Diversity of lactic acid bacteria from modified atmosphere packaged sliced cooked meat products at sell-by date assessed by PCR-denaturing gradient gel electrophoresis.

The predominant lactic acid bacteria (LAB) microbiota associated with three types of modified atmosphere packaged (MAP) sliced cooked meat products (i.e. ham, turkey and chicken) was analyzed at sell-by date using a combination of culturing and molecular population fingerprinting. Likewise routine analyses during industrial MAP production, meat samples were plated on the general heterotrophic Plate Count Agar (PCA) and on the LAB-specific de Man, Rogosa, Sharpe (MRS) agar under different temperature and atmosphere conditions. Subsequently, community DNA extracts were prepared from culturable bacterial fractions harvested from both media and used for PCR targeting the V3 hyper-variable region of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) of PCR amplicons (PCR-DGGE). Irrespective of aerobic or anaerobic incubation conditions, V3-16S rDNA DGGE fingerprints of culturable fractions from PCA and MRS medium displayed a high level of similarity indicating that LAB constituted the most dominant group in the culturable bacterial community. Comparison of DGGE profiles of fractions grown at 20, 28 or 37 degrees C indicated that part of the culturable community consisted of psychrotrophs. Four DGGE bands were common among cooked ham, turkey and chicken products, suggesting that these represent the microbiota circulating in the plant where all three MAP product types were sliced and packaged. Based on band sequencing and band position analysis using LAB reference strains, these four bands could be assigned to Lactobacillus sakei and/or the closely related Lactobacillus fuchuensis, Lactobacillus curvatus, Carnobacterium divergens and Leuconostoc carnosum. In conclusion, the PCR-DGGE approach described in this study allows to discriminate, identify and monitor core and occasional LAB microbiota of MAP sliced cooked meat products and provides valuable complementary information to the current plating procedures routinely used in industrial plants.

A Combined RNA Preservation and Extraction Protocol for Gene Expression Studies in Cacao Beans.

Despite the high economic importance of cacao beans, few RNA-based studies have been conducted on this plant material and hence no optimal RNA-extraction has been reported. Moreover, extraction of high-quality RNA from recalcitrant cacao bean tissue has shown many difficulties and requires optimization. Furthermore, cacao beans are mostly found at remote and under-resourced locations, which pressures the outsourcing of such analysis and thereby demands RNA-stable preservation and transportation of cacao beans. This study aims to select an appropriate RNA extraction and preservation/transportation method for cacao beans. For this purpose, three sample homogenization and five extraction protocols on cacao beans were compared. In addition, 13 preservation conditions-differing in tissue crushing degree, preservation method, duration, and temperature-were compared and evaluated. A comparative analysis revealed that CTAB-based homogenization and extraction outcompeted all tested commercial protocols in RNA yield and integrity, respectively. Preservation at -80°C affected RNA quality the least, whereas freeze-drying was most suitable for transportation at room temperature for maximum 1 week. The cacao bean RNA obtained from the selected methods were compatible for downstream applications. The results of this study will facilitate on-field sampling and transportation of genetically sensitive cacao material prior to cacao bean transcriptomic studies. In addition, valuable insights on sample homogenization, extraction, preservation, and transportation have been provided, which is of interest to every plant geneticist.

Pod storage with roasting: A tool to diversifying the flavor profiles of dark chocolates produced from 'bulk' cocoa beans? (Part II: Quality and sensory profiling of chocolates).

The impact of pod storage (PS) and roasting temperature (RT) on the quality parameters and the sensory profiles of dark chocolates were evaluated. Dark chocolates (70%) from ten liquors of different PS and RT combinations as well as six liquors of different origins (Ecuador, Ghana, Ivory Coast, Madagascar, Venezuela and Vietnam) with variable genetic groups were produced under identical conditions and compared. To a greater extent, the range of chocolate quality attributes underscored the generally minimal effects of PS, RT and origin of liquor on the processing conditions. Although with a few exceptions, generally, chocolate acidity (pH and TA) decreased with increasing PS and vice versa in the case of RT. Furthermore, results from a balanced incomplete block design (BIBD) involving a 16-member expert panel also revealed the impact of the applied treatments (PS and RT) on the final flavor profiles of the chocolates irrespective of the origin or genetic groups of the cocoa beans. The same was confirmed when instrumental aroma results were correlated with the sensory data using partial least squares (PLS) regression models. Thus, this study demonstrates the possibility of creating diverse flavor profiles (even towards 'fine' flavor) from 'bulk' cocoa beans through an optimized combination of PS and RT. The findings are therefore expected to challenge the status-quo, especially in the way 'bulk' cocoa is currently processed and consequently priced, thereby, possibly fostering a win-win situation between cocoa producers and industries.

Non-volatile and volatile composition of West African bulk and Ecuadorian fine-flavor cocoa liquor and chocolate.

Cocoa products are obtained from the seeds of Theobroma cacao L. In this research, cocoa liquor and chocolate produced from cocoa beans from West Africa (Forastero, "bulk" cacao) and Ecuador (Nacional variety, "fine-flavor" cacao), were investigated, using a novel approach in which various analytical techniques are combined in order to obtain in-depth knowledge of the studied cocoa samples. The levels of various classes of primary metabolites were determined and a wide range of secondary metabolites, including volatile organic acids, aldehydes, esters, pyrazines, polyphenols, methylxanthines and biogenic amines, were identified and/or quantified by HS-SPME GC-MS (headspace-solid phase microextraction gas chromatography - mass spectrometry). and UPLC-HRMS (ultra-performance liquid chromatography - high resolution mass spectrometry). Odor Activity Values (OAV) were calculated to assess the contribution of individual volatiles on the final aroma. Various volatile aroma compounds were more abundant in the West African cocoa liquor and chocolate, while the Ecuadorian samples were richer in most quantified non-volatile metabolites. Principal component analysis (PCA) confirmed that the four samples can be clearly distinguished. Alcohols, pyrazines, amino acids and biogenic amines were found to be highly influential in causing this differentiation. The proposed approach can be useful in future studies on more extensive cocoa sample collections, in order to highlight similarities and pinpoint typical differences in chemical composition among these samples.

The development of a novel SNP genotyping assay to differentiate cacao clones.

In this study, a double-mismatch allele-specific (DMAS) qPCR SNP genotyping method has been designed, tested and validated specifically for cacao, using 65 well annotated international cacao reference accessions retrieved from the Center for Forestry Research and Technology Transfer (CEFORTT) and the International Cocoa Quarantine Centre (ICQC). In total, 42 DMAS-qPCR SNP genotyping assays have been validated, with a 98.05% overall efficiency in calling the correct genotype. In addition, the test allowed for the identification of 15.38% off-types and two duplicates, highlighting the problem of mislabeling in cacao collections and the need for conclusive genotyping assays. The developed method showed on average a high genetic diversity (He = 0.416) and information index (I = 0.601), making it applicable to assess intra-population variation. Furthermore, only the 13 most informative markers were needed to achieve maximum differentiation. This simple, effective method provides robust and accurate genotypic data which allows for more efficient resource management (e.g. tackling mislabeling, conserving valuable genetic material, parentage analysis, genetic diversity studies), thus contributing to an increased knowledge on the genetic background of cacao worldwide. Notably, the described method can easily be integrated in other laboratories for a wide range of objectives and organisms.

Tuning the aroma profiles of FORASTERO cocoa liquors by varying pod storage and bean roasting temperature.

The unique impact of roasting conditions on the aroma quality of cocoa beans has been demonstrated in many studies. However, information on the additional impact of pod storage (PS) and its combined effect with roasting temperature (RT) is unknown. Hence, this study sought to elucidate the collective contribution of these post-harvest/process parameters on the aroma profiles of cocoa liquors produced from Forastero cocoa beans. The beans had been subjected to different treatments following a 3 × 4 full factorial experiment, consisting of PS (0, 3, 7 days) and RT (100, 120, 140, 160 °C). Statistical analysis of the results from HS-SPME-GC-MS revealed significant (p < .05) impact of both PS and RT as well as their interaction effects on the ten groups of volatiles (acids, alcohols, esters, terpenes, aldehydes, ketones, pyrazines, furans, pyrroles and others) and their overall aroma concentration. An exception was however noted for aldehydes, where the total concentration was only significantly (p < .05) influenced by the individual effects of PS and RT. A subsequent clustering of the liquors, first on the basis of all identified volatiles, then, on the basis of the odor-active volatiles, also revealed similar pattern where liquors with high RT's possessed more volatiles with higher concentrations and vice versa. More so, it seemed that no or very minimal PS treatment was necessary for preserving more aromatic volatiles with typically fruity, floral or spicy flavor notes, whereas, for liquors with volatiles exhibiting more cocoa, chocolate, nutty and roasted flavor notes, prolonged PS (> 3 days) treatment was required. These findings are expected to challenge the status-quo, specifically in the conventional ways through which the aroma potential of 'bulk' cocoa may be steered. On the one hand, the idea of manipulating PS treatment and roasting conditions may indeed consolidate the possibility of creating diverse and/or distinct aroma profiles from the same 'bulk' cocoa beans, whereas, on the other hand, it raises the question whether the Ghanaian cocoa beans - being described as 'bulk' cocoa - could be a consequence of prolonged pod storage treatment.

Pod storage with roasting: A tool to diversifying the flavor profiles of dark chocolates produced from 'bulk' cocoa beans? (part I: aroma profiling of chocolates).

The impact of pod storage (PS) and roasting temperature (RT) on the aroma profiles of dark chocolates were evaluated. Cocoa liquor samples comprised of ten different combinations of PS and RT, whilst keeping the roasting time fixed at 35 min. Additionally, commercial cocoa liquors from renowned origins (Ecuador, Madagascar, Venezuela, Vietnam, Ivory Coast and Ghana) were acquired for comparison. From these, 70% dark chocolates were produced under the same conditions after which they were subjected to headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis. Although both PS and RT were found to influence the aroma volatile concentrations, the impact of RT over PS seemed to be greater. An agglomerative hierarchical clustering (AHC) of all chocolates on the basis of their aroma profiles revealed a similar impact as earlier observed, where major clustering of the chocolates was in accordance with the intensity of the roasting process applied. However, within each group, the dissimilarities owing to PS among the chocolates was clearly depicted. Comparatively, chocolates with low (100-120 °C), instead of moderate to high (135-160 °C) RT's, rather showed a low dissimilarity with those from the commercial cocoa liquors of the different origins. Although from the same beans, the diversity of aroma profiles of these chocolates as well as the similitude of some treatments to some chocolates from commercial grade cocoa liquors, unequivocally underscores the possibility for steering diverse distinct flavors from 'bulk' cocoa through PS and roasting, with beneficial implications, both from an application and an economic point of view.

Assessing the influence of pod storage on sugar and free amino acid profiles and the implications on some Maillard reaction related flavor volatiles in Forastero cocoa beans.

The practice of pod storage (PS) has been applied in many cocoa producing countries, especially by Ghanaian farmers over the years. However, the study of PS has not received much attention, hence, until now, its potential impact on specific flavor precursor development and implications on the flavor of cocoa beans still remains uncovered. The study was therefore aimed at exploring this possibility through physico-chemical and flavor precursor analyses, carried out on equally fermented and dried pod stored (0, 3 and 7 days) Ghanaian cocoa beans. Flavor analysis was also conducted on equally roasted pod stored cocoa beans. Through visual assessment of the pods, pulp and beans, the compelling impact of PS on fermentation index (FI) and nib acidity could be demonstrated by the various biochemical and physical changes such as respiration, moisture reduction, and cellular degradation, occurring during the process. Whereas the entire reaction of sugar degradation may be deemed complex, a clear relationship between the FI, nib acidity and the glucose content was observed. Also, PS was found to increase with marginal increase in total reducing sugars (glucose and fructose). Although the concentration of free amino acids was directly proportional to the duration of PS, within the framework of this study, a significant difference (p < .05) was only observed in the case of extended duration (7 days). Overall, 7 PS seemed to have enhanced the formation of more volatiles. This was followed by 0 PS and finally 3 PS. Suggestively, these findings could provide some indications in explaining the typical flavor profiles of the Ghanaian cocoa beans, considering the fact that 87.8% of Ghanaian farmers adhere to this practice. Meanwhile, for the chocolate industry, the surging demand for cocoa/chocolate products exhibiting unique flavors, could be partly addressed by adopting PS as a tool for varietizing the flavor capacity of "bulk" cocoa through the expression or suppression of specific flavor precursors in the raw material on the farm level, which comes with almost no additional cost.

Quantitative and Functional Requirements for Bioluminescent Cancer Models.

BACKGROUND: Bioluminescent cancer models are widely used but detailed quantification of the luciferase signal and functional comparison with a non-transfected control cell line are generally lacking. In the present study, we provide quantitative and functional tests for luciferase-transfected cells. MATERIALS AND METHODS: We quantified the luciferase expression in BLM and HCT8/E11 transfected cancer cells, and examined the effect of long-term luciferin exposure. The present study also investigated functional differences between parental and transfected cancer cells. RESULTS: Our results showed that quantification of different single-cell-derived populations are superior with droplet digital polymerase chain reaction. Quantification of luciferase protein level and luciferase bioluminescent activity is only useful when there is a significant difference in copy number. Continuous exposure of cell cultures to luciferin leads to inhibitory effects on mitochondrial activity, cell growth and bioluminescence. These inhibitory effects correlate with luciferase copy number. Cell culture and mouse xenograft assays showed no significant functional differences between luciferase-transfected and parental cells. CONCLUSION: Luciferase-transfected cells should be validated by quantitative and functional assays before starting large-scale experiments.

DTREEv2, a computer-based support system for the risk assessment of genetically modified plants.

Risk assessment of genetically modified organisms (GMOs) remains a contentious area and a major factor influencing the adoption of agricultural biotech. Methodologically, in many countries, risk assessment is conducted by expert committees with little or no recourse to databases and expert systems that can facilitate the risk assessment process. In this paper we describe DTREEv2, a computer-based decision support system for the identification of hazards related to the introduction of GM-crops into the environment. DTREEv2 structures hazard identification and evaluation by means of an Event-Tree type of analysis. The system produces an output flagging identified hazards and potential risks. It is intended to be used for the preparation and evaluation of biosafety dossiers and, as such, its usefulness extends to researchers, risk assessors and regulators in government and industry.