Comparison of vegetable shortening and cocoa butter as vehicles for cortisol manipulation in Salmo trutta

This study demonstrates that vegetable shortening and cocoa butter are two effective vehicles for intraperitoneal cortisol implants in juvenile teleosts, specifically brown trout Salmo trutta, residing in north temperate freshwater environments. Each vehicle showed a different pattern of cortisol elevation. Vegetable shortening was found to be a more suitable vehicle for long‐term cortisol elevation [elevated at 3, 6 and 9 days post treatment (dpt)], while cocoa butter may be better suited for short‐term cortisol elevation (only elevated at 3 dpt). Additionally, plasma cortisol levels were higher with cortisol–vegetable shortening than with cortisol–cocoa butter implants. Plasma glucose levels were elevated 6 and 9 dpt for fishes injected with cortisol–vegetable shortening, but did not change relative to controls and shams in cortisol–cocoa butter fishes. In conclusion, vegetable shortening and cocoa butter are both viable techniques for cortisol manipulation in fishes in temperate climates, providing researchers with different options depending on study objectives.     

Pure yeast culture fermentation of cocoa (Theobroma cacao L): effect on yield of sweatings and cocoa bean quality

Cocoa sweatings, the pale yellowish liquid that drains off during cocoa fermentation, is the breakdown product of the mucilage surrounding the fresh cocoa bean, and constitutes about 10% of the weight of the cocoa fruit. On average, about 1.9 million l of sweatings are produced annually in Ghana during the cocoa harvesting season. It has been shown to be a suitable medium for the production of wines, alcohol, marmalade, jam and syrup. Its rapid collection in high yields and quality is the first step to its utilization on a commercial scale. Thus pure yeast culture fermentation of cocoa under controlled temperature conditions and its effect on yield of sweatings and final cocoa bean quality was investigated. Cocoa fermentations employing Saccharomyces chevalieri or Kluyveromyces fragilis alone gave significantly higher yields of sweatings (p 0.05) than controls. The initial rates of sweating by the two strains were also very high but dropped to a constant minimum value after 12h of fermentation. In contrast, fermentations employing Torulopsis candida or Candida norvengensis alone as well as different combinations of all the yeast strains did not give any significant difference in yield compared to controls (p 0.05). Fermentations using S. chevalieri alone or other combinations in which S. chevalieri was present gave beans with acceptable quality based on different quality indices used for grading cocoa beans commercially.     

The cocoa bean fermentation process: from ecosystem analysis to starter culture development

Cocoa bean fermentation is still a spontaneous curing process to facilitate drying of nongerminating cocoa beans by pulp removal as well as to stimulate colour and flavour development of fermented dry cocoa beans. As it is carried out on farm, cocoa bean fermentation is subjected to various agricultural and operational practices and hence fermented dry cocoa beans of variable quality are obtained. Spontaneous cocoa bean fermentations carried out with care for approximate four days are characterized by a succession of particular microbial activities of three groups of micro‐organisms, namely yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB), which results in well‐fermented fully brown cocoa beans. This has been shown through a plethora of studies, often using a multiphasic experimental approach. Selected strains of several of the prevailing microbial species have been tested in appropriate cocoa pulp simulation media to unravel their functional roles and interactions as well as in small plastic vessels containing fresh cocoa pulp‐bean mass to evaluate their capacity to dominate the cocoa bean fermentation process. Various starter cultures have been proposed for successful fermentation, encompassing both cocoa‐derived and cocoa nonspecific strains of (hybrid) yeasts, LAB and AAB, some of which have been implemented on farms successfully.     

Mapping of Quantitative Trait Loci for Butter Content and Hardness in Cocoa Beans (Theobroma cacao L.)

Cocoa butter is an important raw material for the chocolate, pharmaceutical, and cosmetic industries. The butter content and quality in cocoa beans are genetically controlled characteristics, and affect its commercial value and industrial applicability. In the present work, an F₂ population derived from the cross between the ICS-1 and Scavina-6 cocoa clones was used for molecular mapping. A linkage map was constructed based on amplified fragment length polymorphism, random amplified polymorphic DNA, and simple sequence repeat markers, resulting in a total of 273 markers, distributed in 14 linkage groups (LGs). Phenotyping of butter content was performed after ether extraction and butter hardness was determined by sweeping differential calorimetry. One quantitative trait locus (QTL) associated to butter content was mapped at linkage group 9 (LG9) and two QTLs for butter hardness were identified at linkage groups 9 and 7 (LG9 and LG7). The two QTLs mapped at the LG9 explained 51.0% and 28.8% of the phenotypic variation for butter content and hardness, respectively. These QTLs were concentrated in the same map region, suggesting a close genetic linkage or pleiotropic effect. The QTLs identified may be useful in further marker-assisted selection breeding programs aimed at cocoa butter quality improvement.     

Partial replacement of local common bean mixtures by high yielding angular leaf spot resistant varieties to conserve local genetic diversity while increasing yield

The effect of replacing proportions of local farmer bean (Phaseolus vulgaris) mixtures with varieties resistant to angular leaf spot on grain yield was evaluated under local disease pressure in the Kivu region of Zaire. Local bean mixtures in on-station and in multi-locational trials containing respectively 25%, or 50% of Centro Internacional de Agricultura Tropical (CIAT) breeding lines BAT76, or A285, resistant to angular leaf spot yielded significantly more than the local mixture alone. Yields exceeded expected values in three seasons of on-station testing and in two seasons of multi-locational trials. Yield over the expected was found to be a property of new mixtures not protected with fungicide and were attributed to disease control. Relative to expected yields non-protected farmer mixture components performed 17% and 16% better than in protected plots and A285 components yielded 24% and 16% better at respectively 25% and 50% A285 supplementation levels. A285 increased yields of the local mixture components and benefitted from the local mixture when not protected by fungicide. Yield increases in multi-locational trials were largely attributed to the higher yield potential of the resistant variety A285, although angular leaf spot severity was significantly reduced. It was concluded that high yielding, resistant varieties were able to increase bean yield in the region, but probably at a substantial cost in genetic diversity in farmer bean mixtures. That said, a partial replacement strategy is preferable to strategies which encourage complete replacement of local germplasm with one or few high yielding varieties.     

Phase Transition Stability of Cocoa Butter Emulsions

The impact of tempering-crystallization on microstructure and stability of water-in-cocoa butter (w/o) emulsions was analyzed using differential scanning calorimetry (DSC). The type and volume fraction of the disperse phase, and cooling rate during DSC analysis were systematically varied. Freshly prepared emulsions were additionally characterized by microscopy and laser diffraction. Fresh cocoa butter emulsions were composed of small and well dispersed droplets of an average size of 2.24 μm and 1.96 μm for water and 50 % sucrose solution as disperse phase, respectively. The thermograms revealed that the dissolved sugar lowered freezing and melting temperature and, dependent on volume fraction, the dispersion in the oil phase led to a change in solidification behavior. The temperature at the solidification peaks gives qualitative information about droplet size whereas width and number of exothermic events are related to particle size distribution (mono/polydispersity and mono/multimodality) and microstructure. Emulsions with water as dispersed phase show a clear shift of the freezing peaks of the disperse phase which points on modified emulsion microstructure because of droplet coalescence, which is more pronounced at higher volume fraction and lower cooling rate. Emulsions with sucrose solution as dispersed phase showed the greatest stability, wherein the volume fraction and the cooling rate does not matter. The results allow conclusions about the mechanisms of crystallization processes in cocoa butter emulsions resulting as network crystallization.     

Effect of starch- and lipid-based encapsulation on the culturability of two Bifidobacterium longum strains

AIMS: To assess the applicability of starch- and lipid-based encapsulation methods for improving the viability and culturability of two Bifidobacterium longum strains stored in fermented and nonfermented foods. MATERIALS AND RESULTS: Cells were encapsulated with partially hydrolysed potato starch granules combined with amylose coating, or entrapped in cocoa butter matrix. The tested B. longum strains were not adherent to the starch granules, and the culturability of the cells stored in fermented and nonfermented foods was not improved by starch-based encapsulation. Encapsulation of the cells in cocoa butter was found to increase the plate counts during storage. In addition to plate counts, viability of the cells was measured by fluorescent microscopy using LIVE/DEAD BacLight viability assay. Microscopic counts of the viable cells did not change significantly during storage, suggesting that the cells remained alive despite becoming unable to grow on nutrient agar plates. CONCLUSIONS: Encapsulation with cocoa butter increased the culturability of the cells, but encapsulation with hydrolysed potato starch had no effect. Culture-independent viability assay suggested that cells remained viable despite being unable to grow on agar plates. SIGNIFICANCE AND THE IMPACT OF THE STUDY: This study indicates that encapsulation techniques may be useful in improving the culturability of bacteria, but the plate counts may yield insufficient data on the actual viability of the cells.     

Effect of dietary n-3 fatty acids on HMG-CoA reductase and ACAT activities in liver and intestine of the rabbit

The regulation of hepatic and intestinal 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and acyl-CoA; cholesterol acyltransferase (ACAT) activities by dietary fish oil was examined in the rabbit. Rabbits were fed 10% menhaden oil or menhaden oil plus 1% cholesterol for 14 days. They were compared with animals fed a control diet or one enriched with long-chain saturated fats consisting of 10% cocoa butter oil or cocoa butter oil plus 1% cholesterol. Plasma cholesterol was increased in rabbits fed the fish oil and the two cholesterol-containing diets. In the liver, ACAT activity was increased and HMG-CoA reductase activity was decreased in rabbits ingesting the fish oil. The same was true for animals ingesting both cholesterol-containing diets. In the intestine, ACAT activity was not affected by the ingestion of the fish oil compared to control rabbits; however, it was significantly higher in animals fed the fish oil compared to animals ingesting the cocoa butter. HMG-CoA reductase activity was decreased in the distal two-thirds of the intestine in animals fed the menhaden oil compared to activities observed in controls. In animals ingesting the cholesterol diets, intestinal reductase was significantly decreased, whereas intestinal ACAT activity was increased in rabbits ingesting the cocoa butter and cholesterol diet when compared to their controls. Lipid analysis of hepatic and intestinal microsomes demonstrated an enrichment of n-3 polyunsaturated fatty acids in membranes from rabbits ingesting the menhaden oil.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Incorporation of l-Alanine into Uridine-diphospho N-acetylmuramic Acid by Glycine

Lipases from Aspergillus niger and Rhizopus delemar hydrolyzed triolein and produced l,2 (2,3)-diolein and 2-monoolein. These two lipases appears to have strong specificity towards the outer chains of the triglyceride. Comparing the proportions of fatty acids in position 1 (3) of cocoa butter with proportions of fatty acids liberated after limited hydrolysis of cocoa butter, it becomes clear that these two lipases do not hydrolyze the ester bond in position 2 of the triglyceride.

On the other hand, lipases from Geotrichum candidum Link and Penicillium cyclopium Westring attacked the fatty acid chains regardless of their positions. Geotrichum candidum lipase liberated oleic acid and palmitic acid in preference to stearic acid from cocoa butter.     

Effect of natural antioxidants on the lipase activity in the course of batch and continuous glycerolysis of babassu oil

The effects of several natural antioxidants (copaiba oil, buriti oil, cocoa butter, tucuman butter, oregano and white thyme) were assessed in the enzymatic synthesis of monoglycerides (MAG) from the glycerolysis of babassu oil. The reactions were catalyzed by Burkholderia cepacia lipase immobilized on SiO₂–PVA and the assays carried out in batch and continuous runs. Results were compared with those attained in the control reactions (without any strategy to avoid oxidation), and the best approach was tested in a continuous packed-bed reactor. The best performance was obtained using N₂ in the reaction medium (60 % of MAG) followed by buriti oil (57.6 % of MAG) and cocoa butter (56.6 % of MAG), preventing the oxidation of babassu oil in batch reaction. However, the incorporation of buriti oil in the medium influenced the MAG profile, leading to the largest formation of monoolein, unlike other runs. Similar results were obtained in continuous reactions, using inert atmosphere and cocoa butter (24–25 % of MAG). Thereby, among the tested antioxidant agents, cocoa butter was the most effective in both systems, because it did not interfere in the MAG profile and also reduced the cost of the process.     

Cocoa butter biosynthesis. Purification and characterization of a soluble sn-glycerol-3-phosphate acyltransferase from cocoa seeds

Glycerol-3-phosphate acyltransferase has been purified from the post-microsomal supernatant of cocoa seeds using differential ammonium sulfate solubility along with anion exchange and gel filtration chromatography. Chromatofocusing and isoelectric focusing revealed a series of proteins with acyltransferase activity having isoelectric points close to 5.2. Gel filtration on Sephacryl S-300 in 500 mM NaCl, along with polyacrylamide gel electrophoresis (denaturing and non-denaturing) and immunochemical analysis, gave evidence that the native enzyme has a molecular weight of 2 X 10(5) and consists of an aggregate of 10 Mr 20,000 subunits. The highly purified enzyme carries an acyl donor, probably acyl-CoA, although this is not firmly established. The hydrophobic nature of the purified enzyme was demonstrated by its firm binding to octyl-Sepharose. Mass spectrometric analysis of reaction products revealed the presence of both palmitic and stearic acids. Considering that 1) the fatty acids were derived from the purified enzyme; 2) they were found exclusively in the 1-position of glycerol 3-phosphate; 3) the fatty acid positioning and composition is consistent with that found in cocoa butter, the major storage product of cocoa seeds; and 4) the enzyme is found in the post-microsomal supernatant, it seems reasonable to conclude that the first step in cocoa butter biosynthesis is catalyzed by glycerol-3-phosphate acyltransferase in the cytoplasm of cocoa cotyledon cells.     

Yeast diversity of Ghanaian cocoa bean heap fermentations

The fermentation of the Theobroma cacao beans, involving yeasts, lactic acid bacteria, and acetic acid bacteria, has a major influence on the quality of the resulting cocoa. An assessment of the microbial community of cocoa bean heap fermentations in Ghana resulted in 91 yeast isolates. These were grouped by PCR-fingerprinting with the primer M13. Representative isolates were identified using the D1/D2 region of the large subunit rRNA gene, internal transcribed spacer sequences and partial actin gene sequences leading to the detection of 15 species. Properties of importance for cocoa bean fermentation, namely sucrose, glucose, and citrate assimilation capacity, pH-, ethanol-, and heat-tolerance, were examined for selected isolates. Pichia kudriavzevii ( Issatchenkia orientalis ), Saccharomyces cerevisiae , and Hanseniaspora opuntiae formed the major components of the yeast community. Hanseniaspora opuntiae was identified conclusively for the first time from cocoa fermentations. Among the less frequently encountered species, Candida carpophila, Candida orthopsilosis, Kodamaea ohmeri, Meyerozyma ( Pichia ) caribbica, Pichia manshurica, Saccharomycodes ludwigii , and Yamadazyma ( Pichia ) mexicana were not yet documented from this substrate. Hanseniaspora opuntiae was preferably growing during the earlier phase of fermentation, reflecting its tolerance to low pH and its citrate-negative phenotype, while no specific temporal distribution was recognized for P. kudriavzevii and S. cerevisiae .     

Modification of the fatty-acid composition in lipids of the oleaginous yeast Apiotrichum curvatum by intraspecific spheroplast fusion

Summary Modification of the fatty-acid composition in lipids of the oleaginous yeast Apiotrichum curvatum has been achieved by means of spheroplast fusion between a methionine auxotrophic mutant and an unsaturated fatty acid mutant. Fusion products were a result of nuclear hybridization as determined by flow-cytometric DNA-content analyses. A broad range of fatty acid composition was observed in lipids of different hybrids. In general the level of saturated fatty acids in lipids of hybrid strains is higher than in wild-type A. curvatum and in some hybrids even approaches cocoa butter. Intraspecific spheroplast fusion seems a promising approach for the production of cocoa butter equivalents.     

Insect pest damage increases faba bean (Vicia faba) yield components but only in the absence of insect pollination

Identifying and quantifying crop stressors interactions in agroecosystems is necessary to guide sustainable crop management strategies. Over the last 50 years, faba bean cropping area has been declining, partly due to yield instabilities associated with uneven insect pollination and herbivory. Yet, the effect of interactions between pollinators and a key pest, the broad bean beetle Bruchus rufimanus (florivorous and seed predating herbivore) on faba bean yield has not been investigated. Using a factorial cage experiment in the field, we investigated how interactions between two hypothesized stressors, lack of insect pollination by bumblebees and herbivory by the broad bean beetle, affect faba bean yield. Lack of bumblebee pollination reduced bean weight per plant by 15%. Effects of the broad bean beetle differed between the individual plant and the plant‐stand level (i.e., when averaging individual plant level responses at the cage level), likely due to high variation in the level of herbivory among individual plants. At the individual plant level, herbivory increased several yield components but only in the absence of pollinators, possibly due to plant overcompensation and/or pollination by the broad bean beetle. At the plant‐stand level, we found no effect of the broad bean beetle on yield. However, there was a tendency for heavier individual bean weight with bumblebee pollination, but only in the absence of broad bean beetle herbivory, possibly due to a negative effect of the broad bean beetle on the proportion of legitimate flower visits by bumblebees. This is the first experimental evidence of interactive effects between bumblebees and the broad bean beetle on faba bean yield. Our preliminary findings of negative and indirect associations between the broad bean beetle and individual bean weight call for a better acknowledgment of these interactions in the field in order to understand drivers of crop yield variability in faba bean.     

Cocoa butter-like lipid production ability of non-oleaginous and oleaginous yeasts under nitrogen-limited culture conditions

Applied Microbiology and Biotechnology - Cocoa butter (CB) extracted from cocoa beans is the main raw material for chocolate production. However, growing chocolate demands and limited CB production...     

Effects of long-term cortisol treatments on gonadal development, sex steroids levels and ovarian cortisol content in cultured great sturgeon Huso huso

The objective of this study was to examine the effect of cortisol implantations on gonadal development, sex steroid levels, and ovarian cortisol content in cultured great sturgeon Huso huso. Three groups of 5 fish for each treatment were considered. The experimental groups included: control (capsules containing cocoa butter alone), low cortisol (C(5); 5mg cortisol/kg body mass+cocoa butter) and, high cortisol (C(50); 50mg cortisol/kg body mass+cocoa butter). The capsules containing hormones and cocoa butter were intraperitoneally implanted into 3-year-old female fish at pre-vitellogenic stage (mean initial body mass 6809.7 ± 73 g) every 6 weeks over a 6-month period from January to June. The serum levels of cortisol, glucose, cholesterol and sex steroids (testosterone and 17β-estradiol) were determined at the initial time and three weeks after each implantation. Oocyte histological characteristics (the diameter and area of the oocyte, the diameter and area of the nucleus and the ratio of the nucleus area to the oocyte area) were measured at the end of the experiment and compared to those at the initial time. Ovarian cortisol content was measured at the end of the experiment. The results showed that serum cortisol levels varied in a dose-independent manner, so that the highest cortisol concentrations were observed in C(5)-treated fish throughout the experiment. Serum glucose levels were significantly higher in cortisol-treated groups than those in the control group. The high dose of cortisol elicited a significant constant increase in serum cholesterol concentrations. Fish implanted with the high cortisol dose showed significant declines in serum testosterone and 17β-estradiol concentrations throughout the experiment. No significant differences were found in oocyte histological characteristics among experimental groups. The cortisol implants elicited a dose-dependent increase in ovarian cortisol content. At the end of trial, body-growth indices were the lowest in C(50)-implanted fish, while the low cortisol dose had no effect on growth relative to the controls. These results indicated that chronic stress induced by cortisol implantation in great sturgeon suppressed gonadal steroidogenesis and somatic growth but had no effect on ovarian growth and development.     

两种赋形剂载体中姜黄素干预慢性应激大鼠抑郁样行为和肌肉炎性反应的作用研究

目的:从抑郁样行为和肌肉炎性反应角度,研究姜黄素在由可可脂和花生油两种赋形剂载体时,对慢性应激大鼠的干预作用。方法:分别制备以可可脂和花生油为赋形剂的姜黄素制剂,使用SD雄性大鼠50只随机分为空白对照组、模型对照组、姜黄素可可脂组、姜黄素花生油组、阳性对照组,除空白对照组外,各组均采用束缚、噪音声刺激、冰水浴刺激、夹尾疼痛刺激等方法持续6周建立慢性应激大鼠模型,在维持模型刺激条件的基础上分别给予各种设计药物,连续用药7 d后在考察各组模型动物悬尾挣扎时间、机械痛阈、糖水偏好的行为学变化,然后使用Elisa试剂盒法检测各组大鼠脑中5五羟色胺(5-HT)和多巴胺(DA)含量,制作大鼠咬肌组织的HE染色切片观察肌肉病理变化,检测咬肌组织中高迁移率族蛋白B1(HMGB-1)、白介素1β(IL-1β)、白介素6(IL-6)、肿瘤坏死因子α(TNF-α)水平。结果:与模型组相比,两种姜黄素均能显著降低应激大鼠抑郁样行为,改善脑内5-HT和DA含量的作用较阳性对照组弱,咬肌病理切片中两种姜黄素组可明显减少应激大鼠咬肌组织血管周围的炎性反应。两种姜黄素能显著降低咬肌组织中的IL-1β、IL-6、TNF-α水平(P<0.05),其中姜黄素可可脂组作用最明显。结论:两种姜黄素制剂对抗慢性应激大鼠的抑郁样症状均有治疗作用,均可降低咬肌中炎性因子HMGB-1、IL-1β、IL-6、TNF-α水平,其中可可脂作为赋形剂时作用较显著,在姜黄素的抗抑郁制剂研究中具有较好的开发潜力。     

Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis

The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions.     

Composition of by-products of the essential-oil industry and their potential as feeds for ruminants

Eight plant-residues from the essential-oil industry were assayed for chemical composition. Digestibility of the plant residues was determined by rumen fermentation in vitro and in vivo. Dry-matter digestibility of plant residues from black pepper, vanilla bean, ginger root, cocoa bean and celery seed ranged from 43–65%. Only 15–30% of the residues from nutmeg hulls and cinnamon bark were degraded. These data indicate that anaerobic microbes are capable of degrading significant quantities of the plant residues without further processing of these materials. Long-term feeding studies need to be conducted to determine if ruminants will consume these plant materials, and to investigate the effect of these plant residues on rumen microbial- and host cellular-metabolism.     

Analysis of cocoa products for ochratoxin A and aflatoxins

Eighty-five samples of cocoa products sampled in Canada were analysed for ochratoxin A (OTA) and aflatoxins in 2011–2012. Inclusion of the aflatoxins in this survey required additional method development. Chocolate was extracted with methanol–water plus NaCl, while for cocoa two successive extractions with methanol and methanol–water were made. Extracts were cleaned on an AflaOchra immunoaffinity column (IAC). Determination was by reversed phase high performance liquid chromatography (HPLC). Detection of the aflatoxins was with a post-column photochemical reactor and of OTA by fluorescence detection. Mean limits of quantification (LOQ) of chocolate and cocoa powders were 0.16 ng/g (OTA) and 0.07 ng/g (aflatoxin B₁), respectively. Survey results showed that the incidences of OTA above the LOQ in natural cocoa were 15/15 (mean 1.17 ng/g), 20/21 for alkalized cocoa (mean 1.06 ng/g), 9/9 for baking chocolate (mean 0.49 ng/g), 20/20 for dark chocolate (mean 0.39 ng/g), 7/10 for milk chocolate (mean 0.19 ng/g), 5/5 for cocoa liquor (mean 0.43 ng/g), and 0/5 for cocoa butter. These results confirm our previous work with OTA. In the same samples, incidences of aflatoxin B₁ above the LOQ were 14/15 for natural cocoa (mean 0.86 ng/g), 20/21 for alkalized cocoa (mean 0.37 ng/g), 7/9 for baking chocolate (mean 0.22 ng/g), 16/20 for dark chocolate (mean 0.19 ng/g), 7/10 for milk chocolate (mean 0.09 ng/g), 4/5 for cocoa liquor (mean 0.43 ng/g), and 0/5 for cocoa butter. Both aflatoxins and OTA were confirmed by HPLC-MS/MS when OTA or aflatoxin levels found were above 2 ng/g in cocoa.