Aflatoxigenic fungi and aflatoxin in cocoa

This paper reports the occurrence of aflatoxigenic fungi and the presence of aflatoxins in 226 cocoa samples collected on Brazilian farms. The samples were taken at various stages of fermentation, drying and storage. A total of 819 potentially aflatoxigenic fungi were isolated using Dichloran 18% Glycerol agar after surface disinfection, and identified by standard techniques. The ability of the fungi to produce aflatoxins was determined using the agar plug technique and TLC. The presence of aflatoxins in cocoa samples was determined by HPLC using post-column derivatization with bromide after immunoaffinity column clean up. The aflatoxigenic fungi isolated were Aspergillus flavus, A. parasiticus and A. nomius. A considerable increase in numbers of these species was observed during drying and storage. In spite of the high prevalence of aflatoxigenic fungi, only low levels of aflatoxin were found in the cocoa samples, suggesting the existence of limiting factors to the accumulation of aflatoxins in the beans.     

Behavior of Salmonella during fermentation,drying and storage of cocoa beans

Due to cocoa being considered a possible source of Salmonella contamination in chocolate, the behavior of Salmonella during some cocoa pre-processing stages (fermentation, drying and storage) was investigated. The fermentation process was carried out on a pilot scale (2 kg beans/box) for 7 days. Every day a fermentation box was inoculated with a Salmonella pool (ca. 4 log MPN/g). The results showed that Salmonella did not affect (P > 0.05) the growth of the main microorganism groups involved in cocoa fermentation. On the other hand, the pathogen was influenced (P < 0.05) by yeast, acetic acid bacteria and pH. In spite of Salmonella showing counts ≤ 1 log MPN/g in the first days, at the end of fermentation it grew in all samples, reaching counts as high as 7.49 log MPN/g. For drying and storage, cocoa beans were inoculated during the fermentation (experiment A) or during the drying (experiment B). In these stages the decline of the water activity affected the pathogen behavior. In experiment A during the drying, Salmonella count increased in most of the samples. In experiment B either a slight growth or no growth in the samples inoculated up to 48 h was observed, whereas the other samples showed reductions from the initial count. After 30 days of storage at room temperature, the water activity decreased to 0.68, and reductions of Salmonella ranged from 0.93 to 2.52 log MPN/g. Despite the reductions observed during the storage, the pathogen was detected even after 120 days. Therefore, the results showed that Salmonella growth or survival depends on when the contamination occurs.     

Contamination with storage fungi of human food from Cameroon

In a mycological study, a total of 95 human food samples were investigated to evaluate the incidence of fungal contamination in Cameroon by conventional identification method and partly confirmed by DNA sequencing. The isolated fungal spp. were further studied to determine their toxigenic potentials. The investigation revealed the predominance of Aspergillus and Penicillium with 96% of samples contaminated with at least one species of these fungi, whereas the incidence of co-contamination of samples was 85%. Aspergillus flavus and Aspergillus parasiticus (Flavi section) were the most predominant species contaminating mainly maize and peanuts. In addition, P. crustosum and P. polonicum were the most common contaminants belonging to the genus Penicillium. On the other hand, A. ochraceus (Circumdati section) registered a low incidence rate of 5%, including other members of the Aspergillus group. Other members of the genera Rhizopus and Alternaria spp. were also registered in the study. A majority of fungal strains of A. ochraceus, A. parasiticus, P. crustosum and P. polonicum isolated were toxigenic, producing the mycotoxins tested for, while none was detected in cultures of A. fumigatus. The high incidence rate of fungi contamination coupled with their potentials in producing mycotoxins gives a strong indication that the samples tested may likely be contaminated with various mycotoxins. There is need for further study to assess the incidence of mycotoxins contamination in similar food samples.     

The Content of Polyphenolic Compounds in Cocoa Beans (Theobroma cacao L.), Depending on Variety,Growing Region,and Processing Operations: A Review

Polyphenols form the largest group of compounds among natural antioxidants, which largely affect the overall antioxidant and antifree radical activity of cocoa beans. The qualitative and quantitative composition of individual fractions of polyphenolic compounds, even within one species, is very diverse and depends on many factors, mainly on the area of cocoa trees cultivation, bean maturity, climatic conditions during growth, and the harvest season and storage time after harvest. Thermal processing of cocoa beans and cocoa derivative products at relatively high temperatures may in addition to favorable physicochemical, microbiological, and organoleptic changes result in a decrease of polyphenols concentration. Technological processing of cocoa beans negatively affects the content of polyphenolic compounds.     

Study of ochratoxin A-producing strains in coffee processing

Ochratoxin A (OTA) is the main mycotoxin that has been detected in coffee. The occurrence of OTA in coffee beans can be because of environmental conditions and/or processing conditions. Three coffee processes were evaluated (wet, mechanical and dry processes), at different stages from harvesting to storage, and fungi producing OTA were enumerated and identified. The frequency of potential OTA‐producing fungi and their ability to produce OTA was also studied. By direct plating, the levels of contamination found in the coffee processes were 80, 72 and 92%, respectively, for parchment and dry cherry coffee and 20, 34 and 15% for green coffee. Aspergillus ochraceus isolated from the three processes accounted for 6.6, 8.3 and 3.3%, and Aspergillus niger for 15, 13 and 25% of the strains isolated, respectively. The toxigenic potential of five A. ochraceus and two A. niger strains was tested in FDA medium and coffee medium using the HPLC technique. There was no difference between the processes studied in terms of isolation and occurrence of ochratoxigenic fungi.     

Assessment of Thymus vulgaris L. essential oil as a safe botanical preservative against post harvest fungal infestation of food commodities

A total of 14 odoriferous angiospermic essential oils were tested against the toxigenic strain of Aspergillus flavus. The essential oil of Thymus vulgaris L. showed highest antifungal efficacy. The thyme oil absolutely inhibited the mycelial growth of A. flavus at 0.7 μl ml^− 1 and exhibited a broad fungitoxic spectrum against eight different food contaminating fungi viz. Fusarium oxysporum, Cladosporium herbarum, Curvularia lunata, Aspergillus terreus, Aspergillus niger, Aspergillus fumigatus, Alternaria alternata and Botryodiploidia theobromae. The oil also showed significant antiaflatoxigenic efficacy as it completely arrested the aflatoxin B₁ production at 0.6 μl ml^− 1. Thyme oil as fungitoxicant was also found superior over most of the prevalent synthetic fungicides. The LC₅₀ of thyme oil against mice was recorded as 7142.85 μl kg^− 1 body weight indicating its non-mammalian toxicity and strengthening its safe exploitation as preservative for stored food commodities. The findings recommend the thyme oil as potential botanical preservative in eco-friendly control of biodeterioration of food commodities during storage.

Industrial relevance

The thyme essential oil may be recommended for large scale application as a plant based preservative for stored food items because of its strong antifungal as well as antiaflatoxigenic efficacy. Because of broad antimicrobial spectrum, more efficacy over prevalent synthetic preservatives as well as non-mammalian toxicity, the thyme essential oil may be formulated as a safe and economical plant based preservative against post harvest fungal infestation and aflatoxin contamination of food commodities.     

Occurrence of ochratoxin A in cocoa by-products and determination of its reduction during chocolate manufacture

This work reports an investigation carried out to assess the natural occurrence of ochratoxin A in 168 samples from different fractions obtained during the technological processing of cocoa (shell, nibs, liquor, butter, cake and cocoa powder) and the reduction of ochratoxin A during chocolate manufacture. Ochratoxin A analyses were performed with immunoaffinity columns and detection by high performance liquid chromatography. Concerning the natural ochratoxin A contamination in cocoa by-products, the highest levels of ochratoxin A were found in the shell, cocoa powder and cocoa cake. The cocoa butter was the least contaminated, showing that ochratoxin A seems to remain in the defatted cocoa solids. Under the technological conditions applied during the manufacture of chocolate in this study and the level of contamination present in the cocoa beans, this experiment demonstrated that 93.6% of ochratoxin A present in the beans was reduced during the chocolate producing.     

Effect of low pressures on the survival of cocoa pests at 18°C

This study forms part of an effort to eliminate the need for fumigation with methyl bromide to control insect infestations in stored cocoa beans, through development of novel alternative vacuum-hermetic technology. In this communication, the effects of low pressures and exposure time were studied on the mortality of insects at a temperature of 18°C, chosen to simulate cocoa bean storage conditions in temperate climates.Three insect species were used, two of which are major pests of cocoa beans in producer countries, Ephestia cautella (Walker), and Tribolium castaneum (Herbst), while the third, Oryzaephilus surinamensis (L.), is a potential storage pest in temperate climates. For T. castaneum and E. cautella the egg stage was the most resistant to 55±10 mm Hg at 18°C, the times needed to obtain 99% egg mortality were 96 and 149 h, respectively. For O. surinamensis, the adult stage was the most resistant with 164 h being required to obtain 99% mortality.     

Differenzierung von Theobroma cacao und Theobroma grandiflorum mittels PCR

Zusammenfassung:  Die Gattung Theobroma umfasst etwa 20 Arten, von denen insbesondere die Art Theobroma cacao L. mit den beiden Hauptsorten (Variet?ten) „Criollo“ und „Forastero“ wirtschaftliche Bedeutung hat. Bislang erfolgte die Unterscheidung der Kakaosorten und -arten nur morphologisch und, dadurch bedingt, nur in einem sehr frühen Verarbeitungsstadium, d. h. eine Reinheitskontrolle ist zu einem sp?teren Zeitpunkt nicht mehr m?glich. Andererseits erfolgt die Herstellung von Kakaomasse heute zunehmend im Erzeugerland, Schokolade oder Kakaohalberzeugnisse werden vom verarbeitenden Unternehmen zugekauft. Eine genaue und reproduzierbare Spezifikationskontrolle ist aus den skizzierten Gründen bislang nicht m?glich. Im Gegensatz zur alten Kakaoverordnung liefert die neue Kakaoverordnung vom 15. Dezember 2003 keine Definition des Kakaos mehr. Die Kakao- und Schokoladenindustrie geht aber nach wie vor davon aus, dass nur die Kakaoart T. cacao bei der Herstellung von Schokolade zur Anwendung kommt (allgemeine Verkehrsauffassung in Anlehnung an die alte Kakaoverordnung). Die Kakaoart T. grandiflorum (Cupuassu) gewinnt auf dem südamerikanischen Markt immer mehr an Bedeutung. Beispielsweise werden die Bohnen vermehrt zu Schokolade-artigen Produkten (Cupu-lade) verarbeitet. Im Rahmen dieser Arbeit wurden eine PCR- und eine PCR-RFLP Methode entwickelt, die eine Detektion beider Theobroma-Arten in Kakao und Schokoladenprodukten erlauben.

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The genus Theobroma comprises about 20 species. The highest economic attention is paid to the cultivars (varieties) “Criollo” and “Forastero” of the species Theobroma cacao L., which are used for chocolate production. Up to now, the differentiation of cocoa species and cultivars is performed morphologically. Hince, a purity control in late processing steps of chocolate production is not possible. However, most of the cocoa mass production takes place in the countries of cocoa origin and is then bought as semi-finished products by the chocolate industry. Species identification is not possible at this stage due to the manufacturing process. In the former “Kakaoverordnung” a definition of the cocoa species (T. cacao) used for chocolate production was given. The current “Kakaoverordnung” (passed in December 2003) lacks such a definition. Nevertheless, it is general acceptance in the chocolate industry to exclusively use the species T. cacao for chocolate production. On the other hand, the species T. grandiflorum (Cupuassu) is getting more and more important on the South American market where beans are used for the creation of chocolate-like products e.g. Cupu-late. This work describes the development of a PCR and a PCR-RFLP method for the detection of cocoa species in chocolate products.

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Eingegangen: 20. Juli 2007; angenommen: 8. August 2007     

Isolation, enumeration and PCR characterization of aflatoxigenic fungi from food and feed samples in India

A total of 54 market samples comprising nine different food and feed commodities from Mysore city were examined in order to isolate aflatoxin-producing fungi as well as to assess aflatoxins in the commodities. Thirty-two samples were contaminated with aflatoxigenic fungi and the total mycoflora and aflatoxigenic fungi in different food and feed commodities were in the range of 0.2–260 and 0–100 cfu×10³/g, respectively. In total, 136 fungi were isolated, of which 32 were Aspergillus flavus strains and 26 of them were found to produce aflatoxins. A. flavus group of fungi comprising A. flavus, A. parasiticus, A. oryzae, A. sojae were characterized by using Aspergillus differential medium and PCR. The PCR was performed using two different sets of primers specifically targeted to aflR and omt genes of aflatoxin biosynthesis pathway. Most of the fungi belonging to A. flavus group reacted positively with the primers resulting in expected size amplicons of 796 bp for aflR and 404 bp for omt. Among the nine commodities screened for aflatoxin only, groundnut and groundnut cake were contaminated with aflatoxins B₁ and B₂. The aflatoxin contamination in these commodities exceeded the Indian regulatory limit of 30 μg/kg.     

Analysis and occurrence of deoxynivalenol (DON) in cocoa

The primary objective of this study was to establish a current situation assessment of the possible occurrence of deoxynivalenol in cocoa and cocoa products. Since there was no analytic method for determining DON in cocoa and cocoa products, a special method was developed. The applicability and consistency of the method was confirmed by performing recovery assays on various cocoa products. A special post-column derivatisation procedure was used to increase selectivity and raise sensitivity by a factor of 80. The method’s limit of detection (LOD) was thereby reduced to 7 μg/kg; the limit of quantification (LOQ) was 14 μg/kg. The method was used to test 230 samples for possible DON content, ranging from cocoa beans to cocoa bean shells, nibs, cocoa liquor and cocoa powders through to finished cocoa-based products. In the case of cocoa beans and cocoa bean shells, DON content close to the detection limit was only determined in isolated cases. No DON content was detected in nibs, cocoa liquor, cocoa powders and finished cocoa-based products. Analogous to ochratoxin A and aflatoxins, the results show DON is more likely to be found in cocoa bean shells. Separation of shells during cocoa processing can reduce potential DON contents. Since no DON was determined in the fractions relevant for chocolate production, these assays show it does not represent a considerable issue for the cocoa and chocolate industry.     

Evaluation of chemically characterised essential oils of Coleus aromaticus,Hyptis suaveolens and Ageratum conyzoides against storage fungi and aflatoxin contamination of food commodities

The result of the present investigation explores the efficacy of chemically characterised essential oils (EOs) of Coleus aromaticus, Hyptis suaveolens and Ageratum conyzoides as antifungal and antiaflatoxigenic agent against some storage fungi and the toxigenic strain of Aspergillus flavus (Saktiman 3NSt). Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of these EOs were also determined against the toxigenic strain of A. flavus (Saktiman 3NSt). The EO from C. aromaticus was found to be most effective exhibiting MIC and MFC at 0.1μL mL^?1. The EOs also completely checked aflatoxin B₁ synthesis in concentration‐dependent manner. In addition, fumigation of stored wheat samples with EOs exhibited remarkable protection (>80%) from fungal infestation showing their efficacy during in vivo storage conditions. Based on the results of the present investigation, the EOs of C. aromaticus, H. suaveolens and A. conyzoides may be recommended as novel plant‐based antifungal and aflatoxin B₁ suppressor over the synthetic preservatives.     

ITS-RFLP characterization of black <Emphasis Type="Italic">Aspergillus</Emphasis> isolates responsible for ochratoxin A contamination in cocoa beans

In the present study, ochratoxigenic mycobiota in cocoa beans was identified at species level by digestion of the ITS products using the endonucleases HhaI, NlaIII and RsaI. Of the 132 isolates of Aspergillus section Nigri collected from cocoa beans, 89 were identified as A. tubingensis, 27 as A. niger, 10 as A. tubingensis-like and 6 as A. carbonarius. No variation was observed between RFLP patterns (C, N, T1 and T2) described previously for grape isolates and those of the cocoa isolates analysed. With respect to OTA-producing fungi, a high percentage of black aspergilli (50.7%) was able to produce OTA. Additionally, most of the OTA-producing isolates were of moderate toxigenicity, producing amounts of OTA from 10 μg g⁻¹ to 100 μg g⁻¹. Percentages of OTA-producing isolates in the A. niger aggregate were higher than in other substrates, ranging from 30% to 51.7%. Furthermore, the detected levels of OTA production in the A. niger aggregate, particularly in A. tubingensis species was higher than in A. carbonarius, ranging from 0.7 μg g⁻¹ to 120 μg g⁻¹ (mean 24.55 μg g⁻¹). Due to the high occurrence, percentage of ocratoxigenic isolates and their ability to produce OTA, isolates belonging to the A. niger aggregate could be considered as the main cause of OTA contamination in cocoa beans used for manufacturing cocoa products.     

Chemical composition of Ocimum basilicum L. essential oil and its efficacy as a preservative against fungal and aflatoxin contamination of dry fruits

The study presents fungal and aflatoxin contamination of some dry fruits and Ocimum basilicum essential oil (EO) as a plant‐based preservative. During mycoflora analysis, 2045 fungal isolates were recorded from dry fruits and 40% isolates of Aspergillus flavus were toxigenic in nature. The EO of O. basilicum exhibited strong fungitoxicity against toxigenic strain of A. flavus. Its minimum inhibitory concentration (MIC) was recorded at 1.0 μL ml^?1, and it completely inhibited aflatoxin B₁ production at 0.5 μL ml^?1. The oil exhibited broad fungitoxic spectrum and considerably reduced A. flavus isolates from dry fruits when used as fumigant in closed storage containers at 1.0 μL ml^?1. The chemical profile of the EO was standardised through GC–MS analysis. Based on antifungal potency, antiaflatoxigenicity and efficacy as fumigant during storage conditions, O. basilicum EO may be recommended as a botanical preservative for enhancing the shelf life of dry fruits and edible products during storage.     

Spread of Aspergillus flavus and aflatoxin accumulation in postharvested maize treated with biocontrol products

Maize is a major staple crop and calorie source for many people living in Sub-Saharan Africa. In this region, Aspergillus flavus causes ear rot in maize, contributing to food insecurity due to aflatoxin contamination. The biological control principle of competitive exclusion has been applied in both the United States and Africa to reduce aflatoxin levels in maize grain at harvest by introducing atoxigenic strains that out-compete toxigenic strains. The goal of this study was to determine if the efficacy of preharvest biocontrol treatments carry over into the postharvest drying period, the time between harvest and the point when grain moisture is safe for storage. In Sub-Sahara Africa, this period often is extended by weather and the complexities of postharvest drying practices. Maize grain was collected from fields in Texas and North Carolina that were treated with commercial biocontrol products and untreated control fields. To simulate moisture conditions similar to those experienced by farmers during drying in Sub-Sahara Africa, we adjusted the grain to 20% moisture content and incubated it at 28 °C for 6 days. Although the initial number of kernels infected by fungal species was high in most samples, less than 24% of kernels were infected with Aspergillus flavus and aflatoxin levels were low (<4 ppb). Both toxigenic and atoxigenic strains grew and spread through the grain over the incubation period, and aflatoxin levels increased, even in samples from biocontrol-treated fields. Our molecular analysis suggests that applied biocontrol strains from treated fields may have migrated to untreated fields. These results also indicate that the population of toxigenic A. flavus in the harvested grain will increase and produce aflatoxin during the drying period when moisture is high. Therefore, we conclude that preharvest biocontrol applications will not replace the need for better postharvest practices that reduce the drying time between harvest and storage.     

Early detection of toxigenic fungi on maize by hyperspectral imaging analysis

Fungi can grow on many food commodities. Some fungal species, such as Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Fusarium spp., can produce, under suitable conditions, mycotoxins, secondary metabolites which are toxic for humans and animals. Toxigenic fungi are a real issue, especially for the cereal industry. The aim of this work is to carry out a non destructive, hyperspectral imaging-based method to detect toxigenic fungi on maize kernels, and to discriminate between healthy and diseased kernels. A desktop spectral scanner equipped with an imaging based spectrometer ImSpector- Specim V10, working in the visible-near infrared spectral range (400-1000 nm) was used. The results show that the hyperspectral imaging is able to rapidly discriminate commercial maize kernels infected with toxigenic fungi from uninfected controls when traditional methods are not yet effective: i.e. from 48 h after inoculation with A. niger or A. flavus.     

The mycobiota of coffee beans and its influence on the coffee beverage

The quality of coffee beverage is influenced by several factors, including the species or botanical variety of the beans, agricultural practices, harvesting, drying and storage techniques and also the preparation of the beverage. Apart from these, there is the input of microbial contamination during the processing of the beans. Numerous studies have demonstrated that fungi are important contaminants of coffee beans, especially just after harvesting and drying. However, the relationship between fungal contamination and the sensorial characteristics of the beverage has yet to be described. The aim of this research was to analyze the mycobiota of coffee beans collected from different stages of the coffee production chain and to correlate these data with the sensorial characteristics of the final beverage. Fungal infection of 22 coffee bean samples from the southwest of São Paulo state was analyzed. Samples were collected from the tree (mature cherries), from the ground, from the patio (mature, immature and dried floaters or overripe cherries from the tree) and from storage facilities. In general, coffee samples from this region showed high fungal infection and contamination was higher than 70% in about 45% of the samples. A high diversity of fungi was isolated from all the coffee samples analyzed and the most common were Penicillium brevicompactum, Aspergillus section Nigri, Penicillium sp. nov. (closely related to Penicillium crustosum) and Fusarium sp. Both P. brevicompactum and Penicillium sp. nov. were found at all processing stages, including in the cherries, showing that these fungi are naturally found in the coffee beans from this region. Floater coffee and coffee from the ground showed negative sensorial evaluation with attributes such as moldy, dirty and fermented and presented a high contamination by Aspergillus section Nigri and Aspergillus westerdijikiae.     

Reprint of “The mycobiota of coffee beans and its influence on the coffee beverage”

The quality of coffee beverage is influenced by several factors, including the species or botanical variety of the beans, agricultural practices, harvesting, drying and storage techniques and also the preparation of the beverage. Apart from these, there is the input of microbial contamination during the processing of the beans. Numerous studies have demonstrated that fungi are important contaminants of coffee beans, especially just after harvesting and drying. However, the relationship between fungal contamination and the sensorial characteristics of the beverage has yet to be described. The aim of this research was to analyze the mycobiota of coffee beans collected from different stages of the coffee production chain and to correlate these data with the sensorial characteristics of the final beverage. Fungal infection of 22 coffee bean samples from the southwest of São Paulo state was analyzed. Samples were collected from the tree (mature cherries), from the ground, from the patio (mature, immature and dried floaters or overripe cherries from the tree) and from storage facilities. In general, coffee samples from this region showed high fungal infection and contamination was higher than 70% in about 45% of the samples. A high diversity of fungi was isolated from all the coffee samples analyzed and the most common were Penicillium brevicompactum, Aspergillus section Nigri, Penicillium sp. nov. (closely related to Penicillium crustosum) and Fusarium sp. Both P. brevicompactum and Penicillium sp. nov. were found at all processing stages, including in the cherries, showing that these fungi are naturally found in the coffee beans from this region. Floater coffee and coffee from the ground showed negative sensorial evaluation with attributes such as moldy, dirty and fermented and presented a high contamination by Aspergillus section Nigri and Aspergillus westerdijikiae.     

Solid Fat Content,Pre-Crystallization Conditions,and Sensory Quality of Chocolate with Addition of Cocoa Butter Analogues

The objective of this study was to determine how the addition of two cocoa butter equivalents and cocoa butter improver affect the physical and sensory properties of chocolate. The laboratory-made chocolate samples were tempered at three different pre-crystallization temperatures (25, 27, and 29°C), using different concentrations (3, 5, and 7%) of two commercial cocoa butter equivalents as well as commercial cocoa butter improver of the chocolate. The nucleation time of the chocolate mass primarly depended on pre-crystallization temperature while the value of maximum torque of chocolate mass were influenced by both, pre-crystallization temperature and concentration of fats. Sensory evaluation revealed that cocoa butter equivalents were acceptable in chocolate formulation without producing a negative impact on the sensory quality, while usage of improver required adjustment of raw formulations or process parameters. The results of the instrumentally measured hardness revealed that addition of cocoa butter improver significantly (p > 0.05) increased hardness of chocolate samples.     

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.