Flow properties of single origin chocolates: Effect of product formulation and particle size.

The objective of this research was to evaluate changes in flow behavior of chocolate during chocolate grinding using a stone grinder as affected by chocolate formulation. Three different types of chocolates were evaluated. Two chocolates without milk added (70% chocolate) and two chocolates with milk added and with different amounts of cocoa nibs (30% chocolate and 14% chocolate) were tested. For the 70% chocolates, nibs of two different origins were used; therefore, a total of four samples were evaluated. Chocolates were processed in a stone grinder, and samples were taken as a function of grinding time. For each timepoint, the flow behavior of the samples was measured using a rotational rheometer and fitted to the Casson model. Particle size was measured using a laser scattering instrument. Results showed that yield stress increased linearly while the Casson plastic viscosity decreased exponentially with grinding time (smaller particles). Particle size distribution of the chocolates showed a prominent bimodal distribution for short grinding times (∼9 h) with small (∼15 µm) and large (∼100 µm) particles; with longer grinding time, the population of larger particles decreased. Yield stress values were higher for the 70% chocolate, but they were not very different between the two milk chocolates tested. The Casson plastic viscosity was greatest for the 70% chocolate, followed by the 30% chocolate. The 14% chocolate had the lowest Casson plastic viscosity. Changes of Casson plastic viscosity with particle size were more evident for the dark chocolates compared to the milk ones. These results are helpful to small chocolate producers who need better understanding of how the formulation and grinding of chocolate affect its flow behavior, which will ultimately affect chocolate handling during production.

Research on Application of Japanese Quince (Chaenomeles L.) and Pork Collagen in Dark Chocolate-Benefits in Prevention of Inflammation In Vitro Model.

In the present study, the effect of the addition of quince and collagen type I and III to dessert chocolate on its functional properties was determined. The study evaluated the antioxidant potential of the tested formulations using the FRAP method and the linoleic acid oxidation test and beta-carotene bleaching test. The tested samples were also evaluated for inhibitory activity against enzymes important in preventive health (inflammation and neurodegenerative disorders) namely: AChE, BChE, GR, GPx, COX, and SOD. The addition of quince and collagen to the chocolate samples resulted in higher activity compared to the control sample, as indicated by the FRAP test. The experiment highlighted the impact of including quince fruit on the antioxidant activity of the chocolate samples. Interestingly, merely increasing the quince fruit amount did not consistently enhance antioxidant potential. Specifically, chocolate samples with a lower proportion of quince fruit (2 g/100 g) exhibited greater antioxidant activity when supplemented with collagen I. Conversely, in samples with higher quince percentages (3 g and 4 g), those enriched with collagen III showed higher antioxidant activity. Similar correlations were observed in the linoleic acid oxidation test. Notably, samples containing 3 g and 4 g of quince and type III collagen demonstrated statistically similar highest antioxidant properties. Regardless of the collagen type used, there was no observed increase in activity towards the tested enzymes for samples with the lowest percentage of quince fruit. Both collagen types exhibited the highest activity in the inhibition assay against acetylcholinesterase and butyrylcholinesterase when combined with 3 g and 4 g of quince. Overall, the experimental incorporation of both fruit and collagen enhanced the chocolates' activity. Similarly to the antioxidant activity findings, chocolates with lower quince fruit quantities showed increased activity when supplemented with collagen III, while those with higher quince content (3 g and 4 g) displayed higher activity with collagen I. Bitter chocolate by itself is an attractive food product, rich in many bioactive compounds. However, enriching it with other attractive raw materials can make its properties and taste even more attractive.

Evaluating the galactooligosaccharide stability in chocolate milk beverage submitted to ohmic heating.

Among the emerging prebiotics, galactooligosaccharide (GOS) has a remarkable value with health-promoting properties confirmed by several studies. In addition, the application of ohmic heating has been gaining prominence in food processing, due to its various technological and nutritional benefits. This study focuses on the transformative potential of ohmic heating processing (OH, voltage values 30 and 60 V, frequencies 100, 300, and 500 Hz, respectively) in prebiotic chocolate milk beverage (3.0 %w/v galactooligosaccharide) processing. Chemical stability of GOS was assessed along all the ohmic conditions. In addition, microbiological analysis (predictive modeling), physical analysis (color and rheology), thermal load indicators assessment, bioactivity values, and volatile compound was performed. HPAEC-PAD analysis confirmed GOS stability and volatile compound evaluation supported OH's ability to preserve flavor-associated compounds. Besides, OH treatments demonstrated superior microbial reduction and decreased thermal load indicators as well as the assessment of the bioactivity. In conclusion, OH presented was able to preserve the GOS chemical stability on chocolate milk beverages processing with positive effects of the intrinsic quality parameters of the product.

Glyoxal and methylglyoxal formation in chocolate and their bioaccessibility.

The objective of this study was to assess the effects of simulated digestion on the formation of α-dicarbonyl compounds (α-DCs) in chocolates. For that purpose, the concentrations of glyoxal and methylglyoxal in chocolates were determined through High-Performance Liquid Chromatography (HPLC) analysis before and after in vitro digestion. The initial concentrations ranged from 0.0 and 228.2 µg/100 g, and 0.0 and 555.1 for glyoxal and methylglyoxal, respectively. Following digestion, there was a significant increase in both glyoxal and methylglyoxal levels, reaching up to 1804 % and 859 %, respectively. The findings indicate that digestive system conditions facilitate the formation of advanced glycation end product (AGE) precursors. Also, glyoxal and methylglyoxal levels were found to be low in chocolate samples containing dark chocolate. In contrast, they were found to be high in samples containing hazelnuts, almonds, pistache, and milk. Further studies should focus on α-DCs formation under digestive system conditions, including the colon, to determine the effects of gut microbiota.

A systematic review of randomized controlled trials on the health effects of chocolate enriched/fortified/supplemented with functional components.

The most significant contributor to global mortality are cardiovascular diseases. Dietary factors significantly impact the risk, advancement, and treatment of cardiometabolic conditions. Chocolate, known for its adaptability and capacity to stimulate pleasure centers, emerges as a promising vehicle for integrating different bioactive elements. This systematic review analyzed 10 randomized controlled trials investigating the health effects of consuming enriched, fortified, or supplemented chocolate. These trials varied in chocolate intake amounts (ranging from 5 to 101 g day-1), incorporated bioactive components (co-crystalized astaxanthin, lycopene, wood-based phytosterol-phytostanol mixture, canola sterol esters, etc.), and duration (from 2 weeks to 1 year). Some enriched chocolates were found to reduce total and LDL cholesterol and influence markers of oxidative damage, inflammation, immune function, and skin parameters. However, certain trials showed a minimal impact on health outcomes. Therefore, while enriched chocolate holds promise as a carrier for beneficial bioactive compounds, rigorous scientific inquiry and methodological rigor are crucial to fully substantiate these claims. Comprehensive evaluations covering cardiovascular health, metabolic function, immune response, and other aspects are needed to understand its potential benefits and limitations. Advancing robust research initiatives could help realize the full potential of enriched chocolate in promoting human health and well-being.

Occurrence and Synthesis Pathways of (Suspected) Genotoxic α,ß-Unsaturated Carbonyls in Chocolate and Other Commercial Sweet Snacks.

α,ß-Unsaturated carbonyls are highly reactive and described as structural alerts for genotoxicity. Ten of them (either commercially available or synthesized here by combinatorial chemistry) were first investigated throughout the chocolate-making process by solvent-assisted flavor evaporation (SAFE) coupled to GC-MS/SIM. Monitored α,ß-unsaturated aldehydes were formed during chocolate production, primarily through aldol condensation of Strecker aldehydes triggered by bean roasting. Notably, levels of 2-phenylbut-2-enal (up to 399 µg·kg-1) and 5-methyl-2-phenylhex-2-enal (up to 216 µg·kg-1) increased up to 40-fold. Dry conching caused evaporation of α,ß-unsaturated carbonyls, while wet conching partially restored or increased their levels due to cocoa butter addition. Further analyses showed that α,ß-unsaturated aldehydes also occurred in most commercial sweet snacks (up to 16 µg·kg-1), although often at lower concentrations than in roasted cocoa or derived chocolates. In the end, none of the monitored α,ß-unsaturated aldehydes did raise a health concern compared to current maximum use levels (2-5 mg·kg-1). On the other hand, much higher levels of genotoxic furan-2(5H)-one were found in crepe and cake samples (up to 4.3 mg·kg-1).

Occurrence of heavy metals coupled with elevated levels of essential elements in chocolates: Health risk assessment.

The presence of contaminants in cacao-derived products, especially in chocolates, has raised concerns regarding food safety and human health. The study assessed the concentration variation of 16 elements in 155 chocolate samples from the US market by cacao content and country of geographic origin. The study further examined the potential health risks posed by toxic metals and determined the contribution of essential elements to the Daily Recommended Intake (DRI), estimated based on an ounce (∼28.4 g) of daily chocolate consumption. Dark chocolates with ≥50 % cacao exhibited consecutively increasing mean levels from 1.2 to 391 µg/kg for U, Tl, Th, As, Pb, Se, Cd, and Co. Similarly, Ni, Sr, Cu, Mn, Zn, Fe, Ca, and Mg had mean concentrations from 4.0 to 1890 mg/kg. Dark chocolates sourced from Central and South America exhibited the highest mean levels of Cd, and South America samples also contained elevated Pb, whereas those from West Africa and Asia had low Cd and Pb, respectively. Cacao contents showed increasingly strong association with Cd, Co, Mn, Sr, Ni, Cu, Zn, and Mg (r = 0.60-0.84), and moderately with Se, Fe, As, and Tl (r = 0.35-0.49), indicating these elements are primarily derived from cacao beans. Weak association of cacao contents with Pb, Th, and U levels (r < 0.25), indicates post-harvest contaminations. Hazard Quotient (HQ) > 1 was found only for Cd in 4 dark chocolates, and Hazard Index (HI) > 1 for cumulative risk of Cd, Pb, Ni, As, and U was found in 33 dark chocolates, indicating potential non-carcinogenic risks for 15 kg children but none for 70 kg adults. Dark chocolate also substantially contributed to 47-95 % of the DRI of Cu for children and 50 % for adults. Dark chocolates also provided notable Fe, Mn, Mg, and Zn contributions to the DRI. These essential elements are recognized to reduce the bioavailability of toxic metals such as Cd, Pb, or Ni, thereby potentially lowering associated health risks. This study informs consumers, food industries, and regulatory agencies to target cacao origins or chocolate brands with lower toxic metal contents for food safety and minimizing adverse health effects.

Valorization of cocoa pod side streams improves nutritional and sustainability aspects of chocolate.

Chocolate production faces nutritional, environmental and socio-economic challenges present in the conventional cocoa value chain. Here we developed an approach that addresses these challenges by repurposing the often-discarded pectin-rich cocoa pod endocarp and converting it into a gel. This is done using cocoa pulp juice concentrate to replace traditional sugar from sugar beets. Although swelling of fibres, proteins and starches can limit gel incorporation, our proposed chocolate formulation contains up to 20 wt% gel. It also has comparable sweet taste as traditional chocolate while offering improved nutritional value with higher fibre and reduced saturated fatty acid content. A cradle-to-factory life cycle assessment shows that large-scale production of this chocolate could reduce land use and global warming potential compared with average European dark chocolate production. The process also provides opportunities for diversification of farmers' income and technology transfer, offering potential socio-economic benefits for cocoa-producing regions.

Trace elements in bean-to-bar chocolates from Brazil and Ecuador.

BACKGROUND: The high quality and unique flavor and aroma of bean-to-bar chocolates have resulted in an increase in the consumption of these products. Nevertheless, cocoa beans may present inorganic contaminants from environmental and anthropogenic sources which can contribute to contamination of the chocolates, despite the fewer processing steps and few ingredients used in bean-to-bar manufacturing process compared to the industrial one. Therefore, this study aimed to evaluate the content of trace elements (As, Cd, Co, Cu, Hg, Pb, Se) in bean-to-bar chocolates and traceable cocoa beans from Brazil and Ecuador. METHODS: Bean-to-bar chocolate samples were acquired in Brazil (n=65) and Ecuador (n=10), considering the main products available: white, milk, semisweet and dark chocolate. Cocoa samples from dedicated farms (n=23) were analyzed for trace elements and inorganic contaminants regulated by Brazil and European agencies. Samples were mineralized using acid digestion (nitric acid and hydrogen peroxide) in a closed microwave-assisted system. Quantification of trace elements was performed using Inductively coupled plasma mass spectroscopy (ICP-MS) and Inductively coupled plasma optical emission spectroscopy (ICP OES) in optimized conditions. The analytical control was performed with certified reference materials (ERM BD512 - Dark Chocolate, Tort-2 and Tort-3 - Lobster Hepatopancreas and SRM 1547 - Peach leaves) and recoveries ranged between 84% and 105% for all elements. RESULTS: The trace element levels in the bean-to-bar chocolates were (mg/kg): As (<0.022-0.023), Cd (<0.002-0.74), Cu (0.11-21.2), Co (<0.003-1.88), Hg (<0.010-<0.010), Pb (<0.007-0.22), and Se (<0.029-0.35). The exposure assessment from inorganic contaminants in chocolates revealed up to 93% of provisional tolerable monthly intake (PTMI) for Cd and 123% of tolerable upper intake level (UL) for Co for children. Inorganic contaminants were also analyzed in cocoa beans from dedicated farms and Cd and Pb levels were found above the thresholds established by Brazil health agency. CONCLUSION: The results observed for both bean-to-bar chocolates and raw materials (cocoa beans from dedicated farms) indicated a need for monitoring these trace elements.

Sensory attributes of fine flavor cocoa beans and chocolate: A systematic literature review.

Fine flavor cocoa (FFC) is known for its unique flavor and aroma characteristics, which vary by region. However, a comprehensive overview of the common sensory attributes used to describe FFC beans and chocolate is lacking. Therefore, a systematic review was conducted to analyze existing literature and identify the most commonly used sensory attributes to describe FFC beans and chocolate. A systematic search of the Web of Science and Scopus databases was conducted in May 2023, and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed to ensure transparency and reproducibility. This review summarizes the origins of cocoa and explores their unique flavor profiles, encompassing caramel, fruity, floral, malty, nutty, and spicy notes. Although some origins may exhibit similar unique flavors, they are often described using more specific terms. Another main finding is that although differences in sensory attributes are anticipated at each production stage, discrepancies also arise between liquor and chocolate. Interestingly, fine chocolate as the final product does not consistently retain the distinctive flavors found in the liquor. These findings emphasize the need for precise descriptors in sensory evaluation to capture flavor profiles of each origin. As such, the exploration of attributes from bean to bar holds the potential to empower FFC farmers and chocolate producers to effectively maintain quality control.

One-bite-sized 3D printed finger foods, oriented to malnutrition, sarcopenia and frailty prevention in the older people.

BACKGROUND: In this study, innovative chocolate, citrus and mixture flavoured tofu-based nutritionally customised, dysphagia-oriented, comfortably consumed, appetising, one-bite-sized finger foods, oriented to malnutrition, sarcopenia and frailty prevention in older people were created by using 3D printing technology. Developed products were characterised by evaluating chemical composition and physical properties and performing sensory evaluation among geriatric clinic residents (≥60 years). RESULTS: The dietary composition of the developed foods was: 19-21 g (100 g)-1 protein, 6-8 g (100 g)-1 fibre, 8-9 g (100 g)-1 fat, 11 mg (100 g)-1 iron, 14 mg (100 g)-1 zinc, 70 µg (100 g)-1 selenium. Foods were also enriched with branched-chain amino acids, such as leucine, isoleucine and valine. All formulated foods were classified as level 6 by International Dysphagia Diet Standardisation Initiative classification. Chocolate-flavoured food was much harder (4914 g) with lower adhesiveness value (-33.6 g s), compared to the citrus- or mixture-flavoured foods. Older people evaluated all finger foods as very easy handled by hand, soft, easy to swallow, having a moderate flavour intensity and a weak afterfeel. Despite the fact that the chocolate food was evaluated as having the highest hardness and gumminess values by the instrumental method, this difference was not noticeable to the evaluators. However 7% of the participants said that 3D printed foods were sticky to dentures. CONCLUSION: The results suggest that it is possible to create nutrient-dense comfortably consumed 3D printed foods, oriented to malnutrition, sarcopenia and frailty prevention in older people. © 2024 Society of Chemical Industry.

Determination of the process effect on cocoa butter crystallization by rheometer: Kinetic modeling by Gompertz equation.

In this study, the effects of temperature (22, 24, 26, 28, and 30°C) and strain (0.1%, 1%, and 5%) on cocoa butter (CB) crystallization were investigated by oscillatory test, and the four-parameter Gompertz model was used to interpret the effect of parameters on pre-crystallization, nucleation, and crystal growth stages of CB. Lag time and growth rate were calculated using the Gompertz model using time-dependent storage modulus (G') data. According to the results, CB crystallization at 26°C with a 1% strain value had the highest growth rate value, the shortest lag time, and the formation of ßv polymorph type. Followingly, polymorphic types of the CB crystals were determined based on the melting points of polymorphs via the temperature ramp step, and the results obtained were correlated with a polarized light microscope. In conclusion, using a rheometer in both the observation of the pre-crystallization process and the determination of polymorph types is very important for research and development studies in the chocolate industry for process and formulation optimization. PRACTICAL APPLICATION: This study demonstrates the feasibility of a novel approach for investigating crystallization and oscillatory shear of CB using a rheometer, both for observing crystallization kinetics and determining polymorph type, accompanied by the Gompertz equation to model the crystallization kinetics. According to the results, the effect of process parameters (temperature and shear) on the crystallization behavior of CB can be observed by rheometer, which can provide a detailed perspective for chocolate manufacturers and researchers in research and development studies.

Influence of process conditions of alkalization on quality of cocoa powder.

In this study, the effects of independent variables such as alkaline (NaOH) salt concentration (3.0-6.0 g/100 mL), alkalization temperature (60-90 °C), and time (20-40 min) on cocoa powder (low-fat) properties were investigated by using Central Composite Design. The physicochemical and color properties of samples, powder characteristics, volatile component profile, total polyphenol content (TPC), as well as antioxidant activity potentials using different methods (DPPH and ABTS) were determined. Significant models were identified for the effects on major alkalization indicators (L*, a*/b*, pH), as well as TPC and antioxidant activity potential (DPPH), which are the main motivators for the preference and consumption of cocoa products (p < 0.05). The established model was validated, and their predicted values were found to be very close to real results. It was determined that the alkali concentration had a more significant effect on dependent variables, especially on alkalization indicators, compared to the other independent variables. Furthermore, strong correlations were determined between TPC and antioxidant activity potential and color properties (L*, a*, b*, and a*/b*). Optimum concentration, temperature and time were found to be 5.3 %, 84 °C and 35.7 min for maximizing a*/b* value. The establishment of such models lead to optimizing process conditions of alkalization with minimum effort and labor force for obtaining cocoa powder with desired quality depending on the usage purpose.

Phenolic composition and sensory dynamic profile of chocolate samples enriched with red wine and blueberry powders.

Cabernet Sauvignon (CS) and a combination of Cabernet Sauvignon with blueberry extract (CS + B), were spray dried (using maltodextrin DE10, 13.5% w/w as a carrier) to obtain two types of phenolic-rich powders. The addition of blueberry to CS increased phenolic compounds content by 16%. Eight chocolate formulations were obtained by modifying concentrations of cocoa solids, cocoa butter, and sugar. Six of the samples were added with 10% w/w of phenolic-rich powder, while two of them remained as powder-free controls. The anthocyanin and flavan-3-ol profiles of chocolates were determined by HPLC-DAD-MS and HPLC-MS, respectively. In addition, the sensory dynamic profile of samples was assessed by Temporal Dominance of Sensations with a consumer panel. Results showed that the addition of phenolic-rich powders produced a significant increase in the anthocyanin composition obtaining the highest anthocyanin content in the white chocolate added with CS + B powder. On the other hand, adding 10% of CS powder to dark chocolate (55% cocoa pellets) did not result in a significant increase in phenolic compounds. The addition of phenolic-rich powders to chocolates influenced visual color, texture, and taste, leading to new products with distinctive characteristics and increasing the possibility of using phenolic-rich powders as innovative and healthy ingredients.

Challenges and potential for detecting and quantifying titanium dioxide in food.

BACKGROUND: Titanium dioxide (TiO2) is banned in some countries but its use is still permitted in others. The global food supply chain is therefore challenged with the need to use rapid and reliable testing methods to either detect the presence of TiO2 or to quantify its concentration. The goal of this study was to determine the feasibility of using color, texture profile analysis, Raman microscopy, and X-ray fluorescence (XRF) spectroscopy to detect and quantify TiO2 in fillings used in the pastry and confectionery industry. In this study, two types of fillings were investigated: vanilla based and chocolate based. All fillings were prepared in four different variations - without TiO2 and with three concentrations as follows: 0.25 g*kg-1, 0.5 g*kg-1, or 0.75 g*kg-1 TiO2 per sample. The methods were selected for their ability to analyze the samples in a short period of time. RESULTS: All of the methods showed moderate to high potential for detecting TiO2 in the samples. The results reveal how TiO2 affects the food matrix color and texture. Use of Raman microscopy confirms its detectability, although concentrations of TiO2 do not follow a pattern. X-ray fluorescence spectroscopy showed the greatest potential as it can not only detect TiO2 but can also quantify its concentration in the samples. CONCLUSIONS: The highest potential for quantifying the concentration of this food additive was achieved with XRF. © 2024 Society of Chemical Industry.

Chocolates, compounds and spreads: A review on the use of oleogels, hydrogels and hybrid gels to reduce saturated fat content.

This study is a bibliometric analysis and literature review on the use of oleogels (OGs), hydrogels (HGs) and hybrid gels (HYGs) in chocolate, compounds and spreads with the aim of reducing the saturated fat in these products. The articles were selected by analyzing titles, keywords and abstracts in the Web of Science (WoS), Scopus and Google Scholar databases. Supplementary documents were obtained from government sources, including patent registrations. The theoretical and practical aspects were critically analyzed, highlighting the main points of agreement and disagreement between the authors. The results revealed a lack of regulations and official guidelines that widely allow the use of OGs, HGs and HYGs in chocolate confectionery products. The type and characteristics of raw materials affect the properties of products. Replacing cocoa butter (CB) with OGs, HGs or HYGs also affects texture, melting point and behavior, and nutritional aspects. These substitutions can result in products with better sensory acceptance and health benefits, such as reducing saturated fat and promoting cardiovascular health. However, it is important to find the ideal combination and proportions of components to obtain the desired properties in the final products.

Determination of Acrylamide in Coffee, Cereals, Baby Food, Cocoa, Dry Pet Food, Potato Products, Vegetable Crisps, Biscuits, Tea, Nuts, and Spices by LC-MS/MS in a Single-Laboratory Validation: First Action 2023.01.

BACKGROUND: Acrylamide (AA) is a process contaminant naturally formed during the cooking of starchy food at high temperatures. Considering existing risks of misquantification inherent to the analysis of AA, an AOAC initiative raised the need for a consensus standard to determine AA in a broad variety of food. OBJECTIVE: A quantitative LC-MS/MS method for AA determination in food was validated in a single-laboratory study. Targeted performance requirements in terms of target matrixes, limit of quantification, recovery, and precision were as defined per Standard Method Performance Requirement (SMPR®) 2022.006. METHOD: The proposed method derives from EN 16618:2015 standard pending modifications brought to the (1) sample preparation (simplified, potentially automated); (2) scope of application (significantly extended); and (3) LC conditions (improved selectivity). Confirmatory detection of AA is conducted by LC-MS/MS in the Selected Reaction Monitoring mode (SRM), and isotopic dilution was applied for quantification approach using either 2,3,3-d3-acrylamide (d3-AA), or 13C3-2,3,3-d3-acrylamide (13C3-d3-AA) as labeled internal standard. RESULTS: A total of 16 laboratory samples from nine matrix categories were included in the validation process. A full validation was conducted on coffee (instant, roast), infant cereal, cocoa powder, pet food (croquettes), tea (green tea), spices (black pepper), and nuts (roasted almonds) with satisfactory performances both in terms of recovery (97-108%) and precision (RSDr and RSDiR <12%). The method applicability was further demonstrated through the analysis of quality control materials and reference materials including French fries, potato crisps, vegetable crisps, instant coffee, infant food, and biscuits (cookies), with accuracy values determined within a 94-107% range. CONCLUSIONS: The performances of the presented method are in agreement with the acceptance criteria stipulated in SMPR 2022.006. HIGHLIGHTS: The Expert Review Panel for acrylamide approved the present method as AOAC Official First Action 2023.01.

In-house validation of an LC-MS method for the multiplexed quantitative determination of total allergenic food in chocolate.

Mass spectrometry has been widely accepted as a confirmatory tool for the sensitive detection of undeclared presence of allergenic ingredients. Multiple methods have been developed so far, achieving different levels of sensitivity and robustness, still lacking harmonization of the analytical validation and impairing comparability of results. In this investigation, a quantitative method has been validated in-house for the determination of six allergenic ingredients (cow's milk, hen's egg, peanut, soybean, hazelnut, and almond) in a chocolate-based matrix. The latter has been produced in a food pilot plant to provide a real and well-characterized matrix for proper assessment of method performance characteristics according to official guidelines. In particular, recent considerations issued by the European Committee for Standardization have been followed to guide a rigorous single-laboratory validation and to feature the main method performance, such as selectivity, linearity, and sensitivity. Synthetic surrogates of the peptide markers have been used both in native and labelled forms in matrix-matched calibration curves as external calibrants and internal standards, respectively. A two-order of magnitude range was investigated, focusing on the low concentration range for proper assessment of the detection and quantification limits (LOD and LOQ) by rigorous calibration approach. Conversion factors for all six allergenic ingredients have been determined for the first time to report the final quantitative information as fraction of total allergenic food protein (TAFP) per mass of food (µgTAFP/gfood), since such a reporting unit is exploitable in allergenic risk assessment plans. The method achieved good sensitivity with LOD values ranging between 0.08 and 0.2 µgTAFP/gfood, for all ingredients besides egg and soybean, whose quantitative markers reported a slightly higher limit (1.1 and 1.2 µgTAFP/gfood, respectively). Different samples of chocolate bar incurred at four defined concentration levels close to the currently available threshold doses have been analyzed to test the quantitative performance of the analytical method, with a proper estimate of the measurement uncertainty from different sources of variability. The sensitivity achieved resulted in compliance with the various threshold doses issued or recommended worldwide.

Toward Unified Flavor Quantitation in Cocoa-Based Products.

Because food flavor is perceived through a combination of odor and taste, an analytical method that covers both dimensions would be very beneficial for mapping the consistent product quality over the entirety of a manufacturing process. Such a method, so-called "unified flavor quantitation", has been successfully applied to several different food products in recent years. The simultaneous detection of aroma and taste compounds by means of ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) enables the analysis and quantification of an enormously large number of compounds in a single run. To evaluate the limits of this method, chocolate, a high-fat, complex matrix, was selected. In 38 distinct commercial chocolate samples, 20 flavor-active acids, aldehydes, and sugars were analyzed after a simple, rapid extraction step followed by derivatization with 3-nitrophenylhydrazine using a single UHPLC-MS/MS method. The results obtained highlight the great potential of the "unified flavor quantitation" approach and demonstrate the possibility of high-throughput quantitation of key aroma- and taste-active molecules in a single assay.

Honey contamination from plant protection products approved for cocoa (Theobroma cacao) cultivation: A systematic review of existing research and methods.

The main component of chocolate, cocoa (Theobroma cacao), is a significant commercial agricultural plant that directly sustains the livelihoods of an estimated forty to fifty million people. The economies of many cocoa producing nations, particularly those in the developing world, are supported by cocoa export revenue. To ensure satisfactory yields, however, the plant is usually intensely treated with pesticides because it is vulnerable to disease and pest attacks. Even though pesticides help protect the cocoa plant, unintended environmental contamination is also likely. Honey, produced from nectar obtained by honeybees from flowers while foraging, can serve as a good indicator for the level of pesticide residues and environmental pesticide build-up in landscapes. Here, we use a systematic literature review to quantify the extent of research on residues of pesticides used in cocoa cultivation in honey. In 81% of the 104 studies examined for this analysis, 169 distinct compounds were detected. Imidacloprid was the most frequently detected pesticide, making neonicotinoids the most frequently found class of pesticides overall. However, in cocoa producing countries, organophosphates, organochlorines, and pyrethroids were the most frequently detected pesticides. Interestingly, only 19% of studies were carried out in cocoa producing countries. We recommend prioritizing more research in the countries that produce cocoa to help to understand the potential impact of pesticide residues linked with cocoa cultivation in honey and the environment more generally to inform better pesticide usage, human health, and environmental policies.