Optimization of bitterness in chocolate through roasting with analysis of related changes in important bitter compounds

Chocolate is made from the fermented, dried, and roasted seeds of the Theobroma cacao tree, an important agricultural food crop which contains bioactive flavonoid polyphenols with beneficial health effects. Such effects include improvement of antioxidant status, positive impacts on cardiovascular health and endocrine system function, association with cancer prevention, LDL cholesterol reduction, and reduction of obesity and related conditions. However, products which have the highest levels of cacao flavonoids of all eating-chocolate, such as high-cacao-percentage dark chocolate, are known to be quite bitter, a taste modality that is not readily appreciated by humans. Though the complex causes of bitterness in cacao are still not completely understood, it has long been known that the methylxanthines theobromine and caffeine impart bitterness, as do certain flavan-3-ols, sometimes called catechins, which are a class of the aforementioned healthy bioactive polyphenolic flavonoids, also found in tea. Yet, what else is known of bitterness in cacao is sparse and even contradictory. Work on cacao bitterness has described the importance of cyclic dipeptides called 2,5-diketopiperazines (DKPs), while suggesting some form of interaction between theobromine and DKPs as well. Yet these earlier assertions have only been confirmed with mixed results by others, in part due to the incredible complexity of bitterness in roasted cacao, which has been said to require further sensory evaluation. More recent work on bitterness in cacao suggested for the first time that a DKP called cyclo(Pro-Val) is the most important bitter compound. However, even while seeming to confirm the importance of previously known important bitter compound classes, this research was based upon only a single cacao sample from a single origin of cacao, and with an undefined roasting treatment, even though previous work had noted that differences in DKP formation are dependent upon roast profile. Additionally, sensory work was based in part on recombinants of bitter compounds in aqueous solution, allowing for potentially biased estimation of the contribution of the different compounds to finished chocolate bitterness, since the varying kinetics of dissolution of the diverse bitter compounds from low-moisture, high-fat cacao matrix into saliva were not considered, nor were interactions with aroma compounds present in chocolate. Therefore, much was still to be learned about the variation in bitter-compound composition in cacao and related sensory characteristics, within and between different cacao origins and across different roast profiles. This fact, combined with a growing desire for healthy, functional versions of foods such as chocolate makes research into the impact of cacao roasting on consumer perceptions of bitterness and overall liking in chocolate, and the underlying chemical changes, all the more timely. This research project has resulted in findings covering a significant range of chocolate topics. First of all, a new efficient method for extraction and analysis of important bitter compounds in cacao and chocolate was developed. A custom response-surface methodology (RSM)-based design for the roasting treatments, with emphasis on I-optimality for minimizing prediction variance,was created. Chemical and sensory analysis of the roasted chocolate treatments were carried out, followed by in-depth data analysis and interpretation in the context of current chocolate science. Specifically, the aqueous 70% N,N-Dimethylformamide solvent system and HPLC method developed for fast and efficient extraction, followed by analysis, of important bitter compounds from three different chemical classes (i.e., methylxanthines, flavan-3-ols, and 2,5-diketopiperazines) simultaneously, functioned successfully, resulting in acceptable standard curves,% RSD values, and% recovery values. As for quantitative chemical findings, our work generally supports previous studies as regards changes in chemical concentrations during roasting. However, even with the large number of roasting treatments (i.e. 24, or 8 for each of 3 origins) across a reasonably large experimental region, we did not confirm the presence of concentrations of cyclo(Pro-Val) similar to that of previous research. As for sensory evaluation findings, we discovered that reduction of bitterness, sourness, and astringency are all correlated with increased liking in our chocolates. We also noted that consumers appear to have a preference for increased cocoa intensity. Roast profiles that minimize and maximize these characteristics respectively can vary by origin, but temperature and time combinations such as 20 minutes/171[degrees]C, 80 minutes/135[degrees]C, and 54 minutes/151[degrees]C were generally effective, whereas, raw and lightly roasted treatments (i.e., 0 minutes at 24[degrees]C, 11 minutes at 105[degrees]C, or 55 minutes at 64[degrees]C) were not, resulting in the lowest liking ratings. As with any complex food system, caveats do exist. Additions of sugar, salt, and other ingredients would likely introduce significant effects relevant to overall sensory characteristics and consumer liking, and intensity of various other aroma profiles not yet analyzed could do the same (e.g., floral, fruity, nutty). One additional sensory finding is that we can now say that perception of chocolate aroma is likely to play a large role in the perception of taste modalities (i.e., bitterness, sourness, sweetness), and astringency, as well as liking in chocolate. Finally, regarding the relationship of bitter chemical concentrations in the treatments, and consumer bitterness perception thereof, while the analysis is somewhat complicated by the stability of theobromine and caffeine during roasting, we can say that we have little evidence to suggest that theobromine concentration is strongly correlated to bitterness in chocolate. There is far more evidence that caffeine may play a role in the increase of bitterness in cacao, though the magnitude of its importance is not yet known, and to better understand the impact of both theobromine and caffeine, study of many more origins will be required. As for epicatechin and procyanidin B2, as already known, they are quite well correlated, and of all the chemicals we studied, they were, as a pair, the most correlated with changes in bitterness in our data across all treatments, including all three origins. Given that epicatechin has previously been shown to be a more important contributor to bitterness than higher molecular weight procyanidins (e.g., procyanidin B2), the overall importance of epicatechin could be the greatest of all the compounds that we studied. In contrast, catechin and cyclo(Pro-Val), do not appear to be particularly important for changes in bitterness. More specifically, we have found no evidence supporting the claim that the DKP cyclo(Pro-Val) is the most important bitter compound in cacao or chocolate. This does raise additional questions about the importance of diketopiperazines (DKPs) as a class as they relate to bitterness in chocolate.Includes bibliographical references (pages 353-374)

Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes

Background Staphylococcal toxicity and antibiotic resistance (STAAR) have been menacing public health. Although vancomycin-resistant Staphylococcus aureus (VRSA) is currently not as widespread as methicillin-resistant S. aureus (MRSA), genome evolution of MRSA into VRSA, including strains engineered within the same patient under anti-staphylococcal therapy, may build up to future public health concern. To further complicate diagnosis, infection control and anti-microbial chemotherapy, non-sterile sites such as the nares and the skin could contain both S. aureus and coagulase-negative staphylococci (CoNS), either of which could harbour mecA the gene driving staphylococcal methicillin-resistance and required for MRSA-VRSA evolution. Results A new heptaplex PCR assay has been developed which simultaneously detects seven markers for: i) eubacteria (16S rRNA), ii) Staphylococcus genus (tuf), iii) Staphylococcus aureus (spa), iv) CoNS (cns), v) Panton-Valentine leukocidin (pvl), vi) methicillin resistance (mecA), and vii) vancomycin resistance (vanA). Following successful validation using 255 reference bacterial strains, applicability to analyse clinical samples was evaluated by direct amplification in spiked blood cultures (n = 89) which returned 100 % specificity, negative and positive predictive values. The new assay has LoD of 1.0x103 CFU/mL for the 16S rRNA marker and 1.0x104 CFU/mL for six other markers and completes cycling in less than one hour. Conclusion The speed, sensitivity (100 %), NPV (100 %) and PPV (100 %) suggest the new heptaplex PCR assay could be easily integrated into a routine diagnostic microbiology workflow. Detection of the cns marker allows for unique identification of CoNS in mono-microbial and in poly-microbial samples containing mixtures of CoNS and S. aureus without recourse to the conventional elimination approach which is ambiguous. In addition to the SA-CoNS differential diagnostic essence of the new assay, inclusion of vanA primers will allow microbiology laboratories to stay ahead of the emerging MRSA-VRSA evolution. To the best of our knowledge, the new heptaplex PCR assay is the most multiplexed among similar PCR-based assays for simultaneous detection of STAAR

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase 1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age  6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score  652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc = 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N = 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

Exponential growth, high prevalence of SARS-CoV-2, and vaccine effectiveness associated with the Delta variant

SARS-CoV-2 infections were rising during early summer 2021 in many countries associated with the Delta variant. We assessed RT-PCR swab-positivity in the REal-time Assessment of Community Transmission-1 (REACT-1) study in England. We observed sustained exponential growth with average doubling time (June-July 2021) of 25 days driven by complete replacement of Alpha variant by Delta, and by high prevalence at younger less-vaccinated ages. Unvaccinated people were three times more likely than double-vaccinated people to test positive. However, after adjusting for age and other variables, vaccine effectiveness for double-vaccinated people was estimated at between ~50% and ~60% during this period in England. Increased social mixing in the presence of Delta had the potential to generate sustained growth in infections, even at high levels of vaccination

Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes

Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission

AbstractUnderstanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.</jats:p