A Rapid Size-Exclusion Solid-Phase Extraction Step for Enhanced Sensitivity in Multi-Allergen Determination in Dark Chocolate and Biscuits by Liquid Chromatography–Tandem Mass Spectrometry

Enhanced sensitivity for the simultaneous determination of five nut allergens in biscuit and in dark chocolate complex matrices was obtained by introduction of a rapid size-exclusion solid-phase extraction-based step before liquid chromatography–electrospray ionization-tandem mass spectrometry (LC-ESI-MS²) analysis. A very fast and efficient separation (<12 min) of marker peptides with selected reaction monitoring detection was obtained. Limits of detection in the 0.1–1.3 mg nut/kg and 5–15 mg nut/kg ranges for biscuit and dark chocolate samples as well as high recoveries (84(±6)–106(±4)% for biscuits and 98(±5)–108(±6)% for dark chocolate) proved the excellent capabilities of the exploited sample treatment method combined with the LC-MS² analysis. Good precision in terms of intra- and inter-day repeatability was calculated, being always lower than 19 % (n?=?75). Linearity was demonstrated up to four and three orders of magnitude for biscuit and dark chocolate, respectively. Finally, the validated method was successfully applied to the investigation of hidden nut trace allergens in commercially available biscuits and chocolates of different brands aiming to ascertain possible discrepancies between allergen content and food allergen labelling.     

Tempering of Chocolate in a Scraped Surface Heat Exchanger

Crystallization of dark chocolate during different tempering processes has been studied in-situ in a lab-scale Scraped Surface Heat Exchanger. The linear relationship between torque and viscosity made possible the control of chocolate crystallization during tempering by following torque variations vs. time and converting them to equivalent viscosity values. These variations of equivalent viscosity of chocolate observed during tempering were correlated with temper-meter measurements which had been related to the content of cocoa butter seeding crystals. A temperature cycle (50-26.1-30.5-33.3°C) enabled preparation of a well-tempered chocolate stable for ≥30 min at that temperature. It was characterized by a seed crystals content of about 1.15 ± 0.1% of cocoa butter, crystallization temperature of 23.9 ± 0.2°C in the temper-meter and an equivalent viscosity of 3.0 ± 0.4 Pa.s.     

Effect of post-harvest treatments on the occurrence of ochratoxin A in raw cocoa beans

Cocoa beans are the principal raw material for chocolate manufacture. Moulds have an important place in the change in the quality of cocoa beans due to their role in the production of free fatty acids and mycotoxins, namely ochratoxin A (OTA). This study investigated the impact of the key post-harvest treatments, namely the fermentation and drying methods on OTA contamination of raw cocoa beans. Analytical methods for OTA detection were based on solid–liquid extraction, clean-up using an immunoaffinity column, and identification by reversed-phase HPLC with fluorescence detection. Of a total of 104 randomly selected cocoa samples analysed, 32% had OTA contents above 2 µg kg^–1. Cocoa sourced from pods in a bad state of health had a maximum OTA content of 39.2 µg kg^–1, while that obtained from healthy pods recorded 11.2 µg kg^–1. The production of OTA in cocoa beans increased according to the pod-opening delay and reached 39.2 µg kg^–1 after an opening delay of 7 days after harvest, while 6.1 and 11.2 µg kg^–1 were observed when pods were opened after 0 and 4 days. OTA production also seemed to depend considerably to the cocoa fermentation materials. When using plastic boxes for bean fermentation, the OTA production was enhanced and reached an average OTA content of about 4.9 µg kg^–1, while the raw cocoa treated in banana leaves and wooden boxes recorded 1.6 and 2.2 µg kg^–1 on average respectively. In parallel, the OTA production was not really influenced by either the mixing or the duration of the fermentation or the drying materials.     

A rapid method to evaluate the chocolate smoothness based on the tribological measurement

Cocoa is the primary ingredient in chocolate, and cocoa concentration attributes to the smoothness of the chocolate. Supporting this statement, we assessed the smoothness and melting properties of five chocolates of different cocoa concentration from three brands using the professional sensor evaluation and weight method, respectively. Artificial saliva was added to obtain the mixed chocolate solutions, and their viscosity and coefficient of friction (CoF) were measured for three rubbing pairs. The correlation of chocolate smoothness with their viscosities and average CoF (aCoF) was also discussed. The results indicated that the cocoa concentration significantly affected the smoothness of the chocolates among the three brands. The mixed solution of 50% chocolate could be a representative of oral processing, but their viscosities were not proficient enough to effectively characterize their smoothness. The aCoF of 50% chocolate solution could be rapid and effective to evaluate their smoothness. In addition, the Two-GCTPE rubbing pair was more suitable way to evaluate the smoothness of similar foods in the oral environment. The study results will provide a better insight into characterizing certain attribution of similar foods.     

Use of Palm Mid-Fraction in White Chocolate Formulation

Samples of two types of palm mid-fraction (PMF I, a commercial sample and PMF II, from a laboratory-scale acetone fractionation of PMF I) and a Malaysian deodorised cocoa butter sample were used as the main components in the fat phase for white chocolate formulation. The monounsaturatedtriacylglycerol contents of these fats were 853, 899 and 903 g kg⁻¹, respectively.All the fats had free fatty acid contents of less than 10 g kg⁻¹ and melting points in the range of 34·0–34·5°C. The solid fat content profiles for the three fats were very steep. Differential scanning calorimeter analyses showed that all the fats had two melting peaks, T1 and T2. Results of the study showed that the tempering time to produce a well-tempered chocolate using PMF I was longer than that using PMF II, whereas, the time to produce a well-tempered cocoa butter chocolate increased with increase in the tempering temperature. Chocolates made with PMF I and II were well tempered between 17 and 19°C and with cocoa butter at 23°C. Thermal analyses, carried out on the chocolate showed that PMF I and II produced three melting peaks, T1, T2′ and T2 whereas most of the cocoa butter chocolates exhibited only one melting peak, T2. Storage studies showed that most of the chocolates had good bloom resistance for up to 12 weeks storage.     

Analytical Method Validation and Rapid Determination of Polycyclic Aromatic Hydrocarbons (PAHs) in Cocoa Butter Using HPLC-FLD

A simple, fast and ecological analytical method using a semi-automatic fat extractor and HPLC-FLD (fluorescence detection) for determination of polycyclic aromatic hydrocarbon markers i.e. benzo(a)anthracene (BaA), chrysene (Chr), benzo(a)pyrene (BaP) and benzo(b)fluoranthene (BbF) in cocoa butter has been validated. Validation’s procedure performed out in concordance with French standard NF V03-110 (2010) was based on existing polycyclic aromatic hydrocarbon (PAH) determination methods in various smoked foodstuffs and edible vegetable oils. Determination of correlation coefficients for specific PAHs ranged from 0.9992 to 0.9998. Respective values of limits of detection were 0.010, 0.011, 0.033 and 0.029 μg kg^?1 and those of quantification were 0.035, 0.038, 0.111 and 0.098 μg kg^?1 for BaA, Chr, BbF and BaP. Both values of repeatability and intermediary precision tests coefficients of variation were less than 5%. Recovery scores of four PAH markers matched EU standard 836/2011 recommendations. Sum of four PAH markers (BaA, Chr, BbF, BaP) contents varied from 5.42?±?0.58 to 11.37?±?0.01 μg kg^?1 whereas those of BaP was comprised between 0.26?±?0.00 and 1.75?±?0.13 μg kg^?1 in 20 cocoa butter samples extracted from raw cocoa bean stored at Ivorian cocoa farmer levels.     

低场核磁共振在线检测巧克力的熔化过程

构建了低场核磁共振(LF-NMR)在线控温分析系统,对不同品种巧克力的熔化过程进行了表征,研究了可可脂和粗脂肪含量对巧克力熔化温度的影响。通过对样品的程序升温,持续测定了巧克力的横向弛豫时间(T₂),对四个不同品牌巧克力的熔化过程进行了评价。结果表明,巧克力熔化过程中T₂值随温度增大,巧克力中液体脂肪的运动性增强,且熔化温度随可可脂和粗脂肪含量的增加而升高。本文首次利用LF-NMR在线控温分析系统实时在线检测了巧克力熔化过程,并直观准确地测定了巧克力的熔化温度,为改善巧克力的加工工艺和配方提供了理论指导。     

Analysis of the isothiocyanates present in three Chinese Brassica vegetable seeds and their potential anticancer bioactivities

Epidemiological studies give evidence that high dietary intake of cruciferous vegetables has been associated with lower risk of cancer, this activity due to their content in glucosinolates (GL), which upon myrosinase hydrolysis release the corresponding isothiocyanates (ITCs). In this study, the isothiocyanates contents and the anticancer activity of ethyl acetate extracts from three kinds of Chinese Brassica vegetable seeds have been examined and analyzed. A correlation was found between the potency of anticancer activity and the amount of isothiocyanates in their extracts. Extracts followed by GC–MS analysis revealed that 3-BITC (3-butenyl isothiocyanate) was mainly found in Chinese kale contained 77.58 ± 7.28 mg/g (dry weight); AITC (allyl isothiocyanate), 3-BITC and iberverin were the predominant isothiocyanates in Oxheart cabbage contained 33.57 ± 3.94, 26.50 ± 1.92 and 22.77 ± 0.00 mg/g, respectively; 3-BITC and sulforaphane were found as the major isothiocyanates in broccoli contained 138.52 ± 3.42 and 49.77 ± 0.18 mg/g, respectively. The amount of total isothiocyanates in broccoli was twice higher than that in other two species. The anticancer activity of broccoli was the highest, and the IC₅₀ value of the extract inhibiting on the growth of A549, LAC, HELA and HepG₂ were 14.38 ± 0.35, 10.38 ± 0.34, 19.45 ± 1.72 and 26.75 ± 0.82 mg/g, respectively. The extracts of Chinese Brassica vegetable seeds have the potential in inducing cancer cells apoptosis by morphology observation. The results of this study got a new kind of Brassica vegetable seeds that could produce high content of isothiocyanates, which is important to preparation of anticancer food additives.     

Monitoring Tempered Dark Chocolate Using Ultrasonic Spectrometry

Ultrasonic spectrometry was used to distinguish between properly tempered and untempered commercial dark chocolate. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) were used to characterize the polymorphic state of tempered and untempered chocolate, results that were correlated to those of ultrasonic spectrometry. Four different kinds of dark chocolate samples with different amounts of sugar (7.5–50 %), fat (30–50 %), and cocoa mass (70–90 %) were subjected to two different tempering protocols. The tempering was achieved using cocoa butter seeds under static cooling from 50 to 14 °C. The ultrasonic generator and analyzer SIA-7 (V.N. Instruments) was used to monitor the crystallization process. The instrument generated a chirp signal with a bandwidth of 0.5 to 3 MHz and was set to work in a four-pathway configuration with two transducers and a center frequency of 2.25 MHz. Spectrometric analysis was carried out with chocolate samples containing 2, 4, and 6 % solid fat content (SFC). The SFC was obtained from DSC measurements. Ultrasonic signals for attenuation, reflection, and velocity were compared between tempered and untempered chocolates. It was shown that seed-tempered chocolate with 30 % sugar and 47.5 % fat attenuated 2.5 MHz of ultrasonic waves by 0.8, 1.7, and 2.0 dB/cm at 2, 4, and 6 % SFC, respectively. On the other hand, untempered chocolate attenuated the ultrasound signal by 3.5, 3.6, and 4.3 dB/cm. Furthermore, it was found that ultrasound reflection signals were stronger and ultrasonic velocity was higher in chocolates with high sugar content.     

Evaluation of extraction methods for ochratoxin A detection in cocoa beans employing HPLC

Cocoa is an important ingredient for the chocolate industry and for many food products. However, it is prone to contamination by ochratoxin A (OTA), which is highly toxic and potentially carcinogenic to humans. In this work, four different extraction methods were tested and compared based on their recoveries. The best protocol was established which involves an organic solvent-free extraction method for the detection of OTA in cocoa beans using 1% sodium hydrogen carbonate (NaHCO₃) in water within 30 min. The extraction method is rapid (as compared with existing methods), simple, reliable and practical to perform without complex experimental set-ups. The cocoa samples were freshly extracted and cleaned-up using immunoaffinity column (IAC) for HPLC analysis using a fluorescence detector. Under the optimised condition, the limit of detection (LOD) and limit of quantification (LOQ) for OTA were 0.62 and 1.25 ng ml^–1 respectively in standard solutions. The method could successfully quantify OTA in naturally contaminated samples. Moreover, good recoveries of OTA were obtained up to 86.5% in artificially spiked cocoa samples, with a maximum relative standard deviation (RSD) of 2.7%. The proposed extraction method could determine OTA at the level 1.5 µg kg^–1, which surpassed the standards set by the European Union for cocoa (2 µg kg^–1). In addition, an efficiency comparison of IAC and molecular imprinted polymer (MIP) column was also performed and evaluated.     

Cadmium and lead in cocoa powder and chocolate products in the US Market

Cocoa powder and chocolate products are known to sometimes contain cadmium (Cd) and lead (Pb) from environmental origins. A convenience sample of cocoa powder, dark chocolate, milk chocolate, and cocoa nib products was purchased at retail in the US and analysed using inductively coupled plasma mass spectrometry to assess Cd and Pb concentrations. Cd and Pb were evaluated in relation to the percent cocoa solids and to the reported origin of the cocoa powder and chocolate products. Cd ranged from 0.004 to 3.15 mg/kg and Pb ranged from <LOD to 0.38 mg/kg. Cd and Pb were significantly correlated with percent cocoa, with correlations varying by product type and geographic origin. Geographic variation was observed for Cd, with higher Cd concentrations found in products reported as originating from Latin America than from Africa. The influence of percent cocoa solids and cocoa origin on Cd levels are relevant to international standards for Cd in chocolate products.     

Physiochemical properties and probiotic survivability of symbiotic oat-based beverage

The objectives of this study were to develop a symbiotic oat-based beverage (SOB) and to analyze its physiochemical properties and the probiotic survivability. The beverage base was prepared by fermenting a mix containing oat flour (10%, w/w), sugar (4%, w/w), and inulin (1%, w/w) with a commercial Lactobacillus plantarum (0.003%, w/w) at 30 °C for 12 h. The SOB was formulated using the fermented oat base, sugar, stabilizers (pectin and λ-carrageenan), vitamin C, and citric acid. The beverage was analyzed for total solids (11.65 ± 0.22%), protein (0.58 ± 0.02%), fat (0.37 ± 0.02%), carbohydrate (10.70 ± 0.33%), ash (0.14 ± 0.01%), and dietary fiber (0.70 ± 0.05%). The pH value of the beverage was stable at about 3.60 during 7-week storage. Lactobacillus plantarum population in the beverage remained above 10⁷ CFU/g throughout the storage. Oat-based beverage is a low fat and high dietary fiber symbiotic food.     

The application of Borneo tallow in dark chocolate shell

Dark chocolate shells, formulated using cocoa butter (CB shell), and fat blends containing 15% Borneo tallow (IP) in cocoa butter (CBIP shell), were filled with truffles formulated using white chocolate (W truffle), milk chocolate (M truffle) and dark chocolate (D truffle). Anhydrous milk fat (AMF) content of the W truffles, M truffle and D truffle were 26·3%, 13·7% and 9·2%, respectively. Degree of tempering was determined using a tempermeter. The chocolates were kept at ambient temperature (25·5±0·5°C) for 3 months. Physical changes of chocolate shells and centres were monitored using DSC and a texture analyser. Results showed that the CBIP shell had to be tempered at 32·5%, ie 1°C higher than the normal CB shell. Physical measurements indicated the occurrence of fat migration. The presence of IP reduced the effect and increased the bloom resistance of the chocolate. © 1998 SCI.     

In Situ Ultrasonic Characterization of Cocoa Butter Using a Chirp

Differences between tempered and untempered cocoa butter were investigated by an ultrasonic signal “chirp” generated by contact transducers. Polarized light microscopy and powder X-ray diffraction were used to characterize the morphology and polymorphism of tempered and untempered cocoa butter, whereas pulsed nuclear magnetic resonance was used to determine the amount of crystalline solids present. Ultrasonic wave velocity and attenuation data were collected simultaneously throughout the 5-h crystallization process for cocoa butter. Ultrasonic velocity and attenuation changed at the different solid fat contents (SFC): 4, 8, and 11 %. Untempered cocoa butter showed an attenuation of 3 dB/cm at 1.7 MHz and 4 % SFC, whereas tempered cocoa butter showed an attenuation of 4.5 dB/cm at 1.7 MHz and 4 % SFC. At 3 MHz, the attenuation was 2 dB/cm for untempered and 6 dB/cm for tempered cocoa butter. Under these conditions (4 % SFC, 3 MHz), the chirp wave of tempered sample showed a phase angle change of 0.5 rad, whereas the untempered sample showed ?0.5 rad relative to the canola oil that was taken as 0. The study suggests that an ultrasonic chirp can be effectively used to detect differences between tempered and untempered cocoa butter when measuring attenuation and ultrasonic wave phase angle changes as a function of frequency. The in-line characterization of chocolate “temper” using such nondestructive ultrasonic measurements could be applied to industrial chocolate manufacturing.     

Structural characteristics of cocoa particles and their effect on the viscosity of reduced fat chocolate

Aimed at the manufacture of reduced fat chocolates, a novel method of trapped fat reduction was assessed: Manipulation of the cocoa ingredient. Cocoa mass was replaced with cocoa powder (11 g/100 g or <1 g/100 g fat) and added ‘free’ cocoa butter. A cocoa solids approach to design reduced fat chocolates with satisfactory flow properties is attractive to industry since it circumvents introduction of ingredients not commonly used in chocolate manufacture. Results showed that the cocoa mass chocolate had a higher viscosity than cocoa powder chocolates of the same total fat content due to the presence of trapped fat globules as identified by confocal laser scanning microscopy. The chocolate prepared with standard defatted cocoa powder containing 11 g/100 g fat had a lower viscosity than the chocolate containing highly defatted cocoa powder (<1 g/100 g) due to particle shape and fat diffusion into the particles as revealed by microscopy analyses. Based on the evidence presented, it can be concluded that standard defatted cocoa powder, as widely used by the industry, is indeed the best compromise in terms of free fat, particle size and morphology attempting to formulate fat reduced chocolate of acceptable molten state viscosity.     

Production,nutritional and biological value of bambara groundnut (<Emphasis Type="Italic">Vigna subterranea</Emphasis>) milk and yoghurt

The purpose of this study was to assess the nutritional properties of Bambara milk and yoghurt. The milk was produced by aqueous extraction of flour obtained by dehulling the seeds followed by parboiling. It was then fermented using a mix culture of L. Bulgaricus, S. thermophilus and L. plantarum, followed by the evaluation of the protein biological value on rats. During the process of flour production, from the whole seeds to the flour, a significant drop in total polyphenol content (1.00?±?0.10–0.41?±?0.01 mg/100 g) and phytates (1.18?±?0.03–0.32?±?0.01 mg/100 g) was observed while the protein content increased (19.7?±?1.2–25.47?±?2.28 g/100 g). During the fermentation of the milk into yogurt, a significant decrease in phytate content (0.29?±?0.01–0.03?±?0.01 g/100 g), an increase in the protein content (1.8?±?0.1–2.6?±?0.1 g/100 g) and the protein digestibility (91.5–96%) were equally observed. Red blood cell, glycaemia, the ASAT and ALAT contents of rat bloods fed Bambara milk or yoghurt were not significantly different to rats fed casein as protein reference. In conclusion Bambara groundnut is a source of protein which the quality may be enhanced through processing of high value yogurt.     

Gluten-free cookies added with fibre and bioactive compounds from blackcurrant residue

A blackcurrant by-product of juice production was dehydrated and added to a gluten-free chocolate cookies formulation as a source of dietary fibre and antioxidants. The blackcurrant ingredient showed high bioactive compounds content: total phenolics (TPC) = 37.5 ± 0.9 mg GAE per g d.b., total monomeric anthocyanins = 18.0 ± 0.4 mg cyanidin-3-glucoside per g d.b., antioxidant activity (AA) = 22.1 ± 0.3 mg GAE per g d.b. and total dietary fibre (TDF) = 32.3 ± 0.4% d.b. The cookie formulation was added with 3.75% blackcurrant powder without causing severe organoleptic changes. The cookies presented 62% and 70% increase of TPC and AA, respectively. The in vitro digestion showed that a relevant level of AA could reach the large intestine. Also, the TDF increased 2.5 times, reaching a final content that would enable the use of the claim 'source of dietary fibre' in the labelling according to the Codex Alimentarius. The obtained cookies showed a high level of acceptance among coeliac and non-coeliac consumers in the sensory analysis.     

Quality of Malaysian cocoa butter during storage

Malaysian pure prime pressed and deodorised cocoa butters were obtained from local cocoa grinders and analysed for quality deterioration. Approximately 5 kg of each sample was packed in corrugated paper boxes lined with low‐density polyethylene plastic material of thickness 73.2 µm. The water vapour transmission rate and gas transmission rate (for oxygen) of the plastic material were 3.21 g m^?2 day^?1 (37.8 °C, 90 ± 2% RH) and 71.57 cm³ m^?2 day^?1 (23.0 °C, 0% RH) respectively. The butters were stored at 15 ± 4 °C and 70 ± 4% RH for up to 24 months. Samples were taken every 2 months and analysed for free fatty acid content, saponification value, iodine value, refractive index and peroxide value. The findings indicated that the free fatty acid content, iodine value and peroxide value of both types of cocoa butter increased with storage duration. The other parameters showed no significant changes. However, the free fatty acid content and peroxide value after 24 months of storage were still below the limits specified in Malaysia. Therefore we conclude that both types of Malaysian cocoa butter are still in good condition after 24 months of storage, with minimal quality deterioration. Copyright © 2004 Society of Chemical Industry     

Fluidized bed roasting of cocoa nibs speeds up processing and favors the formation of pyrazines

Roasting is an important step in cocoa processing causing water loss and generating volatile compounds responsible for chocolate aroma like nitrogen-heterocycles. In this study, the comparison of two techniques, oven roasting, and fluidized bed roasting, in terms of effective water diffusivity (De) and activation energies of formation (Ea) of nitrogen-heterocycles was achieved with cocoa nibs. Fluidized bed roasting, recognized for its energy efficiency and low-footprint synthesis, was 16 times faster than oven roasting. The order of magnitude of De in fluidized-bed-roasted nibs was −8, while it was −9 in the oven-roasted nibs. Moreover, the a_w was 50% higher in fluidized-bed-roasted nibs than in the oven-roasted ones. The Ea of nitrogen-heterocycles ranged roughly between 40 and 80 kJ/mol. Those values were lower under fluidized bed roasting than under oven roasting. The more effortless water mobility within fluidized-bed-roasted cocoa demanded lower Ea, and favored the formation of nitrogen-heterocyclics.Industrial relevanceThis study can inspire cocoa manufacturers and equipment designers to pursue the formation of nitrogen-heterocycles during the roasting process of cocoa. It can be done either by adapting and scaling the current fluidized bed coffee roasters to cocoa beans or nibs; or by exploring other alternatives capable of leading enough water diffusivity and water activity in the cocoa nibs, as reported here. These physicochemical conditions undoubtedly boosted the formation of volatile compounds responsible for chocolate aroma, e.g., the pyrazines, without carrying the formation of typical-burn volatile compounds. This natural way of favoring the generation of pyrazines in cocoa nibs could contribute to clean labels by reducing or avoiding the subsequent use of flavorings. The implementation of efficient heat-transfer techniques during roasting, e.g., fluidized bed roasting, could reduce the processing cost and improve sustainability. Studies in the matter of sensory profile, and energy consumption/conversion are called for future research.     

Comparison of Whiteness Index vs. Fractal Fourier in the Determination of Bloom Chocolate Using Image Analysis

Modeling of bloom is essential to evaluate the effectiveness of processes in chocolate and to determine its shelf life. Computer vision and the fractal kinetic method were used to quantify bloom in samples of chocolate coating and cocoa chocolate subjected to a 3- or 6-h temperature cycle. Bloom was also determined by classical methods (L* lightness and whiteness index). In the results, the kinetic bloom rate derived by the fractal kinetic method was higher than that obtained with the L* or white index. Fractal method can be used to determine kinetic bloom and to detect differences in chocolate coating affected by 3 or 6 h of temperature cycle. The bloom rate derived by the fractal method was higher that the mean L* value and whiteness index methods; no differences could be registered between the last two methods. In general, the fractal method can be seen as a new means of quantifying bloom in chocolate and allows registering the bloom faster than the L* value and white index methods.