Determination of aflatoxins in by-products of industrial processing of cocoa beans

This study has examined the occurrence of aflatoxins in 168 samples of different fractions obtained during the processing of cocoa in manufacturing plants (shell, nibs, mass, butter, cake and powder) using an optimised methodology for cocoa by-products. The method validation was based on selectivity, linearity, limit of detection and recovery. The method was shown to be adequate for use in quantifying the contamination of cocoa by aflatoxins B₁, B₂, G₁ and G₂. Furthermore, the method was easier to use than other methods available in the literature. For aflatoxin extraction from cocoa samples, a methanol–water solution was used, and then immunoaffinity columns were employed for clean-up before the determination by high-performance liquid chromatography. A survey demonstrated a widespread occurrence of aflatoxins in cocoa by-products, although in general the levels of aflatoxins present in the fractions from industrial processing of cocoa were low. A maximum aflatoxin contamination of 13.3?ng?g^?1 was found in a nib sample. The lowest contamination levels were found in cocoa butter. Continued monitoring of aflatoxins in cocoa by-products is nevertheless necessary because these toxins have a high toxicity to humans and cocoa is widely consumed by children through cocoa-containing products, like candies.     

Effect of farm and industrial processing on the amino acid profile of cocoa beans

An investigation into the amino acid profiles of unfermented and fermented cocoa nibs, as well as process-line cocoa nibs (P-LCN) and processed cocoa cake samples (PCCS) was carried out. In the unfermented cocoa nibs, Glu (128 mg/g crude protein i.e. 128 mg/gcp) was the most abundant amino acid whilst the most concentrated essential amino acid in the same sample was Leu (72.2 mg/g crude protein); in the fermented cocoa nibs, a similar trend was observed, with respective values of Glu (153 mg/gcp) and Leu (62.4 mg/gcp). Lys (181 mg/gcp) was most abundant amino acid in P-LCN and Ile (63.3 mg/gcp) was the second most abundant; also, in PCCS, Lys (52.7 mg/gcp) was the most abundant amino acid but Asp (43.7 mg/gcp) was the second most abundant. The total amino acid content was (mg/gcp), 641 (unfermented nibs), 708 (fermented nibs), 635 (P-LCN) and 368 (PCCS), with corresponding essential amino acids of 300, 287, 478 and 185 (all with His), respectively. Based on whole hen’s egg, the limiting amino acids for the samples were: Ser (unfermented nibs), Met (fermented nibs), Ala (P-LCN) and Val (PCCS), whereas under provisional amino acid scoring pattern, they were: Met + Cys (unfermented nibs), Met + Cys (fermented nibs), Thr (P-LCN) and Val (PCCS). Prolonged and high heat treatments appeared to have reduced the essential amino acids of the PCCS as compared to the P-LCN. Significant differences existed between contents of essential amino acids and non-essential amino acids at p < 0.05 in unfermented cocoa nibs, fermented cocoa nibs and P-LCN.     

Changes of phenolic compounds and antioxidant capacity in cocoa beans processing

A number of studies have shown that the consumption of cocoa and chocolate products has positive health effects on humans. The object of this research was to monitor changes of total and individual phenolics and antioxidant capacity during the cocoa bean manufacturing. The loss of cocoa phenolics and their antioxidant activity vary with the degree of technological process. The process of roasting and cocoa nib alkalisation had the major influence on phenolic compounds as well as on antioxidant capacity. The roasting treatment resulted in 14% loss of the total phenolics content, while alkalisation resulted in 64% loss of total phenolics content. Procyanidins B1 and B2 as well as (?)‐epigallocatechin were the unstable components, while caffeic acid derivate showed the greatest stability in all technological process. Furthermore, PCA showed that phenolic contents, antioxidant capacity and non‐fat cocoa solids parts of the samples were classified in groups according technological conditions.     

Impact of industrial treatments on ochratoxin A content in artificially contaminated cocoa beans

Ochratoxin A (OTA) is a mycotoxin mainly produced by mould species of the genera Aspergillus and Penicillium, which grow on a variety of agricultural products. OTA-contaminated foodstuffs pose a major health hazard to consumers, including human and animal. In Côte d’Ivoire, numerous studies are being carried out to find the best way of preventing OTA contamination of cocoa raw material. The objectives of this investigation were to assess the impact of industrial treatment on OTA content in cocoa-derived products. Samples of cocoa pods were prepared under specific conditions promoting fungal proliferation on cocoa beans before processing. The beans underwent the usual industrial treatments – roasting, shelling, crushing, pressing and additive addition – and samples were taken at each stage. OTA was extracted with a methanol/3% sodium hydrogen carbonate solution and purified using an immunoaffinity column prior to HPLC analysis with fluorescence detection. OTA was detected in artificially contaminated cocoa beans at levels ranging from 3.4 to 44.7 µg kg^?1 with a mean value of 22.9 ± 3.6 µg kg^?1. OTA was mainly concentrated in the shell (93%). Roasting, shelling and additive addition significantly decreased levels of OTA by 24–40, 76 and 52%, respectively, with an overall reduction of ～91%. These results indicate that industrial processing of cocoa has a real impact on the reduction of OTA in final cocoa products.     

Microflora of imported cocoa beans

A representative sample of cocoa beans from each of 9 countries was examined microbiologically by dilution plating of the ground-up beans. Three of the four samples from the Caribbean gave counts of over 10⁶/g. In contrast, samples from Nigeria, New Guinea and Malaysia gave counts of about 10²/g. Two fungi, Aspergillus fumigatus and Rhizomucor pusillus were present in all the samples. Total numbers of bacteria and actinomycetes ranged from about 10⁶ to over 9.0 × 10⁷/g of beans.     

鱼类下脚料在工业中应用的研究进展

水产品加工业每年产生大量的下脚料,但下脚料的有效利用率很低,随意丢弃这些下脚料既会对环境造成危害也会使资源大量浪费。所以从这些下脚料中提取具有生物活性的功能性成分不仅可以减少污染而且可以再生产出高附加值的产品应用到整个工业领域。本文综述了鱼加工下脚料研究现状,尤其是其工业应用,希望能对鱼加工下脚料的高效开发利用提供有益借鉴。      

Distribution of fumonisins and aflatoxins in corn fractions during industrial cornflake processing

The aim of this study was to investigate the distribution of fumonisins (B(1) B(2,) and B(3)) and total aflatoxins (B(1), B(2), G(1), and G(2)) in various corn processed fractions. 92 batches of whole corn and derived dry-milled fractions (animal feed flour, flaking grits, corn flour and corn meal) and cooked and roasted cornflakes fractions were industrially obtained. Samples were analyzed for both groups of mycotoxins by enzyme-linked immunosorbent assay (ELISA). Dry milling of corn led to a heterogeneous distribution of the two groups of mycotoxins in the different parts of the grain, with increased levels in fractions processed from outer layers (animal feed flour and corn flour) and decreased levels in fractions processed from inner portions, such as corn meal and flaking grits. Levels of fumonisins in cornflakes were lower than 400 microg/kg, the maximum tolerable limit set by the EU. By contrast, three samples of final product were found to exceed the aflatoxin maximum tolerable limit of 4 microg/kg. Animal feed flour showed concentration factors of 317 and 288% for fumonisins and aflatoxins, respectively. Food traceability system was used by the industrial companies which processed corn into breakfast cereals. Nevertheless, even though the use of food traceability, which is defined as the ability to trace any food, feed, food-producing animal or substance that will be used for consumption through all stages of production, processing and distribution, only initial fumonisin contamination of whole corn and contamination of animal feed flour and corn flour were found to be correlated.     

Enzyme activities in cocoa beans during fermentation

The activities of endoprotease, aminopeptidase, carboxypeptidase, invertase (cotyledon and pulp), polyphenol oxidase and glycosidases were studied during heap fermentation of ICS-95 cocoa beans. These enzymes are of key importance in flavour precursor formation and in pigment degradation during cocoa fermentation. Optimal extraction and assay conditions were established to characterise the enzymes reactions and to quantify and compare enzyme activities during cocoa fermentation. The enzymes exhibited large differences in pH optima and stability during fermentation. Aminopeptidase, invertase (cotyledon and pulp) and polyphenol oxidase were strongly inactivated, carboxypeptidase was partly inactivated, whereas endoprotease and glycosidases remained active throughout the fermentation. Since many enzymes are inactivated during fermentation, it is generally recognised that the actual period of enzyme action is short. Although our results confirmed total inactivation for some enzymes, we show that several key enzymes are not completely inactivated during fermentation. Therefore, some enzyme reactions can continue throughout the whole fermentation process. Only polyphenol oxidase was strongly inactivated during sun and artificial drying of the beans. The other enzymes were stable during the drying process. Enzymes like endoprotease and glycosidases are still active in properly fermented and dried beans. © 1998 SCI.     

Site of decomposition of methyl bromide in cocoa beans

The main site of decomposition of methyl bromide in cocoa beans was shown to be in the alcohol-insoluble proteins of the shells. The methyl group of the fumigant becomes covalently bonded to the α-amino group of the various amino acids, the imidazole ring of histidine and the ε-amino group of lysine. An amino acid analysis of cocoa beans shows that methionine is the limiting essential amino acid and that cysteine is concentrated in the alcohol-insoluble proteins of the nib.     

Antioxidant capacity and phenolic content of cocoa beans

This study investigated the antioxidant capacity and total phenolic content of cocoa beans from different countries, namely Malaysia, Ghana, Ivory Coast and Sulawesi. The antioxidant capacity of water and ethanolic extracts prepared from cocoa beans was measured by three different assays. To estimate the total phenolic content, the assay using Folin–Ciocalteu reagent was used. The water extract showed the higher value of antioxidant activity based on β-carotene bleaching assay, while the ethanolic extract showed the highest scavenging and ferric reducing activities. Ghanaian cocoa beans showed the highest antioxidant and scavenging activities, followed by Ivory Coast, Malaysian and Sulawesian. However, Malaysian and Sulawesian beans exhibited the highest ferric reducing activity, compared to the other beans. The highest phenolic content was found in Malaysian beans, followed by Sulawesian, Ghanaian and Ivory Coast. A positive correlation existed for both ethanolic (r = 0.76) and water extracts (r = 0.78) between phenolic content and ferric reducing activity. Our results showed that antioxidant capacity and phenolic content of Malaysian cocoa beans were comparable to Ghanaian, Ivory Coast, and Sulawesian beans.     

Short-term toxicity study of irradiated cocoa beans in rats

Groups of rats (30 males and 30 females) were fed stock diet and diets containing 350 g kg^?1 of cocoa beans (non-irradiated or irradiated) for 18 weeks and observed for food consumption, haematological variations, changes in growth pattern and gross pathology. Even though there was no significant difference among the groups in haematological or gross pathological examinations, consistently less food was consumed by rats fed on diets containing either non-irradiated or irradiated cocoa beans. Furthermore, weight gain and terminal body weights of rats fed on cocoa-containing diets were less than those fed on the stock diet. Such differences, may at least in part, be due to alkaloids in the cocoa-containing diets and not as a result of irradiation of the beans.     

Identification of novel cocoa flavonoids from raw fermented cocoa beans by HPLC–MSn

Using a detailed HPLC–ESI-MS analysis including both high resolution and tandem MS analysis, a series of fourteen novel flavonoids were identified from alcoholic extracts of raw fermented cocoa beans from Cameroon. The novel compounds include a series of isomeric hexosides of flavan-3-ols and novel proanthocyanidine dimers and trimers and a sulfated flavan-3-ol. Additionally a jasmonic acid sulfate derivative was identified in the cocoa bean extracts as one of the major metabolites produced under fermentation stress. Therefore, the chosen HPLC–ESI-MS analysis method revealed a greater complexity of the cocoa bean polyphenolic extract than previously reported and allowed individual assignment of novel compounds.     

Prevention of condensation damage to cocoa beans shipped in containers

When cocoa is shipped in unlined general purpose shipping containers condensation on the internal surfaces of the containers frequently leads to deterioration. Two trial shipments of cocoa were made between Papua New Guinea and Australia to evaluate single-trip insulating linings of 12 mm insulating board and aluminium-faced buiding paper, and also the use of an additional drying process before loading, as means of minimizing condensation damage. Evaluation was based on visual examination for the presence of condensate, temperature and humidity measurement in the head space, measurement of the moisture content of samples of cocoa beans, and on sensory evaluation of chocolate prepared from cocoa samples.

Condensation was found to occur only after arrival in Australia. Building paper was found to be ineffective, and re-drying was accompanied by the formation of off-flavours. However, the insulating board was found to give adequate protection against the damage while introducing no off-flavours.     

Some quality characteristics of solar-dried cocoa beans in St Lucia

A field comparison of solar drying and open-air sun-drying of cocoa (Theobroma cocoa L) beans was carried out in St Lucia. Four methods of drying (indirect solar drier, direct solar drier, open air/perforated steel surface and open air/non-perforated wooden surface) were examined at three loading rates: 13·7, 26·9 and 40·4 kg m⁻². Beans from the open air had a higher incidence of external mould and poorer external appearance, though differences were minor. Beans dried at the lower rate of 13 kg m⁻² showed the best colour, but the highest titratable acidity. Conversely, beans dried at the higher loading rate of 40·4 kg m⁻² showed significantly lower titratable acidity, but poorer colour. Differences in cut-test score, colour, pH and titratable acidity between the open air and closed driers were small or not significant. While not significant, the indirect drier did show the highest cut-test score and the direct drier the poorest. Beans from the indirect drier were darker and more purple, while those from the direct drier were lighter coloured and less purple. The beans from the direct drier, dried to 6% moisture (WB) were, though not significantly so, more brittle and higher in titratable acidity than those from either the open air or indirect drier. Overall the beans from the indirect drier showed the highest quality and those from the direct drier the poorest. Whether the modest improvement of the indirect drier over the open air driers is sufficient to warrant investment in such a drier is unclear. © 1998 SCI.     

Oxidation of polyphenols in unfermented and partly fermented cocoa beans by cocoa polyphenol oxidase and tyrosinase

Unfermented and partly fermented dried cocoa beans have an excessively astringent and bitter flavour owing to their high polyphenol content. Studies on the remaining polyphenol oxidase activity and the oxidation of polyphenols in these beans have been conducted in relation to two factors, ie incubation time (0, 1, 2, 4, 8 and 16 h) and pH of incubation (3.5, 4.5, 5.5 and 6.5). Owing to unsuccessful polyphenol oxidation during incubation of partly fermented beans, incubation–enrichment treatments were carried out by combining incubation time (0, 8, 16, 24 and 32 h) and addition of crude cocoa polyphenol oxidase and purified tyrosinase from mushroom at 88 and 8800 units g^?1 and pH 5.5. Results showed that the remaining polyphenol oxidase activities of unfermented and partly fermented dried beans were 1 and 0.08% with specific activities of 9 and 1% respectively. The polyphenols of unfermented beans were effectively oxidised by incubation at 45 °C and pH 3.5–6.5 without enzyme enrichment; while those of partly fermented beans required enzyme enrichment. Both crude cocoa polyphenol oxidase and tyrosinase could be used for enzyme enrichment, but tyrosinase seemed to be more effective. © 2002 Society of Chemical Industry     

Extraction,identification and semi-quantification of oligopeptides in cocoa beans

Peptides and amino acids generated during cocoa bean fermentation are known to be the most important precursors for the development of cocoa aroma. Although cocoa fermentation and aroma development have been extensively studied, the cocoa oligopeptide fraction is under-investigated. In particular, the identification of specific peptide sequences and the quantification of cocoa peptides are scarce in the literature. The aim of this study was to investigate the presence of oligopeptides in fermented cocoa beans. Peptides were analysed by reversed phase LC/ESI-MS and LC/ESI-MS/MS, and the molecular masses of 44 different peptides were obtained by analysing the mass spectra associated with the most intense chromatographic peaks. Peptides were identified based on the exact molecular masses, mass fragmentation patterns and by comparison with vicilin and 21 kDa cocoa seed protein sequences. Semi-quantitative data on peptide presence in fermented cocoa samples of different geographic origin, different fermentation levels and on roasted products were also provided.     

Acidification,proteolysis and flavour potential in fermenting cocoa beans

Cocoa fermentations in Ghana and Trinidad as well as anaerobic fermentation-like incubations of fresh cocoa beans in Germany were carried out under controlled conditions. Samples of beans were taken during the course of these treatments and determinations were made as to acidification (pH, acetic acid content), proteolysis (free α-amino nitrogen, peptide nitrogen and SDS electrophoresis of the protein peptides) and flavour potential (gas chromatography of the highly volatile compounds, in particular isopentanal and organoleptic analysis after thin layer roasting). A positive correlation between acidification, proteolysis and the development of flavour potential during anaerobic fermentation can be demonstrated in principle. However, the flavour potential is increased if the temperature rise is comparatively slow in both normal fermentation and laboratory incubation. Strong acidification and high accumulation of amino acids and peptides were not essential for a good flavour potential. The isopentanal content proved to be a useful indicator of the progress of normal fermentation in the tropics. These findings can be interpreted on the basis of earlier results about germination-like processes in the protein vacuoles, pre- and post-mortem subcellular structures and the special characteristics of acetic acid diffusion. Conclusions which are relevant to the practice of cocoa fermentation are discussed in more detail.     

The moisture content-equilibrium relative humidity relationship of cocoa beans

The moisture content-equilibrium relative humidity relationships for cocoa beans grown in nine different countries were similar to each other, and the maximum safe storage moisture content in equilibrium with 70% relative humidity was between 6.0 and 7.9% at 25°C. There was a large variation between the moisture content of individual beans within each sample, the maximum standard deviation observed in a sample of thirty beans being 0.97, after the moisture content had been increased by adding water.