coffee and cocoa, and 9 in cooked meat. Oxazolines have been found in cooked meat and roast peanuts, but not to any extent in other foods. 2,4,5-Trimethyl-3-oxazoline has been regularly detected in cooked meat [26], and when it was first identified in boiled beef [27] it was thought that the compound possessed the characteristic meat aroma however, on synthesis it was shown to have a woody, musty, green flavour with a threshold value of 1 mg/kg [28]. Other 3-oxazolines have nutty, sweet or vegetable-like aromas and the oxazoles also appear to be green and vegetable-like [28]. The contribution of these compounds to the overall aroma of heated foods is probably not as important as the closely related thiazoles and thiazolines. 

A-Nitrosodiethanolamine at levels of 600-7386 ppb was detected in 11 samples of cosmetics in the United States which included hand creams, face creams, shampoos, cocoa butter cream, moisturizing lotion and a make-up remover (all products listed diethanolamine and/or triethanolamine as ingredients) (Tunick etal., 1982). TV-Nitroso-diethanolamine was found in all seven cosmetic formulations one with traces (< 10 ng/g) and six at levels of 41 7 000 ng/g and in 12 of 13 lotions (seven with traces <10 ng/g and five with 14-140 ng/g) and in eight of nine hair shampoos (three with traces < 10 ng/g and live with 17-280 ng/g) in the United States (Fan et al., 1977b). Of 191 cosmetics analysed, 77 contained from 10 to more than 2000 ppb N-nitrosodiethanolamine (Elder, 1980). Westin etal. (1990) analysed 20 different suntan lotions in Israel and found that three were contaminated with 17-27 ppb TV-nitroso-diethanolamine (with traces 5-10 ppb). 

Catechin and the proanthocyanidin prodelphinidin B3 are, respectively, the major monomeric and dimeric flavan-3-ols found in barley and malt where prodelphinidin B3 is the main contributor for the radical scavenging activity [Dvorakova et al., 2007], Proanthocyanidins have also been detected in nuts. Hazelnuts (Corylus avellana) and pecans (Carya illinoensis) are particularly rich in proanthocyanidins containing ca. 5 g kg, whereas almonds (Prunus dulcis) and pistachios (Pistachio vera) contain 1.8-2.4 mg kg 1, walnuts (Juglans spp.) ca. 0.67 g kg, roasted peanuts (Arachis hypgaea) 0.16 g kg, and cashews (Anarcardium occidentale) 0.09 g kg 1 [Crozier et al., 2006c]. Dark chocolate derived from the roasted seeds of cocoa (Theobroma cacao) is also a rich source of procyanidins [Gu et al., 2004], Monomeric flavan-3-ols and the proanthocyanidin B2, B5 dimers, and Q trimer are found in fresh cocoa beans (Fig. 1.13). Flavan-3-ols have also been detected in mint... 

Ca. davidsoni (James et al., 1997). Abreu (1997) demonstrated in Brazil that Ca. dimid-iatus and Ca. obsoletus could be detected in cocoa warehouses with pheromone traps. Monitoring traps have also been used to survey beetles in different habitats for infestation by natural enemies such as nematodes (Dowd et al., 1995) these efforts resulted in the discovery of a new nematode species (Poinar and Dowd, 1997). Dowd (2000) used the pheromone of Ca. lugubris to compare infestations of these beetles in Bt and non-Bt sweet com in Illinois. Dispersal of oak wilt fungus by Co. truncatus and Ca. sayi has been investigated with the aid of pheromone traps (Amboum et al., 2005). 

With its development, HPLC was found to be useful in many authenticity determinations, either for the same or different components to those detected by GC. Triglycerides were the most immediate application. With the exception of milk fat, now that the major components of commercial fats can be completely separated by HPLC, the patterns of components can be analysed to detect adulteration. Cocoa butter adulteration with palm fractions can be detected by the presence of excess monounsaturated and diunsaturated components from the palm fraction, while more sophisticated products may be detected by measuring dipalmitoyl-monooleoyl glycerol (POP), palmitoyl-oleoyl-stearoyl glycerol (POS) and distearoyl-monooleoyl glycerol (SOS) components. In other oils, apart from the pattern of components, the presence of any significant level of... 

Many consumers also wish to be made aware of any genetic modification to the crop. Genetic modification might be beneficial for cocoa butter production as the characteristics of the butter could be modified in the growing bean, and also resistance to pests and diseases might be introduced to the plant. Analytical methods are required to detect such modification, in both the raw materials and the processed product. 

All of the classical analytical techniques applied to oils analysis have been applied to studying the authenticity of cocoa butter and considerable attention has been paid to the issue of detecting and quantifying non-cocoa fats in mixtures with cocoa butter and as incorporated into chocolate. A substantial review of these methods has been published (Lipp and Anklam, 1998b). 

Much of the sterol component of shea butter is present as esters of cinnamic acid, which are less readily saponified than esters with glycerol (Peers, 1977) published data for this fat might therefore underestimate the hue value. Surprisingly measurement of cinnamic acid has not been used to test for the presence of shea butter in mixtures. Triterpene alcohols similar to those found in shea also occur in sal fat. Homberg and Bielefeld (1982) showed the presence of triterpene alcohols in illipe and sal fats and in commercial CBEs, and their analysis was proposed as a qualitative measure to detect cocoa butter adulteration. 

Reaction of shea triterpene alcohols with acetic anhydride and sulphuric acid to produce coloured products (Fitelson s reaction) was the basis of a sensitive early test for the presence of shea butter in cocoa butter (Fincke, 1975). Analysis of the triterpene fraction of a commercial cocoa butter by TLC fractionation followed by GC (Fincke, 1976), or argentation TLC followed by GC (Gegiou and Staphylakis, 1985), have been shown to have potential for detecting CBEs in chocolate based on the difference in levels of P-amyrin, butyrospermol and... 

Detection of stigmastadienes in virgin olive oil at levels in excess of 0.15mg/kg is regarded under EC regulations as evidence of the presence of refined oils. The test is highly sensitive stigmastadiene levels in unrefined cocoa butter are well below 0.1 mg/kg whereas in refined butters up to several hundred mg/kg may be present. 

The quantitative determination of sterenes has been used to detect added CBEs or refining in cocoa butter and to establish the presence of CBAs in retail chocolates (Crews et al., 1997). The high quantity of triterpene alcohols in shea butters means that dehydration products of these sterols are formed in detectable levels when shea is bleached. These triterpene sterenes, which have not be characterized, can be detected in the stearin fraction used in CBEs (Crews etal., 1999). 

Pyrolysis of food samples provides a large number of products, which, after detection by MS and analysis by advanced statistical treatment, can be used to compare different samples. The method is very rapid and does not require chromatographic separation or MS identification of the pyrolysis products. Pyrolysis MS, coupled with multivariate data analysis procedures, has been used to discriminate between cocoa butters of three different continental areas (Radovic et al., 1998). The technique could in some cases separate deodorized from non-deodorized cocoa butters and also show those that have had alkali treatment. The presence of non-cocoa fats did not affect the assay. 

Kohiyama et al. (1992) reported mean levels of nickel, iron and copper of 0.03, 0.30 and 0.04 mg/kg, respectively, in 10 samples of cocoa butter. Baxter et al. (2001) found comparable results from the determination of 23 elements in 42 cocoa butters and 22 CBA fats, mostly of known geographical origin and processing history, by the sensitive multi-element technique of inductively coupled MS. No distinction could be made between the cocoa butters based on geographical origin or deodorization, and the very low levels of most elements in the CBA fats meant that their presence in mixtures with cocoa butter could not be detected. 

A fluorescence under UV illumination of an unidentified compound separated by TLC has been used to detect 5% kokum (possibly unrefined) in mixtures with cocoa butter (Deotale el al., 1990). Identification of this compound and its analysis by more specific techniques might be used to improve current methods of quantifying CBEs in chocolate. 

Nuclear magnetic resonance (NMR) can be used as a rapid alternative to differential scanning calorimetry in the determination of the solid fat content and studies on the melting behaviour. The determination is based on detection of the different populations of protons in solid and liquid phases, which indicates the hardness of the fat. Hernandez and Rutledge (1994b) used low resolution pulse NMR to compare melting curves of roasted and non-roasted cocoa butters from Africa, Indonesia and South America. Discriminant analysis techniques showed... 

The advent of relatively inexpensive computers has enabled the accumulation and rapid analysis of large sets of data. By this means patterns and trends not always apparent from visual inspection of chromatograms or tables of data can be discriminated by being sorted into recognizable patterns. This approach is essential for some techniques such as pyrolysis where the quantity of data produced would otherwise be overwhelming. Several statistical approaches to exploit the information content of fatty acid and triacylglycerol patterns for the detection and quantification of CBEs in cocoa butter have been reported (Lipp et al., 2001 Simoneau et al., 1999). 

Deotale, M.Y., Patil, M.N. and Adinarayaniah, C.L. (1990) Thin layer chromatographic detection of kokum butter in cocoa butter. J. Food Sci. Technol. (Mysore), 27, 230. 

Derbesy, M. and Richert, M.T. (1979) Detection of shea butter cocoa butter. Oleagineux, 34, 405 109. 

Fincke, A. (1975) Detection of shea fat in cocoa butter and cocoa butter substitutes. 1. Detection by Fitelson s reaction. Deut. Lebensm.-Rundsch., 71(8), 284-286. 

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